Chapter 1: The Microscope - Introduction

Figure 1.1 Magnification vs. Resolution Since its invention, the microscope has been a valuable tool in the development of scientific theory. Magnifying lenses have been known for as long as recorded history, but it was not until the advent of the modern compound light microscope that the device was used in biology. A compound microscope is composed of two elements a primary magnifying lens and a secondary lens system, similar to a telescope. !ight is caused to pass through an ob"ect and is then focused by the primary and secondary lens. #f the beam of light is replaced by an electron beam, the microscope becomes a transmission electron microscope. #f light is bounced off of the ob"ect instead of passing through, the light microscope becomes a dissecting scope. #f electrons are bounced off of the ob"ect in a scanned pattern, the instrument becomes a scanning electron microscope. $he function of any microscope is to enhance resolution. $he microscope is used to create an enlarged view of an ob"ect such that we can observe details not otherwise possible with the human eye. %ecause of the enlargement, resolution is often confused with magnification, which refers to the si&e of an image. #n general, the greater the magnification, the greater the resolution, but this is not always true. $here are several practical limitations of lens design which can result in increased magnification without increased resolution. Figure 1.1 illustrates this point. #f an image of a cell is magnified from 1'( to )*(, the image gets larger, but not necessarily any clearer. $he image on the left is magnified with no increase in resolution. $he image on the right is magnified the same, but with increasing resolution. +ote that by the time the image is magnified 1'( ,from 1'( to 1''(-, the image on the left is completely unusable. $he image on

the right, however, presents more detailed information. .ithout resolution, no matter how much the image is magnified, the amount of observable detail is fi/ed, and regardless of how much you increase the si&e of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. $his property of the lens is fi/ed by the design and construction of the lens. $o change the resolution, a different lens is often the only answer. $he reason for a dichotomy between magnificatioin and resolution is the ability of the human eye to see two ob"ects. #t is necesarry that two ob"ects be about '.1 mm apart when held 1'0 from the face in order for us to detect them as two ob"ects. #f they are closer than '.1 mm, we will perceive them as a single ob"ect. #f two ob"ects are '.'1 mm apart, we can not detect them unless we magnify an image of them by 1'(. .hat has happened is that we have effectively altered our resolution ability from '.1 mm to '.'1 mm through the use of a magnifying lens. .e would say that our limit of resolution has changed from '.1 mm to '.'1 mm, or inversely, our resolving power ,resolution- has increased by a factor of 1'. 1nfortunately, a lens can magnify an image without increasing the resolution. Several artifacts can be inherent in the lens design which causes the ob"ects to become blurry at the edges. $hus, even though they can be made to appear '.1 mm apart, the edges are so blurry that we lose the ability to see them as two ob"ects. $hink of a standard eye chart2 you can see the increased si&e of a letter, but may be unable to tell what letter is pro"ected. Figure 1.1 illustrates what can be seen with increased magnification and resolution. #f we were to look only at the left side of the figure, we could get the impression that the cell is filled with a homogeneous fluid ,cytoplasm-. #f, however, we look at the right side of the figure, it becomes apparent that the cytoplasm is actually composed of smaller particulate components ,chloroplasts, ribosomes, membranes-. As we increased the resolution of our microscopes we changed our concepts from protoplasm ,the fluid of life- to cytoplasm ,the fluid of the cell outside of the nucleus- to a highly ordered machine full of individual organelles. #t is readily apparent that while microscope lenses are usually discussed in terms of their magnification, the most important value is their resolution. All microscopes will come with a lens that can magnify )' times the normal si&e, but only a 3uality lens will allow you to see more than you would with a good hand4held magnifying lens.

As mentioned, the value for resolution may be determined in one of two ways. #t can be measured as the smallest distance between two points, which allows us to see the points as distinct. .ith this measurement, resolution increases as the distance decreases44that is, there is an inverse correlation between the limit of resolution and what you actually resolve. Equation 1.1b '.51 / !imit of Resolution 6 44444444444 +.A. $o change this to a direct correlation, one need only use the reciprocal of the limit of resolution. Resolution is the recriprocal of the limit of resolution. For measures of resolution then, as the value increases, resolution increases. 7onse3uently, most microscopists today use resolution rather than limit of resolution to measure the 3uality of their lenses. Equation 1.1a +.A. Resolution 6 4444444444 '.51 ( $he resolution of a lens is a property of its physical properties and of the wavelength of light that is passed through the lens. $he physical properties are summed up in a value known as the numerical aperture while the wavelength is determined by the color of light. Equation 1.2 +.A. 6 ( $he numerical aperture of a lens is dependent upon two parameters, the angle of the incidence of light onto the lens, and the refractive inde/ of the glass of which the lens is composed. $he angle of incidence is also known as the cone angle and 189 of this value is designated by the symbol . :alf the cone angle is used to calculate the angle the light subtends relative to the light a/is. $he cone angle and thus can be altered by inclusion of a substage condenser. #f the condenser is

moveable, the cone angle can be varied the closer the substage condenser is to the ob"ect, the greater is the cone angle. $his is a relatively ine/pensive means of effecting the resolution of the microscope and thus nearly all microscopes are e3uipped with substage condensers.

Figure 1.9 7one Angle and +umerical Aperture $he refractive properties of a lens are summed up in a measurement known as the refractive inde/ ,R.#. or -. $he refractive inde/ is a function of the bending of light from air through glass and back again. #n a microscope, the glass of the lens is specially formulated to increase its refactive inde/. ;nce manufactured, however, this property can not be changed. $he media around the lens can be altered, however, by removing air from between the ob"ective and the slide, and replacing it with immersion oil. 1 <utting all of this to practical use, it is apparent that resolution can be increased in three ways. $he easiest method is to increase the angle of light incidence, by altering the position and8or design of the substage condenser. Second, the refractive inde/ can be ma/imi&ed by using specially manufactured lenses, and by controlling the medium through which the light travels, i.e. using immersion oil with lenses designed for this purpose. $he third method is to decrease the wavelength of light used. For practical purposes, the wavelength has a larger effect on resolution than either changes in the angle of incidence or the refractive inde/. For ma/imum resolution, all three properties must be optimi&ed. For routine bright field microscopy, it is more convenient to work in the visible light range, and the shortest wavelength of visible light is blue. $hus, even ine/pensive microscopes have incorporated a blue filter into their design, which is often referred to as a daylight filter. As a rule, the cheaper the microscope the thicker and darker this filter. 9 More e/pensive and higher 3uality lenses manipulate the light source to enhance the 3uality of the light and to correct for lens aberrations inherent in their design. Resolution can be enhanced by reducing the wavelength to the ultraviolet range and yet again by levels of magnitude to the wavelengths electrons have in motion. $he use of electrons as the light source gives rise to the electron microscope. 1= light can not be seen directly by the human eye

,it will in"ure the retina of the eye- nor can we see electron beams. $hus, these forms of microscopy rely on photography, or upon fluorescent screens. =isible light ranges in wavelength from the long red waves , 6 >5' nm- to the short blue8violet waves , 6 )'' nm-. 1ltraviolet waves can be as short as 9?' nm. $he wavelength of an electron beam depends upon its acceleration voltage, with the wavelength being given by <lanck@s law. For an electron of charge e, accelerated by a potential difference of =, is given by the formula2 Equation 1.3 h 6 44444444 ,9m =e$his is made simpler by the appro/imation2 Equation 1.4 6 ,1.*8=- nm For an electron microscope with )',''' volts accelerating voltage, the wavelength of the electron would be '.''5 nm ,,1.*8)','''- -. +ote that 1= light increases the resolution by a factor of 9 over visible light, while the electron microscope has the potential to increase it by a factor of 1' to 1' over visible light. Ma/imum resolution is not attainable, however, unless the lenses are corrected for problems of lens design. Modern microscope lenses are not single lenses, but highly comple/ collections of lenses assembled to enhance the refractive inde/ while minimi&ing chromatic and spherical distortions of the image. Aberrations 7hromatic and Spherical distortions ,0aberrations0- are inherent in the design of a lens. %ecause the lens is a sphere, it pro"ects an image that is spherical, while optical theory is based on images that are flat. Moreover, because different wavelengths of light are refracted differently, the

spherical image is even further distorted into multiple images, as each wavelength of light forms a separate image. A lens that is corrected to yield flat fields rather than curved is known as a plan lens, while one corrected for flat field and color aberrations is termed a plan achromat lens. #f the lens is corrected for chromatic aberrations for red and blue, while correcting spherically for green, the lens is an achromat lens. #ncreased resolution and increased cost of the microscope are primary factors in correction of these aberrations. ;ther than adding colored filters to create monochrome light, there is little or no alteration possible once the system has been built. $hus, we will continue to discuss only those parameters that can be controlled by the user. Angle of #ncidence

Figure 1.? ;ptical path through a light microscope Refer to Figure 1.? for the location of typical microscope components. .hile the angle can be altered, there is a theoretical limit to this angle which would still allow

light to pass into a lens. For any given lens, there should be an ideal or ma/imum position of the substage condenser which would present light to the lens at the appropriate angle, which in turn would allow a ma/imum light intensity, while maintaining as large as possible. <ractically, for

most student microscopes at anything above the lowest power ,9.*4)(-, this is usually in the uppermost position of condenser travel. Aood microscopes allow you to see the condenser diaphragm in the field of light and allow precise ad"ustment of the condenser to its ideal location. Most student microscopes do not allow this, although they will often allow movement of the condenser in a vertical direction, without the ability to ad"ust the alignment. Since iris diaphragms are ine/pensive, virtually all condensers are e3uipped with these. #ris diaphragms are

used to correct for spherical aberrations of the lenses and should be ad"usted for each ob"ective. $hey should not be used to control light intensity, unless resolution is unimportant to the user. Alignment <roper use of a microscope demands that the optics and light source be aligned on the optical a/is. All of the corrections for aberrations depend on proper alignment of the microscope components. $here are two general techni3ues used for proper alignment of the microscope. $he first, and perhaps best, is known as critical illumination. #n this process an image of the light source ,bulb filament- is pro"ected into the plane of the ob"ect, thus superimposing the light source onto the ob"ect. #t has a distinct disadvantage, however, in that it calls for a flat even light source, not really possible with a tungsten filament bulb. $he second alignment procedure is known as Boehler illumination after a pioneer in light optics, August Boehler. #n this procedure, an image of the field diaphragm is pro"ected onto the ob"ect plane. $his procedure re3uires a field condenser lens e3uipped with a moveable ,centerable- iris or diaphragm. Boehler illumination is the most commonly used alignment procedure. %right Field, Cark Field, <hase 7ontrast All microscopes actually allow visuali&ation of ob"ects through minute shifts in the wavelength phase as the light passes through the ob"ect. Further image forming can be had through the use of color, or through a complete negative image of the ob"ect. #f the normal phase shift is increased ,usually by 18) wavelength-, then the microscope becomes a phase contrast microscope. <hase contrast microscopes can be designed to have medium phase or dark phase renditions, by altering the degree of additional shift to the wavelength from 18) to 189 wavelengths, respectively. #f the beam of light is shifted in phase by a variable amount, the system becomes a differential interference contrast microscope. $he most commonly used system of interference microscopy is known as a +ormarski #nterference Microscope, named for the optical theoretician, Aeorge +omarski. ;nce used nearly e/clusively by parasitologists, this type of microscopy has increased in use because of the work currently done on the nematode C. elegans interference microscopes are superb for both observation and measuring thickness of embryos within specimens with little or no contrast. #f the light image is reversed, then the microscope becomes a dark field microscope. All standard bright field microscopes can be readily converted to dark field by inserting a round opa3ue disk beneath the condenser. Cark field microscopy was first utili&ed to e/amine trans4filterable

infectious agents, later to be termed viruses, and to determine that they were particulate in nature. Small ob"ects, even those below the limits of resolution, can be detected easily with dark field, as the ob"ect appears to emit light on a dark field. !ook at the sky for a comparison. #t is fairly easy to see stars in a dark sky, but impossible during the day. $he same is true for dark field vs bright field microscopy. Finally, if the normal light microscope is functionally turned upside down, the microscope becomes an inverted microscope. $his is particularly useful in tissue culture since it allows observation of cells through the bottom of a culture vessel, without opening the container, and without the air interface normally present between the ob"ective and the surface of the culture. %y adding phase contrast optics to the inverted microscope, it is possible to monitor tissue cultures directly, without the aid of stains or other enhancements. $he Dlectron Microscope $he transmission electron microscope ,$DM- is the workhorse of histology primarily because of its resolving power ,?41' E-, and its similarity to traditional light microscopy and histotechni3ue. $he scanning electron microscope ,SDM- is becoming increasingly popular with cell biologists because of its remarkable ability for 3uantifiable mapping of surface detail, along with improved resolution ,?'41'' E - and its ability to show ?C structure. $he transmission electron microscope is identical in concept to the modern binocular light microscope. #t is composed of a light source ,in this case an electron source-, a substage condenser to focus the electrons on the specimen, and an ob"ective and ocular lens system. #n the electron microscope, the ocular lens is replaced with a pro"ection lens, since it pro"ects an image onto a fluorescent screen or a photographic plate. Since the electrons do not pass through glass, they are focused by electro4magnetic fields. #nstead of rotating a nose4piece with different fi/ed lenses, the DM merely changes the current and voltage applied to the electromagnetic lenses. $he si&e of an electron microscope is dependent upon two factors. <rimary is the need for a good vacuum through which the electrons must pass ,it takes less than 1 cm of air to completely stop an electron beam-. <eripheral pumps and elaborate valves controls are needed to create the vacuum. A substantial electrical potential ,voltage- is also needed to accelerate the electrons out of the source. $he source is usually a tungsten filament, very much like a light bulb, but with )'4 1*' Billivolts of accelerating voltage applied to an anode to accelerate the electrons down the microscope column. ? Modern electronics have produced transformers which are reasonably

small but capable of generating 5',''' volts. $ransformers used to be room si&ed ,and a large room at that-. $he million4volt electron microscopes have transformers as large as buildings 4 in fact, the building housing such a microscope is for the most part a cover for the transformer. .hile electron microscopes work with high voltage, they use only milliamps of current through the electron gun. .ith the need for mechanical stability that is imposed by resolution in the Angstrom range, electron microscopes are usually formidable instruments. #deally they lie on their own shock mountings to isolate the instrument from building and ground vibrations. $he si&e and cost of an electron microscope is due primarily to the need for stability and maintenance of the vacuum. .ith advances in vacuum technology ,similar to the microchip revolution in electronics-, and perhaps with the ability to manufacture devices in deep space, the si&e ,and cost- of electron microscopes should decrease. Another characteristic of electron microscopes is that they are usually designed upside4down, similar to an inverted light microscope. $he electron source is on top, and the electrons travel down the tube, opposite to light rays traveling up a microscope tube. $his is merely a design feature that allows the operator and technicians ease of access to its various components. $he newer electron microscope is beginning to look like a desk with a $= monitor on it. 1ntil recently, the ma"or advantage of an electron microscope also has been its ma"or disadvantage. #n theory, the transmission electron microscope should be capable of giving a resolution of several angstroms. $his would give e/cellent molecular resolution of cell organelles. :owever, as the resolution increases, the field of view decreases and it becomes increasingly difficult to view the molecular detail within the cell. Dlectron microscopes designed to yield high resolution have to be compromised to view larger ob"ects. 7ell structures fall within the si&e range that was most problematic for viewing. For e/ample, if we wished to resolve the architecture of an entire eucaryotic chromosome, not "ust the chromosome, but the cell itself was too large to be seen effectively in an electron microscope. Fooming in on paired chromosomes was impossible. Modern electron microscope design allows for this &ooming, and the observation of whole tissues while retaining macromolecular resolution.) $he Scanning Dlectron Microscope

Figure 1.) 1seful emission from electron bombardment Figure 1.* Secondary electron and /4ray images from SDM $he scanning electron microscope works by bouncing electrons off of the surface and forming an image from the reflected electrons. Actually, the electrons reaching the specimen ,the 1 G electrons- are normally not used ,although they can form a transmitted image, similar to standard $DM-, but they incite a second group of electrons ,the 9 G electrons- to be given off from the very surface of the ob"ect. $hus, if a beam of primary electrons is scanned across an ob"ect in a raster pattern ,similar to a television scan-, the ob"ect will give off secondary electrons in the same scanned pattern. $hese electrons are gathered by a positively charged detector, which is scanned in synchrony with the emission beam scan. $hus, the name scanning electron microscope, with the image formed by the collection of secondary electrons. #t is possible to focus the primary electrons in e/actly the same manner as a $DM. Since the primary electrons can be focused independently of the secondary electrons, two images can be produced simultaneously. $hus, an image of a sectioned material can be superimposed on an image of its surface. $he instrument then becomes a S$DM, or Scanning4$ransmission Dlectron Microscope. #t has the same capabilities of a $DM, with the added benefits of an SDM. SDM allows a good deal of analytical data to be collected in addition to the formed image. As the primary electrons bombard the surface of an ob"ect, they interact with the atoms of the surface to yield even more particles and radiations other than secondary electrons. Among these radiations are Auger electrons, and characteristic (4rays. $he (4rays have uni3ue, discreet energy values, characteristic of the atomic structure of the atom from which they emanated. #f one collects these (4rays and analy&es their inherent energy, the process becomes Dnergy Cispersive (4ray Analysis. 7ombining the scan information from secondary and Auger electrons, together with the 3ualitative and 3uantitative (4ray information allows the complete molecular mapping of an ob"ect@s surface. Figure 1.) presents a diagram of the principal emissions from an electron bombardment, while Figure 1.*compares a secondary electron image to an image recreated from /4ray data.

Finally, the scanning microscope has one further advantage that is useful in cell structure analysis. As the electron beam scans the surface of an ob"ect, it can be designed to etch the surface. $hat is, it can be made to blow apart the outermost atomic layer. As with the emission of characteristic /4rays, the particles can be collected and analy&ed with each pass of the electron beam. $hus, the outer layer can be analy&ed on the first scan, and subse3uently lower layers analy&ed with each additional scan. Dlectrons are relatively small, and the etching can be enhanced by bombarding the surface with ions rather than electrons ,the e3uivalent of bombarding with bowling balls rather than %%@s-. $he resultant Secondary Dmmisions4#on Scanning data can finally be analy&ed and the three4 dimensional bit4mapped atomic image of an ob"ect reconstructed.

Exercise 1.1 - The Bright Field Microscope

!D=D! #

Figure 1.5 +ikon S47b

Figure 1.5a +ikon %inocular Microscope Refer to Figure 1.5 and Figure 1.5a for the names of the various components of a +ikon binocular microscope. Materials

%inocular Microscope * Microscope slide with letter e

Procedure
1. <ick up a microscope from the cabinet by placing one hand under the base and the other

on the arm of the microscope. Most microscope damage is due to careless transport. #t is important that you carry the microscope securely, with two hands, and in an upright position. Remember that you are handling H1,''' of precision instrumentation.
2. <lace the microscope in front of you, unwind the power cord and plug it in. $he

microscope is normally provided in its storage position, that is, with its eyepieces pointed back over the arm. $his takes less room in a cabinet, but is not the position for which it was designed to be used. #f your instructor approves, slightly loosen the screw holding the binocular head and rotate the entire binocular head 1I'G. 7arefully ,and gentlytighten the screw to prevent the head from falling off. Jou will notice that all parts of the microscope are now conveniently located for your use, with an uninterrupted view of the stage, and substage. $he focus controls are conveniently at arms4length.
3. +ote the magnification power and the numerical aperture of the lenses which are on your

microscope@s nose4piece. $hese values are stamped or painted onto the barrels of the ob"ectives. Record the magnification power and numerical aperture of each lens in the space provided below. Magnification (x ! "u#erical $perture ("$!

Dnter the numerical aperture of the condenser KKKKKKKKKK Dnter the magnification of the oculars and whether they are normal or widefield KKKKKKKKKK Jour ma/imum resolution will depend upon the highest effective numerical aperture of the system. $he highest value is normally given by the 1''(, or oil immersion lens.

#ndicate the numerical aperture of the 1''( lens #ndicate the numerical aperture of the condenser $he numerical aperture for an air interphase 6 $he numerical aperture for oil interphase 6

KKKKKKKKKK KKKKKKKKKK 1.' 1.? 4 1.*

$he ma/imum effective numerical aperture is the lowest of those listed. #t depends on the angle and thus on ma/imum positioning of the condenser. 1sing the lowest +A value

from above as the working numerical aperture, calculate the limit of resolution for your microscope, assuming violet light with a wavelength of )'' nm. From D3uation 1.1b, the limit of resolution 6 '.51 / value for your microscope is2 !imit of resolution 6 KKKKKKKKKKKKKKKKKKKK L
4. ;btain a prepared microscope slide with the letter e. <lace the slide on the stage and

8 +A, and therefore, the calculated

ensure that it is locked in place with the slide holder. Rotate the condenser focusing knob to move the condenser to its highest position of travel. Although there is an ideal location for the condenser, the correct position of the condenser will vary slightly for each ob"ective. 1nless directed otherwise, it will not be necessary to move the condenser during any of the intended uses in this course. #f, however, you wish to find the ideal location, focus the microscope on any portion of a slide, and then simply close down the condenser aperture and move the condenser until you have a sharply focused view of the condenser aperture ,usually with a slight blue ha&y edge-. #f you do this, you can then open the aperture until it "ust fills the field of view ,different for each ob"ective-. $his is the correct location and use of the condenser and aperture and the condenser should not be moved from this position. +ever use the condenser aperture for control of light intensity. 7ontrol of light intensity is the purpose of the variable rheostat ,dimmer switch, or voltage regulator- on the light source. 5
5. $urn on the microscope by rotating the dimmer switch and ad"ust the light intensity to a

comfortable level. %e sure that the condenser aperture is open if you have not set it as directed in the previous paragraph ,slide the condenser diaphragm lever back and forth to check-.

6. !ooking down into the microscope, ad"ust the eyepieces to your interpupillary distance

and diopter. $he +ikon microscope is e3uipped with a knob between the eye tube e/tensions for this ad"ustment. Many microscopes simply re3uire pushing the eye tubes together or apart directly. Move the eye tubes back or forth until you see one uniform field of view. $he first time you use the microscope, ad"ust the eyepieces for your personal comfort. +ote that modern microscopes have :B ,high eye point- eyepieces and conse3uently you need not remove eyeglasses if you are wearing them. Muite the contrary, they should be worn to prevent eyestrain while you constantly shift from looking through the microscope to reading the lab manual. %egin by focusing the microscope on any ob"ect within the field of view. >
o

Find a suitably contrasty location in the center of the field of view and close your left eye. 1sing the coarse and fine ad"ustments, focus until you obtain a sharp image with your right eye onlyN +ow close your right eye and ad"ust the focus of the left eyepiece by rotating the diopter ad"usting ring located on the left eyepiece. Co not read"ust the focus of the left eye with the coarse or fine ad"ustments of the microscope 4 use the ad"ustment ring on the eye tube.

All subse3uent uses of the same microscope will involve use of the coarse and fine focus ad"ustments, without reference to the procedures in step 9. $hat is, step 9 need only be performed once at the beginning of your lab. #t may, of course, be checked periodically if desired, and will need to be read"usted if someone else uses your microscope.
7. ;ptional2

Familiari&e yourself with the operation of any tension ad"ustment options or pre4set devices that may be attached to the microscope. I
o

7oarse Ad"ustment $ension2 $he coarse ad"ustment may be eased or tightened by the ad"usting ring. #f the rotation of the coarse focus knob is too loose, turn the ad"usting ring counterclockwise. $oo much tension may be ad"usted by turning clockwise. Avoid e/cessive rotation as it will place undo stress on the internal gears. Ad"ust the tension so that the stage will remain stationary after focusing but

can be moved with relative ease by turning the coarse ad"ustment knob. Some microscopes re3uire turning the two coarse ad"ustment knobs in opposite directions, while others re3uire the use of a screwdriver. %e sure to check with your instructor or the manufacturer@s directions before ad"usting this feature.
o

<reset Cevice2 ;n the +ikon S47b, the right4hand focus knob has a preset lever on its drum. .hen the lever is turned clockwise, it will lock the stage so that it can not be moved closer to the ob"ective. $hat is, the stage can be moved away from the ob"ective, but not closer. $he preset device is used to rapidly change slides, but has a distinct disadvantage for the neophyte, since it can be locked and the operator might proceed to force the focus. Forcing the focus will result in immediate and costly damage to the microscopeN

%nless other&ise instructed' do not use the preset de(ice) #f asked to use the device, refer to the specific directions from the manufacturer. O
8. Always begin focusing the microscope with the 1'( magnification. Dven if you are going

to use the 1''(, it is more efficient to begin with the 1'( and then move up to the power desired. $he ob"ective lenses are parfocal, which means that if one is focused, each of the others is appro/imately in focus when revolved into position. .ith the slide from Step ) in place, rotate the coarse focus control until the slide is as close to the 1'( ob"ective as possible. Move the stage manipulators until a portion of the slide is directly under the ob"ective and focus carefully on the ob"ect in view. After ad"usting the focus at 1'(, center the ob"ect to be viewed, and rotate the nosepiece to the ne/t highest magnification. 1se the fine focus control only once the )'( or 1''( ob"ectives are in place. Manipulate the fine focus to obtain the sharpest image. Curing use of the microscope, one hand should remain on the fine focus as constant read"ustment will be called for. 1se the other hand to manipulate stage movements. +ote that the microscope is typically designed so that one revolution of the fine focus knob raises or lowers the microscope stage '.9 mm. $his permits direct readings on the fine focus knob scale to '.''9 mm ,9 microns- and can be used to determine the thickness of materials being e/amined.

9. Return to the 1'( ob"ective and move the slide around until you locate the letter e in the

view. +ote the orientation of the letter e on your slide and in the field of view.
10. $o use the )'( ob"ective, center the ob"ect you wish to view ,the )'( will have a smaller

field of view- and rotate the ob"ective turret ,referred to as the nosepiece- to bring the )'( ob"ective into position. #s there any change in the orientation of the letter eP *o not rotate the turret in such a #anner as to +ring the 1,,- into position.

11. Craw the image of the letter e at 1'(. 12. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK 13. Q 14. Q 15. Q 16. Q 17. Q 18. Q 19. Q 20. Q 21. Q 22. Q 23. Q 24. Q 25. Q 26. Q 27. Q

Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of letter e ,1' ( magnification-

28. QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

Exercise 1.. - %se of /il I##ersion (1,,x!

!D=D! # Materials

Microscope e3uipped with 1''(, oil immersion lens #mmersion oil <repared slide of bacteria or suitably small ob"ects

Procedure
1. <lace the prepared slide on the microscope stage. 1sing the 1'( ob"ective, focus the

microscope on an appropriate field containing bacteria. 7enter the bacteria in the field of view, by manipulating the lateral movement knobs.
2. Rotate the nosepiece to the )'( ob"ective and refocus with the fine focus. Again, center

the ob"ect which you wish to e/amine.

3. Rotate the nosepiece so that an intermediate position between the )'( and 1''(

ob"ectives is obtained.
4. <lace a small drop of immersion oil 1' on the center of the viewing area of the slide. 5. 7ontinue to rotate the nosepiece so that the 1''( ob"ective is rotated into the oil.

*o not under an0 circu#stances place the 1,x o+2ecti(e in the oil 1se only the fine focus and refocus your specimen.
6. Cetermine the shapes of the bacteria and draw them in the place provided. 7. #mmediately after using the oil, remove any residual oil from the slide and from the front

of the 1''( ob"ective by gently rubbing with lens paper dipped in /ylol, toluene, or better, a /ylol substitute designed for this purpose. "earl0 all organic sol(ents' and especiall0 x0lol and toluene' are potentiall0 ha3ardous. The0 are fla##a+le and readil0 enter the +odd0 +0 direct a+sorption through the s4in or lungs. Consistent high exposure to thses sol(ents has +een lin4ed &ith li(er da#age and potential carcinogenesis. %se all sol(ents sparingl0 &hen needed and al&a0s in a &ell- (entilated area. ;nce oil is placed on the slide, the 1'( and )'( ob"ectives will no longer be useful until the oil is removed. $he oil will blur any image attempted with the lower magnifications. 1se oil only when necessary and after completing all work at the lower magnifications.

#n order to return to work at the lower magnifications, the slide must be completely cleaned of any residual oil and dried. For this reason, oil immersion is employed only when necessary, and only after thorough observations at the high dry magnification ,)'(-. 11 KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of bacterial cell types Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

Exercise 1.5 - Measure#ents: /cular and 6tage Micro#eters

!D=D! #

Figure 1.> Superimposed ocular and stage micrometers Materials

Microscope

;cular micrometer Stage micrometer Millimeter ruler <repared slide with letter e

Procedure
1. <lace a stage micrometer on the microscope stage, and using the lowest magnification

,)(-, focus on the grid of the stage micrometer.

2. Rotate the ocular micrometer by turning the appropriate eyepiece. Move the stage until

you superimpose the lines of the ocular micrometer upon those of the stage micrometer. .ith the lines of the two micrometers coinciding at one end of the field, count the spaces of each micrometer to a point at which the lines of the micrometers coincide again ,Figure 1.>-.
3. Since each division of the stage micrometer measures 1' micrometers, and since you

know how many ocular divisions are e3uivalent to one stage division, you can now calculate the number of micrometers in each space of the ocular scale.
4. Repeat for 1'( and )'(, and 1''(. Record your calculations below.

Microscope =alue for each ocular unit at )( =alue for each ocular unit at 1'( =alue for each ocular unit at )'8)*( =alue for each ocular unit at 1''(
5. 1sing the stage micrometer, determine the smallest length ,in microns- which can be

resolved with each ob"ective. $his is the #easured limit of resolution for each lens. 7ompare this value to the theoretical limit of resolution calculated on the basis of the numerical aperture of the lens and a wavelength of )*' nm ,blue light-.
6. 1sing the calculated values for your ocular micrometer, determine the dimensions of the

letter e found on your microscope slide, and add the dimensions to your drawing in D/ercise 1.1. 1se a millimeter ruler to measure the letter e directly and compare with the calculated values obtained through the microscope. "otes

$o measure an ob"ect seen in a microscope, an ocular micrometer serves as a scale or rule. $his is simply a disc of glass upon which e3ually spaced divisions are etched. $he rule may be divided into *' subdivisions, or more rarely 1'' subdivisions. $o use the ocular micrometer, calibrate it against a fi/ed and known ruler, the stage micrometer. Stage micrometers also come in varying lengths, but most are 9 mm long and subdivided into '.'1 mm ,1' micrometerlengths. Dach ob"ective will need to be calibrated independently. $o use, simply superimpose the ocular micrometer onto the stage micrometer and note the relationship of the length of the ocular to the stage micrometer ,Refer to Figure 1.>-. +ote that at different magnifications, the stage micrometer changes, but the ocular micrometer is fi/ed in dimension. #n reality, the stage micrometer is also fi/ed, and what is changing is the power of the magnification of the ob"ective.

Exercise 1.1 - Measuring *epth

!D=D! # Materials

Microscope <repared slide with three colored, crossed threads

Procedure
1. <lace a slide containing three colored and crossed threads on the microscope stage. 2. Cetermine the width ,diameter- of the threads using the procedures from D/ercise 1.?. 3. !ocate a spot where all three threads cross each other at the same point. 1se the fine

focus control to focus first on the lowermost thread, then the middle thread and finally, the uppermost thread. !ist the order of the threads from the top to the bottom, by indicating their color.
4. Focus on the top of the uppermost thread. +ote the scale markings on the fine focus knob

and record the calibrated reading directly from the fine focus control. 7arefully rotate the fine focus and stop when the microscope is "ust focused on the upper edge of the ne/t thread. Record the reading from the focus control below. $he difference between the two readings is the depth ,thickness- of the upper thread. Position Color *ia#eter *epth

$op Middle %ottom

Exercise 1.7 - Measuring $rea

!D=D! # Materials

Microscope S3uare ocular grid <repared slide of blood smear

Procedure
1. ;btain an ocular grid etched with a s3uare, and insert it into an ocular of the microscope

,or use a microsocope previously setup by the instructor-.

2. 7alibrate the ocular grid in a manner similar to that outlined in D/ercise 1.?, and

determine the area of each marked grid section. Craw the grid in the space below and add all pertinent dimensions. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

Q Q Q Q

Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ
3. <lace a prepared slide of a blood smear on the microscope and focus on the slide using

the )'( ob"ective. 7ount the number of cells within the four margins of the grid area. 7ount only cells which touch the top and left margins of the grid. Co not count any cell which touches the right or bottom margin of the grid. Record the number in the bo/ on the ne/t page.
4. Select four additional random fields of view with appro/imately the same density of cells

and count the number of cells per grid. Record the numbers in the space provided.
5. Average the results and, based on the known dimensions of your grid, calculate the

number of cells per mm for the blood smear. 19 Area of the grid ,)'(- KKKKKKKKKKKKKKKKKKKK Cell Count "u#+er of cells8##

Average number of cells per mm KKKKKKKKKKKKKKKKKKKK

Exercise 1.9 - Measuring :olu#e

!D=D! #

Figure 1.I #mproved +eubauer hemacytometer Materials

Microscope :emacytometer and coverslip Suspension of yeast 1?

Procedure
1. Make a serial dilution series of the yeast suspension, from 181' to 181'''. 2. ;btain a hemacytometer and place it on the desk before you. <lace a clean coverslip over

the center chamber. 1)

3. Starting with the 181' dilution, use a pasteur pipette to transfer a small ali3uot of the

dilution to the hemacytometer. <lace the tip of the pipette into the = shaped groove of the hemacytometer and allow the cell suspension to flow into the chamber of the hemacytometer by capillary action until the chamber is filled. Co not overfill the chamber.
4. Add a similar sample of diluted yeast to the opposite side of the chamber and allow the

cells to settle for about 1 minute before counting.

5. Refer to the diagram of the hemacytometer grid in Figure 1.I and note the following2

$he coverslip is '.1 mm above the grid, and the lines etched on the grid are at preset dimensions. $he four outer s3uares, marked 14), each cover a a volume of 1' $he inner s3uare, marked as *, also covers a volume of 1' ml.

ml, but is further subdivided ml.

into 9* smaller s3uares. $he volume over each of the 9* smaller s3uares is ).' ( 1'

Dach of the 9* smaller s3uares is further divided into 15 s3uares, which are the smallest gradations on the hemacytometer. $he volume over these smallest s3uares is .9* ( 1 ml.

Aiven these volumes, the number of cells in a sample can be determined by counting the number of cells in one or more of the s3uares. .hich s3uare to use depends on the si&e of the ob"ect to be counted. .hole cells would use the larger s3uares, counted with 1'( magnification. #solated mitochondria would be counted in the smallest s3uares with at least )'( magnification.
o

For the s3uares marked 14), the area of each is 1 mm , and the volume is .1 mm . Since .1 mm e3uals 1' ml, the number of cells8ml 6 Average R of cells per 1

mm times 1' times any sample dilution.

o

For the 9* smaller s3uares in the center of the grid marked *, each small s3uare is '.9 / '.9 mm , and the volume is thus '.'') mm . For small cells, or organelles, the particles8ml e3uals the Average R of particles per small s3uare times 9* ( 1' times any sample dilution.

Arids 14* are all 1 mm . Arids 14) are divided into 15 smaller s3uares ,'.9* mm on each side-, grid * is divided into 9* smaller s3uares ,'.9 mm on each side-. Arid * is further subdivided into 15 of the smallest s3uares found on the hemacytometer.

For the yeast suspension, count the number of cells in * of the intermediate, smaller s3uares of the hemacytometer. For statistical validity, the count should be between 1' and 1'' cells per s3uare. #f the count is higher, clean out the hemacytometer and begin again with step ?, but use the ne/t dilution in the series. Record the dilution used, and the five separate counts.
6. Average your counts, multiply by the dilution factor, and calculate the number of cells8ml

in the yeast suspension. Record this information in the space provided. 6;uare 1 9 ? ) Cell Count

* Area of each s3uare 6 KKKKKKKKKKKKKKK mm / '.1 mm depth 6 volume of each s3uare. =olume of each s3uare 6 KKKKKKKKKKKKKKK mm Average number of cells per mm 6 KKKKKKKKKKKKKKK +umber of cells per cm ,1''' / above- 6 KKKKKKKKKKKKKKK +ote2 +umber of cells per cm is also number per ml. +umber of cells per ml KKKKKKKKKK / dilution factor ,9''- 6 KKKKKKKKKK cells per ml of whole blood.

Exercise 1.< - Measure#ent of Cell /rganelles

!D=D! #

Figure 1.O <hoto of various cells showing organelles Materials

<repared Slides of the following2

o o o o o o

<aramecium Duglena Spirogyra ;nion Root $ip $rachea Mitochondria

Cicot !eaf /.s.

Microscope

Procedure
1. Dach of the slides presents a representative cell, with various organelles of particular

interest. Figure 1.O gives a sample of some of these cell types. ;bserve each slide, and make a drawing of each cell and its representative organelle,s-. Dach drawing should meet the following guidelines2
o

All drawings must be completed in pencil. A R? pencil is preferred since it will not smudge as readily as the standard R9. 7olored pencils are optional. #nk is unacceptable. Dach drawing must be a minimum of )0 / *0 in si&e. Smaller drawings will not demonstrate sufficient detail, while much larger drawings re3uire e/cessive time to fill in detail. Crawings should be completed during the lab period and where applicable, on any forms provided. All labels should be added and appropriate indications of si&e should be made. Si&e is best indicated by including a ruler bar at the bottom of the drawing, drawn to scale and with the dimensions added.

2. Measure the appropriate dimensions of all structures. !abel the drawings, and indicate the

magnification used to view each.

3. ;n each drawing, place a line ,bar- to indicate a length of 1' microns.

Exercise 1.= - %se of *ar4field Illu#ination

!D=D! ## Materials

Microscope with dark field stop Suspension of Amoeba proteus $ransfer pipette Slides, coverslips

Procedure Make a simple wet mount of the Amoeba by placing a drop of the culture on a slide and placing a cover slip on the slide. ;bserve and measure the Amoeba using normal bright field optics. <lace a dark field stop 1* in the filter holder below the microscope@s substage condenser and and continue to monitor the movements of the amoeba. +ote the differences in observable structure possible with dark field microscopy when compared to bright field. Craw and label the amoeba viewed in both ways. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of ameoba ,bright and dard field microscope views-.

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

Exercise 1.> - The Phase Contrast Microscope

!D=D! ## Materials

<hase contrast microscope $elescopic ocular for centering phase rings 7ulture of Amoeba proteus $ransfer pipettes, slides, coverslips <repared, prestained slide of Amoeba proteus

Procedure Dstablish Boehler illumination on the microscope. #f the instructor approves, center the phase annular ring and its corresponding phase plate.
o

<lace a slide on the stage of the microscope, move the condenser to its highest position and focus at 1'( magnification. ;pen the condenser diaphragm to its ma/imum setting, and close the field diaphragm completely 1sing the condenser movement control, move the condenser until a sharp image of the field diaphragm is observed. $o determine that the focus is indeed the field diaphragm, slightly open and close the field diaphragm to see if its movement can be detected in the field of view. .hen focused, there will be a slight blue ha&e on the edge of the diaphragm. ;pen the field diaphragm until it nearly fills the field, but can be still seen. 7enter the field diaphragm in the field of view using the centering screws on the substage condenser. ;pen the field diaphragm to completely fill the field of view. Remove one of the oculars from its tube and while peering down the tube, open the condenser diaphragm until it "ust fills the field of view at the bottom of the tube. Replace the ocular in the tube. 7ompleting steps a4d establishes Boehler illumination, where the field diaphragm is superimposed onto the ob"ect and centers the ma"or optical components of the microscope. $o check on the phase annulus and its corresponding phase plate, remove an ocular and replace it with a telescopic ocular designed to focus on the rear lens of

a phase ob"ective. Match the phase ob"ective with its corresponding setting on the phase condenser and visually verify that the phase annulus ,a clear ring- is perfectly matched to the phase plate ,a darker ring-. #f it is not, ask the instructor for assistance in centering the phase annulus. $his is most often accomplished by ad"usting a second set of centering screws attached to the phase condenser. Replace the normal ocular before using the phase contrast optics. Return the phase condenser setting to the normal bright field position. Make a simple wet mount of the amoeba and observe under bright field microscopy at 1'(. !ocate an active amoeba and center it in the field of view. Rotate the condenser phase ring to match the 1'( phase with the 1'( ob"ective. ;bserve the difference in the appearance of the amoeba between normal bright field and phase contrast. Craw the amoeba viewed under phase contrast. !abel organelles which are more clearly visible with phase contrast than with bright field microscopy. Return the phase control on the condenser to the normal bright field setting, switch to a higher magnification ,9'( or )'(- and observe the amoeba at the higher magnifications with and without phase enhancement. 7ompare the view of the amoeba under phase contrast, normal unstained bright field and darkfield ,D/ercise 1.I- with the view of the prestained commercial preparation of Amoeba proteus. !ist the organelles and8or structures which are more clearly demonstrated by each optical techni3ue. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

Q Q Q Q Q Q

Q Q Q Q Q Q Crawing of amoeba observed with phase contrast optics

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

Exercise 1.1, - The In(erted Phase Microscope

!D=D! ##

Figure 1.1' ;lympus 7B19 inverted microscope Materials

#nverted phase microscope Monolayer cultured cells or a suspension of proto&oa in a plastic disposable petri plate

Procedure
1. 1sing Figure 1.1' as a guide, familiari&e yourself with the components of the inverted

microscope. +ote that the operation of the microscope is the same as in D/ercise 1.1, the

only e/ception being that the components are inverted. $he phase condenser may be an annular ring or a phase slider as depicted in Figure 1.1'.
2. <lace either a culture flask containing a monolayer of cells or a plastic petri

plate 15 containing a suspension of proto&oa on the stage of the microscope.

3. ;bserve the culture with normal bright field and with phase contrast. +ote any

movements of the cells and over a period of time, note any changes in the nuclear material of the monolayer cells.
4. Craw and label your observations in the space provided.

KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of cells observed with inverted phase microscope

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

Exercise 1.11 - The Trans#ission Electron Microscope

!D=D! ###

Figure 1.11 Feiss DMO4S transmission electron microscope

Materials

$ransmission electron microscope facility

Procedure
1. =isit an electron microscope facility. 2. Familiari&e yourself with the basic operations of Fi/ation, embedding and sectioning of

materials for viewing in an electron microscope. #f permitted, identify the primary parts of the $DM, including is various controls and operation. Refer toFigure 1.11 for details of the Feiss DMO4S microscope.
3. 7omplete the following operations.
o o o o o o

#nsert and Remove a Specimen from the chamber. $urn on the high voltage, check current levels and turn on the beam. Find and center the beam. 1se magnification controls and condenser. 1se binocular viewing screen $ake an electron photomicrograph.

Exercise 1.1. - Co#parison of Electron Micrographs

!D=D! ### Procedure
1. 7ompare the transmission electron microscope image of the cell organelles in Figure

1.19 to the image of that organelle in the light microscope ,D/ercise 1.>-.

2. !ist the organelles observed with each type of microscope, indicate their si&e and

diagram a comparison of each organelle observed at the two levels.

Chapter 1: The Microscope - Endnotes

1.

For absolute ma/imum resolution, oil should be used between the condenser and the slide as well, although in practice this is rarely done. %lue has an additional advantage in that the eye perceives light blue as white . that is the reason for adding bluing to white laundry, to make it look brighter. green light, is nearly as good for resolution as blue and is more pleasing to the eyes. we can withstand longer and more intense observations with green light ,note the color of computer monitors and most automobile dash lights-. if a green filter is available, replace the blue filter with the green 44 be sure to remove the blue 44 and note any differences. Since the electrons are shot down the column, the electron source is known as the electron gun, and the electrons are drawn off of the tungsten wire by a low voltage gun cap, then accelerated by the high voltage anode. #t should be noted that for most routine cell biology, a resolution of about ?' E is all that is re3uired. Dlectron microscopes will give resolutions of around *4> E, and the top research microscopes are beginning to approach the theoretical limit of "ust above 1 E. as we approach this theoretical limit, however, we enter the can@t see the forest for the trees syndrome, or in more perspective, can@t see the forest for the chlorophyll molecules syndrome. $he cost of the instrument increases e/ponentially with increased resolution and e/tremely high resolution electron microscopes are used for research on electron optic theory, rather than for biology. $he one advantage of high voltage DM for biologists is the ability to view thicker specimens, even hydrated living cells. $he directions are given for the +ikon Model S47b, but will apply to most microscopes, provided allowances are made for variations in manufacturer design. D/ceptions to this occur when you are not attempting to use ma/imum resolution and are willing to trade resolution for some other purpose, such as ease of operation, or to compensate for the lack of a rheostat.

2.

3.

4.

5.

6.

7.

$he +ikon S47b is provided with coa/ial, coarse and fine focus knobs, both of which are located near the base. 7lockwise rotation of either of the focus knobs by the operator lowers the microscope stage. other microscopes may have two separate focus knobs, and may alter the position of the upper lens tubes and8or the ob"ectives rather than the stage. ;n the +ikon S47b models, the most confusing and potentially damaging part of the microscope is the tension ad"ustment device for the coarse ad"ustment, and the preset device. incorrect ad"ustment of these will result in certain damage to the focus ad"ustment of the microscope, necessitating complicated repair. For the +ikon microscopes e3uipped with a pre4set device, the lever should remain in its counter4clockwise position 44 be sure to check this position periodically during the use of the microscope. this device has proven to cause enough problems that many laboratories are resorting to removing the lever from the microscope, to prevent damage to the focusing gears caused by improper use.

8.

9.

10. $ype A immersion has a lower viscosity than $ype % and is much preferred. Some

laboratories will mi/ A and % to obtain intermediate viscosities. #n any case, the oil should be a non4drying and with low fluorescence.
11. #f the image observed with the )'( appears dull and with a milky halo to it, the first

thing to suspect is oil on either the front of the lens or the slide. the second thing to check is the presence of oils and8or make4up from the eye lids on the oculars.
12. $he average is statistically more valid, since there may be differing concentrations

within various areas of the slide. A blood smear will definitely have variations since it will be normally a gradient from one end of the slide to the other, due to the means of preparing the smear.
13. #f desired, whole blood may be substituted, diluted 129'' with saline prior to use. 14. $he coverslip used for the hemacytometer is especially thick, in order to prevent sagging

in the middle. A regular microscope coverslip should never be used as a substitute, as it will yield incorrect volumes, often varying with each count.
15. Most bright field microscopes come with or can be easily fitted with a dark field stop. #f

your microscope does not have the disk supplied, try taping an ordinary dime into the filter holder below the condenser, and moving the condenser to a position where light can move in a ring around the coin. For many microscopes, this is an economical method of

producing a dark field microscope. Alternatively, copy the image of a darkfield disk, si&e to fit your filter holder and cut it out of cardboard, construction paper or aluminum foil. <lace it or tape it in the filter holder, or directly onto the condenser. $he important feature is to keep the light moving in a cone around the periphery of the lens, and not through the center of the lens.
16. <lastic plates are preferred for this purpose, since the plastic has a more uniform

thickness than ordinary glass petri plates. Special glass plates are available with ground optically flat glass in the bottom, but these are both e/pensive and fragile.

Chapter .: ?istoche#istr0 - Introduction

A few cell types are thin enough to be viewed directly in a microscope ,algae, proto&oa, blood, tissue cultures-, but most tissues ,kidney, liver, brain- are too thick to allow light to be transmitted through them. $he tissues can be sliced into very thin sections provided they are first processed to prevent cell damage. $he processing involves a series of steps fi/ation, dehydration, embedment and subse3uent sectioning with amicrotome. $hese steps, e/plained in detail later in this chapter, are time consuming and often alter the cell structure in subtle ways. Fi/ing cells with formaldehyde, for e/ample, will preserve the general organelle structure of the cell, but may destroy en&ymes and antigens which are located in the cell. <athologists routinely e/amine tissues which have been fi/ed in formaldehyde and embedded in paraffin wa/ prior to sectioning. $he process re3uires a minimum of 9) hours, and usually more if automated e3uipment is not available. $his time delay can be crucial when a diagnosis of benign or malignant cancer is at stake. =aluable time can be saved by skipping the fi/ation and dehydration steps re3uired for paraffin embedding, and free&ing the tissue in a modified microtome, the cryostat. Sections can be prepared within minutes and diagnoses made while the patient remains on the operating table. Additionally, fro&en sections will more often retain their en&yme and antigen functions. $he use of fro&en sections can reduce the processing time, but it is not a panacea. Free&ing is not ade3uate for long term preservation of the tissues and the formation of ice crystals within the cells destroys subcellular detail. Fro&en sections are also thicker since ice does not section as thin as paraffin. $his results in poor microscopic resolution and poor images of what subcellular structures remain. #f time or en&yme function is critical fro&en sections are the preferred process. #f subcellular detail is important, other procedures must be used. Selection of the correct procedure depends on what the cell biologist is looking for and

to a point, becomes an art form. $he histologist must choose among hundreds of procedures to prepare tissues in a manner that is most appropriate to the task at hand. Fi/ation Since cellular decomposition begins immediately after the death of an organism, biologists must fi/ the cells to prevent alterations in their structure through decomposition. Routine fi/ation involves the chemical cross4linking of proteins ,to prevent en&yme action and digestion- and the removal of water to further denature the proteins of the cell. :eavy metals may also be used for their denaturing effect. A typical laboratory procedure involves the use of an aldehyde as the primary fi/ative. Alutaraldehyde is used for transmission electron microscopy ,$DM-, and formaldehyde is used for routine light microscopy. $he formaldehyde solution most often employed was originally formulated by %aker in 1O)). %aker@s Formalin Fi/ative contains2 calcium chloride cadmium chloride 1.' g 1.' g

formalin, concentrated 1'.' ml distilled water 1''.' ml

%locks of tissue ,liver, kidney, pancreas, etc.- of appro/imately 1 cm are rapidly removed from a freshly killed organism and placed in the fi/ative. $hey are allowed to remain in the fi/ative for a minimum of four hours but usually overnight. $he longer the blocks remain in the fi/ative, the deeper the fi/ative penetrates into the block and the more protein cross44linking occurs. $he fi/ative is therefore termed progressive. %locks may remain in this fi/ative indefinitely, although the tissues will become increasingly brittle with long e/posures and will be more difficult to section. .hile it is not recommended, sections have been cut from blocks left for years in formalin. Formalin has lately been implicated as a causative agent for strong allergy reactions ,contact dermatitis with prolonged e/posure- and may be a carcinogen 4444 it should be used with care and always in a well ventilated environment. Formalin is a ?OS solution of formaldehyde gas. $he fi/ative is generally used as a 1'S formalin or the e3uivalent )S formaldehyde solution. $he

key operative term here is gas 44 formaldehyde should be handled in a hood, if possible. As a gas, it is 3uite capable of fi/ing nasal passages, lungs and corneas. Cehydration Fi/atives, such as formaldehyde, have the potential to further react with any staining procedure which may be used later in the process. 7onse3uently, any remaining fi/ative is washed out by placing the blocks in running water overnight or by successive changes of water and8or a buffer. $here are myriad means of washing the tissues ,using temperature, p: and osmotically controlled buffers-, but usually simple washing in tap water is sufficient. #f the tissues are to be embedded in paraffin or plastic, all traces of water must be removed2 water and paraffin are immiscible. $he removal of water is dehydration. $he dehydration process is accomplished by passing the tissue through a series of increasing alcohol concentrations. $he blocks of tissue are transferred se3uentially to ?'S, *'S, >'S, I'S, O'S, O*S, and 1''S alcohols for about two hours each. $he blocks are then placed in a second 1''S ethanol solution to ensure that all water is removed. +ote that ethanol is hydroscopic and absorbs water vapor from the air. Absolute ethanol is only absolute if steps are taken to ensure that no water has been absorbed. #t is important to distinguish between dehydration and drying. $issues should +D=DR be allowed to air dry. Cehydration involves slow substitution of the water in the tissue with an organic solvent. For comparative purposes, consider the grape. A properly dehydrated grape would still look like a grape. A dried grape is a raisin. #t is virtually impossible to make a raisin look like a grape again, and it is e3ually impossible to make a cell look normal after you allow it to dry.

Dmbedding After dehydration, the tissues can be embedded in paraffin, nitrocellulose or various formulations of plastics. <araffin is the least e/pensive and therefore the most commonly used material. More recently, plastics have come into increased use, primarily because they allow thinner sections ,about 1.* microns compared to *44> microns for paraffin-. Paraffin For paraffin embedding, first clear the tissues. 7learing refers to the use of an intermediate fluid that is miscible with ethanol and paraffin, since these two compounds are immiscible. %en&ene,

chloroform, toluene or /ylol are the most commonly used clearing agents, although some histologists prefer mi/tures of various oils ,cedarwood oil, methyl salicylate, creosote, clove oil, amyl acetate or 7ellosolve-. Cio/ane is fre3uently used and has the advantage of short preparation times. #t has the distinct disadvantage of inducing liver and kidney damage to the user and should only be used with ade3uate ventilation and protection. %e wary of all organic solvents. Most are implicated as carcinogenic agents. :eed all precautions for the proper use of these compounds. $he most often used clearing agent is toluene. #t is used by moving the blocks into a *'2*' mi/ture of absolute ethanol2toluene for two hours. $he blocks are then placed into pure toluene and then into a mi/ture of toluene and paraffin ,also *'2*'-. $hey are then placed in an oven at *5 4 *IG 7 ,the melting temperature of paraffin-. $he blocks are transferred to pure paraffin in the oven for 1 hour and then into a second pot of melted paraffin for an additional 944? hours. Curing this time the tissue block is completely infiltrated with melted paraffin. Subse3uent to infiltration, the tissue is placed into an embedding mold and melted paraffin is poured into the mold to form a block. $he blocks are allowed to cool and are then ready for sectioning.

<lastic More recent developments in the formulation of plastic resins have begun to alter the way sections are embedded. For electron microscopy that re3uires ultrathin sections, paraffin is simply not suitable. <araffin and nitrocellulose are too soft to yield thin enough sections. #nstead, special formulations of hard plastics are used, and the basic process is similar to that for paraffin. $he alterations involve placing a dehydrated tissue sample of about 1 mm into a li3uid plastic which is then polymeri&ed to form a hard block. $he plastic block is trimmed and sectioned with an ultramicrotome to obtain sections of a few hundred Angstroms. $able 9.1 presents a comparison of paraffin embedding with the typical Dpon embedment for $DM. Softer plastics are also being used for routine light microscopy. $he average thickness of a paraffin4sectioned tissue is between > and 1' microns. ;ften this will consist of two cell layers and, conse3uently lack definition for cytoplasmic structures. .ith a plastic such as <olysciences

T%44) it is possible to section tissues in the 144? micron range with increased sharpness. $his is particularly helpful if photomicrographs are to be taken. .ith the decrease in section thickness, however, comes a loss of contrast, and thin sections ,1 micron- usually re3uire the use of a phase contrast microscope as well as special staining procedures. $he sharp image makes the effort worthwhile. $able Sample Si&e Fi/ative <ost4Fi/ation Cehydration 7learing Agent !ight 1 cm Formaldehyde +one Dlectron 1 mm Alutaradehyde ;smium $etro/ide

Araded Alcohol Alcohol or Acetone (ylol8$oluene <ropylene ;/ide =arious <lastics 5'4O' nm :eavy Metals

Dmbedding Material <faffin Section $hickness Stains *41'L 7olored dyes

$able 9.1 !ight and electron microscopy preparations. $hese soft plastics can be sectioned with a standard steel microtome blade and do not re3uire glass or diamond knives, as with the harder plastics used for DM work. Sectioning

Figure 9.1 A; microtome for paraffin sectioning Figure 9.1 presents a photograph of the A; standard microtome. $his device is found universally in cell biology laboratories and remains a fundamental instrument for histology.

$his rather simple device consists of a stationary knife holder8blade and a specimen holder which advances by pre4set intervals with each rotation of the flywheel mounted on the right hand side. #n operation, it is similar to the meat and cheese slicers found within delicatessans. A control knob ad"usts internal cams which advance the paraffin block with each stroke. #t is relatively easy to section paraffin at 1' microns but re3uires a lot of skill and practice to cut at * microns. Since each section comes off of the block serially, it is possible to align all of the sections on a microscope slide and produce a serial section from one end of a tissue to the other. .hile virtually anyone can cut a section within minutes of being introduced to the microtome, proper use of the microtome is an art form and re3uires practice and inventiveness. Many a cell biology research pro"ect has depended on the skills inherent in the use of this instrument. A microscope is nearly useless without a good thin, flat, and undistorted section from properly fi/ed, dehydrated and embedded tissue. The Ultramicrotome

Figure 9.9 Sorvall ultramicrome Figure 9.9 presents a view of an ultramicrotome. #n principle, it is the offspring of the standard microtome, in that it also is a mechanical device that involves a stationary knife ,glass or diamond- and a moving specimen. $he specimen, or block, is a plastic embedded tissue that advances in nanometers rather than microns. ;perationally, the only difference is that smaller samples are handled, which in turn re3uires a binocular dissecting microscope mounted over the blade. $he tissue sections are too thin to see their thickness with the naked eye, one usually estimates thickness by the color of the diffraction pattern on the section as it floats off the knife onto the surface of a water bath. $he sections are also too thin to be handled directly, and they are therefore transferred with wire loops, or picked off the water directly onto an DM grid.$his process re3uires a good light source mounted to cast the light at "ust the correct angle to see the color pattern.

Since the plastics are hard enough to break steel knives, freshly prepared glass knives or commercially available diamond knives are used. A glass knife costs several dollars each, while a good diamond knife will cost in e/cess of H?,'''. Dither can be permanently damaged with a single careless stroke by the operator. Ciamond knives are used in research laboratories by trained technicians because they have the advantage of a consistent knife edge ,unlike glass which varies with each use- and can last for years if treated properly. $hey can usually be resharpened several times before discarding. $o minimi&e vibrations ,which lead to uneven sections- ultramicrotomes are cast in heavy metal, are mounted on shock absorbent tables and, preferrably, kept in draft free environments of relatively constant temperature. $o further minimi&e vibrations, some manufacturers have replaced the block@s mechanical advance mechanism with a thermal bar, which advances the tissue by heating a metal rod. $his can be e/3uisitely precise and is the ultimate in thin sectioning. ;f course with this advancement comes increased cost and maintenance, and decreased ability to withstand rough treatment. $he mechanically advanced ultramicrotome remains as the workhorse of the cell biology laboratory. The Cryostat

Figure 9.? Modern cryostat .hether the sectioning is performed with a microtome or an ultramicrotome, one of the ma"or delays in preparing a tissue section is the time re3uired to dehydrate and embed the tissue. $his can be overcome by direct sectioning of a fro&en tissue. $ypically a piece of tissue can be 3uick fro&en to about 41* to 49' G7 ,for light microscopic work- and sectioned immediately in a device termed a cryostat. $he cryostat is merely a microtome mounted within a free&er bo/. Figure 9.? presents a modern cryostat. A piece of tissue is removed from an organism, placed onto a metal stub and covered with a viscous embedding compound to keep it in a form convenient for sectioning. $he stud and tissue are placed within the cryostat and 3uick fro&en.

$his method has the advantage of speed, maintenance of most en&yme and immunological functions ,fi/ation is unnecessary- and relative ease of handling ,far fewer steps to manipulate-. #t has the disadvantage that ice crystals formed during the free&ing process will distort the image of the cell ,bursting vacuoles and membranes for e/ample- and the blocks tend to free&e4dry or sublimate. $hus, the blocks must be used immediately and great care must be taken to guard against induced artifact from the free&ing process. .hen temperature4sensitive ,or lipid4soluble- molecules are to be studied, or where speed is of the essence ,such as pathological e/amination during an operation- this is the preferred method. Sectioning operation with the cryostat is similar to that of the microtome, with the e/ception that one handles single fro&en sections and thus all operations must be handled at reduced temperatures.

Exercise ..1 - 6electi(e 6taining: Prepared 6lides

!D=D! # Materials

<repared slides of2

o o o o o o o o

C+A by Feulgen reaction C+Aase treated Feulgen reaction R+A by methyl green 44 pyronin R+Aase treated methyl green 44 pyronin <AS or %est carmine positive granules Amylase treated <AS or %est carmine sections Sudan black or osmium stained lipid !ipase treated sudan black sections

Microscope

Procedure
1. ;btain one of the prepared slides which has not been treated with an en&yme. D/amine

the slide carefully and draw a representative field of view.

2. Select a corresponding en&yme treated section and draw a representative field. +ote any

structures present in the untreated slide which are absent in the en&yme treated slide.

Figure 9.)a Feulgen stained, <hase contrast, liver cells "otes Dach preparation in this e/ercise has a control ,untreated- and en&yme treated slide. Dach pair should be e/amined and compared. $he pairs represent a view of similar cells which have had something removed by en&ymatic digestion. Jou should determine which structures were removed by the en&yme treatment. Figure 9.)a presents an image of feulgen stained liver cells ,left- and a similar section ,right- stained identically, but pretreated with the en&yme C+Aase. $here are prominent nuclei present before C+Aase treatment, but they are absent after treatment with the en&yme. $hus, the conclusion that nuclei contain significant amounts of C+A.

Exercise ... - Basophilia

!D=D! # Materials

<araffin sections of 7arnoy fi/ed tissue ,liver, kidneyAraded series of ethanol, *', >', I', O* and 1''S ,v8vMayer hemato/ylin Scott solution Dosin $oluidine blue (ylol 7overslips and <ermount Microscope

Procedure
1. ;btain a slide from the instructor. <reviously cut paraffin sections have been mounted on

slides, and deparaffini&ed with /ylol. $hey will be brought to the lab in 7oplin "ars filled with /ylol. Jou will remove the slides from the /ylol and transfer them to the indicated solutions which follow2

*o not let the slides dr0 at an0 point in the procedure

2. $ransfer the slides se3uentially to 7oplin "ars containing ,leave in each solution for the

time indicated in parentheses-2

o o o o o o o o o o o o o o o o o o o o

Absolute Dthanol ,1 minuteO*S ethanol ,1 minuteI'S ethanol ,1 minute>'S ethanol ,1 minute*'S ethanol ,1 minuteCistilled water ,1 minuteMayer hemato/ylin ,? minutesRunning water ,? minutesScott solution ,? minutesDosin ,?' secondsCip 9( in O*S ethanol Cip 9( in Absolute ethanol Cip *( in tap water Stain in toluidine blue ,1' secondsCip rapidly in distilled water Cip once in O*S ethanol. Cip *( each in two changes of absolute ethanol <lace in Absolute ethanol ,9' seconds<lace in (ylol ,9 minutes<lace in Second (ylol ,9 minutes-

3. Remove the slides from /ylol, add a drop of permount and a cover slip. 4. D/amine the slides with the low ,1'(- and high dry ,)'(-ob"ectives of a light

microscope. Co not use the oil immersion lens 44 the permount will take several days to dry sufficiently and the slides should be treated carefully. #f you wish to keep your slides, ask your instructor how to dry them. Be particularl0 careful not to get per#ount on the lenses.

Figure 9.)b :yperbasophilic region of rat liver.

5. Craw each slide and compare to the commercial preparations of basophilic stained

material. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of cells stained with basophilic dyes "otes $his techni3ue was originally designed for the analysis of Mast 7ells to demonstrate the differential staining of cells. #t will result in cells with pink cytoplasm and blue4 4purple nuclei. $his is the standard color given to animal cells, "ust as plant cells are usually dyed green with red nuclei. ;f course, the choice of color is purely a matter of personal preference. Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

.ith the basophilic stain used here, collagen ,an e/tracellular material- will stain pink, and erythrocytes ,red blood cells- will stain red. $he procedure is a modification of the standard :UD stain ,hemato/ylin8eosin- used by most cytologists. $he procedures adds the use of toluidine blue, a metachromatic dye. $hat is, the dye has different colors depending upon the chemical nature of its polymeri&ation. $oluidine blue is a basophilic dye, implying that it stains acid compounds ,such as C+A and R+A and acid mucopolysaccharides-. Staining with basophilic dyes such as toluidine blue ,or methylene blue- is known as basophilia. #ntensive staining with these dyes is known as hyperbasophilia and is often used in clinical pathological diagnoses.

Exercise ..5 - Periodic $cid 6chiff (P$6! @eaction

!D=D! # Materials

7arnoy fi/ed paraffin sections 1 Dthanol series, *', >', O* and 1''S ,v8v1S Amylase in '.1 M sodium phosphate buffer, p: >.' '.1 M Sodium phosphate %uffer, p: >.' #ncubator at ?>G 7 <eriodic acid Schiff@s Reagent Sulfurous acid (ylol, <ermount and coverslips Microscope

Procedure
1. Ceparaffini&e the sections by passing them through two changes of /ylol ,9 minutes

each-.
2. Rehydrate the sections by passing them through 1''S, O*S, >'S and *'S alcohols ,1

minute each-.
3. Rinse gently in distilled water ,9 minutes4. <ass the slides se3uentilly through the following2

<lace a slide in 1S buffered amylase at ?>G 7 ,1 hour-. <lace another slide in buffer at ?>G 7 ,1 hour;/idi&e both slides in periodate ,* minutesRinse gently in fresh distilled water ,1 minuteStain in Schiff@s reagent ,1*44?' minutes%leach in sulfurous acid to remove any non44specific stain ,1*4?' minutesRinse gently in fresh distilled water ,* minutes>'S alcohol ,* minutesO*S alcohol ,* minutes1''S alcohol ,* minutes(ylol ,* minutes-

o o o o o o o o o

5. Mount in <ermount 6. 7ompare the amylase treated slides with those not so treated. Amylase removes amylose,

animal starch. $hose structures present without en&yme treatment, but missing after treatment are composed of amylose. Remember that the staining reagent is for aldehydes. Any compound with an aldehyde will turn pink with this reaction. 7areful controls must always be used and even greater care taken in the final analysis of stained structures. "otes $he ma"or carbohydrates which remain in cells affi/ed to slides are the rather insoluble glucose polymers2 glycogen, starch, and cellulose. All three polysaccharides give a positive periodic acid44Schiff ,<AS- reaction. $his staining procedure can be used on a variety of plant and animal material to determine the presence and intracellular locali&ation of these polysacchrides. <eriodic acid is an o/idi&ing agent which breaks the 74 47 bond between two ad"acent hydro/yl groups. $he 1,944diol group in glucose is converted into a dialdehyde and any carbonyl groups are converted to carbo/ilic acids. $he advantage of periodic acid lies in the specificity of its o/idation. #t forms aldehydes within the polysacchride molecule but it does not continue the o/idation of the polymers to low molecular weight water soluble forms. $hus, glycogen, starch, and cellulose contain dialdehyde groups after the periodate treatment and are left in the cell in

insoluble forms which can then be treated with Schiff@s aldehyde reagent to form a purple colored product.

Exercise ..1 - Meth0l Areen-P0ronin 6taining of *"$

!D=D! # Materials

7ryostat 7old subbed slides Acid alcohol n4butanol 1 + :7l Methyl green <yronin J in acetone #ncubator at ?>G 7 ;ven at 5'G 7 '.1S ,w8v- R+Aase in '.1 M sodium phosphate buffer, p: >.' '.1 M Sodium phosphate buffer, p: >.' (ylol, <ermount and coverslips Microscope

Procedure
1. Remove a sample of tissue from a freshly sacrificed animal and free&e immediately for

use in a cryostat.
2. 7ut >41' sections and immediately fi/ in acid44alcohol ,9 min.3. Rinse the fi/ed sections gently in distilled water ,9 min.- and pass the sections through

the following2
o o o o o

Cigest one slide in buffered '.9S R+ase at ?>G 7 ,1 hourCigest second slide in 1+ :7l at 5'G 7 ,1 hourCigest third slide in buffer only at ?>G 7 ,1 hourRinse all slides gently in fresh distilled water Stain in '.9S methyl green ,) min.-

o o o o

%lot e/cess methyl green from slides Rinse in n4butanol ,* minStain in '.5S pyronin J in acetone ,1 min.(ylol ,* min.-

4. Mount in <ermount 5. ;bserve and draw the tissues in the space provided. 7ompare the cells treated with

R+Aase to those treated with :7l and buffer only. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of methyl green 4 pyronin stained cells "otes $he methyl green4pyronin procedure uses the high net negative charge of nucleic acids. Methyl green is a cation which binds rather specifically to C+A and thus serves as a convenient means of staining nuclei in both f i/ed material and living cells. <yronin, a red dye, is fairly specific for R+A with some binding to protein. Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

7ontrol slides are important in interpreting the results of methyl green4pyronin staining, since the procedure is readily susceptible to artifact. ;ne or both of the nucleic acids should be removed either en&ymatically or by acid e/traction.

Exercise ..7 - The Feulgen Procedure for *"$

!D=D! # Materials

7arnoy fi/ed, paraffin sections of tissue 9 ;ven at 5'G 7 1 + :7l Schiff Reagent Sulphurous acid Araded ethanol series, *', >', O* and 1''S ,v8v(ylol, <ermount and coverslips Microscope

Procedure
1. Ceparaffini&e slides by passing through two changes of /ylol. Rehydrate the slides by

passing through 1''S, O*S, >'S and *'S alcohols.

2. Rinse gently in distilled water ,9 min.3. <ass the slides se3uentially through the following2
o o o o o o o o o o

D/tract one slide in 1+ :7l at 5'G 7 ,19 min.D/tract second slide in 1+ :7l at 5'G 7 ,1 hourD/tract third slide in water at 5'G 7 ,1 hourRinse gently in fresh distilled water ,1 min.Stain in Schiff@s reagent ,?' min.%leach with sulphurous acid ,1' min.Rinse gently in fresh distilled water ,1 min.>'S alcohol ,* min.1''S alcohol ,* min.(ylol ,* min.-

4. Mount in <ermount. 5. Craw representative cells from each slide in the bo/ provided. #f Figure 9.) has been

completed, compare the cells stained with methyl green4pyronin to those stained with the feulgen reaction. %e sure to include a comparison of any en&yme or acid hydroly&ed controls. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Crawing of feulgen stained cells "otes $he Feulgen procedure is undoubtedly the most widely used and most 3uantitative of all the cytochemical methods. Schiff@s aldehyde reagent is the stain used. $he great value of the Feulgen procedure is the manner in which cells are pretreated so that C+A furnishes the only available aldehyde to react with Schiff@s reagent. D/traction of cells with 1 + :7l at 5'G 7 for 19 minutes provides optimum hydrolysis of purines from C+A, e/posing the 7144aldehydes of deo/yribose. $he feulgen reaction is a semi443uantitative techni3ue. #f the only aldehydes remaining in the cell are those produced from the hydrolysis of C+A, then the techni3ue is 3uantitative for C+A. #t is possible to use an instrument known as a microdensitometer or microscpectrophotometer to actually measure the intensity of the pink feulgen reaction for a given organelle. 1sing this procedure, it was early determined that interphase cells were composed of two populations, those with 97 level of C+A and those with )7. $he nuclei looked identical, but one contained twice as much C+A within it. Q Q Q Q Q Q Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

$his gave rise to the division of the interphase period of the cell cycle to a A1, an S, and a A9 based on the synthesis of that e/tra C+A. For now, see if you can visually discern differences in the intensity of the feulgen stain reaction within the nuclei.

Exercise ..9 - $l4aline Phosphatase Bocali3ation

!D=D! ## Materials

Bidney sections ,preferrably fro&en sections7old acetone 7hloroform #ncubating Solution '.IS ,w8v- paranitrophenyl phosphate 9S ,w8v- sodium barbitol distilled water 9S ,w8v- calcium chloride *S ,w8v- magnesium sulfate 9.' ml 9.' ml 1.' ml ).' ml '.9 ml

#ncubator at ?>G 7 9S ,w8v- cobalt nitrate 9S ,w8v- ammonium sulfide Acetone solutions 1'', I' and *'S ,v8v- in water Mayer@s hemato/ylin Scott solution Dthanol series, *', >', O* and 1''S ,v8v(ylol, <ermount, slides, coverslips Microscope

Procedure
1. 1se a cryostat to cut 1'41*m fro&en sections of kidney at 49G 7. Mount on gelatin coated

slides and dip in cold acetone to fi/. Alternatively cut 1'm paraffin sections of acetone or formaldehyde fi/ed materials. Acetone fi/ation will result in better en&yme preservation

than formaldehyde. $issues should be fi/ed in chilled acetone for 9) hours prior to paraffin embedding. !ow temperature paraffin ,*5' melting temperature- works best and 1'm sections need to be deparaffini&ed with chloroform before hydration through a series of acetones to water.
2. #ncubate in incubating solution, ?>G 7 for ?' minutes. 3. .ash in distilled water. 4. <lace the slides in 9S cobalt nitrate for * minutes. 5. Rinse in distilled water. 6. <lace the slides in 9S ,w8v- yellow ammonium sulfide for 9 minutes. $his step should be

done in a ventilated hood to avoid the sulfide fumes.

7. .ash in distilled water.

$he slides can be viewed at this point with phase contrast or as a simple wet mount. #f there is not sufficient black sulfide precipitate, the slides can be returned to the ammonium sulfide for an additional 9 minutes. #f further contrast is needed, the cells can be counterstained with any basophilic stain. $he following uses Mayer
o o o

<lace in Mayer@s hemato/ylin ,? minutes<lace in running water ,? minutes<lace in Scott solution ,? minutes-

8. Cehydrate by passing through *', >', O* and 1''S ethanols, clear with /ylol and mount

a coverslip with permount.

9. D/amine all slides and compare the distribution of alkaline phosphatase on the surface of

the cells. Sites of alkaline phosphatase will be black, sharp and clear, nondiffuse. $he reaction precipitates calcium phosphate as the phosphate is released by the en&yme. $he calcium phosphate in turn is turned to black ,or brown- prepicipate in the presence of the cobalt nitrate and ammonium sulfide.

Exercise ..< - I##unofluorescent Bocali3ation of Tu+ulin

!D=D! ## Materials

7overslip cultures of an appropriate monolayer cell line

<hosphate buffered saline ,<%SAcetone8Methanol ,absolute- in a *'2*' volume mi/ture Rabbit anti4tubulin ,or other primary antibody to tubulinF#$74labeled goat anti4rabbit ,or secondary antibody to match the primaryFluorescence Mounting Media Fluorescent Microscope e3uipped with )O' nm e/citation filter and *1* nmbarrier filter Bodachrome film or e3uivalent color slide film ,Bodak $ri4( or #lford :<)'' may be substituted for black and white photography-

Procedure
1. Set up a coverslip culture of an appropriate cell line 9) hours prior to the lab. $his is best

accomplished by dry sterili&ation of R1 coverslips which are subse3uently placed in plastic tissue culture plates. 7ells are placed on the coverslips with sufficient media to cover and allowed to grow for 9) hours. $here should be sufficient cells to view comfortably, but they should not be crowded on the slide.
2. Remove the coverslip from the culture plate and dip several times in a beaker of

phosphate buffered saline ,<%S- to rinse off the culture media. Crain, but do not allow to dry.
3. #mmediately immerse in a *'2*' mi/ture of acetone8methanol at room temperature.

Allow the coverslips to remain in the acetone8methanol for 9 minutes.

4. Remove the coverslips from the acetone mi/ture and rinse 9( with <%S. 5. <repare a 18)' anti4tubulin dilution using <%S. <bS alone may be used or better, augment

the <%S with ?S ,w8v- %ovine Serum Albumin ,%SA-. #t may be necessary to check the appropriate antibody dilution. #f so, make 181', 181'', 181,''' and 181',''' dilutions to establish the correct titer. .orking dilutions also may vary with the manufacturer 4 check the literature that accompanies your primary antibody.
6. <lace the coverslip in a petri plate containing filter paper moistened with <%S. Make sure

the cells are pointed up when placed in the petri plateN Flood the coverslip with *' ml of 18)' dilution of the primary antibody ,or as determined in step *-.
7. #ncubate at room temperature for 14) hours. $he incubation may be left overnight if

necessary.

8. .ash ?( with <%S. <lace coverslips in a new petri plate containing <%S moistened filter

paper.
9. Apply *' ml of F#$74labeled second antibody. A 181'' dilution usually is satisfactory.

Jou may need to determine the appropriate dilution based on manufacturer directions or through trial and error dilutions in the range of 181' to 18?''.
10. #ncubate for ?' minutes at room temperature. 11. .ash ?( with <%S. 12. <lace a drop of glycerin or appropriate commercial fluorescent mounting media on a slide

and place the coverslip onto the slide with the cells facing down into the glycerin.
13. ;bserve immediately with a fluorescent microscope ad"usted for fluorescein ,)O' nm

e/citation and *1* barrier filter-. $he slides are best photographed using Bodak Dktachrome or e3uivalent with an ASA or 9''4)''. An e/posure of 149 seconds is usually sufficient, although for low light, ?' seconds may be re3uired. #t is best to make a test e/posure roll if a photometer is not available.

Figure 9.> Microtubules observed via fluorescent labeling "otes: $he procedure works for most primary antibodies merely by replacing the anti4tubulin with another appropriate antibody ,anti4actin, anti4laminin, etc.-. Tust be sure to keep the secondary antibody appropriate to the host for the primary.

Exercise ..= - $utoradiograph0

!D=D! ### Materials

:4$hymidine, specific activity of 9.' curie8millimole ;nion sets, "ars and toothpicks Alcohol4acetic acid fi/ative

Materials for feulgen reaction ,D/ercise 9.)<araffin embedding, sectioning e3uipment Bodak +uclear $rack Dmulsion ,or e3uivalentCarkroom .ater bath at )9G 7 Bodak C1O Ceveloper Bodak Fi/er (ylol, <ermount and coverslips Microscope

Procedure
1. 7arefully read and follow all precautions for the safe handling of radioactive materials

,Appendi/ :-.
2. <lace toothpicks into the sides of an onion and set the onion into the top of a small "ar or

beaker. Fill the "ar with water and allow the onion to begin root development ,?4 > days-. Mild aeration of the water will assist in the growth of the roots.
3. Set up another beaker, containing a 1' L 8ml solution of :4thymidine such that there are

appro/imately 1' Lc8ml, transfer the onion and its growing roots to the "ar containing the radioactive thymidine.
4. Allow the roots to stay in the radioactive solution for 1 hour at room temperature. 5. Remove the onion and hold the bulb over a beaker containing water. Rinse the onion

roots by dipping several times in the beaker. $ransfer the bulb to yet another beaker of aerated water and allow to remain in this beaker for four hours.
6. 7ut off the roots tips and fi/ in alcohol4acetic acid ,?21- overnight. 7hange the fi/ative

after the first four hours.

7. .ash the roots in water for several minutes and place in 1+ :7l at 5'G 7 for 19 minutes. 8. Stain the root tips with the Feulgen Reaction as given in Steps ?.b4?.e of D/ercise 9.*. 9. Cehydrate the root tips, embed in paraffin and section at 1'41* microns. Mount on

microscope slides.

10. Ceparaffini&e in /ylol and rehydrate sections by passing first through a series of alcohols

and finally in two changes of water.

11. #n a dark room ? melt some li3uid autoradiographic emulsion at a water bath at )9G 7. 12. <lace two slides back4to4back and dip slowly into the melted emulsion. Remove, allow to

drain and place in an appropriate light4proof container and allow them to dry in a vertical position.
13. .hen dry, place the slides into an opa3ue slide bo/ containing drierite. .rap in

aluminum foil and place in a refrigerator.

14. $he slides must stay in the refrigerator until a proper e/posure has been made. $his can

vary from * days to over two weeks. After one week, a trial slide should be developed using Steps 1* and 15 below. D/amine the slide at 1'( with a bright field microscope and look for the presence of black silver grains located over the cells. A correct e/posure is determined by the appearance of silver grains over the cells, but few or no silver grains located in areas without cells. #f the appearance of the trial slide is correct, then the remainder should be processed immediately. Repeat an additional trial slide in 94? days, and repeat every three days thereafter until an ade3uate e/posure is obtained.
15. Cevelop the autoradiogram emulsion in the darkroom as follows2
o o o o o o o

Cevelop in Bodak C41O Ceveloper at 9'G 7 for ? minutes. .ash for 1' seconds in distilled water. Fi/ with Bodak Fi/er for ? minutes. .ash in running water for 1* minutes. Cehydrate by placing in O*S alcohol for ? minutes. <lace in 1''S alcohol for ? minutes. 7lear in two changes of /ylol for ? minutes each.

16. Mount coverslip with <ermount. 17. Craw and label the autoradiograms in the space provided on the following page.

7alculate the percent of cells actively undergoing C+A synthesis during the time of e/posure to radioactive thymidine. +ote that the tissue and the silver grains are in different planes of focus and you will need to constantly switch focus from one plane to the other.

KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Crawing of thymidine incorporation "otes $his procedure involves the incorporation of a radioactive substance into a cell, and subse3uent detection of that material through the use of a photographic emulsion. $he primary source used for cell biology is an organic molecule containing tritium, the radioactive form of hydrogen. Radioactive carbon, phosphorous and iodine are occasionally utili&ed, but tritium has inherently more resolution than any of the others. $ritiated thymidine , :4thymidine- is often used, for e/ample, to study the synthesis and location of C+A. $hymidine is a soluble base which is specific to C+A. #t is incorporated into the macromolecular structure of C+A during synthesis and replication of the chromosomes. 1pon fi/ing the cells for standard histological e/amination, the C+A molecules ,with their incorporated, radioactive, thymidine- are precipitated or cross4linked as permanent parts of the cell. 1n4incorporated thymidine is removed from the cell, as it remains soluble and is disposed of in the tissue washing procedures. Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

.hen the tissues are sectioned and applied to a glass slide, they will contain radioactive nuclei, but only those nuclei that were in the S phase of division during the e/posure of the cells to :4 thymidine. Radioactive sources can not be detected directly, but if a photographic emulsion is applied directly over the section, it will become e/posed by the radioactive source. .hen the photographic emulsion is subse3uently processed ,i.e. developed-, the e/posed portions of the emulsion will contain reduced silver grains in direct proportion to the amount of radiation being given off beneath it ,in the nuclei of our e/ample-. #f the e/posed, developed slides are now e/amined with a microscope, there will be two layers of interest. Focusing the microscope on the tissue itself will give a view of the tissue and cell architecture. #f the focus of the microscope is moved upward, however, the cells will go out of focus, e/actly as the photographic emulsion comes into focus. .ithin the emulsion will be areas of reduced silver grains and clear areas containing no silver grains. %y alternately focusing on the tissue and the emulsion, those nuclei that are radioactive can be readily identified. #f desired, the number of silver grains could be counted to give a 3uantitative measure of :4 thymidine incorporation ,and thus C+A synthesis-, although this is a rather comple/ procedure to control, with any significant accuracy. Chapter .: ?istoche#istr0 - Endnotes

1.

Sections may be cut directly from fro&en blocks, if a cryostat is available. $he sections should be fi/ed for 9 minutes in acid4alcohol, rehydrated and the procedure then begun with the amylase treatment ,Step )-. Fro&en sections fi/ed in acid4alcohol may be substituted. %egin processing with distilled water wash ,Step 9-. A Series 9 .ratten Filter with 1* watt bulb, which is at least * feet from the work area may be used, but reference should be made to the emulsion manufacturer@s directions for safelight.

2.

3.

Chapter 5: Cell Fractionation - Introduction

All of the procedures given in 7hapter ;ne have in common the use of a microscope. $he basic principle for all microscopes is that the cell is composed of smaller physical units, the organelles. Cefinition of the organelles is possible with microscopy, but the function of individual organelles

is often beyond the ability of observations through a microscope. .e are able to increase our chemical knowledge of organelle function by isolating organelles into reasonably pure fractions. A host of fractionation procedures are employed by cell biologists. Dach organelle has characteristics ,si&e, shape and density for e/ample- which make it different from other organelles within the same cell. #f the cell is broken open in a gentle manner, each of its organelles can be subse3uently isolated. $he process of breaking open cells is homogeni&ation and the subse3uent isolation of organelles is fractionation. #solating the organelles re3uires the use of physical chemistry techni3ues, and those techni3ues can range from the use of simple sieves, gravity sedimentation or differential precipitation, to ultracentrifugation of fluorescent labeled organelles in computer generated density gradients. omogeni!ation Figure ?.1 Cevices used for homogeni&ation ;ften, the first step in the preparation of isolated organelles is to obtain a 0pure0 sample for further analysis. 7ells which are not attached to others ,such as blood or suspension tissue cultures- can be separated if they have distinct shapes, densities or characteristics which can be marked ,such as charge, antigen or en&yme presence-. 7ells which are part of a more solid tissue ,such as liver or kidney- will first need to be separated from all connections with other cells. #n some cases this can be performed by simply chelating the environment ,removing 7a Mg -, but in most instances the cells will need to be en&ymatically or mechanically and8or

disaggregated. $his often results in subtle changes to the cells, and at a minimum will disrupt such cell4cell communications as CDSM;S;MDS and $#A:$ T1+7$#;+S. :omogeni&ation techni3ues can be divided into those brought about by osmotic alteration of the media which cells are found in, or those which re3uire physical force to disrupt cell structure. $he physical means encompass use of mortars and pestles, blenders, compression and8or e/pansion, or ultrasonification. "smotic alterations Many organelles are easier to separate if the cells are slightly swollen. $he inbibition of water into a cell will cause osmotic swelling of the cell and8or organelle, which can often assist in the rupture of the cell and subse3uent organelle separation. $he use of a hypo4osmotic buffer can be

very beneficial, for e/ample, in the isolation of mitochondria and in the isolation of mitotic chromosomes. #ortars$ Pestles <erhaps the most common procedures use $en %roeck or Counce homogeni&ers, both of which are glass mortar and pestle arrangements with manufactured, controlled bore si&es. $he addition of a motor driven teflon pestle creates the <otter4Dlvi"em homogeni&er. 1ltrasonification is a useful ad"unct to this procedure, but is often sufficient by itself. $o obtain pure organelles, the cells must be ruptured, so that the cell membrane is broken, but the organelle to be studied is not. $he process of rupturing a cell is known as homogeni&ation of the cell. #t also varies from simple mortar8pestle grinding ,with the aid of sand or glass beads- for many plant materials, to repeated high velocity compression and e/pansion in what is known as a 0French <ress.0 $he French <ress is very powerful and can disrupt bacteria and viral particles as well. #t is favored for use when molecular dissociation is re3uired, such as in the separation of C+A from the nematode worm C. elegans. ;ften, cell rupture is aided by rapid free&ing ,in li3uid nitrogen- and subse3uent application of mechanical forces. .ith all forms of homogeni&ation, the shear force must be carefully controlled. $oo little and the organelles will not be separated, too much and even the molecules can be broken. %len&ers For molecular separations, mechanical blenders are often used, varying in sophistication from household blenders to high speed blenders with specially designed blades and chambers ,e.g. a =irtis $issue :omogeni&er-. $he mechanical procedures are augmented by various organic solvents ,for phase separations- and8or detergents to assist the denaturation and separation of molecules ,e.g. C+A from histones-. .hen specific molecules are sought, care must be taken to inhibit powerful degradation en&ymes ,such as R+ase when e/tracting R+A-. $his can be accomplished by sub"ecting the specimen to cold temperature, or by adding specific organic inhibitors ,Ciethylpyrocarbonate for R+ase-, or both. Compression'E(pansion For cellular material which is difficult to shear by the above mentioned techni3ues ,plant cells and bacteria-, a device known as a 0French <ress0 is ocassionally used. $his device forces a slurry of the cells through an orifice ,opening- at very high pressures. $he rapid e/pansion of the pressure from within literally 0blows0 the cells apart. .hile this techni3ue is not often re3uired,

it is the only way to break open some materials. $he units have capacities from 1 to )' ml and can reach pressures of 9','''4)',''' pounds per s3uare inch ,psi-. Ultrasonification 1ltrasonicators have been used with increasing popularity to separate organelles from cells, particularly from tissue culture cells. !ight use of an ultrasonic wave can readily remove cells from a tissue culture substrate ,such as the culture flask-. #t can also be ad"usted to merely separate cells, or to break open the plasma membrane and leave the internal organelles intact. Figure ?.1 presents a few of the various devices used for homogeni&ation. Fractionation

Figure ?.9 <hysical <roperties of %iological Materials )ra*ity +e&imentation ;nce the cells have been homogeni&ed, the various components must be separated. For some materials ,whole blood, cells in suspension-, this can be accomplished by the simple use of gravity sedimentation. #n this procedure, the samples are allowed to sit, and separation occurs due to the natural differences in si&e and shape ,density- of the cells. Red blood cells are denser than white cells, and thus whole blood separates into an R%74rich bottom layer, an intermediate 0buffy coat0 layer of .%7@s and an upper plasma portion of settled blood samples ,an anti4 coagulant is added to prevent coagulation, which would interfer with the separation-. Centrifugation .ithout 3uestion, however, the most widely used techni3ue for fractionating cellular components is the use of centrifugal force. <rocedures employing low speed instruments with greater volume capacity and refrigeration are known as 0preparative0 techni3ues. Analytical procedures, on the other hand, usually call for high speed with a corresponding lower volume capacity. A centrifuge working at speeds in e/cess of 9',''' R<M is an ultracentrifuge. ;rganelles may be separated in a centrifuge according to a number of basic procedures. $hey can be part of a moving boundary, a moving &one, a classical sedimentation e3uilibrium, a preformed

gradient isodensity, an e3uilibrium isodensity or separated at an interface. $hese are briefly diagrammed in Figure ?.9. <hysical <roperties of %iological Materials

Figure ?.? Methods for centrifugal separations %efore undertaking the centrifugal separation of biological particles, let@s discuss the particle behavior in a centrifugal force. <articles in suspension can be separated by either sedimentation velocity, or bysedimentation e3uilibrium. Sedimentation velocity is also known as &one centrifugation and has the advantage of low speed centrifugation and short times, but yields incomplete separations. Sedimentation e3uilibrium is also known as isopycnic or density e3uilibration and re3uires specimens to be sub"ect to high speeds for prolonged periods of time. #t has the advantage of separating particles completely. +e&imentation ,elocity <articles in solution will accelerate and attain a terminal velocity when sub"ected to a centrifugal force. $his velocity is determined in part by the si&e, weight, density and shape of the particle, as well as the viscosity of the medium through which it must travel, and, of course, the centrifugal force generated. $he terminal velocity is referred to as the sedimentation velocity of the particle and can bew used to measure the si&e, weight or density of the particle. +e&imentation Coefficient $he sedimentation velocity ,as terminal velocity- is measurable. $he terminal velocity is dependent upon the relative centrifugal force ,R7F- applied to the particle and is related to a mathematical factor, thesedimentation coefficient or sedimentation constant. $his coefficient is given in Svedberg ,S- units, so named for the Swedish pioneer of centrifugation theory and operation, $. Svedberg. $he S units are measured as fractions of time, specifically 1' sec. $he

sedimentation coefficient is determined by dividing the terminal velocity by the centrifugal force

field strength. $he relationship of sedimentation coefficient to the diameter of a particle is visually presented in Figure ?.9. -iffusion Coefficient .hile a particle is reaching its terminal velocity, it is also effected by diffusion, usually in a direction opposite to its movement under force. $his is a movement by %rownian motion and can be mathematically stated as a constant, the diffusion coefficient ,C-, which is the spread of the molecule divided by time. #t is e/pressed in units, Ficks, which are 1' cm 8sec. As noted

in Figure ?.9, there is a direct relationship between the diffusion coefficient of a particle and its diameter. %oth, in turn, are related to the sedimentation coefficient of the particle. %oth the sedimentation coefficient and the diffusion coefficient are corrected mathematically to e/press their values at 9'G 7, with water as the medium through which they move. $hey are almost never measured in water nor at this temperature, but formulas e/ist for the conversion to standard conditions. .hat is important is that the S value can be measured and will give an important clue as to the physical structure and si&e of the particle. #n practice, the S value is reasonably easy to determine. $he sedimentation coefficient is given by the formula2 S 6 18 r V dr8dt ,D3uation ?.1where 6 angular velocity of the rotor in radians8sec calculated as '.1')>9 / R<M r 6 the distance between the particle and the center of rotation ,mmdr8dt 6 the rate of movement of the particle ,cm8sec$he value of the sedimentation coefficient can be determined by timing the movement ,velocity and distance- of a particle in a medium of known viscosity. $he simplest means to do this is to centrifuge for a specific time with a known force, and to calculate the distance moved. Far more e/pensive, but 3uicker would be to monitor the movement of the particles while they actually are in the centrifugal field. $his monitoring can be accomplished through the use of an 0Analytical 1ltracentrifuge0 e3uipped with Schlierien ;ptics. %asically, this procedure takes stroboscopic photos of the advancing molecules within the centrifugal field. #t is far too e/pensive an instrument to have in all but the best e3uipped molecular laboratories, however. +e&imentation equilibrium

#f a sample contains many different particles with differing densities and si&es, they will begin to separate on the basis of those parameters. $he large particles will settle to the bottom of a tube faster than the smaller ones. #f the relative centrifugal force is gradually increased, the time for the conse3uent separation of particles can be decreased. %y varying centrifugation force ,speed- and time, while maintaining a continous media density, different si&es of particles can be separated on the basis of their si&e. !arge particles, such as whole cells and nuclei are sedimented at low speeds. Mitochondria and chloroplasts re3uire higher speeds and8or longer times of centrifugation. Ribosomes re3uire even greater forces and longer times. $hus, it is possible to design a protocol which first sediments large organelles, and then by increasing the centrifugation time or speed to sediment smaller particles from the same tube. $his protocol is known as differential centrifugation, and the process makes use of both time and speed. Since the procedure sediments large organelles first, they are often contaminated by the smaller organelles which start at the bottom of the centrifuge tube. At the beginning, the pellet area ,bottom of tube- will contain both small and large randomly distributed organelles. As the centrifuge is run, larger particles move down the tube, but smaller ones do not move up the process is based on sedimentation, not flotation. As the process continues, the larger particles are removed and thus the smaller the particle, the purer the isolated fraction will be at later centrifugation steps. ;f course, the smaller organelles are separated as both contaminants of the larger organelles, and as sediments of subse3uent centrifugations. $hus, if you wish to ma/imi&e the collection of smaller organelles, or minimi&e the presence of smaller organelles in the large organelle fraction, it is necessary to recentrifuge the larger fractions several times and to collect and pool the resulting smaller units. #t is possible, however, to sediment and float the particles simultaneously. #f the particles to be separated have differing densities ,gm8ml- they can be separated through a medium that allows particles of one density to 0float0 and particles of higher density to sink to the bottom. Such media can be layered into the centrifuge tube in step gradients or linear gradients. #n either gradient, the particles are centrifuged until they reach a density e3ual to the media, thus the namee3uilibrium density separations. $his process has been greatly utili&ed in the analysis of molecular weights for proteins and nucleic acids, since to a great e/tent, the density of these molecules can be directly related to their si&e. D3uilibrium density is also used to successfuly isolate membranes and other high lipid4containing organelles.

Exercise 5.1 - 6ucrose Fractionation of Castor Bean

!D=D! #

Figure ?.) :omemade gradient forming device Materials

7astor beans 1 Aradient former Sucrose solutions in '.'1 M DC$A, p: >.* ??S, ))S, *'S, *>S, 5'S ,w8v- 9

Arinding Medium
o o o o o o

'.) M sucrose '.15* M $ris4:7l buffer, p: >.* '.'1 M B7l '.'1 M Mg7l '.'1 M DC$A ad"usted to p: >.* '.'1 M dithiothreitol

7hopping board and knife or ra&or blade Mortar and pestle 7heesecloth Refrigerated centrifuge ,ultracentrifuge preferred- with swinging bucket rotor Spectrophotometer and tubes :emacytometer and phase contrast microscope

Procedure ?
1. Aerminate castor beans by soaking overnight, followed by germination in moist

vermiculite, or paper towels, at ?'G 7. Castor +eans are poisonous and #a0 +e fatal if ta4en internall0. Cear glo(es &hile handling.

2. After appro/imately * days, remove the embryos and cotyledons and discard. .ash the

endosperm in distilled water and chill on ice.

3. <repare two centrifuge tubes for sucrose gradients one for a linear ??45'S gradient and

the other for a stepped gradient. ) $he volume of each solution will depend on the centrifuge rotor to be used and its corresponding centrifuge tube capacity. $he following are directions for ?* ml centrifuge tubes. 6tepped Aradient 7arefully layer, in order, the following solutions into a centrifuge tube
o o o o o

? ml of 5'S sucrose 5 ml of *>S sucrose O ml of *'S sucrose O ml of ))S sucrose ? ml of ??S sucrose

Binear Aradient Add * ml of 5'S sucrose to the bottom of a centrifuge tube and form a linear 5' to ??S sucrose gradient on top of that. 1sing a gradient former, as shown in Figure ?.), place 19 ml of 5'S sucrose in the right chamber and 19 ml of ??S sucrose in the left chamber.
4. 7ombine 5' gm of endosperm tissue with O' ml of grinding medium and chop

vigorously.
5. $ransfer the coarse material to a cold mortar and pestle and continue to grind until a fine

paste is formed.
6. Filter the B@EI ,the paste from step *- through two layers of cheesecloth. 7ollect the

fluid into a beaker, transfer it to a centrifuge tube, and centrifuge the filtrate for 1' minutes at 9>' /g to remove unbroken cells and large debris.
7. Cecant the supernatant into a clean, cold centrifuge tube and recentrifuge for ?' minutes

at 1',I'' 9'/g. Resuspend the pellet in * ml. of grinding medium and hold on ice for further analysis.
8. 7arefully decant the supernatant and save for subse3uent analysis. Aently resuspend the

pellet in 5 ml. of grinding medium.

9. 7arefully layer 9 ml of the pellet onto a stepped sucrose gradient and 9 ml of the pellet

onto a linear gradient.

10. $eam up with another laboratory section and carefully balance your corresponding tubes.

$hat is, be sure that the stepped gradients for both sections are e/actly the same weight. Add grinding media to balance, where appropriate. :ave the instructor check the balance before proceeding.
11. 7entrifuge the tubes at )G 7 for ) hours at 9*,''' R<M in a %eckman S.9> rotor or

e3uivalent. *
12. 1pon completion of the centrifugation, fractionate the samples into appro/imately 9'4?'

e3ual portions. #t is convenient to collect samples of 1.* ml. $his yields a volume suitable for subse3uent spectrophotometric analysis without the use of small volume cuvettes. $his is accomplished most rapidly by gently inserting a long 1I gauge needle, with the tip ground off, into the centrifuge tube so that it rests on the bottom of the tube. .ithout moving the needle ,so as not to stir the contents-, attach a 9.' ml syringe and pull out the bottom 1.* ml of the sample. Remove the syringe and place the 1.* ml fraction into a test tube marked as R1. Re4attach the syringe without disturbing the gradient, and repeat as often as need to totally fractionate the gradient. Alternatively, the gradient can be fractionated by puncturing the bottom of the tubes with a needle, and collecting the fractions in 1.* ml portions by counting the appropriate number of drops that drip from the punctured tube. $here are several commercially available fraction devices designed for this purpose. #f one is available, follow the manufacturer@s directions.
13. Read the optical density ,Absorbance- of each fraction in a spectrophotometer at *)' nm.

<lot a double graph, with fraction number on the / a/is ,bottom to top- vs ;C a/is, and fraction number vs S sucrose on the y a/is. 5

on the y

14. 7arefully e/amine all fractions with a phase contrast microscope. #dentify and count ,use

a hemacytometer where appropriate- all structures found in each fraction. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q

Q Q Q Q Q Q Q Q Q Q Araph of fraction number vs Absorbance

Q Q Q Q Q Q Q Q Q QQ

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

Exercise 5.. - 6edi#entation *ensities

!D=D! # Materials

$able ?.9 Censity and refractive inde/es of sucrose $able ?.1 Censities of cell structures in sucrose Sucrose density fractions from D/ercise ?.1

Procedure
1. 1sing the densities for sucrose given in $able ?.9 >, compute the density of each

organelle found within the fractions collected by differential centrifugation.

2. 7ompare these values with those listed in $able ?.1. I

"otes #t is possible to appro/imate the densities of the particles separated above, based on the linear gradient separation data. #t would be better to perform an additional centrifugation using 7s7l as the viscous media, but for our purposes, the density of the sucrose will be sufficient.

Exercise 5.5 - E;uili+riu# *ensit0 Aradient - Percoll

!D=D! ##

Figure ?.* Svedberg =alues in <ercoll and Sucrose $able ?.? <hysical characteristics of gradient media Materials

1ltracentrifuge and swinging bucket rotor <ercoll +a7l solutions with the following osmolarities2
o

9'', ?'' and )'' milliosmols

.hole blood :emacytometer $est tubes 7olored density marking beads O

Procedure 1'
1. For your ultracentrifuge, obtain a series of centrifuge tubes containing appropriate

volumes of <ercoll starting at 1.'I g8ml density as follows2 a. <ercoll W colored marking beads. b. <ercoll W 9'' m;sm +a7l c. <ercoll W ?'' m;sm +a7l d. <ercoll W )'' m;sm +a7l
2. 1se a hemacytometer to calculate the number of cells per ml of your blood sample.

7arefully layer a suspension containing about 1'' / 1' blood cells onto the gradients in tubes b4d. 7entrifuge the four tubes for the e3uivalent clearance of %eckman ?'.9 rotor at ?*,''' /g for 1* minutes at 9'G 7.
3. Remove the tubes, fractionate into '.* ml fractions and count the number of cells in each

fraction with a hemocytometer.

4. Araph the distance from the bottom of the tube vs. the number of cells in each fraction.

5. ;verlay this graph with a comparison of distance vs. density of the medium, as

determined by the position of the colored beads in your control tube.

6. 7ompare your results to Figure ?.*.

"otes <ertoft and coworkers developed a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. $his material is marketed under the trade name of <ercoll. $able ?.? give the characteristics of this medium, compared to several other density media, namely sucrose, metri&amide and Ficoll . will

;f particular interest is the fact that during centrifugation in a fi/ed angle rotor, <ercoll of centrifugation. $hus, it becomes a simple matter to establish a linear density gradient. Figure ?.* demonstrates a comparison of <ercoll

spontaneously form linear gradients, the shape of which is dependent upon rotor speed and time

fractionation of cellular components to

sucrose fractionation. $his figure also presents, a standard way to comare components, by defining the relationship of density to the sedimentation coefficient for a cell or organelle. +ote, that lymphocytes, granulocytes and erythrocytes have very similar sedimentation coefficients, but can be separated on the basis of density. ;rganelle separations are much easier to accomplish on <ercoll density gradients than on sucrose gradients.

Exercise 5.1 - Bo& 6peed 6eparaton of Cells

!D=D! ## Materials

Sepracell4M+ tubes 11 7linical centrifuge with fi/ed angle rotor <hase contrast microscope .hole %lood 19 <hosphate %uffered Saline ,<%S- W '.1S ,w8v- %ovine Serum Albumin ,%SA-

Procedure
1. 7ollect at least *.' ml of blood into a =acutainer containing DC$A as an anticoagulant.

2. Add *.' ml of DC$A anti4coagulated whole blood to a Sepracell4M+ tube. Mi/ gently by

inversion.
3. 7entrifuge at 9,''' /g for 1' minutes at room temperature with a fi/ed angle rotor. 1?

Cecelerate the centrifuge slowly ,do not use a brake-.

4. Beeping the Sepracell4M+ tube upright, insert a Seprapette into the tube and push until

the M+7 band ,mononuclear cells- is displaced into the Seprapette. Co not collect more than 9.* ml into the Seprapette. After the centrifugation of whole blood, there should be an opalescent compact band "ust below the meniscus of the tube. $his bands contains the mononuclear cells ,lymphocytes and monocytes- and platelets. %elow this band is the plasma, while the erythrocytes and polymorphonuclear cells will form a dark band at the bottom of the tube.
5. $ransfer the contents within the Seprapette by inverting it into a 1* ml conical centrifuge

tube. Rinse the inside of the Seprapette with 9.* ml of <%S and transfer the washing to the conical centrifuge tube.
6. .ash the mononuclear cells in the conical tube by adding * ml of <%S containing '.1S

%SA for a total volume of 1' ml and mi/ by inversion. 7entrifuge at ?'' /g for 1' minutes at room temperature.
7. Resuspend the mononuclear cells in *.' ml of <%S. Make a simple wet mount of the

suspended cells and e/amine the purity of the separation using a phase contrast microscope.

Exercise 5.7 - 6eparation of Cells +0 :elocit0 6edi#entation

!D=D! ### #t is possible to combine variations in density and si&e to allow for separation of cell populations within the earth@s gravitational field, i.e. without a centrifuge. A techni3ue devised by Miller and <hillips 1) uses a ?4?'S gradient of fetal calf serum in phosphate buffered saline, coupled to the use of a specially designed sedimentation chamber. $he techni3ue substitutes time for the e/pense of a centrifuge, but also keeps the cells from being sub"ected to increased gravitational forces. Several commercial applications of this techni3ue are now available for the separation of blood cells within reasonable times ,9) hours-.

Miller and <hillips used the techni3ue to separate spleen cell suspensions and sheep erythrocytes. $he techni3ue is particularly valuable for the separation of cells of the hemopoietic system, prior to sub4culture or analysis. Chapter 5: Cell Fractionation - Endnotes

1.

%ean sprouts or alfalfa sprouts from a local supermarket may be substituted. $hey are ine/pensive, non4poisonous and contain immature chloroplasts which separate well upon sucrose gradient centrifugation. For optimal performance, the sucrose concentration should be determined by use of a refractometer. <ure sucrose, manufactured for use in molecular biology should be used, or it may be necessary to treat the sucrose for contaminating en&ymes. Modified from $errance A. 7ooper. The Tools of %iochemistry. Tohn .iley U Sons, +ew Jork, 1O>>. pp ?)>4?*9. #f only one laboratory section is used, two of each gradient should be prepared. $he e/tra are used to balance the tubes in the centrifuge. #t is not good practice to balance an ultracentrifuge with water or differing gradients since the density distribution will not be balanced, even if the total weight is. $his rotor has served the author well over many years, but is no longer available. $he S.9> rotor has a k 6 ??> X 9*,''' R<M. Refer to Appendi/ F for conversion directions for other rotors. 7ontinuous flow spectrophotometry may be substituted, if available. Cata from Techniques of Preparati*e$ .onal$ an& Continuous /lo0 Ultracentrifugation by ;wen Mitch Ariffith, <h.C. Applications Research Cepartment, Spinco Civision, %eckman #nstruments, #nc. 1O>O, page O. #bid, page ). $hese beads are a 3uick means of visually checking the density separation during formation of the <ercoll gradient. $hey are available from <harmacia, but are e/pensive. $he use of the beads may be eliminated, with the corresponding elimination of Step 1a.

2.

3.

4.

5.

6. 7.

8. 9.

10. As reported in <ertoft, :., et al. 0$he 1se of Censity Aradients of <ercoll for the

Separation of %iological <articles.0 in +eparation of Cells an& +ubcellular Particles ,:. <eeters, Dd.- <ergamon <ress, ;/ford, 1O>O.
11. Sepratech 7orporation, ;klahoma 7ity, ;B >?19>. 12. Sepracell4M+ tubes were devised for human whole blood, but will work well on rabbit

or other animal blood. $he blood needs to be less than 19 hours old for best results, and no older than ?5 hours.
13. #f a fi/ed angle rotor is unavailable, use a swinging bucket, but increase the

centrifugation time to 9' minutes.

14. Miller, R.A. and R.A. <hillips. 0Separation of 7ells by =elocity Sedimentation0 T. 7ell

<hysiol., >?21O149'9. 1O5O.

Chapter 1: Electrophoresis - Introduction

Figure ).1 :oefer SD )'' Sturdier Dlectrophoresis units Dlectrophoresis may be the main techni3ue for molecular separation in today@s cell biology laboratory. %ecause it is such a powerful techni3ue, and yet reasonably easy and ine/pensive, it has become commonplace. #n spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated. 1 Dlectrophoresis can be one dimensional ,i.e. one plane of separation- or two dimensional. ;ne dimensional electrophoresis is used for most routine protein and nucleic acid separations. $wo dimensional separation of proteins is used for finger printing , and when properly constructed can be e/tremely accurate in resolving all of the proteins present within a cell ,greater than 1,*''-. $he support medium for electrophoresis can be formed into a gel within a tube or it can be layered into flat sheets. $he tubes are used for easy one dimensional separations ,nearly anyone can make their own apparatus from ine/pensive materials found in any lab-, while the sheets

have a larger surface area and are better for two4 dimensional separations. Figure ).1 shows a typical slab electrophoresis unit. .hen the detergent SCS ,sodium dodecyl sulfate- 9 is used with proteins, all of the proteins become negatively charged by their attachment to the SCS anions. .hen separated on a polyacrylamide gel, the procedure is abbreviated as SCS44<AAD ,for Sodium Codecyl Sulfate <olyAcrylamide Ael Dlectrophoresis-. $he techni3ue has become a standard means for molecular weight determination. <olyacrylamide gels are formed from the polymeri&ation of two compounds, acrylamide and +,+ 4methylene4 bis4acrylamide ,%is, for short-. %is is a cross4linking agent for the gels. $he polymeri&ation is initiated by the addition of ammonium persulfate along with either 4dimethyl amino4propionitrile ,CMA<- or +,+,+ ,+ ,4 tetramethylethylenediamine ,$DMDC-. $he gels are neutral, hydrophillic, three4dimensional networks of long hydrocarbons crosslinked by methylene groups. $he separation of molecules within a gel is determined by the relative si&e of the pores formed within the gel. $he pore si&e of a gel is determined by two factors, the total amount of acrylamide present ,designated as S$- and the amount of cross4linker ,S7-. As the total amount of acrylamide increases, the pore si&e decreases. .ith cross4 linking, *S7 gives the smallest pore si&e. Any increase or decrease in S7 increases the pore si&e. Aels are designated as percent solutions and will have two necessary parameters. $he total acrylamide is given as a S ,w8v- of the acrylamide plus the bis4acrylamide. $hus, a > 189 S$ would indicate that there is a total of >.* gms of acrylamide and bis per 1'' ml of gel. A gel designated as >.*S$2*S7 would have a total of >.*S ,w8v- acrylamide W bis, and the bis would be *S of the total ,with pure acrylamide composing the remaining 9.*S-. <roteins with molecular weights ranging from 1',''' to 1,''',''' may be separated with > 189S acrylamide gels, while proteins with higher molecular weights re3uire lower acrylamide gel concentrations. 7onversely, gels up to ?'S have been used to separate small polypeptides. $he higher the gel concentration, the smaller the pore si&e of the gel and the better it will be able to separate smaller molecules. $he percent gel to use depends on the molecular weight of the protein to be separated. 1se *S gels for proteins ranging from 5',''' to 9'',''' daltons, 1'S

gels for a range of 15,''' to >',''' daltons and 1*S gels for a range of 19,''' to )*,''' daltons. ? Cationic *s anionic systems #n electrophoresis, proteins are separated on the basis of charge, and the charge of a protein can be either W or 44 , depending upon the p: of the buffer. #n normal operation, a column of gel is partitioned into three sections, known as the Separating or Running Ael, the Stacking Ael and the Sample Ael. $he sample gel may be eliminated and the sample introduced via a dense non4 convective medium such as sucrose. Dlectrodes are attached to the ends of the column and an electric current passed through the partitioned gels. #f the electrodes are arranged in such a way that the upper bath is 44 ,cathode-, while the lower bath is W ,anode-, and 44 anions are allowed to flow toward the anode, the system is known as an anionic system. Flow in the opposite direction, with W cations flowing to the cathode is a cationic system. $ube vs Slab Systems

Figure ).9 Dlectrophoretic separations of proteins $wo basic approaches have been used in the design of electrophoresis protocols. ;ne, column electrophoresis, uses tubular gels formed in glass tubes, while the other, slab gel electrophoresis, uses flat gels formed between two plates of glass. $ube gels have an advantage in that the movement of molecules through the gels is less prone to lateral movement and thus there is a slightly improved resolution of the bands, particularly for proteins. #t is also more economical, since it is relatively easy to construct homemade systems from materials on hand. :owever, slab gels have the advantage of allowing for two dimensional analysis, and of running multiple samples simultaneously in the same gel. Slab gels are designed with multiple lanes set up such that samples run in parallel. $he si&e and number of the lanes can be varied and, since the samples run in the same medium, there is less likelihood of sample variation due to minor changes in the gel structure. Slab gels are un3uestionably the the techni3ue of choice for any blot analyses and for autoradiographic analysis. 7onse3uently, for laboratories performing routine nucleic acid analyses, and those employing antigenic controls, slab gels have become standard. $he availability of reasonably

priced commercial slab gel units has increased the use of slab gel systems, and the use of tube gels is becoming rare. $he theory and operation of slab gel electrophoresis is identical to tube gel electrophoresis. .hich system is used depends more on the e/perience of the investigator than on any other factor, and the availability of e3uipment. Figure ).9 presents a typical protein separation pattern. 7ontinuous vs discontinuous gel systems

Figure ).? Schematic diagram of electrophoresis $he original use of gels as separating media involved using a single gel with a uniform p: throughout. Molecules were separated on the basis of their mobility through a single gel matri/. $his system has only occasional use in today@s laboratory. #t has been replaced with discontinous, ) multiple gel systems. #n multiple gel systems, a separating gel is augmented with a stacking gel and an optional sample gel. $hese gels can have different concentrations of the same support media, or may be completely different agents. $he key difference is how the molecules separate when they enter the separating gel. $he proteins in the sample gel will concentrate into a small &one in the stacking gel before entering the separating gel. $he &one within the stacking gel can range in thickness from a few microns to a full millimeter. As the proteins are stacked in concentrated bands, they continue to migrate into the separating gel in concentrated narrow bands. $he bands then are separated from each other on a discontinuous ,i.e. disc - p: gel. * ;nce the protein bands enter the separating gel, separation of the bands is enhanced by ions passing through the gel column in pairs. Dach ioin in the pair has the same charge polarity as the protein ,usually negative-, but differ in charge magnitude. ;ne ion will have a much greater charge magnitude than the proteins, while the other has a lesser charge magnitude than the proteins. $he ion having a greater charge will move faster and is thus the leading ion, while the ion with the lesser charge will be the trailing ion. .hen an anionic system is employed, the 7lY and glycinate ,glycine as its acid derivative- ions are derived from the reservoir buffer ,$ris4

Alycine-. $he leading ion is usually 7lY glycinate is the trailing ion. A schematic of this anionic system is shown in Figure ).?. 7hloride ions enter the separating gel first and rapidly move down the gel, followed by the proteins and then the glycinate ions. $he glycinate ions overtake the proteins and ultimately establish a uniform linear voltage gradient within the gel. $he proteins then sort themselves within this gradient according to their charge and si&e. Agarose Aels

Figure ).) Agarose separation of cC+A .hile acrylamide gels have become the standard for protein analysis, they are less suitable for e/tremely high molecular weight nucleic acids ,above 9'',''' daltons-. #n order to properly separate these large molecules, the acrylamide concentration needs to be reduced to a level where it remains li3uid. $he gels can be formed, however, by the addition of agarose, a naturally linear polysaccharide, to the low concentration of acrylamide. .ith the addition of agarose, acrylamide concentrations of '.*S can be used and molecular weights of up to ?.* / 1' daltons can be separated. $his is particularly useful for the separation of large se3uences of C+A. 7onse3uently, agarose4 acrylamide gels are used e/tensively in today@s genetic laboratories for the determination of gene maps. $his chapter will concentrate on the separation of proteins, but Figure ).) demonstrates the separation of C+A fragments on an agarose gel.

Exercise 1.1 - Preparation of 6*6-Pol0acr0la#ide Aels

!D=D! # Figure ).* Formed slab gel in casting unit Materials

7asting gel unit for electrophoresis Siliconi&ed pasteur pipettes

Syringes e3uipped with blunt, stub nosed, needles 5 =acuum chamber for degassing gels. Micropipettes ,1'4?'' LlStock ?'S$2'.IS7 Acrylamide monomer 1.* M $ris4:7l buffer, p: I.I 1'S ,w8v- SCS 1'S ,w8v- Ammonium persulfate Te#ed acr0la#ide is a po&erful neurotoxin. *o not +reathe po&der or other&ise co#e in contact &ith the #ono#er. Cear glo(es at all ti#es. Separation Ael Mi/ed Tust <rior $o 1se
o o o o

9' ml of Acrylamide monomer 1* ml of $ris4:7l %uffer, p: I.I '.5 ml of 1'S ,w8v- SCS 9).1 ml of : ;

Stacking gel mi/ed "ust prior to use

o o o o

9.55 ml of Acrylamide monomer *.' ml of $ris buffer, p: I.I '.9 ml of 1'S ,w8v- SCS 19.9 ml of : ;

Procedure
1. Assemble your slab gel unit with the glass sandwich set in the casting mode, and with 1.*

mm spacers in place. Refer to Figure ).*.

2. <repare a separating gel from the ingredients listed above. 3. Add the separating gel to a side arm flask, stopper the flask and attach to a vacuum pump

e3uipped with a cold trap. $urn on the vacuum and degas the solution for appro/imately 1' minutes. Curing this period, gently swirl the solution in the flask. >
4. $urn off the vacuum, open the flask and add 9'' Ll of ammonium persulfate and 9' Ll of

$DMDC to the solution.

5. Add the stopper to the flask and degas for an additional 9 minutes while gently swirling

the solution to mi/ the two accelerators. 1se this solution within a few minutes of mi/ing, or it will gel in the flask.
6. $ransfer the degassed acrylamide solution to the casting chamber with a pasteur pipette.

Aently fill the center of the glass chamber with the solution by allowing the solution to run down the side of one of the spacers. %e careful not to introduce air bubbles during this step.
7. Ad"ust the level of the gel in the chamber by inserting a syringe e3uipped with a 99 gauge

needle into the chamber and removing e/cess gel.

8. #mmediately water layer the gels to prevent formation of a curved meniscus.

1sing a second syringe and needle, add appro/imately '.* ml of water to the chamber by placing the tip of the needle at an angle to a spacer and gently allowing the water to flow down the edge of the spacer and over the gel. Add an additional '.* ml of water to the chamber by layering it against the spacer on the opposite side of the chamber. Cone appropriately, the water will form a layer over the gel, and a clear line of demarcation will be observed as the gel polymeri&es. I
9. After ?' minutes, the gel should be polymeri&ed. #f degassing was insufficient, or the

ammonium persulfate not fresh, the polymeri&ation may take an hour or more. .hen the gel is polymeri&ed, lift the gel in its casting chamber and tilt to decant the water layer.
10. <repare a stacking gel from the listed ingredients. 11. Cegas the stacking gel as in step ?. 12. Add >* Ll ammonium persulfate and 1' Ll $DMDC to the stacking gel and degas for an

additional 9 minutes.
13. Add appro/imately 1 ml of stacking gel to the gel chamber and gently rock back and

forth to wash the surface of the separating gel. <our off the still li3uid stacking gel and dispose of properly. Remember that li3uid acrylamide is e/tremely ha&ardousN
14. Add fresh stacking gel to nearly fill the chamber, but allow room for the insertion of a

teflon comb used to form sample wells. 7arefully insert a teflon comb into the chamber. O Ad"ust the volume of the stacking gel as needed to completely fill the spaces in the comb.

%e careful not to trap any air bubbles beneath the combs. ;/ygen inhibits polymeri&ation, and will subse3uently result in poor protein separations.
15. Allow the gels to polymeri&e for at least ?' minutes prior to use.

Exercise 1.. - 6eparation of Protein 6tandards: 6*6-P$AE

!D=D! # Figure ).5 <rotein standards and SCS4<AAD Figure ).> Rf vs log molecular weight ,!aemmliMaterials

1'S SCS4polyacrylamide gel from D/ercise ).1 <rotein standards 1' 9(4SCS Sample %uffer 1(4SCS Dlectrophoresis Running %uffer ,$ris4Alycine W SCS'.''1S ,w8v- %romophenol %lue Micropipettes with flat tips for electrophoresis wells

Procedure
1. Remove the teflon combs from prepared gels by gently lifting the combs from the

chamber. Rinse the wells ,formed by the removal of the combs- with distilled water and drain off the water.
2. Fill the wells and the chamber with running buffer. 3. <repare ali3uots of a known protein standard by mi/ing e3ual parts of the protein

standard with 9( sample buffer.

4. 1sing a micropipette, add the sample to the bottom of a well. 11 5. Add the same volume used in step ) of SCS4sample buffer or bromophenol blue to a

separate well.19
6. Remove the gel from its casting stand and assemble it into the appropriate slab unit for

running the electrophoresis. %e sure to follow the manufacturer@s directions for assembly.

7. <our a sufficient 3uantity of running buffer into both the lower and upper chambers of the

electrophoresis apparatus until the bottom of the gel is immersed in buffer, and the top is covered, while the electrodes reach into the buffer of the upper chamber. %e careful not to disturb the samples in the wells when adding buffer to the upper chamber.
8. Assemble the top of the electrophoresis apparatus and connect the system to an

appropriate power source. %e sure that the cathode ,W- is connected to the upper buffer chamber.
9. $urn on the power supply and run the gel at 9' mA constant current per 1.* mm gel.

For e/ample, if two gels are run, each having 1.* mm spacers, the current should be ad"usted to )' mA. ;ne gel with 1.* mm spacers should be run at 9' mA, while a gel with '.>* mm spacers should be run at 1' mA.
10. .hen the tracking dye reaches the separating gel layer, increase the current to ?' mA per

1.* mm gel.
11. 7ontinue applying the current until the tracking dye reaches the bottom of the separating

gel layer ,appro/imately ) hours-.

12. $urn off and disconnect the power supply. Cisassemble the gel apparatus and remove the

glass sandwich containing the gel. <lace the sandwich flat on paper towels and carefully remove the clamps from the sandwich.
13. .orking on one side of the sandwich, carefully slide one of the spacers out from between

the two glass plates. 1sing the spacer or a plastic wedge as a lever, gently pry the glass plates apart without damaging the gel contained within.
14. !ift the bottom glass plate with the gel and transfer the gel to an appropriate

container 1? filled with buffer, stain or preservative. $he gel may at this point be used for 7oomasie %lue staining ,D/ercise ).? -, silver staining ,D/ercise ).)-, en&yme detection ,D/ercise ).>-, .estern blots ,D/ercise ).O- or for more advanced procedur es, such as electroblotting or electroelution. #f prestained protein standards were used, the gels may be scanned directly for analysis ,D/ercise ).*or D/ercise ).5-. <lace the gel into *'S methanol and gently rock the container for about ?' minutes prior to scanning. $his can be accomplished by placing the gels into a flat dish and gently lifting the edge of the disk once every ?' seconds. $here are commercially available rocker units for this purpose.

#f the gel is to be dried, use a commercial gel dryer, such as the :oefer SD*)' or SD 115' Slab Ael Cryer. Follow the manufactuer@s directions. Figure ).5 demonstrates a dried and stained gel containing a series of proteins of known molecular weights.
15. <lot the relati(e #o+ilit0 of each protein against the log of its molecular weight.

Relative mobility is the term used for the ratio of the distance the protein has moved from its point of origin ,the beginning of the separating gel- relative to the distance the tracking dye has moved ,the gel front-. $he ratio is abbreviated as Rf. Molecular weight is e/pressed in daltons. Figure ).> presents a plot of the relative molecular weight of protein standards against the log of their molecular weight.

Exercise 1.5 - Coo#asie Blue 6taining of Protein Aels

!D=D! # Materials

<rotein gel from D/ercise ).9 '.9*S ,w8v- 7oomasie %rilliant %lue R 9*' in methanol4water4glacial acetic acid ,*4*41-, filtered immediately before use. >S ,v8v- acetic acid 7ommercial destaining unit ,;ptional-

Procedure
1. <lace a gel prepared as in D/ercise ).9 in at least 1' volumes of 7oomasie %lue staining

solution for 94) hours. Rock gently to distribute the dye evenly over the gel.
2. At the conclusion of the staining, wash the gels with several changes of water. 3. <lace the gels into a solution of >S acetic acid for at least 1 hour. 4. #f the background is still deeply stained at the end of the hour, move the gels to fresh >S

acetic acid as often as necessary. #f a commercial destainer is available, this will decrease the time re3uired for stain removal. Follow the manufacturer@s directions for use of the destainer. 1)
5. <lace the gels into containers filled with >S acetic acid as a final fi/ative. 6. <hotograph the gels or analy&e the gels spectrophotometrically.

"otes

7oomasie %rilliant %lue R 9*' is the most commonly used staining procedure for the detection of proteins. #t is the method of choice if SCS is used in the electrophoresis of proteins, and is sensitive for a range of '.* to 9' micrograms of protein. .ithin this range, it also follows the %eer4!ambert law and thus can be 3uantitative as well as 3ualitative. $he ma"or drawback is the length of time for the procedure and a re3uirement for destaining. ;verstaining results in a significant retention of stain within the gel, and thus a high background stain, which might obliterate the bands. $he length of time for staining must be carefully monitored, and can range from 9' minutes to several hours. #f ma/imum sensitivity is desired, one should try 9 hours for a *S gel and ) hours for a 1'S gel. Cestaining must be monitored visually and ad"usted accordingly.

Exercise 1.1 - 6il(er 6taining of Aels

!D=D! #

Figure ).I Silver stained protein gel Materials

<rotein gel from D/ercise ).9 )*S ,v8v- Methanol W 19S ,w8v- acetic acid *S ,v8v- Methanol W >S ,w8v- acetic acid 1'S Alutaraldehyde '.'1M Cithiothreitol Silver nitrate solution Sodium citrate 8 formaldehyde Bodak Farmer@s Reducer or Bodak Rapid Fi/er

Procedure
1. Fi/ gels by gently rocking them in a solution of )*S methanol 8 19S acetic acid until the

gels are completely submerged. Fi/ for ?' minutes at room temperature.
2. Remove the fi/ative and wash 9/ for 1*@ each with *S ethanol 8 >S acetic acid. ,Aels

thicker than 1 mm re3uire longer washing.-

3. Soak the gels for ?' minutes in 1'S glutaraldehyde. 4. .ash ?/ with deioni&ed water, 1' minutes each. 5. <lace in dithiothreitol for ?' minutes. 6. <lace in silver nitrate solution for ?' minutes. 7. .ash for 1 minute with deioni&ed water.

*ispose of used sil(er nitrate solution i##ediatel0 &ith continuous flushing. This solution is potentiall0 explosi(e &hen cr0stals for# upon dr0ing.
8. <lace in sodium citrate 8 formaldehyde solution for 1 minute. 9. Replace the sodium carbonate8formaldehyde solution with a fresh batch, place gels on a

light bo/ and observe the development of the bands. 7ontinue to rock gently as the gel develops.
10. .hen the desired degree of banding is observed ,and before the entire gel turns black-,

withdraw the citrate 8 formaldehyde solution and immediately add 1S glacial acetic acid for * minutes.
11. Replace the glacial acetic acid with Farmer@s reducer or Bodak Rapid Fi/er for 1 minute.

Remove Farmer@s reducer and wash with several changes of deioni&ed water.
12. <hotograph or scan the gel with a densitometer. Figure ).I demonstrates a typical silver

stained protein gel.

13. For storage soak the gel in ?S glycerol for * minutes and dry between dialysis

membranes under reduced pressure at I'4I9G 7 for ? hours. Alternatively, place the wet gel into a plastic container ,a storage bag will do- and store at room temperature. #f desired, the gels may be dried between .hatman ?MM filter paper for autoradiography, or dried using a commercial gel dryer.

Exercise 1.7 - *ocu#entation

!D=D! #

Figure ).I Silver stained protein gel Materials

<olaroid camera ,Fotodyne Foto8<horesis # or e3uivalent- 1* or ?* mm camera e3uipped with macro lens Stained gel

Procedure
1. <hotograph the gels. 2. 1se the photographs or negatives to measure the distance from the point of protein

application ,or for two gel systems, the line separating the stacking and separating gelsto the final location of the tracking dye near the bottom of the gel. 15
3. Measure the distance from the point of origin to the center of each band appearing on the

gel.
4. Civide each of the values obtained in Step ? by that obtained in Step 9 to obtain the

relative mobility ,the Rf value- for each band.

5. 1sing either the graph of Rf values and molecular weights from D/ercise ).9, or Figure

).I, compute the molecular weights of each band. /ptional Scan the negative with a densitometer and compute Rf values based on the distances from the point of origin to the peak tracing for each protein band. #ntegration of the area of each peak will yield 3uantitative data as well as the molecular weight.

Exercise 1.9 - *irect *ensito#etr0 of Aels

!D=D! ##

Figure ).Ia Model 1?19 Censitometer

Figure ).O <rotein standard scan from Model 1?19 Materials

Censitometer 1> .ashed and stained gel from D/ercise ).9

Procedure
1. 1sing the scanning cuvette supplied with your densitometer8scanner, calibrate the scanner

and choose the proper scanning mode and wavelength filter for your application. For e/ample, for the #sco Model 1?19, use the instrument on Automatic Scan Mode with a 59' nm filter for 7oomasie blue, or a *I' nm filter for silver stained gels.
2. <lace the cuvette holder on the lab bench and pour about 1' ml of buffer or acetic acid

into the cuvette. 1nfi/ed gels still in the buffer that you used to wash them will work best for this purpose. #f the gels have been fi/ed, be careful of spills and minimi&e contact with the cuvette to prevent corrosion of the cuvette and long term etching of the glass plates.
3. <lace the stained gel into the buffer in the cuvette and orient the gel so that the lanes are

vertical as you look at them, with the origin at the top and the front at the bottom. %e careful to gently remove any trapped air bubbles beneath the gel by gently lifting the gel with a blunt glass rod.
4. =ery carefully lay the top glass plate onto the gel to hold it flat. $he plate should be

placed on the gel at an angle, with the two black knobs on the right, and slowly lowered onto the gel. Again, be careful to prevent trapping air bubbles beneath the glass plate.
5. Ad"ust the scan rate to match that of the recorder ,try an initial scan rate of ?'' cm8hr-.

<lace the cuvette onto the optical reading device, and manually ad"ust the position of the

gel so that the optical reading head of the scanner is placed "ust before the origin point of the gel lane to be scanned, in an area where there are no bands. Ad"ust the baseline of the scanner and recorder to &ero.
6. <ress and hold the S$AR$8S$;< button until the scan begins. 7ontinue the scan until the

optical head returns to its original position. %e careful that the head does not strike one of the knobs on the top glass plate during operation. $his will result in possible damage to the optical unit.
7. .hen all lanes are scanned, return the gel to storage and carefully clean the gel cuvette. 8. 7alculate the Rf values for each peak and determine the molecular weights of the proteins

located within each peak. Figure ).O presents a typical scan of protein standards. /ptional Modern scanners are e3uipped with capability to output analog data directly to various computer data management systems, while converting the data to digital information. #n addition to storing the scan as a digital reading, these programs can be used to integrate the area under the curves for each peak and thereby yield 3uantitative data. #f such a capability is present on your system, calculate the amount of each protein found within the ma"or peaks. 1I

Exercise 1.< - 6eparation of B*? Iso30#es fro# 6eru#

!D=D! ##

Figure ).1' <reparation rack for tube gels

Figure ).11 Filling gel tubes

Figure ).19 Assembling tubes and bath chambers

Figure ).1? !C: iso&yme separation

Figure ).1) Censitometry tracings of !C: patterns Materials

1.' M $ris4:7l p: I.I Stock ?'S$2'.IS7 Acrylamide 1'S ,w8v- Ammonium persulfate )'S ,w8v- Sucrose $ris4Alycine %uffer !C: Stain2 Must be prepared "ust prior to use. For use, combine I parts A to 1 part %. Solution A2 5'S Sodium !actate +itro blue tetra&olium <hena&ine Methosulfate 9.) ml '.'? gm '.''? gm

Sodium phosphate buffer to make 1'' ml

Solution %2 '.*S ,w8v- +AC ,use * mg8ml : ;

+on4heparani&ed hematocrit tubes and centrifuge %lood lancets and alcohol for swabbing 1O

Procedure $he following directions are given for the preparation of tube gels. Many laboratories continue to use tube gels. $he directions can be readily modified to use slab gels as detailed in D/ercise ).1 andD/ercise ).9
1. Assemble the appropriate si&ed glass tubes and a preparation rack. $hese may be cut from

standard glass tubing or available commercially.

2. #nsert the glass tubes in the preparation rack by laying a strip of <arafilm appro/imately

1)0 long on top of the black grommets located on the base of the preparation rack ,Figure ).1'-. <lace <arafilm so that it "ust covers the first grommet and allow an appro/imate 90 overhang at the opposite end. %eginning at the first grommet, insert the tube through the hole in the upper section of the rack and press the tube down into the grommet, pushing the <arafilm ahead to form a tight leak4proof seal. <lace your finger under the grommet and seat the tube and <arafilm against your finger. Co not stretch or pull the <arafilm while inserting the tubes. !eave a small amount of slack as you proceed from one tube to the ne/t.
3. <repare the separating gel ,*S acrylamide- by mi/ing the following2

Stock Acrylamide $ris4:7l buffer $DMDC

?.' ml ?.' ml 9' Ll

Ammonium persulfate 5.' ml

4. Mi/ slighty more than 1 ml of solution for each tube being prepared. ;nce the reagents

are mi/ed, complete the ne/t two steps 3uickly. $he mi/ture will begin to polymeri&e in appro/imately 1' minutes. Rinse syringe, beakers, and other implements after use to prevent the gel solution from solidifying on them.
5. 1sing a 1' cc syringe with a !oading Syringe Stub +eedle, fill the gel tubes to within

appro/imately 9 cm of the top with the separating gel mi/ture ,Figure ).11-. D/pel any remaining solution from the syringe when done.
6. #nsert the syringe with stub needle into each gel tube as far as it will go draw off as much

gel solution as possible. $his procedure will result in gels of uniform height. After removing e/cess gel solution from the gel tubes, rinse the syringe and stub needle.

7arefully add a small layer of $ris4:7l %uffer to the top of the gel, without disturbing the gel itself.
7. Allow the separating gel solution to stand undisturbed for ?' minutes during

polymeri&ation. The follo&ing step in(ol(es the use of hu#an +lood. Extre#e caution #ust +e ta4en to guard against the dangers of infection. %se disposa+le lancets' &ear glo(es at all ti#es and dispose of sharps as indicated +0 the instructor. $lternati(el0 use +lood fro# a la+orator0 ani#al.
8. 1sing a non4heparani&ed hematocrit capillary tube, obtain a sample of blood. 7entrifuge

the blood sample in a hematocrit head of a clinical centrifuge to separate the formed elements from the serum8plasma. A standard hematocrit tube contains "ust the right serum for a single gel tube analysis. Alternatively, blood can be centrifuged in regular centrifuge tubes, if there is a sufficient 3uantity of blood.
9. For each blood sample, prepare a 121' dilution of the sample by combining 1 part of

sample serum with O parts of sucrose. <repare at least 1'' L l of diluted serum for each specimen, enough for two gels.
10. Remove the storage solution from three gel tubes by inverting the gel tubes over a layer

of absorbent paper. Shake the tubes abruptly once or twice in a downward motion to remove all the buffer. .hile the gel tubes are still inverted, blot the ends of the tubes to remove any remaining li3uid.
11. Apply *' L l of diluted serum to each of two gel tubes. Co not touch the surface of the

gel, but allow the sample to flow onto its surface.

12. !ayer each gel tube with tris4glycine buffer to completely fill the tube. %e e/tremely

careful not to disturb the serum layer.

13. Remove gel tube R1 from the preparation rack. Moisten the upper end of the tube with a

little water and insert sample end up into the bottom of the bath tube adapter ,position 1 44 -. <ush the tube in so that its upper edge is flush with the upper edge of the adapter. <lace all gel tubes into the bath in this manner. ;bserve the numbers on the upper bath lid and use them to identify the corresponding samples from the preparation rack. :andle the gel tubes with care to prevent mi/ing of sample and buffer layer. 1se the hollow plastic bath stoppers to plug any empty tube adapters.

14. <our enough tris4glycine buffer into the lower bath so that the bath is appro/imately half

full.
15. Assemble the upper and lower chambers, ensuring that the lower portions of the gel tubes

are immersed in the lower bath and that all trapped air bubbles are removed from the ends of the tubes.
16. 7arefully pour additional tris4glycine buffer into the upper bath, so that the level is

sufficient enough to make contact with the cathode when it is inserted ,Figure ).19-. %e particularly careful not to disturb the buffer inside the gel tubes. Again, check the gel tubes to insure that there are no air bubbles trapped in the upper part of the gel tubes.
17. <lace the cover onto the upper bath and connect the electrodes to the baths and the power

supply.
18. $urn on the power source and, if necessary, allow it to warm up. 7heck the bath to insure

that the polarity is normal. 7onnect the safety interlock "ack to the pins on the bath. Ad"ust the power source to deliver * milliamps of current per gel tube in the bath. For e/ample, if 5 gel tubes are being run, the total current should be ?' milliamps.
19. 7ontinue electrophoresis for 9' minutes or until a clearly defined albumin band is seen

near the bottom of the tube. #f prestained protein markers are used in the standard, the timing can be precisely controlled by visually checking its progress.
20. .hen electrophoresis is complete, disconnect the safety inter lock and turn the power

source off. <our the upper bath buffer into a storage container. <lace the upper bath on the 14stand and remove the first gel tube.
21. Remove the separating gel for staining. Fit the 1' cc syringe with the blunt4tipped gel

removing needle and half fill the syringe with water. .hile holding the gel tube with one hand, carefully insert the needle at the sample application end of the tube, between the inside wall of the tube and the gel. .hile slowly pushing the needle in and keeping it flat against the tube wall to avoid scratching the gel, force a stream of water through the needle and rotate it completely around the circumference of the gel. Remove the needle and insert it from the other end of the tube ,the separating gel end- using the same techni3ue described above. ;nce the needle is inserted and rotated completely, the gel will come loose and slide from the tube.

22. Rinse the gel with distilled or deioni&ed water to remove any en&ymes on the surface and

place the gel in a stoppered test tube.

23. Repeat steps 91499 for the rest of the tubes. !abel each gel carefully. 24. <lace 9 ml of freshly prepared !C: stain solution into 1' / >* mm amber tubes and add

a gel to each tube. Beep stoppered and away from light.

25. #ncubate the gels in stain solution at ?> G 7 for 5' minutes. 26. .hen the color of the bands has developed, drain off the stain solution from the tubes and

fill them with >S acetic acid. 7ontinue to protect the gels from light for one hour. At the end of this time, the acetic acid will have inhibited further color development and preserved the protein gels.
27. $ransfer each gel to a clear glass test tube containing fresh >S acetic acid and stopper the

tube. $he gels are now ready for photography or densitomer 3uantitation. Figure ).1? and ).1) demonstrate !C: separation patterns for a normal individual and a comparison with a pattern consistent with myocardial necrosis.
28. #f the gels are scanned, integrate the area of each band and calculate the amount of each

iso&yme as a S of the total !C: present. "otes $he term iso&yme was introduced by 7.!. Markert and F. Moller in 1O*O 9' to describe multiple en&yme forms with similar or identical substrate specificity, and occurring within the same organism. Markert later proposed to modify the term by such ad"ectives as allelic, nonallelic, multimeric, conformational, and con"ugated. $hese ad"ectives imply a broader definition of the term iso&yme and thus include many genetic variations, as well as physiological modifications of the protein structures. Serum lactic dehydrogenase ,!C:- is an ideal en&yme for the analysis of iso&ymes, and is also a model system for electrophoretic analysis. $he en&yme actually consists of five electrophoretically separable isoen&ymes, identified as !C:441, !C:449, !C:44?, !C:44), and !C:44* in the order of their relative rates of electrophoretic migration !C:441 migrates most rapidly. Since these isoen&ymes are usually associated with characteristic tissue or organ sources, their relative concentrations in serum may provide useful information in the differentiation of diseases of various body systems, such as myocardial infarction, liver necrosis, pulmonary

infarct, primary muscle dystrophy, pernicious anemia, and malignancy. 7onse3uently several easily available commercial kits can be used for their analysis. !C: iso&ymes are important indicators of cellular differentation as well. Analysis of the peptide synthesis for !C: iso&ymes presents what is now a classical analysis of differential gene activity. !C:1 is composed of a single peptide species A, while !C:* is composed of the single peptide species %. !C:94) are the permutations of combining species A and % into a functional tetramer. $hus, whether !C:1 or !C:* are synthesi&ed depends entirely on the gene transcription and translation for species A and % respectively. #f both are turned on and are e3uimolar within the cell, then !C:? will be the predominant form ,AA%%-. #n short, the !C: tetramer is a self4 assembling molecule that depends upon the concentration of reactants for its tetrameric form. $he distribution of iso&ymes can thus be analy&ed statistically to determine the e/tent to which gene A and8or gene % are functioning.

Exercise 1.= - Cestern Blots

!D=D! ### Figure ).1* Samples of a .estern blot Materials

%lot 7ell %A I? '.9 L m pore nitrocellulose sheets %uffer, <%S4$ween 9' Antigenic proteins, antibodies, and horseradish pero/idase labeled antiglobulins

Procedure 91
1. Run an electrophoretic separation of known antigenic proteins according to the

procedures in D/ercise ).1 and ).9.

2. Craw a line '.* cm from the top edge of an I / 1' cm nitrocellulose sheet and soak it in

blot buffer for about * minutes. "itrocellulose is +oth fragile and fla##a+le and easil0 conta#inated during handling. Cear glo(es &hich are pre&ashed.

.hen soaking the nicrocellulose wet first one side and then turn the sheet over and wet the other, to prevent trapping air within the filter.
3. <lace 9'' ml of blot buffer into a tray and add a piece of filter paper slightly larger than

the electrophoretic gel from Step 1.

4. Remove the gel from the electrophoresis chamber after the proteins have been separated,

and place the gel into the tray containing the filter paper. Co not allow the gel to fall onto the paper, but place it ne/t to the paper in the tray.
5. Aently slide the gel onto the top of the filter paper. Beep the stacking gel off of the paper

until the last moment, since it tends to stick and make repositioning difficult.
6. :olding the gel and the filter paper together, carefully remove them from the tray of blot

buffer and transfer the paper and gel to a pad of the blot cell with the gel facing up.
7. $ransfer the nitrocellulose sheet ,ink side down- onto the top of the gel and line up the

line drawn on the sheet with the top of the stacking gel. /nce the gel and nitrocellulose touch the0 can not +e separates.
8. Roll a glass rod across the surface of the nitrocellulose to remove any air bubbles and

insure good contact between the gel and nitrocellulose.

9. !ay another sheet of wet filter paper on top of the nitrocellulose creating a sandwich of

paper4gel4 nitrocellulose4paper, all lying on the pad of the blot cell.

10. Add a second pad to the top of the sandwich and place the entire group inside of the

support frame of the blot cell, and assemble the blot cell so that the nitrocellulose side of the sandwich is toward the positive terminal.
11. 7heck that the buffer levels are ade3uate and that the cooling water bath is ad"usted to at

least *G 7. Sub"ect the gel to electrophoresis for ?' minutes with the electrodes in the high field4intensity position. Follow the manufacturer directions during this phase. Failure to closely monitor the electrophoresis buffer or temperature can result in a fire. 1se a circulating cold bath appropriate to the apparatus and hold the voltage to a constant 1'' vdc.
12. 1pon completion of the electrophoresis ,timed according to manufacturer@s directions-,

turn off the power and disassemble the apparatus. Remove the blot pads from the sandwich and remove the filter paper from the nitrocellulose side.

13. <lace the sandwich, nitrocellulose side down, onto a glass plate and remove the other

filter paper.
14. 1se a ball point pen to outline the edges of the separating gel onto the nitrocellulose,

including the location of the wells. 7arefully lift the gel away from the nitrocellulose and mark the locations of the pre4stained molecular weight standards as the gel is peeled away. <eel the gel from the separating gel side, not the stacking gel.
15. .ash the blot ,the nitrocellulose sheet- at least four times with 1'' ml of <%S4$ween 9'

for five minutes each on a rocking platform.

16. 7ut the blot into '.* cm strips. 17. #nactivate sera containing positive and negative antibody controls to the antigens under

e/amination by treating at *5 G 7 for ?' minutes. Make dilutions of 121'' and 121''' of the controls with <%S4$ween 9'.
18. <lace ? ml of the diluted sera or controls onto a strip from Step 15 and incubate for 1

hour at room temperature while continuously rocking the sample.

19. .ash the strips four times for * minutes each with 1' ml 3uantities of <%S4$ween 9'.

$he first wash should be done at *'G 7 but the last three may be done at room temperature.
20. Add ? ml of horseradish pero/idase4labeled antiglobulin, optimally diluted in <%S4

$ween and incubate at room temperature for 1 hour with continuous agitation.
21. .ash the strips four times for * minutes each with <%S4 $ween 9' and one more time

with <%S only.

22. Remove the <%S and add * ml of substrate solution. <ositive reaction bands usually

appear within 1' minutes. Stop the reaction by washing with water. Refer to Figure ).1* for a comparison. "otes ;ne of the more difficult tasks of electrophoretic separations is the identification of specific bands or spots within a developed gel. As observed with !C: iso&ymes, one method of doing this is to react the bands with an en&yme substrate that can be detected colorimetrically. As a rule, however, most peptides are denatured during electrophoresis, and, of course, nucleic acids have no en&yme activity. $he methods employed for identifying non4 en&ymatic proteins

and nucleic acids have been termed .estern for immunoblotting of proteins, Southern for techni3ues using C+A probes +orthern when using R+A probes. $he probes are radioactive complimentary strands of nucleic acid. $he first of these techni3ues was the Southern, named for the developer of the procedure, Ddward Southern. +orthern and then .estern blots were named by analogy. %lotting techni3ues first develop a primary gel2 protein on acrylamide or C+A8R+A on agarose. $he gel patterns are then transferred to nitrocellulose membrane filters and immobili&ed within the nitrocellulose membrane. $his process of transfer to an immobili&ing substrate is where the term blotting originated. $he process is widely used in today@s laboratories because the immobili&ation allows for e/tensive biochemical and immunological binding assays that range from simple chemical composition to affinity purification of monospecific antibodies and cell4 protein ligand interactions. #n practice, the electrophoresis gel is sandwhiched between two layers of filters, two foam pads ,for support- and two layers of a stainless steel mesh. $his entire apparatus can be submerged in a buffer and transfer allowed to occur by diffusion ,yielding two blots, one on each filter-, or can be arranged in an electro4convective system so that transfer occurs in a second electrophoretic field. ;nce the transfer has occurred, the blots can be probed with any number of specific or non4 specific entities. C+A can be probed, for e/ample, with cC+A or even a specific messenger R+A to identify the presence of the gene for that message. Chapter 1: Electrophoresis - Endnotes

1.

.e refer to molecular orientation. #t is also possible to separate whole cells or organelles on the basis of their surface charge. Also known as sodium lauryl sulfate or S!S. Auidelines given by Tohn A. Smith, in Current Protocols in #olecular %iology, Section ##, 1'.9.9 0Dlectrophoretic Separation of <roteins0. Areene <ublishing Associates, %rooklyn 1OI>. $he term disc is applied to discontinuous gels, but unfortunately has two meanings when applied to electrophoresis. Some investigators continue to use an older reference to the discoidal front found in tube gels, and thus use disc interchangeably with the terms

2. 3.

4.

column or tube. Modern usage of the term refers to the term being applied to a discontinuous p: system which may be run in a tube or slab configuration.
5.

#f a continuous p: gradient is employed, the stacking gel is eliminated, the buffers are different, and the techni3ue is referred to as &one electrophoresis. #f the molecules being separated are allowed to migrate to a particular &one due to a p: gradient in the gel, the procedure becomes known as electrofocusing. $hese can be easily manufactured from 99 gauge needles with the points ground off. +ever use pointed needles when working with acrylamideN Alternatively, add a stirring bar to the flask and stir on a magnetic mi/er at low speed. #t is important that the solution be stirred slowly so as to not introduce air bubbles. #f problems are encountered using water at this step, water saturated isobutanol may be substituted. $o aid even further, a little ;il Red ; dye can be added to the isobutanol for visual distinction. Since the alcohol is less dense than water, it layers more readily without mi/ing with the gel. #t must be thoroughly rinsed out before adding the stacking gel, however. $eflon combs come in a variety of configurations for sample volume and number of lanes ,?,*,1',1* or 9'-. 1se a comb suitable for the number of samples to be run.

6.

7.

8.

9.

10. <restained molecular weight standards are available from several sources %R!,

Rockville, Md, %io4Rad or Sigma 7hemical 7o, St. !ouis.

11. $he SCS8sample buffer contains glycerol and conse3uently the sample will have a higher

density than the SCS8separating buffer. #t is best to use a flat tipped micropipette designed specifically for this application, but any flat tipped syringe needle will also work. $he volume of sample will depend on the si&e of the wells, but typically is 9*41'' Ll. Some e/perimentation will be necessary to obtain the proper volume.
12. #n some applications, the bromphenol blue is added to the SCS8sample buffer and thus

this step is unnecessary. %romphenol blue is known as 0tracking0 dye and is used to monitor the progress of the electrophoresis separation. A drop of '.1S phenol red may be substituted. Alternatively, prestained protein standards may be used without a tracking dye, although this is not recommended.

13. 1'/1'/9 glass baking dishes are useful for this purpose, or ordinary refrigerator

containers of appropriate si&e. .hile the former will need a covering of plastic wrap, the latter come with lids.
14. #f tube gels are used, the apparatus used for separation can be modified for stain removal

if slightly larger tubes can be fitted. <lace a gel that has been washed in water and >S acetic acid ,1 hr.- into a tube that has had a '.* cm gel plug formed in the tip. <lace the gel into the tube with the origin up, that is away from the plug, and fill the tube with >S acetic acid. #nsert the tube into the appropriate apparatus and fill both upper and lower chambers with >S acetic acid. .ith the upper pole as the cathode, apply a 1* milliamp current per tube. $he stain will wash into the lower bath and the progress can be monitored visually.
15. Fotodyne #nc. +ew %erlin, .isconsin. 16. Measurements can be made easier if the image of the gel is first enlarged such that the

distance from the point of origin to the front is 1' cm on the final print. $his can be done with a photographic enlarger, and a print may not be re3uired.
17. Cirections given for #sco Model 1?19 Ael Scanner ,#sco, #nc. )>'' Superior, !incoln,

+D 5I*')-.
18. Alternatively, a cruder yet effective method, is to cut the peaks from the paper tracing

and weigh the paper for each peak. $his must be done at one period of time to prevent changes in the weight due to humidity. $he techni3ue is surprisingly accurate, however. $his process can also be used with photographs of the gels, without the use of an e/pensive gel scanner.
19. :uman serum works well for this e/ercise, and students may use their own blood. $o

avoid the obvious problems in obtaining safe samples of human blood products, substitute fresh sera from laboratory animals. A rabbit or rat will work, but the !C: patterns will be different than those given in this e/ercise.
20. 7.!. Markert and F. Moller 1O*O. 21. From Dn&yme4!inked #mmunoelectrotransfer %lot $echni3ue ,.estern %lot- for :uman

$4!ymphotropic =irus $ype ###8!ymphadenopathy4Associated =irus ,:$!=4###8!A=Antibodies =7...$sang, B.:anclck, M. .ilson, C.F. <almer, S.C. .haley, T.S.

McCougal, S. Bennedy. #mmunology Series +o. 1* <rocedural Auide. 1.S. Cept. of :ealth and :uman Services. Cecember 1OI5.

Chapter 7: En30#es - Introduction

7ells function largely because of the action of en&ymes. !ife is a dynamic process that involves constant changes in chemical composition. $hese changes are regulated by catalytic reactions, which are regulated by en&ymes. At one time, the cell was actually concieved of as a sac of en&ymes. #t was believed that if we knew all of the reactions and their rates of action, we could define the cell, and indeed, life itself. Few biologists continue to think of this as a simple task, but we know that life as we know it could not e/ist without the function of en&ymes. #deally, we would e/amine en&ymes within an intact cell, but this is difficult to control. 7onse3uently, en&ymes are studied in vitro after e/traction from cells. #n this e/ercise, we will e/tract the en&yme tyrosinase and study its kinetic parameters. #t is only one of thousands of en&ymes working in concert within cells, but it is one but which readily demonstrates the main features of en&yme kinetics. Since all en&ymes are proteins, and proteins are differentially soluble in salt solutions, en&yme e/traction procedures often begin with salt ,typically, ammonium sulfate- precipitation. ;n the simplest level, proteins can be divided into albumins and globulins on the basis of their solubility in dilute salts. Albumins are considered to be soluble while globulins are insoluble. Solubility is relative, however, and as the salt concentration is increased, most proteins will precipitate. $hus, if we homogeni&e a tissue in a solution that retains the en&yme in its soluble state, the en&yme can be subse3uently separated from all insoluble proteins by centrifugation or filtration. $he en&yme will be impure, since it will be in solution with many other proteins. #f ali3uots of a concentrated ammonium sulfate solution are then added serially, individual proteins will begin to precipitate according to their solubility. %y careful manipulation of the salt concentrations, we can produce fractions which contain purer solutions of en&ymes, or at least are enriched for a given en&yme. Fortunately, absolute purity of an en&yme e/tract is seldom re3uired, but when it is, the fractions must be sub"ected to further procedures designed for purification ,such as electrophoresis and8or column chromatography-.

#n order to determine the effectiveness of the purification, each step in the e/traction procedure must be monitored for en&yme activity. $hat monitoring can be accomplished in many ways, but usually involves a measurement of the decrease in substrate, or the increase in product specific to the en&yme. #t is important to remember that en&ymes act as catalysts to a reaction and that they affect only the reaction rate. $he general formula for the action of an en&yme is given by the following2 B1 4444444444Z DWS B9 [4444444444 B? 444444444Z B) DW< [44444444 ,D3uation *.1where D 6 concentration of the en&yme S U < 6 concentrations of substrate and product, respectivelye DS U D< 6 concentration of en&yme4substrate comple/ and en&yme4product comple/ k14k) 6 rate constants for each step. From e3uation *.1, the rates,velocities- of each reaction can be given by2 v1 6 k1,D-,Sv9 6 k9,DSv? 6 k?,DSv) 6 k),D-,<formation of en&yme4substrate comple/ reformation of free en&yme and substrate formation of product and free en&yme reformation of en&yme4product comple/ DS [444444Z D<

#n steady state e3uilibrium, ,v14v9-6,v?4v)- and, if all product is either removed or does not recombine with the en&yme, then k) 6 ', and k1,D-,S-4k9,DS-6k?,DS$his e3uation can then be rearranged to yield2 B9 W B? B1 ,D-,S,D3uation *.9,DS44444444444 6 44444444

where the left side of this e3uation can be given as a single constant, known as B , the rate constant, or the Michaelis constant. +ote that the units for this constant will be those of concentration. ;ne of the important concepts of metabolism is that en&ymes from differing sources may have the same function ,i.e. the same substrate and product-, but possess significantly different B values. Since biological function is as dependent on the rate of a reaction as it is on the direction of a reaction, it becomes necessary to measure the B value for any en&yme under study.

Dn&ymes act as catalysts because of their three dimensional protein structure. $his structure is controlled by many factors, but is particularly sensitive to changes in p:, salts and temperature. Small changes in the temperature of a reaction can significantly alter the reaction rate, and e/tremely high temperatures can irreversibly alter both the three dimensional structure of the en&yme and its activity. #t may even render the en&yme non4functional that is, to denature the en&yme. Salts can also cause denaturation, but the effects of ammonium sulfate are usually reversible. :eavy metal salts, by contrast, usually irreversibly alter the structure of the protein, and thus their routine use as fi/atives in histological work. $cti(e 6ites An en&yme works by binding to a given substrate in such a geometrical fashion that the substrate is able to undergo its inherent reaction at a more rapid rate. $his type of reaction is commonly referred to as the lock and key mode for en&yme action. #t implies that there is a particular part of the en&yme structure, the active site, which specifically binds sterically to a substrate. $he en&yme does not actually react with the substrate but merely brings the substrate into the proper alignment or configuration for it to react spontaneously or in con"unction with another substance. Since a reaction proceeds normally by a random kinetic action of molecules bumping into each other, any time molecules are aligned, they will react faster. $hus, for any given en&yme there will be a best fit configuration to the protein in order to align the substrate and to facilitate the reaction. .hen the en&yme is in its ideal configuration, the reaction will proceed at its ma/imum rate, and the overall rate of activity will be dependent upon substrate concentration.

Figure *.1 :ypothetical effect of temperature and p: Ma/imum reaction rate assumes that an optimal p:, salt environment and temperature have been established.Figure *.1 demonstrates some typical effects of temperature and p: on the rate of an en&yme cataly&ed reaction. Ma/imum rate further assumes the presence of any co4en&ymes and8or cofactors that the en&yme re3uires. 7o4en&ymes are organic molecules which must bind to the protein portion of the en&yme in order to form the correct configuration for a reaction. 7ofactors are inorganic molecules which do the same. +ow, if we measure the concentration of an en&yme via its rate of activity ,i.e. the velocity of the cataly&ed reaction-, we must control the reaction for the effects of temperature, p:, salt concentration, co4en&ymes, cofactors, and substrate concentration. Dach of these parameters affects the rate of an en&yme reaction. $hus, each must be carefully controlled if we attempt to study the affects of changes in the en&yme itself. For e/ample, alterations in the rate of a reaction are directly dependent upon the concentration of functional en&yme molecules only when the en&yme is the limiting factor in the reaction. $here must be sufficient substrate to saturate all en&yme molecules in order for this criterion to be met. #f the substrate concentration is lowered to the point where it becomes rate limiting, it is impossible to accurately measure the en&yme concentration, because there will be two variables at work.

Figure *.9 Michaells4Menten plot of en&yme activity

Figure *.? Dffect of increasing en&yme concentration $he relationship between substrate concentration and en&yme concentration was mathematically established by pioneering work of two biochemists, !. Michaelis and M.!. Menten in 1O1?. #n recognition of their work, the plots of en&yme activity vs. substrate concentration are known as Michaelis4Menten plots. $hese are relatively simple plots ,Figure *.9- in which the substrate concentration is on the /4a/is, and the velocity of reaction is on the y4a/is. $he plot demonstrates that as the substrate increases, the velocity increases hyperbolically, and approaches a ma/imum rate known as = . $his is dependent upon saturation of the en&yme. At = , all en&yme

molecules are comple/ed with substrate, and thus any additional substrate added to the reaction has no effect on the rate of reaction.

:owever, this situation becomes more comple/2 as you change the en&yme concentration, = will also change ,Figure *.?-. $hus, = is not a constant value, but is constant only for a given can not be used directly to infer en&yme

en&yme concentration. 7onse3uently, the value of =

concentration. #t is dependent upon at least two variables, en&yme concentration and substrate concentration ,assuming temperature, p: and cofactors have all been controlled-. .hat Michaelis and Menten discovered was a simple means of solving the e3uations for two variables. #f multiple plots of en&yme activity vs. substrate concentration are made with increasing en&yme concentration, the value of = corresponds to 189 = continues to increase, but the substrate concentration which

remains constant. $his concentration is theMichaelis 7onstant for an and is operationally the concentration of substrate

en&yme. As mentioned, it is designated as B which will give e/actly 189 = and cofactors. According to the Michaelis4Menten e3uation2 = = 6 44444444444 B W ,S,S-

when reacted with an en&yme with ma/imum p:, temperature

,D3uation *.?-

$his e3uation is derived from the formula for a hyperbola ,c6/y- where B .hen v6= 89, B 6 ,S-,= 8,=

6 ,S-,=

8v41-

89-41- 6 ,S- confirming that the units of this constant are

those of concentration. A Michaelis4Menten plot thus can give us an easy way to measure the rate constant for a given en&yme. An immediate difficulty is apparent, however, when Michaelis4Menten plots are used. = is an asymptote. #ts value can only be certain if the reaction is run at an infinite concentration of substrate. ;bviously, this is an impossible prospect in lab. #n 1O?), two individuals, !ineweaver and %urke made a simple mathematical alteration in the process by plotting a double inverse of substrate concentration and reaction rate ,velocity-. $he !ineweaver8%urke e3uation is2 1 B W ,S-

44444 6 44444444444 v =

,D3uation *.),S-

$his e3uation fits the general form of a straight line, y6m/Wb, where m is the slope of the line and b is the intercept ,Figure *.)-. $hus, the !ineweaver8%urke <lot for an en&yme is more useful than Michaelis4Menten, since as velocity reaches infinity, 18= the plot results in a straight line, the slope is e3ual to B 8= approaches '. Moreover, since , the y intercept e3uals 18= ,when 18=6'-.

,18S6'-. <ro"ection of the line back through the / a/is yields the value 418B

$hese values can easily be determined by using a linear regression plot and calculating the corresponding values for /6' and y6'. $he inverse of the intercept values will then yield = and B .

Figure *.) !ineweaver4%urke plot of en&yme activity Remember that the point of all of these calculations is to determine the true activity and thus the concentration of the en&yme. #f the reaction conditions are ad"usted so that the substrate concentration is at B , then alterations in the rate of reaction are linear and due to alterations in en&yme concentration. Binetic analysis is the only means of accurately determining the concentration of active en&yme. 6pecific $cti(it0 $his brings us to a definition for en&yme activity. specific activity is defined in terms of en&yme units per mg en&yme protein. An en&yme unit is the amount of substrate converted to product per unit time under specific reaction conditions for p: and temperature.

As generally accepted, an en&yme unit is defined as that which cataly&es the transformation of 1 micromole of substrate per minute at ?'G 7 and optimal chemical environment ,p: and Substrate concentration-. Specific Activity relates the en&yme units to the amount of protein in the sample. .hile it is relatively easy to measure the protein content of a cell fraction, there may be a variable relationship between the protein content and a specific en&yme function. Remember that the initial e/traction of an en&yme is accomplished by differential salt precipitation. Many proteins will precipitate together due to their solubility, but have no other common characteristics. $o determine both protein content and en&yme activity re3uires two different procedures. .e can measure the amount of protein, or we can kinetically measure the en&yme activity. 7ombining the two will give us the specific activity. En30#e Inhi+ition Finally, before studying a specific en&yme, let@s e/amine the problem of en&yme inhibition. Remember that en&ymes function by sterically binding to a substrate. #f a molecule interferes with that binding, it will hinder or inhibit the activity of the en&yme. #f the inhibitor molecule binds to the same active site as the substrate, then the reaction is known as competitive inhibition because the two molecules, substrate and inhibitor, compete for the same reaction site on the en&yme molecule. .ith this type of inhibition, = because = will not change

is a function of all en&yme molecules uniting with substrate ,thus having no

effective competition-. B , on the other hand, will alter with changes in the concentration of a competitive inhibitor, because it re3uires larger concentrations of substrate to overcome the direct competition of the inhibitor for the active site. #f, however, the inhibitor binds to a site on the en&yme other than the active site, then the inhibition is known as allosteric, or non4competitive inhibition. #n this instance, the substrate and inhibitor bind to different parts of the en&yme molecule and thus are not in competition. An allosteric inhibitor alters the structure of the en&yme or physically blocks access to the active site. .ith non4competitive inhibition, = will change because, in effect, en&yme is being

removed from the reaction. #ts kinetic effects are e3uivalent to lowering the en&yme

concentration. B

will not change, however, since this value is constant regardless of the

effective en&yme concentration. Finally, there is a third class of inhibitor which can best be defined by its effect on = allosteric sites. $he graphic representation of the three types of en&yme inhibition are show in Figure *.* and B .

Bnown as uncompetitive, it alters both of these values. #t has effects on both the active site and

Figure *.* #nhibitor effects on !4% plots T0rosinase $his e/ercise involves the isolation ,e/traction- of the en&yme tyrosinase from potatoes and subse3uent measurement of its activity. $yrosinase is the common name for an en&yme that is formally termed Monophenol Monoo/ygenase and is listed as Dn&yme R1.1).1I.1 in the standard Dn&yme +omenclature. 1 #t is also known as phenolase, monophenol o/idase and as cresolase. #t is, functionally, an o/ygen o/idoreductase en&yme. $his nomenclature points out another difficulty of working with en&ymes. $heir names are derived from the known activities. Dn&ymes isolated from different sources and measured for their catalytic activity with varying substrates can turn out to be the same protein. $hus, the en&yme tyrosinase, discovered in animal systems, was named for its action on the amino acid tyrosine, and specifically for its ability to form dopa3uinone, an intermediate metabolite in the production of melanin. $he same en&yme isolated from plant materials had been e/amined for its ability to o/idi&e phenolic residues, and thus the names phenolase, monophenol o/idase and cresolase. Since it has

been e/tensively studied in melanin production, we will continue to use the common name of tyrosinase. $he en&yme tyrosinase is fairly ubi3uitous that is, it is found in nearly all cells. #n research, it has been purified from the fungus +. crassa 9 by free&ing kilogram 3uantities of the fungal mycelia in li3uid nitrogen, homogeni&ing the fro&en tissue with a French <ress, precipitating the proteins in ammonium sulfate, and purifying the en&yme chromatographically on Sephade/ and 7elite columns. A fairly comple/ undertaking. $yrosinase has been e/tracted from hamster melanomas by modifications of this techni3ue and with the addition of acetone e/tractions as well as CDAD4cellulose chromatography and alumina treatments. ?$yrosinase has also been separated from many plant tissues utili&ing a far simpler techni3ue based principally on ammonium sulfate precipitation of proteins. $he catalytic action of this en&yme is the conversion of tyrosine W ; to yield dihydro/yphenylalanine ,C;<A-, which is then converted to dopa3uinone W : ;. Copa3uinone in turn can be readily converted to dopachrome, an orange to red pigment ,found in human red hair-, which can then be converted to the black8brown melanin pigments ,found in virtually all human pigments-. $yrosine W 189 ; 44444Z C;<A

9 C;<A W ; 4444Z 9 Copa3uinone W 9 : ; Copa3uinone 4444Z !eukodopachrome !eukodopachrome W Copa3uinone 4444Z Copachrome W C;<A $he en&yme cataly&es the first two of these reactions, namely the conversion of tyrosine and the conversion of C;<A. $he formation of dopachrome from dopa3uinone is spontaneous. .e can now monitor the activity of the en&yme by analy&ing the disappearance of tyrosine and8or C;<A as substrates, the appearance of leukodopachrome or dopachrome as products, or by monitoring the use of o/ygen. <hysiologists and chemists have long preferred the manometric determination of gaseous o/ygen e/change, but far simpler is the determination of dopachrome, a natural pigment with an absorbance ma/imum at )>* nm. $his absorbance allows us to use

standard spectrophotometric analysis by analy&ing the formation rate of dopachrome from the substrate C;<A. $he summary reaction for tyrosinase activity, as used in this e/ercise is C;<A W 189 ; 4444Z Copachrome

Exercise 7.1 - Extraction of T0rosinase

!D=D! # Materials

<otatoes <aring Bnife %lender '.1 M +aF Rubber Aloves Saturated Ammonium Sulfate ,).1 M X9*G 7=olumetric 7ylinders ,*' ml, 1'' ml, 9*' ml7heesecloth %eakers ,1'' ml, 9*' ml7hilled 7entrifuge $ubes ,?'4*' mlRefrigerated 7entrifuge '.1 M 7itrate %uffer, p: ).I Alass Stirring Rod

Procedure
1. <eel a small potato and cut into pieces about 1 inch s3uare. 2. Add 1'' grams of the potato to a blender, along with 1'' ml of sodium fluoride ,+aF-.

:omogeni&e for about one minute at high speed. Caution: 6odiu# Fluoride is a poison) Cear ru++er glo(es &hile handling' and &ipe up an0 spills i##ediatel0.
3. <our the homogenate ,mi/ture- through several layers of cheesecloth and into a beaker. 4. Measure the volume of the homogenate and add an e3ual volume of saturated ammonium

sulfate. $hat is, if the fluid volume of your homogenate is 1*' ml, add 1*' ml of

ammonium sulfate. $his will cause a floculent white precipitate to appear as many of the previously soluble potato proteins become insoluble. $he en&yme tyrosinase is one of these proteins and thus will be found in the subse3uent precipitate.
5. Civide the ammonium sulfate4treated homogenate into chilled centrifuge tubes and

centrifuge at 1,*'' /g for * minutes X )G 7.

6. 7ollect the centrifuge tubes, and carefully pour off and discard the fluid

,supernatant-. 6a(e the pellets. 7ombine all of the pellets into a 1'' ml beaker.
7. Add 5' ml. of citrate buffer, p: ).I, to the pooled pellet and stir the contents well. 1se a

glass rod to break up the pellet. 7ontinue to stir for 9 minutes while keeping the solution cool.
8. Again divide the solution into centrifuge tubes and recentrifuge at ?'' /g for * minutes at

)G 7.
9. 7ollect and save the supernatant. This is 0our en30#e extract) <lace it in an erlenmeyer

flask, label it as Dn&yme D/tract and place it in an ice bucket. $he en&yme tyrosinase is insoluble in *'S ammonium sulfate, but is soluble in the citrate buffer. Beep this e/tract chilled for the duration of the laboratory. $yrosinase is stable for about an hour under the conditions of this e/ercise. #f not used within this period, you will need to e/tract more en&yme from a fresh potato.

Exercise 7.. - Preparation of 6tandard Cur(e

!D=D! # Materials

I mM C;<A Dn&yme D/tract 4 From D/ercise *.1 $est tubes * ml <ipette '.1M 7itrate %uffer, p: ).I Spectrophotometer and 7uvettes

Procedure
1. %egin by preparing a standard solution of the orange colored dopachrome from !4C;<A.

$o 1' ml of I mM C;<A, add '.* ml of your en&yme e/tract ) and allow the solution to

sit for 1* minutes at room temperature. Curing this period, all of the C;<A will be converted to dopachrome, and your solution will now contain I mM dopachrome. Copachrome is somewhat unstable in the presence of light and should be stored in an amber bottle or out of the light.
2. <repare a 121 series of dilutions of the I mM Copachrome to yield the concentrations in

the following table2 Add ?.' ml of each indicated concentration to tubes R14I. Tu+e 1 9 ? ) * 5 > I Final Concentration of *opachro#e (#M! ' '.19* '.9* '.* 1.' 9.' ).' I.'

3. .ith these dilutions, you have prepared tubes containing concentrations from ' to I mM

dopachrome ,tubes 14I-. $ube 1 contains no dopachrome and is used for blanking the spectrophotometer.
4. $he units of concentration are millimolar ,mM-. A 1.' mM solution contains .''1 moles

per liter or .'''''1 moles per ml. $hus, with a volume of ?.' ml, there are .'''''? moles of dopachrome, or ? micromoles. 7orrespondingly, tubes 94I contain 1 to 9) micromoles of dopachrome. For the remainder of this e/ercise, be sure to distinguish between concentration ,mM- and total amount of substance present ,micromoles-.
5. $urn on your spectrophotometer and set a wavelength of )>*. 1se tube R1 from the above

dilutions as a blank and ad"ust the spectrophotometer for ' and 1''S $. Read the absorbance ,or read and convert transmittance- of each of the solutions in tubes 94I and complete the following table2 $ube 7oncentration of Absorbance A87

Copachrome ,mMR1 R9 R? R) R* R5 R> RI ' '.19* '.9* '.* 1.' 9.' ).* I.' ' 4444

6. 7alculate the values for the last column of the table. $his column represents the simplest

calculation of the e/tinction coefficient for dopachrome absorbance. Average the values in this column and enter the number at the bottom of the column. $his is the average e/tinction coefficient and can be used in subse3uent determinations of dopachrome concentrations according to the %eer4!ambert law. Jou can more accurately determine the e/tinction coefficient by performing a linear regression analysis of your data, and computing the slope and y intercept. $he slope of the linear regression will represent the e/tinction coefficient for your sample.
7. <lot a scattergram of the absorbance value against the concentration of dopachrome. $he

known concentration of dopachrome should be the / a/is, while absorbance should be the y a/is.
8. <lot the computed slope and intercept of the linear regression as a straight line overlaying

your scattergram. $he e3uation for a straight line is y 6 m/ W b, where m is the slope and b the intercept. * "otes Since tyrosinase cataly&es the conversion of !4C;<A to dopachrome, this e/ercise measures the conversion of colorless C;<A to the dark orange dopachrome. Substrate and product are in a 121 ratio for this reaction, thus the amount of product formed e3uals the amount of substrate used. $he optical density of dopachrome X)>* nm is directly proportional to the intensity of orange color formation in solution ,%eer4!ambert !aw-.

Exercise 7.5 - En30#e Concentration

!D=D! # Materials

Dn&yme D/tract '.1 M 7itrate %uffer, p: ).I 1' ml <ipette I mM C;<A Spectrophotometer and 7uvettes #ce %ath

Procedure
1. $o determine the kinetic effects of the en&yme reaction, first determine an appropriate

dilution of your en&yme e/tract. $his will give a rate of reaction of *41' micromoles of C;<A converted per minute. <repare a serial dilution of your en&yme e/tract. <lace O.' ml of citrate buffer into each of three test4tubes. !abel the tubes 181', 181'' and 181'''.
2. <ipette 1.' ml of your en&yme e/tract into the first of these tubes ,the one labeled 181'-

and mi/ by inversion.

3. <ipette 1.' ml of the 181' dilution into the second tube ,labeled as 181''- and mi/ by

inversion.
4. <ipette 1.' ml of the 181'' dilution into the third tube ,labeled as 181'''- and mi/ by

inversion.
5. <lace all of the dilutions in the ice bath until ready to use. 6. #f not already done, turn on a spectrophotometer, ad"ust to )>* nm and blank with a tube

containing 9.* ml of citrate buffer and '.* ml of en&yme e/tract.

7. Add 9.* ml of I mM C;<A to each of ) cuvettes or testtubes. +ote that each tube

contains .''9* ( .''I moles or 9' micromoles of C;<A.

8. Add '.* ml of undiluted en&yme e/tract to one of the tubes containing the I mM C;<A.

Mi/ by inversion, place into the spectrophotometer and immediately begin timing the reaction. 7arefully measure the time re3uired for the conversion of I micromoles of C;<A. +ote that since the cuvette will contain a volume of ?.' ml, the concentration when I micromoles are converted will be I8?.' or 9.5> mM dopachrome. 1se the data

from the standard curve ,D/ercise *.9- to determine the absorbance e3ual to 9.5> mM dopachrome. $his absorbance value will be the end point for the reaction. $he Absorbance e3ual to ?.?? mM dopachrome ,from D/ercise *.9- 6 KKKKKKK
9. As the reaction takes place within the spectrophotometer, the absorbance will increase as

dopachrome is formed. .hen the absorbance reaches the value above, note the elapsed time from the mi/ing of the en&yme e/tract with the 1' mM C;<A. D/press the time as a decimal rather than minutes, seconds. $he time should be between three and five minutes. #f the end point is reached before three minutes, repeat Step I, but using the ne/t dilution of en&yme ,i.e. the 181' after the undiluted, the 181'' after the 181' and the 181''' after the 181''-. $he rate of activity 6 KKKKKKKKKKKKKKKK micromoles8minute8'.* ml of diluted e/tract. $he dilution factor ,inverse of dilution, 1,1',1'' or 1'''- is KKKKKK. $he activity of the undiluted en&yme is KKKKKKKKKKKK micromoles8minute8'.* ml or KKKKKKKKKKKKK micromoles8minute81.' ml of e/tract.
10. For the en&yme dilution which reaches the end point between ? and * minutes, calculate

the velocity of reaction. Civide the amount of product formed ,1' micromoles- by the time re3uired to reach the end point.

Exercise 7.1 - Effects of p?

!D=D! # Materials

I mM C;<A in citrate buffer ad"usted to p: values of ?.5, ).9, ).I, *.), 5.', 5.5, >.9, >.I Dn&yme D/tract Spectrophotometer and 7uvettes Stopwatch

Procedure
1. Set up a series of test tubes each containing 9.* ml of I mM C;<A, but ad"usted to the

following p: values2 ?.5, ).9, ).I, *.), 5.', 5.5, >.9, and >.I

2. %egin with the tube containing C;<A X p: ?.5, add '.* ml of the diluted en&yme e/tract

which will convert 1' micromoles of C;<A in ?4* minutes as determined in D/ercise *.?. Start timing the reaction, mi/ by inversion and insert into the spectrophotometer. +ote the time for conversion of 1' micromoles of C;<A.
3. Repeat Step 9 for each of the indicated p: values. 7omplete the following table2

p: ?.5 ).9 ).I *.) 5.' 5.5 >.9 >.I

$ime,Minutes-

Micromoles of Copachrome 1' 1' 1' 1' 1' 1' 1' 1'

=elocity ,Micromoles8Minute-

4. <lot p: ,/4a/is- versus Reaction =elocity ,y4a/is-

Exercise 7.7 - Effects of Te#perature !D=D! # Materials

Dn&yme D/tract I mM C;<A p: 5.5 #ncubators or .ater baths ad"usted to 1', 1*, 9', 9*, ?', ?* and )'G 7 5 Spectrophotometer and 7uvettes Stopwatch

Procedure

1. Set up a series of test tubes each containing 9.* ml of I mM C;<A buffered to a p: 6

5.5. <lace one tube in an ice bath or incubator ad"usted to the following temperature 1', 1*, 9', 9*, ?', ?* and )'G 7
2. Add '.* ml of an appropriately diluted en&yme e/tract ,to yield 1' micromoles

dopachrome in ?4* minutes- to each of a second series of tubes. <lace one each in the corresponding temperature baths. Allow all of the tubes to temperature e3uilibrate for * minutes. *o not #ix the tu+es.
3. %eginning with the 1'G 7 tube, and with the spectrophotometer ad"usted to )>* nm and

properly blanked, pour the en&yme ,'.* ml X 1'G 7 - into the tube containing the C;<A and begin timing the reaction. Mi/ thoroughly. +ote the time to reach the end point e3uivalent to the conversion of 1' micromoles of substrate.
4. Repeat Step ? for each of the listed temperatures, complete the following and plot the

data. Te#perature (D C ! 1' 1* 9' 9* ?' ?* )' Ti#e (Minutes! Micro#oles of *opachro#e 1' 1' 1' 1' 1' 1' 1' :elocit0 (Micro#oles8Minute!

Exercise 7.9 - Co#puter 6i#ulation

!D=D! # Jou will note that for the determination of p: and temperature effects, we established conditions that scanned a wide range of the variables. $o refine these procedures, we would add more

values, for e/ample, temperatures from ?'4 )'G 7 at steps of 1.'G 7 or ?54?>G 7 with a step of '.1G 7. $his is time consuming. $o establish the principle, however, let@s shift to computer simulation of en&yme activity. 7omputer simulations will also assist in the rapid accumulation of data for kinetic analysis. $here are several e/cellent commercial programs available for en&yme kinetic analysis, and use of these must be left to the descretion of the instructor. $he author has had e/cellent results with a rather old %AS#7 program, D+FB#+, which is still available from 7onduit. > $his is a simple, straight4forward and ine/pensive program which is available for Apple 7omputers and for main frames. #t can readily be converted to run on other machines with some simple source code changes. 1nfortunately, 7onduit may not continue supplying this program in the future. #f you do not wish to convert programs, or if you prefer a more sophisticated program is desired, the author recommends D+F<A7B =er. 9.'. I #n addition to simulation of en&yme kinetics, this program allows entry of data with subse3uent graphing and analysis. #t also allows for high order kinetic analysis in addition to !ineweaver4%urk plots.

Exercise 7.< - Einetic anal0sis

!D=D! ## Materials

I mM C;<A p: 5.5 Dn&yme D/tract, diluted to yield 1' micomoles of dopachrome in ?4* minutes ,D/ercise *.9Spectrophotometer and 7uvettes Stopwatch

Procedure
1. <repare a reaction blank in a clean cuvette to contain 9.* ml of citrate buffer and '.* ml

of en&yme e/tract. 1se this blank to ad"ust your spectrophotometer for 1''S transmittance. Remove the blank and save.
2. Add 9.* ml of I mM C;<A, p: 5.5 to a clean cuvette.

3. Add '.* ml of appropriately diluted en&yme e/tract. Shake well and immediately insert

the tube into the spectrophotometer. Record the absorbance or transmittance as 3uickly as possible. Cesignate this reading as time '.
4. At ?' second intervals read and record the transmittance until a transmittance value of

1'S ,Absorbance 6 1.'- is reached. 7omplete the following table2 $ime ,Minutes'.* 1.' 9.' 9.* ?.' ?.* ).' ).* *.'
5. <lot time in minutes ,/4a/is- versus the amount of dopachrome formed ,y4a/is-.

Absorbance

7oncentration of

Micromoles of

Copachrome,mM- Copachrome

Exercise 7.= - *eter#ination of E# and :#ax

!D=D! ## Materials

Dn&yme D/tract I mM !4C;<A ad"usted to p: 5.5 Spectrophotometer and 7uvettes Stopwatch

Procedure
1. Cilute the C;<A standard ,I mM- to obtain each of the following concentrations of !4

C;<A2 '.* mM, 1 mM, 9 mM ) mM, and I mM.

2. Repeat D/ercise *.> for each of the substrate concentrations listed, substituting the

change in concentration where appropriate.

3. <lot each set of data and from the data calculate the time re3uired to convert 1'

micromoles of C;<A to dopachrome. 7ompute the velocity of en&yme reaction for each substrate concentration. Fill in the following table2O 6u+strate (*/P$! Concentration (#M! '.* 1.' 9.' ).' I.' :elocit0 Micro#oles8Minute 18s 9.'' 1.'' '.*' '.9* '.19* 18(

4. <lot the rate of C;<A conversion ,v- against substrate concentration in the appropriate

place below. $his is a Michaelis4Menten plot. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q Q Michaelis4Menten plot Q Q Q Q Q Q Q Q Q

QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQ

5. <lot a double reciprocal of the values plotted in step ) that is, 18s versus 18v. $his is a

!ineweaver4%urke plot. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK Q Q Q Q Q Q Q Q QKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK !ineweaver4%urke plot

6. <erform a linear regression analysis on the second plot and compute the slope and both y

Q Q Q Q Q Q Q Q

and / intercepts. +ote that the / intercept is 418B , the negative inverse of which is the Michaelis4Menten 7onstant. $he y intercept is 18= and the slope e3uals Bm8= .

Exercise 7.> - $ddition of En30#e Inhi+itors

!D=D! ## Materials

Dn&yme D/tract I mM !4C;<A I mM %en&oic Acid I mM B7+ '.1 M 7itrate %uffer, p: 5.5

Procedure
1. $yrosinase is inhibited by compounds that comple/ with copper, as well as by ben&oic

acid and cyanide. $o determine the inhibitory effects of ben&oic acid and cyanide, set up a series of tubes as indicated.

2. 1sing one tube at a time, add '.* ml of the en&yme dilution previously calculated to yield

1' micromoles of dopachrome in 94? minutes. For each tube, measure the time re3uired to convert 1' micromoles of C;<A to dopachrome. Dnter those times in the table below. 7ompute the reaction velocity for each substrate concentration2 Tu+e 1 %en&oic Acid 9 #nhibited ? Series ) * 5 > I O 1' 11 Ti#e (Minutes! Final F*/P$G nM 5.5> 5.'' *.?? ).5> ).'' ?.?? 9.5> 9.'' 1.?? '.5> ' :elocit0 Micro#oles8Minute

19 B7+ 1? #nhibited 1) SDR#DS

5.'' *.?? ).5>

1* 15 1> 1I 1O 9' 91

).'' ?.?? 9.5> 9.'' 1.?? '.5> '

3. 7alculate the values for 18s and 18v for each of the corresponding s and v in the table

below. <lot 18v vs 18s for the presence of ben&oic acid and a second plot for the presence of B7+. 7ompute the values of = and B for the presence of each inhibitor.

Cetermine whether these inhibitors are competitive, non4competitive or uncompetitive.

Ben3oic $cid Inhi+ition Tu+e 1 9 ? ) * 5 > = #M */P$ 9.' 1.I 1.5 1.) 1.9 1.' '.I = #M Ben3oic $cid '.* '.* '.* '.* '.* '.* '.* Buffer ' '.9 '.) '.5 '.I 1.' 1.9

I O 1' 11

'.5 '.) '.9 ' EC" Inhi+ition

'.* '.* '.* '.*

1.) 1.5 1.I 9.'

Tu+e 19 1? 1) 1* 15 1> 1I 1O 9' 91

= #M */P$ 1.I 1.5 1.) 1.9 1.' '.I '.5 '.) '.9 '

= #M EC" '.* '.* '.* '.* '.* '.* '.* '.* '.* '.*

Buffer '.9 '.) '.5 '.I 1.' 1.9 1.) 1.5 1.I 9.'

Exercise 7.1, - Protein Concentration8En30#e $cti(it0

!D=D! ## Materials

7ommercially pure tyrosinase 1= Spectrophotometer or Materials for !owry or %radford <rotein determination !4C;<A '.1 M 7itrate buffer, p: 5.5

Procedure
1. <repare a solution of '.> micrograms of commercially pure tyrosinase diluted to ) ml.

with '.1 M 7itrate %uffer, p: 5.5.

2. Measure the ;C

of your sample and prepare a dilution of your en&yme e/tract to a

final concentration of '.> micrograms in ) ml of citrate buffer.

3. <lace both en&yme samples in a water bath X ?'G 7 for * minutes to temperature

e3uilibrate.
4. $urn on the spectrophotometer, set the wavelength to )>* nm and blank the instrument

using citrate buffer as the blank.

5. Select the commercial preparation and add e/actly 1.' ml of !4C;<A ,) mg8ml in citrate

buffer- and immediately read the absorbance at )>* nm.

6. Replace the tube in the water bath and wait e/actly * minutes. Read the ;C

immediately.
7. $he molar absorbance coefficient for dopachrome is ?.> / 1' . 1se this value to compute

the specific activity of the commercial en&yme preparation. 7heck this activity against that listed with the en&yme preparation.
8. Repeat steps * and 5 with your e/tracted en&yme preparation. 7ompute the specific

activity ,en&yme units of activity8 mg protein- of your en&yme preparation. En!yme Unit1 The absorbance rea&ing un&er the con&itions specifie& in this e(ercise is proportional to the en!yme concentration$ 0here 1 unit of en!yme acti*ity yiel& a 2.31 "- change in rea&ings. "otes $he protein content can be measured by the !owry or %iuret procedures found in the Appendi/ A, or more simply by a single spectrophotometric measure of the absorbance of the sample at

9I' nm. .ithout going into mathematical detail, a 1S pure solution of tyrosinase has an ;C e3ual to 1*.58cm. $he %eer4!ambert law can thus be used to determine protein content in a non4 destructive manner.

Exercise 7.11 - $d(anced $nal0sis of En30#e Para#eters

!D=D! ### #n the e/traction procedure for tyrosinase, only one precipitation was employed utili&ing ammonium sulfate. $his procedure yields a rapid means for obtaining the en&yme, but results in increased contamination and lower yield. $he following improved procedure uses a serial increase in ammonium sulfate. After blending the potato, add 189 volume of ammonium sulfate to yield a ??S solution as opposed to *'S. 7ollect the precipitate and label it as <1. Add another 189 volume of ammonium sulfate to raise the concentration to *'S and collect and label the resulting precipitate as <9. Finally add a third 189 volume to raise the concentration to 5>S and collect and label the precipitate as <?. 1se each of these to measure the specific activity as well as the total activity in each fraction. $he step4wise addition of ammonium sulfate will increase the specific activity. #f pure en&yme is desired, the steps can be made even smaller, going from ?'S to >'S in 1'S increments. #f you wish further purification, then sub"ect the resulting precipitated protein to electrophoretic separation and analy&e the resulting bands for their tyrosinase activity. .hile these procedures can yield pure en&yme fractions, they are more pertinent to biochemical studies. A more interesting analysis can be made by comparing tyrosinase e/tracted from differing biological materials ,mushrooms, bananas, melanocytes- for the values of Bm and for their optimum p: and temperature. %ased on those differences, might it be possible to predict the darkening of fruit under varying conditions of p: and temperatureP Finally, the en&yme can be studied in a structure linked mode by combining aspects of cell organelle isolation ,7hapter $hree- to the activity of the en&yme. $he presence ,or absence- of tyrosinase can be monitored within the organelles of various cells. Melanocytes would be an

e/cellent source of cellular tyrosinase. Jou could then correlate the activity of tyrosinase with the production of the pigment melanin. Chapter 7: En30#es - Endnotes

1. 2.

+umerical order or en&ymes as classified by the #nternational 1nion of %iochemistry. +.:. :orowit&, M. Aling and A. :orn. $yrosinase ,4eurospora crassa- in Methods in Dn&ymology, =ol. (=##. ,:. $abor and 7... $abor eds.- p. 51*4 59'. 1O>'. S.:. <omerant& and T.<. !i, $yrosinase ,:amster Melanoma- in Methods in Dn&ymology, =ol (##, p 59'4595. #f the e/traction of tyrosinase is to be skipped, a commercial preparation of '.> micrograms of tyrosinase ,),''' units8mg- in ) ml of citrate buffer may be substituted. $his can be easily accomplished with many standard 0scientific0 calculators, most computer spreadsheets, or with the %AS#7 program listed in Appendi/ C. $he e/ercise is easier to perform if a temperature controlled spectrophotometer is available, since the reaction rate can be monitored at the proper temperature. !acking this piece of e3uipment, however, the reaction can be placed in an ice bath or incubator and monitored every ?' seconds. 7onduit, 1niversity of #owa, ;akdale 7ampus, #owa 7ity, #owa *99)9. %#;S;F$, <; %o/ *I', Milltown, +T 'II*'. +ote that the table lists the concentrations as those of the initial substrate. $his is not technically correct since the addition of '.* ml of en&yme will dilute the substrate. #f D/ercise *.O is to be performed, the values of 18s will need to be ad"usted accordingly.

3.

4.

5.

6.

7. 8. 9.

Chapter 9: Me#+ranes - Introduction

Although not all researchers agree on the fundamental nature of membranes, all agree that membranes take on properties fundamental to the very e/istence of life. Following the theory of Albert S&ent4Ayorgyi, they believe that the understanding of life can be achieved through an understanding of the mechanisms for electron flow through basic organic systems ,photosynthesis and respiration- and that these systems in turn e/ist only

through compartmentali&ation within a cell. .ithout it, there would be no organelles, and indeed, no cell. .hile membranes can be studied within living cells, membrane composition re3uires isolation and subfractionation of the membrane components. ;nce isolated, the membranes can be solubili&ed through the use of detergents and analy&ed for proteins, lipids and carbohydrates. $he study of biomembranes can be done with techni3ues re3uiring minimal e3uipment, such as osmotic shrinking or swelling of erythrocytes. Some techni3ues can be so comple/ as to re3uire a complete organic and physical chemistry lab. $he labs in this e/ercise can be completed with the e3uipment in the average college biochemistry lab. $he earliest investigations of membranes were those concerned with chemical analysis and were based on the observation that lipid solvents readily permeated cells. Membranes were known to contain protein, and from these early studies, it was concluded that lipids were also a significant factor in membrane composition. $he specific nature of the proteins and lipids has been the sub"ect of much research since these early days. As an e/ample of the protein and lipid composition of membranes, as well as the percentage of some other substances, $able 5.1 presents the composition of myelin membranes from the central nervous system and the brain. Myelin is one of the most often studied biomembranes. #t is readily available in 3uantity, and has a relatively simple composition when compared to membranes from other cells. $here are three ma"or types of protein found associated with myelin, basic protein, Folch4 !ees proteolipid, and .olfgram protein. $here is a high percentage of lipid composition and the lipids are reasonably uncomplicated. ;f course, this is a simple view of myelin based on early chemical e/tractions. Anatomists have long recogni&ed the distinction between myelin from the central nervous system ,7+S- and that from the peripheral nervous system ,<+S-. 7+S myelin is formed by oligodendrocytes, while <+S myelin is formed by the Schwann 7ells. $he <+S system is the most often illustrated because of the ease of identification of the laminar membranes of the system. %iochemical analysis of myelin further indicates that there may be subtle differences even within the broad categories of myelin, with, for e/ample,

clear distinction between that found in the brain and the spinal cord of the 7+S, additional distinctions among species. ;ften the chemical composition is of interest by itself, but more intriguing are the dynamic characteristics of membranes, ,such as semi4permeability- and functions ,such as active transport and facilitated transport-. $o study these properties, a relatively new approach has been developed2 the synthesis of artifical membranes in the form of lipid bilayers ,also referred to as bilayer lipid membranes or %!M@s-. $he lipid bilayers are composed of natural or synthetic lipids that are artificially held between two a3ueous environments. $able 5.9 presents a list of the ma"or lipids used for lipid bilayer formation, while $able 5.? indicates some of the membranes these systems have modeled. Artificial membrane research has been significantly advanced by the addition of membrane components e/tracted from biological systems. $he components have come from brain e/tracts, chloroplast or mitochondrial membranes, and have been augmented with o/idi&ed cholesterol and any number of surfactants. Researchers have been able to create fairly comple/ artificial membranes and which can mimic nearly all of the 3ualities of a cell membrane. $hey have formed the lipid membranes as sheets spread across tiny apertures, or as small droplets within an a3ueous environment. Sheets are more relevant to membrane permeability studies, while droplets ,known as liposomesare useful for analy&ing cell fusions and membrane flow. Jet another approach to membrane analysis has been the use of markers or probes. $he work utili&es either en&ymes or immunofluorescenct reagents. More recently, fluorescent dyes have been covalently attached to specific membrane proteins, which can in turn be micro4in"ected into cells and their paths through the cell and monitored by computer aided video systems. .ith this new advancement, 3uantitative as well as 3ualitative analysis of membrane flow in a dynamic living cell is possible. $hese chemical studies augment the abundant data available from the work of virologists on such phenomena as capping, and membrane flow related to viral reproduction. $hey are also supplemented by studies of vessicle8vacuole dynamics during cell endocytosis. Finally, physiologists have studied ion flu/ across membranes. +o view of membranes would be complete without discussion of osmosis, diffusion, active transport and facilitated diffusion. $hese processes of flu/ are mathematically defined by the

movement of ions across the membrane. .hile virtually every type of cell has been studied, the ma"or work has involved the erythrocyte and various components of nerve and muscle function. $hrough all of these approaches, a coherent theory for membrane structure is emerging. Most membrane analysts believe that essentially membranes are lipids in fluid suspension between two a3ueous phases ,inside and outside the cell-, while the proteins are then attached to this lipid bilayer. Some researchers, however, feel that the protein matri/ of the membrane is set and gives substance to the membrane. !ipids are then attached to a protein substrate due to their hydrophobic or hydrophyllic tendencies. $his latter view would account for the more structured nature of polar cellular membranes, which do not appear to behave strictly as fluids.

Exercise 9.1 - Bipid 6olu+ilit0 of Me#+ranes

!D=D! # Materials

Fresh beets Solutions of the following alcohols2

o o o o o

99 M Methanol I.* M Dthanol ?.' M n4<ropanol 1.1 M n4%utanol '.?I M Amyl alcohol ,;ptional-

Ra&or blades Cepression slides Stopwatch Microscope

Procedure 1
1. %eet cells contain a high concentration of the red pigment anthocyanin. .hen e/posed to

a compound which dissolves the cell membranes, the anthocyanin will leak out of the cells and cause a red color to occur in the surrounding media.

7ut thin slices of a beet so that they can be placed on a microscope depression slide and viewed with the lowest power ,)(-.
2. .hile watching the edge of the sliced beet, add appro/imately 1.' ml of each of the

above alcohols to the slide, until the beet section is submerged. %e careful not to allow the alcohol to flow off the slide. Iso-a#0l alcohol has a strong' o+noxious odor and the fu#es are so#e&hat irritating. $de;uate (entilation is re;uired.
3. #mmediately begin to time the dissolution of the beet cell membranes. Mark the time

when a red color is first observed in the surrounding alcohol solution.

4. Repeat the entire series for 189 and 18) dilutions of each of the alcohols. 5. For each dilution of each alcohol, calculate a penetration coefficient by dividing the time

of pigment appearance by the molar concentration of the alcohol. #n the space given on page 1?>, plot this penetration coefficient against the relative miscibility of the alcohol ,known as the partitition or distribution coefficient-. $lcohol Methanol Dthanol n4<ropanol n4%utanol n4Amyl alcohol For#ula 7: ;: 7 : ;: 7 : ;: 7 : ;: 7 : ;: Molecular Ceight ?9.') )5.'> 5'.'O >).19 II.1* Partition coefficient '.'1 '.'? '.1? '.*I 9.''

Exercise 9.. - /s#osis

!D=D! # Materials

Dlodea, guard cells from plant leaves or animal blood

Solutions ranging from '.'* M to '.*' M each of sucrose, sodium chloride, potassium chloride and calcium nitrate Ra&or blades Cepression slides Microscope ;smometer ,;ptional-

Procedure
1. <lace '.* ml of '.*' M +a7l into the center of a depression slide and add either a small

piece of an Dlodea leaf, the stripped lower epidermal layer of a leaf containing guard cells, or a small drop of blood.
2. <lace the slide on the microscope and observe the cells for swelling or shrinking.

Swelling is difficult to determine with Dlodea since the cell wall inhibits swelling. 7ell shrinking can be observed as a pulling away of the cell membrane from the cell wall. #n guard cells, the swelling will open the stomates, while shrinking will cause the stomates to close. Red blood cells react 3uickly to changes in the environmental salt concentrations and will shrink or swell. Shrinking is observed as a wrinkling or crenulation of the cell, and swelling may proceed to the point where the cells burst ,plasmolysis-.
3. Repeat steps 1 and 9 se3uentially with '.), '.?, '.9, '.1 and '.'* M solutions of +a7l.

+ote which solution induces cellular shrinking or swelling. ;ne solution will result in neither shrinking nor swelling. $his is known as the isotonic solution. $hose causing the cell to shrink are known as hypertonic, and those causing swelling are hypotonic.
4. Repeat steps 14? with each of the remaining salts and with sucrose. +ote which

concentration of each is the e3uivalent of the isotonic +a7l. $onic refers to the +a7l e3uivalence of a solution. #t is more correct for other salts and especially for organic non4 electrolytes to use the terms isosmotic rather than isotonic.
5. %ased on the molarity of the salt solutions, calculate the osmolarity of each solution and

compare the osmolarity of each solution that is isosmotic to the cells under study. Multiplying the osmolarity by 99.) will yield the osmotic pressure in atmospheres. /ptional

1se an osmometer to determine the precise osmolarity of each solution. ;smometers measure the free&ing point depression, which in turn can be related to an e3uivalent solution of +a7l. #f the osmometer is available, follow the manufactuer@s instructions for its use.

Exercise 9.5 - Pinoc0tosis

!D=D! # Materials

Actively growing culture Amoeba proteus 9 '.''1 M Alcian blue '.'1 M Sodium a&ide <etroleum "elly Slides, coverslips Microscope

Procedure
1. Form a small ring of petroleum "elly onto a microscope slide. 2. Add a small drop of Amoeba suspension, and add about 1' Ll of alcian blue to the slide.

+ote the time. #mmediately place a coverslip onto the slide and gently press to seal the amoeba within the petroleum "elly ring.
3. ;bserve the Amoeba with 1' ( magnification of the microscope. ? 4. Record the time for each of the following events2

a. Surface staining b. Rounding up of the cell c. Formation of rosette ,crinkling of celld. 7hanneling ,flattened pseudopodia and invagination of membranese. <inosome formation ,small vessicles of dye pinched off from invaginated membranes-.
5. <repare another slide with a "elly ring, but add 1' Ll of sodium a&ide along with the

Amoeba. Allow the Amoeba to remain in the a&ide solution for about * minutes and then add the alcian blue and a coverslip. Repeat steps ? and ).

6. 7ompare the times for each event of step ) for the control and for those Amoeba treated

with the a&ide. "otes Membranes normally do not allow larger molecules ,such as dyes- to enter a cell through simple diffusion. #f a dye is presented to an amoeba, however, the dye will be incorporated as part of what is referred to as receptor mediated endocytosis. #n more general terms, the cell will drink or eat the dye, i.e. pinocytosis ,cell drinking- or phagocytosis ,cell eating-. $he dye will enter into internal vacuoles, known as food vacuoles. .ith some dyes, the cell will actively transport the dye back out of the cell. #t re3uires energy for this dye e/clusion, energy derived from metabolism. Dnergy is only produced in living cells, and the phenomenon can be monitored in what is known as the dye e/clusion test. $he dye e/clusion test becomes a way to monitor cell viability ,7hapter 19-. For now, however, it implies that we can monitor the health of an amoeba by timing the movement of dye, and we can observe alterations in membrane function by alterations in the process of dye movement. A healthy, functioning cell will not stain and will have a minimum number of dyed vacuoles. #ts membrane will remain intact and there will be minimum interruption of its ligand4mediated endocytotic processes. A damaged cell, however, will undergo physiological and morphological changes as the membrane receptor sites become irreversibly bound to dye. As it loses its ability to e/crete the dye, it will become stained, round up and ultimately die.

Exercise 9.1 - Co#puter 6i#ulation of Me#+rane Function

!D=D! # Materials

7D!!M program found in Appendi/ C 7omputer capable of running %AS#7

Procedure
1. $urn on the computer, boot with an appropriate C;S, and load the program 7D!!M.

$he program asks you to make decisions about the amount of water, sugar, ions and wastes that are within an E. Coli cell. 7hanges in the cell due to osmosis and diffusion take place automatically. Dnergy is re3uired for the movement of wastes and for other

functions ,such as preparation for reproduction-. As you utili&e the sugar for energy, wastes will build automatically. $o dispose of waste re3uires ) molecules of sugar. From the time you begin the program, the cell will burn sugar for energy. Ciffusion and osmotic potentials go into effect. #f you successfully manipulate the parameters, the cell will divide. ;therwise, your cell dies.
2. Jour initial concentrations of materials are as follows2

.ater Sugar Sodium <otassium .aste

5>S or 5>'' molecules ?' molecules ' molecules *? molecules '

3. Jour cell is then placed into a solution containing2

.ater Sugar Sodium <otassium .aste

I>S or I>'' molecules 1'S or 1''' molecules trace trace '

4. Jou must ad"ust the five parameters of the cell, while maintaining viability. Jour safe

concentrations are2 .ater Sugar Sodium 5'''4I''' molecules 1'4?* molecules '41? molecules

<otassium .aste

)'4*? molecules '41' molecules

5. .hile you play the role of a membrane, attempt to understand the relationship of the

various components to each other.

6. Submit a single page report on the game, detailing the components, and how they interact

to make the cell divide.

Exercise 9.7 - Extraction of Brain Bipids

!D=D! ## Materials

7alf brain ) 7hloroform Methanol '.1 + :7l %lender or tissue homogeni&er Refrigerated preparative centrifuge !arge bucket rotor Free&e dryer %uchner funnel and filter paper ,with cold trap-

Procedure
1. :omogeni&e 9* g of white matter from calf brain, in *'' ml of chloroform2methanol

mi/ture ,921, v8v-. *o not use an ordinar0 +lender. The sol(ents &ill dissol(e the gas4et and #a0 +urst into fla#es)
2. 7entrifuge the mi/ture in a large scale rotor X 1',''' /g for five minutes at ) G 7 to

clarify the solution.

3. Add 9' ml. of distilled water per 1'' ml of solution from step 9 and shake to form an

emulsion.
4. !yophili&e to dryness.

5. Cissolve the residue in *' ml of chloroform and filter through three layers of filter paper

in a buchner funnel. 1se a water vacuum system and a cold trap to prevent chloroform vapors from entering a pump and causing a fire.
6. Add 1.9 volumes of methanol. 7. Add 1.* volumes of '.1 + :7l and shake vigorously. 8. 7entrifuge and collect the lower phase. Ciscard supernatant and interphase material. 9. Filter the lower phase three times with fresh filter paper. 10. Add 18? volume of methanol to the filtrate and store in 1.' ml lots in a free&er. $he

e/tract will remain stable for appro/imately 9 months.

Exercise 9.9 - For#ation of Bipid Bila0er

!D=D! ## Materials

!ipids e/tracted per D/ercise 5.* 4$ocopherol 7holesterol 1' ml $eflon cup and larger polystyrene beaker Sable paintbrush '.1 M +a7l #nterference scale

Procedure
1. Form a working lipid bilayer solution "ust before use, by adding )'' mg alpha4tocopherol

and ?' mg cholesterol to 1.' ml of the e/tracted lipids from D/ercise 5.5. *
2. Make a chamber from a teflon cup by piercing the cup with a 141.* mm hole, and placing

the cup into a slightly larger polystyrene beaker.

3. Fill both the teflon cup and the outer chamber with '.1 M +a7l. 4. 1se a fine sable brush ,?4* hairs- to apply a small amount of the working solution to the

hole in the teflon cup. %e careful to spread the lipid solution evenly but do not puncture the membrane which will form.
5. ;bserve the characteristics of the membrane formation visually.

$he solution will spread over the hole, then begin to recede and form a gradually thinning membrane. As it does so, the optical properties will change, especially if viewed with reflected light with a simple hand held magnifier ,1'49'(-. $he membrane will demonstrate interference colors as it begins to thin and will ultimately become thin enough to become completely transparent. As it becomes transparent, it becomes known as a black membrane.
6. 1sing an interference scale, plot the thickness of the membrane over time during its

formation.

Exercise 9.< - Electrical Properties of $rtificial Me#+ranes

!D=D! ## Materials

Artificial lipid membrane from D/ercise 5.5 B7l electrodes 5 C7 voltage source .heatstone bridge to monitor resistance >

Procedure
1. #nsert a salt bridge connected to standard electrodes and suitable for measuring both

resistance and potential differences across the membrane.

2. Measure the resistance of the synthetic membrane by passing a small C7 current through

the membrane and measuring the drop in voltage. #f one knows the applied voltage ,1''49'' m=- and the measured voltage , m=-, the following formula will give the resistance ,;hm@s law-2 R 6 ,D 8 ,D 4 D -- \ R

where

R is the series resistance D is the calibrated input voltage

is the voltage appearing across the membrane.

3. 7alculate the area of the membrane based on the diameter of the drilled hole, and

multiply R ,measured resistance- by the area of the membrane. $his will give a normali&ed resistance in ohms4cm .
4. 7ompare your calculated value to the data in $able 5.). 5. Civide your calculated value by the thickness of the membrane I to yield the membrane

resistivity or specific resistance.

Exercise 9.= - Measure#ent of Trans#e#+rane Potential

!D=D! ## Materials

Artificial membrane and recording apparatus from D/ercise 5.5 1.' M +a7l 1.' M B7l

Procedure
1. 1sing the recording device from D/ercise 5.>, measure the voltage across the membrane

with '.1 M +a7l on both sides of the membrane.

2. Add 1.' ml of 1 M +a7l to the e/ternal chamber and mi/ gently. #mmediately record the

changes in voltage. O
3. .hen the change begins to stabili&e, introduce 1.' ml of B7l into the teflon cup and

continue to monitor the voltage.

4. Araph the change in voltage across the membrane with time and the addition of first

+a7l and then B7l.

5. 7ompare your results with the action potential of a typical vertebrate neuron.

"otes $he same apparatus as that used for measurement of resistance can be used to measure a potential, provided there is a difference in the salt concentration in the teflon cup and outside of the cup. $his is best accomplished by the slight addition of a salt solution to the teflon cup,

although rather elaborate procedures e/ist for complete substitution of the solutions ,e/treme care must be taken not to rupture the membrane-. #n vertebrate nerve, the potential that is generated is from an influ/ of +a followed by an efflu/ of B . $hese flu/es are mimiced by the addition of +a7l to the outer chamber, followed by an addition of B7l to the inner chamber.

Exercise 9.> - Che#ical Co#position of M0elin

!D=D! ### Materials

7alf brain 1' '.9* M sucrose '.II M sucrose %lender or tissue homogeni&er Refrigerated centrifuge

Procedure
1. :omogeni&e 1'' gms of calf brain in '.9* M sucrose to make a 1'S brei. 2. 7entrifuge the homogenate at *'' /g for 1' minutes at )G 7. 3. 7ollect the supernatant and recentrifuge the supernatant at 11,''' /g for 1' minutes. 11 4. Ciscard the supernatant and resuspend the pellet in 1' ml. of '.9* M sucrose. 5. 7arefully add 9* ml of '.II M sucrose to the bottom of the tube, so that the resuspended

pellet lies on top of the sucrose.

6. 7entrifuge at 11,''' /g for 1' minutes to separate the mitochondria from the myelin

vessicles. $he myelin will float on the surface, while the denser mitochondria should pellet at the bottom of the tube.
7. 7arefully remove the upper myelin vessicle layer.

Exercise 9.1, - M0elin Bipid Extraction

!D=D! ### Fraction $: Cholesterol Materials

Myelin vessicles from D/ercise 5.O Acetone :omogeni&er manufactured for organic solvents %uchner funnel and water aspirator All organic solvents are flammable and should be handled with e/treme care. +o open flames will be tolerated and all heating should be done with steam baths. Co not use a standard household blender 4 the acetone will dissolve the gaskets, run into the motor and e/plodeN

<rocedure
1. $o the purified myelin vessicles, add ) volumes of acetone. %lend the mi/ture for 1

minute at high speed.

2. Add the blended mi/ture to a beaker and manually continue stirring for * minutes.

Co not use any electric stirring motors. Filter the mi/ture using a %uchner funnel and cold trap attached to a water aspirator for reduced pressure. Save the filtered acetone solution.
3. .ash the residue from the filter paper back into the blender. Add 1'' ml. of fresh acetone

and reblend for 1 minute.

4. Add the second homogenate to the beaker, stir for * minutes and filter.

7ombine the two filtered acetone portions.

5. $he acetone e/tracter material is FRA7$#;+ A. #t contains crude cholesterol.

%efore storing, it should be dried to remove the acetone. $his is best accomplished by flash evaporation or lyophili&ation. Alternatively, the solution can be allowed to evaporate in a fume hood designed to vent organic solvents. $here will, of course, be some decomposition with the latter techni3ue. Dvaporate the acetone, collect the dry crude cholesterol and obtain its dry weight.
6. Beep the acetone insoluble residue ,on the filter paper- for

e/traction of Fractions % and 7. Fraction B: Becithin and Cephalin Materials

Residue from e/traction of Fraction A <etroleum ether %uchner funnel

<rocedure
1. D/tract the acetone insoluble material left over from Step 5 of the e/traction of Fraction

A with 9'' ml of ether. Aently stir for * minutes.

2. 7ollect the filtrate using a %uchner Funnel, as above. 3. <lace the ether insoluble material on the filter into a flask and re4e/tract with an

additional 9'' ml of ether. Repeat the filtration and re4e/traction one final time with a third 9'' ml of ether.
4. 7ombine all three ether filtrates.

Save the residue for Fraction 7.

5. Dvaporate the ether filtrates to *' ml under reduced pressure. <our the concentrated

e/tract into 9'' ml of acetone and stir.

6. 7ollect the precipitate on filter paper and discard the filtrate. $he precipitate left on the

filter paper FRA7$#;+ %, containing lecithin and cephalin.

7. ;btain the dry weight of Fraction %.

Fraction C: 6phingosine Phosphatides and Al0cosides Materials

Residue from e/traction of Fraction % Dthanol

<rocedure
1. D/tract the ether insoluble residue obtained in the steps above with *' ml. of boiling

ethanol. =acuum filter the hot ethanol while it is still hot. Ciscard the residue. 1se steam heat only and work in a laboratory properly e3uipped for organic e/tractions. :ot ethanol has a very low flash point 4 it will e/plodeN
2. 7ool the ethanol filtrate and collect the precipitate by vacuum filtration. $his precipitate

is Fraction 7.
3. ;btain the dry weight of Fraction 7.

4. Cetermine the percentage of each Fraction dry weight relative to the original wet weight

of calf brain. Cissolve each of the Fractions to a final concentration of 1S ,w8v- in chloroform2methanol ,?21- before further analysis.

Exercise 9.11 - Huantitati(e $nal0sis of Bipid Classes +0 TBC

!D=D! ### $wo methods of lipid analysis are commonly used. $he first involves isolation of classes of lipids, followed by $hin !ayer 7hromatographic separation and 3uantitation directly on the chromatographic plates. $he second method involves the isolation of the components on the $!7 plate, followed by conventional 3uantitative methods. $he lipid classes are divided into2
1. +eutral lipids ,triglycerides2. <olar !ipids ,phosphoipids3. 7holesterol

;rdinarily, the neutral lipids are analy&ed first, as they are readily separated with one dimensional $!7 systems. $he polar lipids re3uire two dimensional $!7 analysis, and cholesterol needs to be analy&ed separately. $he lipids may be chromatographed and measured for the following2
1. Melting <oint 2. Salkowski $est ,color developed with : S; 3. !iebermann4%urchard $est 9' ,test for ?4hydro/ysteroids using acetic anhydride4. Cigitonide Cerivative ,precipitates cholesterol5. 7harring ,spraying with an o/idant and heating to carboni&e the lipid-

A typical analysis would involve two dimensional chromatography followed by charring. #ndividual lipids would be identified by the Rf values of the spots on the chromatograms and could be 3uantitated densitometrically. Chapter 9: Me#+ranes - Endnotes
1.

Modified from Aiese, A.7. 1O5). 5aboratory #anual in Cell Physiology Revised ed., p 5>45I. %o/wood <ress, <ittsburgh. 7ollect Amoeba by centrifugation three days prior to use, wash, and resuspend without food. #t is important that the Amoeba be starved for this e/ercise.

2.

3.

#f the dye is too strong, and the Amoeba are stained intensely blue, dilute the dye 181' or even 181''. $his material is readily available from local slaughter houses. #f there is not one convenient to your location, and a local butcher will not help, rat brains can be substituted. Muantities will need to be ad"usted accordingly. $his solution is stable for about * days if refrigerated, and can be prepared ahead and used for multiple lab sessions. A salt bridge is simply a saturated B7l solution ,in 9S ,w8v- agar- placed within glass, polyethylene, or teflon capillary tubes and with platinum electrodes or commercially available calomel electrodes immersed within the agar. $hus, they bridge the gap between the solution being monitored and the rather large wire connections of a recording electrode. A suitable measurement can be made by using the millivolt potential scale on a good p: meter. #t may re3uire some assistance from the local physical chemist, or a physicist to construct the proper electrodes.

4.

5.

6.

7.

8. 9.

Assume a thickness of * / 1'

cm if it was not measured.

$his will model the normal distribution of increased e/ternal +a observed in vertebrate neurons. As the +a diffuses into the center of the teflon cup, it will model the influ/ of ions associated with an action potential.

10. Jields of myelin will vary depending upon the developmental age of the animal and

upon the species. Adult rat brains will yield about )'4O' mg. of pure myelin per brain while the white matter from bovine brain will yield about 1'' mg of pure myelin per gram of white matter. $he purity will depend upon whether whole brain is used or "ust the white matter, with the latter yielding better results. .hole brains would contain a fair amount of microsomal membranes isolated along with the myelin membranes, and thus would have more impurities.
11. $he pellet contains mitochondria and myelin vessicles.

Chapter <: Microso#es

Introduction:

Figure >.1 =acuome concept

Figure >.9 #nternal membranes $able >.1 <hysical properties of microsome $he use of homogeni&ation techni3ues and differential centrifugation has made possible the study of many isolated, membrane bound organelles. $he nucleus, mitochondria and various plastids were obvious targets for analysis, since they are relatively large, and are visible with the light microscope. Smaller structures, and particularly those associated with internal membranes, were more difficult to analy&e. #n 1O?1, ..:. !ewis observed the uptake of neutral red dye into cell vacuoles, 1 and coined the term <#+;7J$;S#S, literally cell drinking. !ewis was interested in the small membrane bound organelles found within plants cells, and named vacuomes by the French %otanist, <.A. Cangeard in 1O1O. $he term vacuome described a structure or structures in plant cells which were associated with the tonoplast ,the membrane surrounding a large central vacuole- and which was apparently part of an internal membrane system of communication and flow. Figure >.1 presents an e/tended view of the vacuome concept, which was developed to e/plain both pinocytosis and its related phagocytosis, cell eating . $he study of vacuomes was an early attempt to define the function of internal membrane bound vessicles as components of membrane flow. Several years later, the concept of an organi&ed system of internal membranes was e/tended to animal cells. ;ur perspective on membrane bound vessicles was altered significantly in 1O)O when 7hristian CeCuve analy&ed the activity of the en&yme acid

phosphorylase during a study of insulin activation. 9Acid phosphorylase is a useful indicator of en&ymatic digestion in cells. CeCuve was interested in how cells digested substances which were either pinocyti&ed or phagocyti&ed, and specifically, what the relationship of digestion was to the small vacuoles, M#7R;S;MDS, which could be isolated via centrifugation. As CeCuve e/plains, the lab purchased an e/pensive and new ultracentrifuge capable of isolating small particles and even molecules. CeCuve discovered that acid phosphorylase was associated with specific microsome fractions and that the activity of the en&yme could be released by the actions of blenders, hypotonic media, free&ing8thawing or detergents. As work progressed, several other en&ymes were locali&ed within these microsomes. #n 1O**, CeCuve et al published the now classic paper describing for the first time, a new organelle, the lysosome . ? CeCuve was later to be awarded a +obel <ri&e for this pioneering work. $he lysosome is one type of microsome its role in cell digestion brought together a story of internal membrane flow which is integrated with the twin processes of D+C;7J$;S#S and D(;7J$;S#S of the plasma membrane. $he lysosome was seen as a part of an elaborate system, that could be fractionated, but which only made sense in the whole. <inocytosis, <hagocytosis, Aolgi 7omple/, Dndoplasmic Reticulum and <lasma Membrane where all part of this system. Figure >.9 presents our current concept of how a cell@s internal membranes are organi&ed. $he e/ercises in this chapter deal with the isolation and analysis of different types of microsomes. 7areful homogeni&ation of the cell is critical for the subse3uent separation of microsome fractions. !ysosomes, for e/ample, can be isolated from rat liver in either an isosmotic sucrose media ,'.9* M-, or in a hyperosmotic sucrose ,'.II M-. .ith the use of isosmotic solutions, mild homogeni&ation is re3uired to prevent rupturing of the lysosome. D/cessive membrane damage also results in the creation of high levels of contaminating vessicles formed from broken plasma membranes, endoplasmic reticulum, and nuclear envelopes. $he physical properties of isolated organelles can be determined and their function inferred from their chemical compositiion. $able >.1 lists some of the properties of microsomes isolated in isosmotic media.

<.1 B0soso#e Isolation in Isotonic 6ucrose

!D=D! # MA$DR#A!S

Rat liver <hysiological saline ,'.I*S w8v +a7l'.9* M sucrose in 1' mM $ris4:7!, p: >.) %rendler teflon homogeni&er Refrigerated preparative centrifuge '.'I M 7a7l in '.9* M sucrose plus 1' mM $ris4:7l 1*' mM B7l in 1' mM $ris4:7l %uffer, p: >.) <hase contrast microscope, slides, coverslips

<R;7DC1RD )
1. Cecapitate and e/sanguinate a rat that has been starved for at least 9) hours prior to the

lab. * Fill a syringe with saline and gently perfuse the liver by forcing the saline through the hepatic portal vein, and through the liver.
2. Remove the liver, place it in a preweighed beaker and weigh the beaker and liver.

7alculate the weight of the liver.

3. <repare a 1'S ,w8v- homogenate or brei. For each gram of liver, add O.' ml of '.9* M

sucrose in 1' mM $ris4:7!, p: >.). to the beaker.

4. Aently chop the liver in the sucrose and transfer the chopped liver to a teflon

homogeni&er. 5 Aently homogeni&e the liver while keeping it chilled.

5. 7entrifuge the brei at 19,''' /g for 1' minutes at ) G 7. Cecant the supernatant into a

chilled beaker and discard the pellet.

6. Add '.'I M 7a7l to the supernatant to yield a final concentration of I mM ,use 1 ml of

7a7l per O ml of supernatant-. Stir gently and recentrifuge at 9*,''' /g for 1* minutes at )G7

7. 7arefully remove and discard the supernatant. > 8. Resuspend the pellet ,containing the lysosomes- in ?' ml of 1*' mM B7l in 1' mM $ris4

:7l %uffer, p: >.).

9. Re4sediment the lysosomes by a final centrifugation at 9*,''' /g for 1* minutes at ) G 7. 10. Remove a small portion of the pellet for D/ercise >.9. Resuspend the remainder of the

pellet in ?' ml of 1*' mM B7l81' mM $ris4:7l %uffer. $his suspension is the lysosome fraction for D/ercise >.?.
11. <repare a wet mount of the resuspended lysosome pellet and observe with a phase

contrast microscope at 1''(. Craw any structures observed.

<.. E# o+ser(ations of #icroso#es

!D=D! #

Figure >.? #solated microsomes

Figure >.) $DM of hepatocyte MA$DR#A!S

1S Alutaraldehye ,A$A1S ;smium tetro/ide Dpo/y or vinyl resin for $DM $DM photomicrograph of liver cells

$ransmission electron microscope

<R;7DC1RD
1. <lace a mm piece of the final pellet from D/ercise >.1 into a small vial containing 1S

A$A. Fi/ the pellet for 9 hours.

2. Rinse the pellet with water and place in three changes of water for ?' minutes each, to

remove any residual A$A.

3. <ost fi/ the lysosome pellet in 1S osmium tetro/ide for 1 hour. .ash thoroughly with ?

changes of water ,?' minutes each-.

4. Cehydrate the tissue by passing through a series of graded alcohols or acetone, and

embed in plastic blocks.

5. Section the plastic blocks and place on coated grid. Couble stain with uranyl acetate and

lead nitrate and e/amine with a transmission electron microscope.

6. 7ompare the view of the compacted lysosome pellet to the structure and distribution of

microsomes in an intact hepatocyte ,liver cell-. 1se figures >.? and >.) for reference.

<.5 Che#ical characteri3ation - acid phosphatase

!D=D! # $able >.9 a list of chemical constituents found in isolated lysosomes MA$DR#A!S

1 M Sodium acetate buffer, p: *.> '.1 M Mg7l '.'* M p4+itrophenyl phosphate Microsome fraction from D/ercise >.1 1*' mM B7l in 1' mM $ris4:7l %uffer, p: >.) '.* + B;: .ater bath at ?' G 7 Spectrophotometer and tubes %radford <rotein assay

<R;7DC1RD

1. <repare a series of si/ test tubes containing '.* ml of each of the following ,total of 1.*

ml-2 1 M Sodium acetate buffer '.1 M Mg7l '.'* M p4+itrophenyl phosphate

2. Add ?.? ml of distilled : ; to each of the si/ tubes. Mi/ well, and place in a ?' G 7 water

bath to temperature e3uilibrate.

3. <repare a serial dilution of your lysosome fraction by adding 1.' ml of lysosome

suspension fromD/ercise >.1 to O.' ml of 1*' mM B7l81' mM $ris4:7l %uffer. Mi/ well and add 1.' ml of the diluted suspension to a new tube containing O.' ml of $ris4 :7l buffer. Mi/ and repeat the dilutions two more times. #n addition to the undiluted lysosome suspension, label the diluted fractions as 181', 181'', 181,''' and 181','''.
4. Add '.9 ml of $ris4:7l buffer and 9.' ml of B;: to tube R1 from step 1. 5. $urn on a spectrophotometer, ad"ust the wavelength to )'* nm and use the sample in tube

R1 to blank the instrument. 5. Add '.9 ml of the undiluted the lysosome fraction to tube R9, mi/ and place in water bath at ?' G 7 for e/actly * minutes.
6. Add 9.' ml of B;: to tube R9, mi/ and immediately read the absorbance of the solution

at )'* nm.
7. $he absorbance should be between '.? and '.). #f not, repeat steps 5 and > for the diluted

samples, starting with the 181' in tube R? and continuing through the 181',''' dilution in tube R5. Dach should be done separately, one at a time to prevent incorrect timing of the reaction.
8. 1sing the absorbance reading for the dilution which yields an absorbance change of '.?4

'.) in five minutes, determine the actual rate of absorbance change for that dilution. 1'. 7onvert the absorbance change to a rate of p4+itrophenyl phosphate conversion to p4 nitrophenol. 1se the %eer4!ambert law, with an e/tinction coefficient 6 1I.I / 1' Abs. 1nits8mole of p4nitrophenol. 7onvert all absorbance readings to micromoles of p4nitrophenol formed in five minutes. Civide by the time ,* minutes- to calculate micromoles formed per minute.

9. Cetermine the concentration of protein in the dilution from step O, using the %radford

protein determination , Appendi/ A - and bovine serum albumin as the standard.

10. Cetermine the rate of en&yme activity per mg. protein present in the diluted fraction.

$ube8Cilution <rotein 7ontent Absorbance,*@- ]p4nitrophenol^ Activity8minute 1. %lank 9. 1' ?. 1' ). 1' *. 1' 5. 1' ;<$#;+A! Dn&yme activity and protein concentration can be measured for each step in the centrifugation and isolation of lysosomes. Assuming that acid phosphatase is an effective measure of the purity of lysosomes, the increased purity during separation can be monitored by measuring the increased en&yme activity per mg of protein. !ysosome purification can be monitored to yield the ma/imum activity per mg. protein. .hile lysosomes are best characteri&ed by the presence of acid pyrophosphorylases, they contain a large number of functional en&ymes. $able >.9 presents a list of chemical constituents found in isolated lysosomes. KKKK ' KKKK KKKK

<.1 Chro#affin granules fro# adrenal glands

!D=D! ##

Figure >.* Schematic membrane of chromaffin granule $able >.? 7omposition of chromaffin granules MA$DR#A!S

%ovine adrenal glands ,preferred fresh from slaughterhouse'.9> M Sucrose, 1' mM $ris4maleate buffer, p: >.' ?'S <ercoll ,v8v- in '.9> M sucrose, 1' mM $ris4maleate, p: >.' Refrigerated, preparative centrifuge with swinging bucket rotor for *' ml tubes Fraction collecting device A$<4luciferase kit I 1=8=#S spectrophotometer ;ptional
o o o

!owry protein assay 7atecholamine assay $DM fi/atives and embedding materials

<R;7DC1RDS O
1. Cissect out the medulla of an adrenal gland, weigh and 3uickly mince. 2. For each gram of tissue, add * volumes of chilled '.9> M sucrose, 1' mM $ris4maleate

buffer, p: >.' and homogeni&e in a <otter4Dlvi"em homogeni&er.

3. 7entrifuge the brei for * minutes X 1,9*' /g X ) G 7 . Ciscard the pellet and

recentrifuge the supernatant XI,5*' /g X) G 7 for ?* minutes.

4. 7ollect the second pellet ,pink, granular- and wash by resuspending in fresh sucrose and

recentrifuging XI,5*' /g as above.

5. Resuspend the washed pellet to yield a final protein concentration of 5'4I' mg8ml ,as

measured by ;C -.
6. Add ?I ml of ?'S <ercoll4sucrose in tris4maleate buffer to a *' ml clear polycarbonate

centrifuge tube and centrifuge the <ercoll for * minutes X9',9'' /g to form a gradient.
7. Add 9.' ml of the resuspended chromaffin granule pellet to the top of the preformed

<ercoll gradient and centrifuge for )' minutes X I,>'' /g X ) G 7.

8. Fractionate the centrifuged chromaffin granules into 1.' ml fractions. Measure the

absorbance of each fraction at 5*' nm.

9. Measure the A$< content of each fraction using a commercially available luciferase

reaction.
10. <lot the fraction number vs the concentration of A$<.

;<$#;+A! Measure catecholamines by spectrophotometric analysis of adrenochrome formation at )I' nm, using norepinephrine as a standard. 1' Repellet the chromaffin granules and prepare a sample for electron microscopic observation. Monitor mitochondrial and lysosomal contamination via the presence of cytochrome o/idase and acid phosphatase, respectively. Cetermine total protein content by the !owry method. +;$DS Adrenal medullary tissue consists of cells containing chromaffin granules ,named for their ability to stain with chromium stains- which are 1''4?'' nm in diameter. $hese granules consist of the catecholamine hormones epinephrine and norepinephrine ,9'S by weight-, A$< ,1*S-, protein ,?*S- and lipid ,9'S-. About I*S of the granules store epinephrine, while the remainder store norepinephrine. $able >.? presents a more detailed chemical composition of chromaffin granules. $he adrenal medulla from cows and pigs has long served as a source of material for the isolation of speciali&ed organelles which were originally believed to be storage vessicles for the catecholamines. $hese organelles are known as chromaffin granules and are embryologically related to the adrenergic neurotransmittor granules of the central nervous system. !ately, the presence of a proton pump has been identified within these granules and e/tensive studies are underway to relate the chemiosmotic concepts of membrane transport to the synthesis and release of the biologically active catecholamines. 11

A schematic of a chromaffin granule membrane is presented in figure >.*.

<.7 Measure#ent of catechola#ines

!D=D! ###

Figure >.5 7atecholamine metabolism $he function of chromaffin granules is the synthesis of the catecholamines. $hus, we can analy&e chromaffin granule function by the level of catecholamines found in isolated granules in particular dopamine, epinephrine and8or norepinephrine can be monitored. 1nfortunately, these compounds are somewhat difficult to measure. .hile there are several means of monitoring these compounds, 19 the primary procedure re3uires the use of radio4immunoassay. 1? As a reference, figure >.5 illustrates the interaction of the catecholamine hormones.. Chapter <: Microso#es - Endnotes

1. 2.

!ewis, ..:. 1O?1. %1!!. T;:+S :;<B#+S :;S<. )O,1>. According to the words of 7. CeCuve in $he !ysosome in Retrospect in !JS;S;MDS #+ %#;!;AJ A+C <A$:;!;AJ =ol. 1 ,T.$. Cingle and :.%. Fell, eds.- +orth4 :olland <ublishing, Amsterdam, 1O5O.

Chapter =: Photos0nthesisI@espiration - Introduction

$he physiological reactions of mitochondria and chloroplasts can be reduced to a series of electron transfers, cataly&ed by specific en&ymes found within the organelles. $hus, we can study the component processes of photosynthesis and respiration by isolating the organelles and measuring specific en&yme activity associated with that organelle. <hotosynthesis re3uires two processes which can be functionally dissociated, although they work as a unit within the chloroplast. $he first process is known as the light reactions, while the

second is known by analogy as the dark reactions. $he light reactions are rapid changes in the subatomic arrangements of molecules that ultimately split water in the presence of light ,photodissociation-. :ydrogen atoms from the water are used to reduce +AC< to +AC<: , and are then used to reduce 7; to 7: ;.

Figure I.1 !ight reactions of photosynthesis $he photodissociation of water in the presence of chloroplast fragments is known as the :ill Reaction. #t results from the physical capture of light 3uanta ,energy- into the electron orbit of chlorophyll molecules and the subse3uent transfer of an 0e/cited0 electron to the orbit of an ad"acent molecule. $he end products of this reaction are free hydrogen, o/ygen and electrons. $he electrons are utili&ed for further chemical reduction reactions, the hydrogen becomes the ultimate hydrogen acceptor in the reactions and o/ygen is a by4product. Figure I.1 presents the light reactions, along with the redo/ potentials of the compounds involved. 7omplete details of the cyclic and noncyclic pathways of the light reactions are beyond the scope of this manual. For our purposes, however, the :ill reaction can be monitored by the addition of an electron accepter to the system, and one which will more readily accept the electron. #f the electron acceptor is a pigment which alters its color when reduced ,by gaining an electron-, a simple colorimetric analysis can be used to monitor the photodissociation of water. %asically, the reducing power generated by splitting the water can be used to reduce a dye such as 9,54 dichloroindophenol, which is blue when o/idi&ed, and colorless when reduced. #n respiration, a similar type of o/idation8reduction occurs, e/cept that the process is not physical, but chemical ,i.e. it is temperature dependent and slower than a physical reaction-. Molecules such as succinic acid are o/idi&ed within the Breb@s 7ycle by the removal of both a

hydrogen atom and a corresponding electron. Succinic acid dehydrogenation produces fumaric acid the en&yme performing this reaction is succinic dehydrogenase, and the hydrogen with its electron is normally transferred to +AC.

Figure I.9 Dlectron transfer system $hese reactions are associated with the inner membranes of the mitochondria, and specifically to structures known as respiratory particles. .ithin these fragments of the inner membrane, the electron transport system functions and the hydrogen and its electron are dissociated. $he electron passes through a series of respiratory pigments ,the cytochromes- and combines with molecular o/ygen through the use of the en&yme cytochrome o/idase. $he electron transfer system is outlined in Figure I.9. $he activities of two en&ymes, succinic dehydrogenase and cytochrome o/idase, can be monitored in a manner similar to that for the :ill reaction of photosynthesis. A dye intermediate can be introduced to intercept the electron from the D$S. $he gain of electrons will reduce the dye which will conse3uently change color. $he color change may be monitored spectrophotometrically.

Figure I.? 7? metabolism ,7alvin %enson 7ycle $he dark reactions of photosynthesis behave differently than the light. $hey are chemical reactions cataly&ed by en&ymes and are thus slower than the physical reactions of the light reactions. As chemical reactions, they are temperature dependent. $he 7? and 7) pathways of the dark reactions are given in Figures Figures I.? andI.), respectively.

$he chemical reactions of respiration are divided into two ma"or pathways, glycolysis and the Breb@s cycle. $hese pathways are presented in Figure I.*. D/amination of Figures I.? through I.* will demonstrate many similarities in the biochemistry of energy metabolism, whether within chloroplasts or mitochondria. +ote that glycolysis occurs within the cytoplasm of cells whereas Brebs reactions occur within the mitochondrial stroma. 7? metabolism is within a single plast cell with reactions occuring in the cytoplasm as well as within the chloroplast. 7) metabolism is a process that re3uires several cells, with distinct compartmentali&ation of function on a tissue level. Mitochondria and 7hloroplasts can be e/tracted by gentle rupture of the pertinent cells and differential centrifugation in a media designed to maintain the osmotic and functional integrity of the organelles. $he activity of the organelles will vary significantly with the source, the age, and such factors as the length of time of storage before e/traction. $he organelles can be kept for several days in cold storage, but will gradually degenerate and decompose. #t is easier to work with isolated intact organelles than directly with 3uantosomes or o/ysomes, which are unstable under laboratory conditions.

Figure I.) 7) metabolism

Figure I.* Alycolysis and the Brebs cycle

Exercise =.1 - Isolation of Chloroplasts fro# 6pinach Bea(es

!D=D! # Materials

Fresh spinach leaves Arinding solution

o o o o o

'.?? M Sorbitol 1' mM Sodium pyrophosphate ,+a < ; ) mM Mg7l 9 mM Ascorbic Acid Ad"ust p: to 5.* with :7l

7hopping board and knife 7hilled mortar and pestle 7heesecloth Refrigerated preparative centrifuge

Suspension solution
o o o o o

'.?? M Sorbitol 9 mM DC$A 1 mM Mg7l *' mM :D<DS Ad"ust p: to >.5 with +a;:

:emacytometer and microscope

Procedure
1. <repare an ice bath and pre4cool all glassware to be used. 2. Select several fresh spinach leaves and remove the large veins by tearing them loose from

the leaves. .eigh out ).' grams of deveined leaf tissue.

3. 7hop the tissue as fine as possible. Add the tissue to an ice4cold mortar containing 1* ml

of grinding solution and grind to a fine paste.

4. Filter the solution through double layered cheesecloth into a beaker and s3uee&e the

tissue pulp to recover all of the suspension.

5. $ransfer the green suspension to a cold *' ml. centrifuge tube and centrifuge at 9'' /g

for 1 minute at )G 7 to pellet the unbroken cells and fragments.

6. Cecant the supernatant into a clean centrifuge tube and recentrifuge at 1''' /g for >

minutes. $he pellet formed during this centrifugation contains chloroplasts. Cecant and discard the supernatant.
7. Resuspend the chloroplast pellet in *.' ml. of cold suspension solution or '.'?* M +a7l.

1se a glass stirring rod to gently disrupt the packed pellet. $his is the chloroplast suspension for use in subse3uent procedures.
8. Dnclose the tube in aluminum foil and place it in an ice bucket. 1 9. Cetermine the number of chloroplasts8ml of suspension media using a hemocytometer.

Record the R of chloroplasts8mlKKKKKKKKK "otes $he isolation procedure used here leaves the chloroplast outer membrane intact.#f you wish to study the en&ymes for photophosphorylation, wash the chloroplasts and rupture the outer

membranes. $o rupture the outer membranes, resuspend the chloroplasts in diluted suspension solution ,129*-. #mmediately centrifuge the chloroplast suspension at I,''' /g for * minutes to collect the chloroplasts. Remove the diluted suspension media and resuspend the chloroplasts in isotonic media ,'.?* M +a7l or undiluted suspension buffer-.

Exercise =.. - Chloroph0ll Content

!D=D! # Materials

7hloroplast suspension from D/ercise I.1 Acetone Spectrophotometer and tubes 7linical centrifuge and tubes

Procedure
1. <ipet 1.' ml of a chloroplast suspension into a 1* ml centrifuge tube and add I.' ml of

acetone. Mi/ thoroughly.

2. Add 1.' ml of distilled water, mi/ again and centrifuge at 1''' /g for ?4* minutes. 3. $urn on a spectrophotometer, ad"ust the wavelength to 5*9 nm and blank the instrument

with water.
4. Measure the absorbance of the supernatant from step 9. 5. 7alculate the amount of total chlorophyll in 1.' ml of the chloroplast suspension. 9 $he

amount of chlorophyll is givenby the e3uation2 mg chlorophyll Absorbance 4444444444444444 6 4444444444444444 ml ?).*
6. 7alculate the dilution factor for your original chloroplast suspension so that when diluted

with suspension solution, the final concentration of chlorophyll will be '.'1 mg8ml. All subse3uent work with chloroplasts will use the diluted suspension so that the criterion of step 5 is met.
7. 1se a hemacytometer to determine the number of chloroplasts per ml of any diluted

suspensions.

Absorbance of acetone e/tracted chlorophyll solution 6 KKKKKKKKKKK 7alculated concentration of chlorophyll 6 KKKKKKKKKKKKmg chlor.8ml Cilution factor for suspension to yield '.'1 mg chlorophyll8ml 6 KKKKKKKKKKK 7hloroplasts8ml of suspension so diluted 6 KKKKKKKKKKK

Exercise =.5 - The ?ill @eaction

!D=D! # Materials

7hloroplast suspension ,D/ercise I.9'.'?* M +a7l %oiling water bath #ce bath * / 1' M ?4,?,)4Cichlorophenyl-41,1,4Cimethylurea ,C7M1- ?

'.1 M B :<; 8B: <; %uffer, p: 5.* 1 / 1' M Cichlorophenolindophenol ,C7<#<- )

Procedure
1. Cilute the chloroplast suspension with '.'?* M +a7l to yield a final chlorophyll

concentration of '.'1 mg8ml ,Refer to D/ercise I.9-.

2. :eat a *.' ml portion of the diluted suspension into a boiling water bath. Remove the

inactivated chloroplasts from the boiling water after five minutes and place in an ice bath to cool. Beep the remainder of the active chloroplast suspension at )G 7 ,ice bath-.
3. <repare five test tubes as follows2

%uffer

?.' ml

?.' ml

?.' ml

?.' ml

?.' ml

:eated chloroplasts

1.' ml

1.' ml

+one

+one

+one

Active chloroplasts

+one

+one

1.' ml

1.' ml

1.' ml

C7M1

+one

+one

+one

+one

1.' ml

.ater

9.' ml

1.' ml

1.' ml

1.' ml

+one

4. $urn on the spectrophotometer and ad"ust the wavelength to 5'' nm. 1se the solution in

tube R1 to blank the spectrophotometer.

5. Add 1.' ml of C7<#< dye to tube R9, mi/ by swirling and immediately measure the

absorbance at 5'' nm.

6. Add 1.' ml of C7<#< dye to tubes R?, R) and R*. Mi/ and immediately measure the

absorbance of each. +ote the time for each reading.

7. .rap tube R) in dark paper, and aluminum foil. $his tube serves as a 0dark0 control and

the absorbance of this solution will be read only at the end of the e/periment. #t is important that no light reach the chloroplasts inside.
8. <lace tubes R9, R?, and R* in a beaker of ice water and illuminate with a strong light

source. *
9. Measure the absorbance of each tube after 1' minutes, 9' minutes and finally after ?'

minutes. %e sure to wipe the tubes dry before inserting the tube into the spectrophotometer. Record your results. $ime ,Minutes9 ? ) *

'

1'

9'

?'
10. 7alculate the total dye reduced at 1' minute intervals using the e3uation2

7o,Ao4Ac7r 6 44444444444 Ao where2 7r is the dye reduced in moles8liter 7o is the original dye concentration in moles8liter Ao is the absorbance of the mi/ture at the start Ac is the absorbance of the mi/ture at the end of the e/periment.
11. 7alculate the percent dye reduced and plot percent dye reduced per minute for tubes 9

through *.

Exercise =.1 - Isolation of Mitochondria

!D=D! # Materials

Rat, mouse or suitable source of fresh liver 5 '.9* M sucrose in 1' mM :D<DS buffer, p: >.* ,:omogeni&ation buffer'.9* M sucrose in 1' mM :D<DS, p: >.* W 1 mM DC$A ,Suspension buffer$eflon homogeni&er Refrigerated centrifuge Tanus Areen % :emacytometer and microscope

Procedure
1. Sacrifice and e/san3uinate a rat that has not been fed for at least 9) hours prior to lab.

2. Remove the liver and weigh it. 3. Add the liver to a beaker, and for each gram of liver, add O.' ml of '.9* M sucrose in 1'

mM :D<DS buffer, p: >.*. $his will produce a 1'S brei, a term used to indicate a homogeni&ed suspension.
4. Add the brei to centrifuge tubes and centrifuge at ),*'' /g for 1' minutes at )G 7. 5. Cecant the supernatant into clean centrifuge tubes and discard the pellet. 6. Recentrifuge the supernatant at 15,''' /g for 9* minutes at )G 7. 7. Cecant and discard the supernatant. Resuspend the pelleted mitochondria in 9' ml of '.9*

M sucrose in 1' mM :D<DS. Skip to step 1'. ;R /ptional: if cleaner mitochondria are desired, resuspend in 9' ml of '.9* M sucrose in 1' mM :D<DS W 1 mM DC$A and perform steps I and O.
8. Recentrifuge the suspended pellet at 15,''' /g for 9* minutes at )G 7. 9. Cecant and discard the supernatant. Resuspend the washed pellet in 9' ml of fresh

sucrose without DC$A and place the suspension in an ice bath until further use is re3uired. $he suspension will remain active for appro/imately )45 hours if kept cold.
10. Mi/ a few drops of Tanus Areen % solution with '.1 ml of mitochondrial suspension.

<lace one drop of this mi/ture in a hemocytometer and determine the number of mitochondria per ml. #f there are too many mitochondria to count, make serial dilutions of 181' to 181''' and recount. Ciluted mitochondria must be counted rapidly. $hey are not stable and will decompose if not counted within a few minutes of the dilution. Record the number of mitochondria8mlKKKKKKKKKKKKKK

Exercise =.7 - @espiration: 6uccinic *eh0drogenase

!D=D! ## Materials

Mitochondrial suspension from D/ercise I.) Sodium dithionite '.9 M Sorenson <hosphate %uffer, p: >.* 1S ,w8v- %ovine Serum Albumin > '.''* M <otassium 7yanide

'.'''9* M Cichlorophenolindophenol '.5 M Sodium succinate, p: >.* '.5 M Sodium malonate, p: >.* '.'??S ,v8v- <hena&ine Methosulfate in <hosphate %uffer I Spectrophotometer and cuvettes

Procedure
1. <lace 1.' ml of mitochondrial suspension in a test tube and place in a boiling water bath

for * minutes. 7ool before using. $his boiled preparation will allow measurement of background absorption due to the turbidity of the mitochondria when used in a spectrophotometer.
2. <repare a series of tubes as follows2

1 <hosphate %uffer %SA B7+ C7<#< Succinate Malonate 9.' ml '.1 ml 1.' ml 1.' ml +one +one

9 9.' ml '.1 ml 1.' ml 1.' ml 1.' ml +one

? 9.' ml '.1 ml 1.' ml 1.' ml +one 1.' ml

) 1.' ml '.1 ml 1.' ml 1.' ml 1.' ml 1.' ml

3. Mi/ 9 ml of '.'''9* M C7<#< with an amount of sodium dithionite sufficient to fully

reduce it. $he amount of sodium dithionite is not crucial. Add a small 0pinch,0 shake to dissolve and continue until the color of the C7<#< becomes clear.
4. 1se the reduced C7<#< from step ? to blank a spectrophotometer at 5'' nm. 5. Add 1.' ml of the boiled mitochondrial suspension and 1.' ml of <MS to tube R1, mi/ by

inversion and immediately place in the previously blanked spectrophotometer. Record the absorbance and continue to record at ?' second intervals for ? minutes.
6. Add 1.' ml of the mitochondria suspension to tube R9 and measure the absorbance

immediately. Read and record the absorbance every ?' seconds for 54> minutes or until no further changes occur.
7. Repeat Step 5 for tube R?. 8. Repeat Step 5 for tube R). 9. 1se a hemacytometer to count the number of mitochondria per ml of suspension used. 10. 7alculate the reaction rate for each tube as millimoles of dye reduced8minute81''

mitochondria. 7onvert the absorbance readings to millimoles of dye by using D 6 1O.1 millimoles for C7<#< X5'' nm. Refer to Appendi/ A for details of the %eer4!ambert law.
11. Record the amount of dye reduced at ?' second intervals. 1se linear regression to plot the

dye reduction over time for each tube. $ime ,Minutes' '.* 1.' 1.* 1 9 ? )

9.' 9.* ?.' ?.* ).' ).* *.' *.* 5.' 5.* >.'

Exercise =.9 - C0tochro#e /xidase Mano#etric $nal0sis

!D=D! ## Materials

'.'1 M <otassium <hosphate %uffer, p: >.) Sat. B Fe,7+'.5 M Sodium Malonate ,Malonic acid, sodium salt* mM B7+ '.'9S ,w8v- p4phenylenediamine o/alate ,<<C;- O

Mitochondria suspension from D/ercise I.) Spectrophotometer and cuvettes

<rocedure
1. <repare a series of * tubes as follows2

Substance

%uffer

?.' ml

?.' ml

9.' ml

9.' ml

?.' ml

B Fe,7+-

'.1 ml

'.1 ml

'.1 ml

'.1 ml

'.1 ml

Malonic Acid

+one

+one

+one

1.' ml

+one

B7+

+one

+one

1.' ml

+one

+one

.ater

1.' ml

+one

+one

+one

+one

2. $urn on a spectrophotometer and ad"ust the wavelength to 5?' nm. 1se tube R1 to blank

the spectrophotometer.
3. Add 1.' ml of the mitochondria suspension to tube R9 and mi/ by gentle inversion. Add

1'' L l of <<C; to the tube and immediately measure its absorbance at 5?' nm.
4. 7ontinue to measure the absorbance every ?' seconds for )4* minutes or until there is no

further change in absorbance. +ote that o/idation of p4phenylenediamine o/alate ,<<C;- changes the dye from its colorless form ,reduced- to purple ,o/idi&ed-. 7onse3uently, there should be a gradual increase in absorbance as the dye is o/idi&ed, corresponding to the activity of cytochrome o/idase.

5. Add 1.' ml of the mitochondria suspension and 1'' Ll of <<C; to tube R?. Measure the

absorbance immediately.
6. 7ontinue to measure the absorbance every ?' seconds for )4* minutes or until there is no

further change in absorbance.

7. Repeat Steps * and 5 with tube R). 8. <lace 1.' ml of mitochondria suspension into a tube, and place in boiling water for *

minutes. 7ool and add to tube R*. Add 1'' Ll of <<C; to tube R*, invert and measure the absorbance as with tubes R94).
9. Record the changes in absorbance for tubes 94* at ?' second intervals. <lot the changes in

absorbance for each tube. $ime ,Minutes9 ? ) *

'

'.*

1.'

1.*

9.'

9.*

?.'

?.*

).'

).*

*.'

Mano#etric $nal0sis Dach of the preceding e/ercises utili&ed the reducing power produced by the reactions of photosynthesis or respiration to monitor the color change in a dye. Another approach to the process would be to monitor the production or utili&ation of o/ygen in the process. Since o/ygen is a gas, it can be monitored through direct measurements of change in volumes of a gaseous phase. $his is known as respirometry, and can be monitored simply ,with testtubes and capillary pipettes- or by more comple/ and more accurate means ,with the use of a Ailson Respirometer or a .arburg Apparatus-. $he respiration of single cells has been measured with 7artesian divers. $hese systems lend themselves primarily to fluid systems with a gas intermediate, an ideal arrangement for isolated organelles in suspension. #t also works well for cells in suspension ,algae, bacteria, yeast, tissue culture- and for moist tissue slices ,leaf disks, liver slices-. %ecause of the nature of gas volume measurements, the primary difficulty with these techni3ues is control over temperature and atmospheric pressure. For simple procedures ,such as the measure of gas evolved from an elodea leaf in a test tube- these are usually ignored or some type of control is attempted ,water baths and heat shields for the lights-. For accurate measurement on the cell level more e/tensive control must be had. $he .arburg Manometer was developed as a system to maintain constant temperature and volume, thus measuring changes in gas e/change by changes in pressure. #t is e/tremely accurate, but it also is tedious and re3uires a good deal of mathematical corrections ,made easier, of course, by using a computer-. $he Ailson Respirometer keeps constant temperature and pressure and monitors the change in gas e/change by changes in volume. $o its advantage, the

volume can be read directly and thus much of the mathematical corrections needed for the .arburg Manometer are eliminated. $he constant pressure respirometer suffers in accuracy when compared to the constant volume manometer of .arburg, but it is sufficient for nearly all cell physiology purposes. #ts inherent ability for easy automation has made it a standard for most cell labs. %y contrast, the manometer remains the tool of choice for physical chemists. $he Ailson 7onstant <ressure Respirometer $he reaction flask consists of a main reaction chamber, a side arm, a center well and connections to a micrometer and a second chamber. $he reaction is started by pouring the contents of the sidearm ,usually a substrate- into the main vessel ,containing the main reactants-. $he center well contains any materials which are accessory to the reaction ,such as B;: to absorb 7; -. 7hanges in volume within the main vessel are altered through the use of a micrometer8piston system, and the micrometer is usually designed to read directly in microliters of gas e/changed. #nterpretation of what gas has been e/changed is a function of the e/periment design.

Exercise =.< - @espiration of Mitochondria

!D=D! ### Materials

Ailson ,or .arburg- Respirometer at ?>G 7 Mitochondria suspension from D/ercise I.) Brebs <hosphate Ringers ,B<R1'S ,w8v- Alucose '.?OS ,w8v- Sodium a&ide 1I.) mgS ,w8v- Cinitrophenol ,C+<5.5)S ,w8v- Sodium Malonate 1'S ,w8v- B;:

Procedure
1. .hen you enter the lab, check that the reference chambers of the Ailson respirometer are

filled with the appropriate solution ,usually formaldehyde-. $urn on the instrument and e3uilibrate the temperature.

2. !ine up fourteen reaction vessels and ensure that each is clean, free of cracks and has all

the necessary springs ,) each-, stoppers and vent tubes handy.

3. 1se a micropipette to carefully place '.9 ml of 1'S B;: in the center well of each. #t is

e/tremely important that no B;: spill into the main reaction vessel,as this will destroy the mitochondria placed in the vessel.
4. <lace 9.' ml of B<R in the main vessel of flasks R1 and R9. 5. <lace '.* ml of B<R in the main vessel of flasks R? and R). 6. <lace '.9 ml of B<R in the main vessel of flasks R* through R19. 7. <lace '.1 ml of B<R in the main vessel of flasks R1? and R1). 8. <lace 1.' ml of mitochondria suspension in the main vessels of flasks R? through R1). 9. Add '.* ml of glucose to the side arm of flasks R? through R19. 10. Finally, add the following to the main vessels of the indicated flasks2

R* and R5 R> and RI RO and R1' R11 and R19

'.? ml of glucose '.? ml of sodium a&ide '.? ml of C+< '.? ml of sodium malonate

11. 7ut fourteen 9 cm s3uares from filter paper and make small fans out of each. <lace one

piece each into the center well. Coing this will increase the surface area available for 7; absorption by the B;:.
12. 7arefully attach the sidearm stopper ,or closed vent tube- to the flasks. Attach the flasks

to the manometers and secure with springs. !ower the flasks into the water bath.
13. Allow all of the reaction flasks to temperature e3uilibrate for * minutes. Curing this time,

set all of the micrometers to a reading of *''.

14. Add the contents of the sidearms to the main vessels by lifting the entire manometer

assembly from the water bath and gently tipping the flask. %D 7ARDF1! +;$ $; S<#!! A+J 7;+$D+$S #+$; ;R ;1$ ;F $:D 7D+$DR .D!!.

15. <lace all of the flasks back into the water bath and close all valves of the respirometer.

$his is the &ero point for your data 4 record the time.
16. $urn on the shaking motor and ad"ust the speed to obtain a gentle swirl of the flask

contents.
17. After 1' minutes, ad"ust the manometer fluid to the original starting line by turning the

appropriate micrometer. Record the micrometer readings for each of the fourteen flasks.
18. Repeat the micrometer readings at 1' minute intervals until a stable slope is obtained for

the respirometers. $his will generally be within )'45' minutes from the &ero point. Record all readings.
19. For each reading, subtract the original value of *''. Average the readings obtained in

flasks R1 and R9 and subtract this average from the averages of R?8R), R*8R5, R>8RI, RO8R1', R118R19 and R1?8R1).
20. <lot the change in gas volume ,o/ygen consumption- for each of the replicate

conditions. 1' "otes $ubes 1 and 9 are thermobarometers. Any changes in gas volume within these tubes reflects changes in the temperature and8or barometric pressure. $ubes ?8) and *85 represent all of the conditions for o/idative phosphorylation ,respiration-. $ubes >8I are inhibited by a&ide which uncouples the electron transport system. $ubes O81' are inhibited by the presence of C+< which effects glucose transport as well as substrate entry into the electron transport system. Sodium malonate in tubes 11819 is a competitive inhibitor of succinic dehydrogenase within the Brebs cycle. #t will not halt the reactions of the D$S, and will only slow them down if glucose is in low concentration relative to the malonate. Finally, tubes 1?81) represent any endogenous reactions of the mitochondria in the sucrose isolation media. $echnically, glucose is not metaboli&ed by the mitochondria. #t must be altered by cytoplasmic en&ymes to form pyruvate before entering the mitochondria. $he isolation procedure used in mitochondrial preparation is important. #f the mitochondria are 0purified0 the measurements will not work. 1sing a rough homogenate in sucrose will maintain enough en&ymes for glycolysis to occur. $he e/periment could be altered by using pyruvic acid rather than the glucose. Alucose is used in the traditional e/periment.

Record the fourteen manometer readings every 1' minutes for at least one hour. Average each pair of readings and substract the reading for 1?81) from each of the averages for ?8), *85, >8I, O81' and 11819. <lot the corrected values against time, using a linear regression plot for each.

Flask R8$ime

1'

9'

?'

)'

*'

>

1'

11

19

1?

1)

Exercise =.= - Photos0nthesis +0 @espiro#etr0

!D=D! ### $able I.1 7haracteristics of isolated chloroplasts Materials

Ailson respirometer e3uipped with photoflood lamps 7hloroplast suspension 11 7hloroplast suspension buffer ,suspension solution from D/ercise I.1 '.1 M +a:7; #nhibitor 1 ,1 / 1' #nhibitor 9 ,* / 1' C7M1C7M1-

Procedure
1. #n general, use the procedure listed in D/ercise I.> for mitochondria respiration, e/cept

that the reactions are run at 9*G 7 and a bank of photoflood lights must be used to ensure photosynthesis.
2. Set up the reactions vessels in the following manner2

$ube 149 ?4)

Side arm

Main =essel 9.' ml 7hloroplast buffer '.* ml buffer 1.' ml 7hloroplast Suspension '.9 ml buffer 1.' ml 7hloroplast Suspension '.9 ml buffer 1.' ml 7hloroplast Suspension '.9 ml buffer 1.' 7hloroplast Suspension '.9 ml buffer 1.' ml 7hloroplast Suspension '.* ml buffer 1.' ml %oiled 7hloroplasts

'.* ml +a:7;

*45

'.I ml +a:7;

>4I

'.* ml +a:7; '.? ml Sodium A&ide '.* ml +a:7; '.? ml #nhibitor 1 '.* ml +a:7; '.? ml #nhibitor 9

O41'

11419

1?41)

'.* ml +a:7;

3. Measure the amount of o/ygen evolved every 1' minutes until a steady rate is reached.

<lot results and compare the effects of the selected inhibitors. Chapter =: Photos0nthesisI@espiration - Endnotes

1.

7hloroplasts are e/tremely light labile. $hey should be kept cool and in reduced light at all times, e/cept when actually being used for analysis of photosynthesis. ;nce diluted, the chloroplasts will be reasonably unstable and you should work as rapidly as possible while keeping all reactants as cool as possible. %e careful to not confuse the pigment 7:!;R;<:J!! with the organelle 7:!;R;<!AS$.

2.

3.

C7M1 is a powerful electron acceptor and will inhibit System ## electron transfers in intact chloroplasts. May substitute 1' mM $etra&olium %lue X *I' nm, 9' mM Methyl Red X ))' nm, or . ''1 M 7 ,7 ,7 ,7 4$etramethyl4p4pheynlenediamine ,CAC- if System # is to be specifically measured. CAC will reverse the uncoupling of System # induced by C7M1. Sunlight is best, but photofloods will do. #f photoflood lights are used, a heat filter must be used. $his can be accomplished by placing a small a3uarium full of water between the light source and the beaker containing your chloroplast suspensions. Fresh cauliflower can be substituted if the homogeni&ation is done with a mortar and pestle. $here will be some plastid contamination, but it should not affect respiration measurements with normal room light e/posure. .eigh cauliflower and begin with step ?. $here will be significantly fewer mitochondria isolated per gram of tissue than with liver. May be omitted. #t works to protect the mitochondria from rapid degradation. <repare "ust before use. A stock solution of '.??S may be stored fro&en in an amber bottle. $haw and dilute "ust before use. ;/idation of p4phenylenediamine o/alate ,<<C;- changes the dye from its colorless form ,reduced- to purple ,o/idi&ed-.

4.

5.

6.

7. 8.

9.

10. $his is easier to do if the data are entered directly into a computer spreadsheet program.

Refer toAppendi/ C.
11. $he isolation of intact chloroplasts is a significant factor in the success of this e/ercise.

Many of the structures and functions of chloroplasts are altered during the e/traction process. Refer to $able I.1.

Chapter >: Tu+ulesIFila#ents - Introduction

Figure O.1 Macrophage stained with F#$7 labeled anti4tubulin

Figure O.9 $DM of striated muscle $able O.1 #ntermediate filaments and matri/ components 7ell shape is determined by microscaffolding created by microtubules and microfilaments. #nternally, these tubules and filaments make up the cytoskeleton. D/ternally they are the components of the e/tracellular matri/ ,glycocaly/ and plant cell wall-. Microtubules have a diameter of about 9) nm and are usually found in groups of 1? protofilaments. $he basic structural subunit is a dimer of two similar proteins, and

tubulin, each with a molecular weight of **,''' daltons. $ubulin and several larger microtubule4associated proteins ,MA<s- interact in the presence of A$< and Mg to

form hollow tubes known as protofilaments which can stretch for long distances across the cytoplasm. $he MA<s function to anchor the tubulin protofilaments together into larger structures ,centrioles, basal bodies, flagella- and to attach the protofilaments to other structures within the cell ,nuclear envelope, chromosomes- as well as to other filamentous molecules ,actin, intermediate filaments-. <olymeri&ed tubules are also important in cell division,motility, communication and differentiation ,morphogenesis-. $hey are believed to be important elements of neural cell function and glandular secretion. .ithin a cell, microtubule integrity is affected by temperature. Microtubules are relatively stable at physiological temperatures, but below 1'G 7 they spontaneously depolymeri&e into constituent subunits, namely tubulin and the microtubule4associated proteins ,MA<@s-. .hen the cells are returned to ?>G 7, microtubules are reformed in about 1* minutes ,providing that sufficient A$< and Mg Microtubules ;ne of the several methods currently used to isolate microtubules takes advantage of the temperature4dependent stability of the microtubule proteins. <olymeri&ed microtubules maintain their structural organi&ation at physiological temperatures, but rapidly depolymeri&e below 1'G 7. A crude e/tract of tissue containing large numbers of are present-.

microtubules is warmed to ?>G 7 in a buffer containing A$<, DA$A ,a chelating agent that binds calcium ions- and Mg . Microtubules will form in appro/imately 1* minutes.

As the proteins polymeri&e into tubules, the viscosity of the solution will increase. #ncreased viscosity can be monitored directly with a viscometer, or indirectly by a change in sedimentation coefficient or by an alteration in the turbidity of the solution. $urbidity is the easiest alteration to monitor, since it can be measured with a 1= spectrophotometer. After the formation of the microtubules, a pellet enriched in microtubules may be prepared by centrifugation of the viscous solution at warm temperatures ,1'G 7-. $he pellet is then depolymeri&ed by incubation at )G 7 in buffer, once again yielding a solution of tubulin, with a slight decrease in yield, but an increase in the purity. $he supernatant containing both tubulin and microtubule4associated protein is again warmed for microtubule polymeri&ation. Several cycles of this temperature dependent, assembly8disassembly procedure results in highly pure preparations of microtubules. $he polymeri&ed microtubule structure can be e/amined by transmission electron microscopy for structural integrity. $he microtubules are negatively stained and dried directly onto a coated DM grid for observation. Alternatively, they can be pelleted, embedded and sectioned for routine $DM analysis. Since the tubulin and MA<s are protein, antibodies can be made against the molecules. $hese can be used either for DM analysis or for immunofluorescent analysis at the light microscope level. Microtubule assembly can be inhibited by several chemical agents. 7olchicine or vincristine depolymeri&e microtubules, and8or prevent their polymeri&ation from the monomer tubulin. 7olchicine is an alkaloid e/tracted from the plant 7olchicum sativum. 7olcemid, a synthetic derivative, is commonly used in tissue cultures to halt mitotic cells at metaphase. .hen colcemid is removed, spindle fibers will form and the cell will divide. #f the colcemid treatment is prolonged, the cell will not divide and will result in polyploid formation.. #mmunofluorescent Analysis 7ells fi/ed at ?>G 7 in their microtubular structure. $o visuali&e the organi&ation of the microtubules, plasma membranes are solubili&ed and made permeable to anti4tubulin

antibodies. #f the antibody has previously been bonded to a fluorescent dye, the organi&ation of microtubules within the cell can be observed with a fluorescent microscope. Microfilaments ;ne of the most studied forms of molecular architecture associated with cellular function has been the relationship of thick and thin filaments of the striated muscle of vertebrates. $he thin actin filaments ,54I nm diameter- are interposed between the thick myosin filaments ,1I nm diameter- and these in turn are often associated with intermediate filaments ,about 1' nm diameter-. Figure O.1 presents a schematic of the relationship of these filaments in a typical striated muscle whileFigure O.9 gives both light and $DM views. #n smooth muscle, the connections are not as clearly defined, but the presence of the three types of filaments can be observed. #n smooth muscle, the intermediate filaments are integrated into the thin filaments. #mtermediate Filaments Any analysis of microfilaments would be incomplete without some study of the host of filaments collectively referred to as intermediate filaments. $here are five types of intermediate filaments2 tonofilaments ,keratins or cytokeratins-, neurofilaments, glial filaments, desmin filaments and vimentin filaments. For further details, refer to $able O.1. AAAs $here are a host of filamentous and globular proteins on the surface of cells which are composed of both proteins and carbohydrates. ;lder terminology referred to these as glycoproteins if there was more protein than carbohydrate and mucopolysaccharides if there was more carbohydrate. $oday, the common term is Alycos Amino Alycans or AAAs. D/amples of AAAs are collagenous and non4collagenous glycoproteins, elastin, sulphated proteoglycans ,heparin- and comple/es with hyaluronic acid. AAAs are involved in the formation of the 0pericellular0 matrices of developing cells. $hese matrices are important for cell4cell contact in early embryogenesis and differentiation, and in the formation of e/tracellular basement membranes. $here is

evidence that they are involved in information processing of R+A within the cell as well as their structural function. Receptor mediated endocytosis is also dependent upon these surface molecules as is normal receptor activities of hormones, neurohormones and the activation of cAM<.

Exercise >.1 - Fluorescent $nal0sis of Microtu+ules

!D=D! #

Figure O.1 Macrophage stained with F#$7 labeled anti4tubulin Materials

<ro"ection slides of tubulin fluorescence1

Procedure
1. Craw the cells represented in Figure O.1. $hese are photographs of macrophages treated

with a fluorescent anti4tubulin antibody.

2. Cescribe the shape and general appearance of the cells observed in the first three slides.

:ow does the organi&ation of the microtubules affect cell shapeP

3. #dentify a place in the cell from which the microtubules appear to e/tend. $his is the

microtubule4organi&ing center. !abel it in the appropriate drawings.

4. $he cells in slide ) were e/posed to colchicine for ?' minutes and then prepared as

above. .hat do you predict will happen to the microtubules after cell treatment with colchicineP .as this prediction fulfilledP Craw a colchicine4treated cell.
5. $he cells in slide * were placed on ice for ?' minutes and then prepared as above. .hat

do you predict will happen to the microtubules after incubation of the cells in the coldP #s the microtubule diameter larger or smaller than the resolving power of the light microscopeP :ow can microtubules be visuali&ed with immunofluorescence if the tubules are less than the resolution of the light microscopeP

Exercise >.. - $ctin J M0osin-Microscopic /+ser(ation of Muscle

!D=D! #

Figure O.9 $DM of striated muscle Materials

Alycerinated rabbit muscle fibers * mM A$< in '.'1 M $ris4:7l buffer, p: >.5 plus '.'* M B7l '.''1 M 7a7l <repared slides of striated muscles ,longitudinal sections<hase contrast microscope, slides, coverslips

<rocedure
1. ;btain a prepared slide of striated muscle cells. Craw the structure of an individual cell,

paying particular attention to the striations. Measure the distance between successive bands of the muscle and indicate these on the drawing.
2. 7ompare the image seen in the light microscopic to that in Figure O.9. 3. $ease a small fiber from the glycerinated rabbit muscle and place in on a microscope

slide. <lace a coverslip gently over the muscle fiber and e/amine with a phase contrast microscope.
4. Measure the distance between the bands and compare to the distance found in the

prepared slide.
5. Aently lift off the coverslip ,or use a second fiber- and add a drop of A$< and a drop of

7a7l . Return the coverslip and immediately monitor the cells through the microscope. ;nce again measure the distance between the bands. ;ptional $o observe the contraction of the muscle, carefully add the A$< and 7a to the edge of the

coverslip and allow it to diffuse under the coverslip. Jou may also time the contraction process with a stopwatch. Finally, variations in the ion concentration as well as alternative ions can be added to other muscle fibers to test the effects of ions on muscle contraction.

Exercise >.5 - ?istolog0 of Extracellular Matrix

!D=D! # Materials

<repared slides of _ _ _ _ _ _ _ #ntestine !oose connective tissue !amina rara of epidermal tissues Skin and8or bone :yaline cartilage Amniotic membrane Dlastic Fibers _ _ _ _ _ _ _ %asement Membranes Fibronectin !aminin 7ollagen $ype # 7ollagen $ype ## 7ollagen $ype ### Dlastin

Microscope

Procedure
1. 7omponents of the e/tra cellular matri/ are too small to be observed directly with a light

microscope, but their presence can be observed in the structures they form. $he list of slides above includes the basic intermediate filament type associated with each tissue structure. D/amine each of the slides, paying particular attention to the location and structure of any e/tracellular matrices.
2. Craw each of the tissues. !abel the location and type of matri/ component e/pected in

each tissue.

Exercise >.1 - Isolation of Microtu+ules (Bo(ine Brain!

!D=D! ## Materials

Freshly removed bovine brain 9 .ire sieve ,tea strainerMicrotubule buffer ,M$ buffero o

'.1 M MDS ,94,+4Morphilino-ethanesulfonic acid1 mM DA$A ,Dthylene Alycol4bis, 4aminoethyl Dther- +,+,+@,+@4tetraacetic acid-

o o

'.* mM Mg7l Ad"ust p: to 5.)

I M Alycerol in M$ buffer =irtis homogeni&er ,or e3uivalent-

Refrigerated centrifuge A$< %radford protein assay

Procedure ?
1. Remove the meninges and peripheral blood vessels from the brain. <uree the brain by

pushing it through a wire sieve and directly into a chilled beaker.

2. :omogeni&e the cerebral hemispheres of the brain at )G 7 in a tissue homogeni&er

capable of homogeni&ing with minimal sheer force. ) 1se the ma/imum speed allowable for your homogeni&er for 1' seconds. <lace the brain hemispheres in the homogeni&er with microtubule buffer at a ratio of '.* ml of M$ buffer to 1 g of wet weight of brain tissue.
3. 7entrifuge the crude brain homogenate at 1O,5'' /g for ?' minutes at )G 7 to remove

connective tissue and cellular debris.

4. Cecant the supernatant and recentrifuge the supernatant at 9>,''' /g for )* minutes at )G

7 for further clarification.

5. 7ollect 1' ml of the supernatant and determine the protein content of your sample using

the %radford protein assay ,Appendi/ A-. 1se bovine serum albumin or lyso&yme for establishing a standard curve.
6. Cecant the remainder of the crude supernatant into a chilled beaker, and add an e3ual

volume of I M glycerol in M$ buffer.

7. Add dry A$< ,M. *9?- to the crude supernatant to make a 1.' mM final concentration

of A$< and incubate the mi/ture at ?>G 7 for ?' minutes.

8. 7entrifuge the mi/ture at 1'',''' /g for 5' minutes at 9*G 7. #t is crucial that the

temperature be maintained at 9*G 7. At a lower temperature the microtubules will not polymeri&e ,thus no pellet- and at a higher temperature the tubulin may be degraded.
9. Remove the pellet and resuspend in )' ml of cold M$ buffer. #ncubate for ?' minutes at

)G 7. ;ccasional homogeni&ation in a ground glass homogeni&er will facilitate depolymeri&ation of the microtubules.

For good tubulin polymeri&ation, a series of polymeri&ations and depolymeri&ations, with subse3uent centrifuge collections is re3uired. Steps > through O should be repeated a minimum of ? times.
10. .ith each successive polymeri&ation with A$< and subse3uent collection of the

precipitated microtubules, repeat the protein determination on each sample.

Exercise >.7 - :iscosit0 J Pol0#eri3tion of Microtu+ules

!D=D! ## Materials

$ubulin ,%rain e/tract from D/ercise O.)A$< A$< =iscometer .aterbath or incubator at ?>G 7

Procedure
1. 7ompute the amount of A$< ,M... *9?- needed to make a final concentration '.* mM

A$< when added to )' ml. of brain e/tract in microtubule buffer. !ikewise, compute the amount of dry A$< ,M.. *'>- needed to make a final concentration of '.* mM A$<.
2. .ith the brain e/tract in an ice bath, add both the A$< and the A$< ,swirl to dissolve-

and immediately transfer the mi/ture to a water bath at ?>G 7.

3. 1se a viscometer to measure the viscosity at ? minute intervals for a period of ?'

minutes, or until change is no longer observed. A viscometer is a device which makes use of capillary flow. #nsert a glass viscometer into a water bath at the appropriate temperature ,?>G 7 for polymeri&ation- and add a ?.' ml sample to the viscometer. Apply suction or pressure as appropriate to the viscometer to pull the sample into the e/panded sample chamber. Release the suction ,or pressure- and measure the time it takes for the sample to move from the upper mark on the viscometer to the lower mark. 1se sucrose solutions of known viscosity to calibrate the viscometer.
4. <lot the change in viscosity with time of incubation.

"otes

$ubulin and MA<@s will polymeri&e in the presence of Mg

and A$<. $he tubulin e/tract is

placed in a buffer that supplies all of the necessary ingredients, e/cept A$<. 1pon the addition of A$<, the tubulin will begin to polymeri&e and can be monitored.

Exercise >.9 - Pol0#eri3ation Induced Changes in $+sor+ance

!D=D! ## Materials

1= spectrophotometer .ater baths set at 1'G 7 and ?>G 7 $ubulin e/tract from D/ercise O.* M$ buffer, A$< and A$<

Procedure
1. $urn on the spectrophotometer and ad"ust the wavelength to ?*' nm. Ad"ust the

spectrophotometer to read absorbance, using microtubule buffer as a blank.

2. Starting with the tubulin e/tract in the water bath and e3uilibrated to 1'G 7, add A$< and

A$< as in steps 1 and 9 of D/ercise O.*. #mmediately measure the absorbance of the solution. Speed is importantN <lace the sample still in the spectrophotometer cuvette back into the 1'G 7 waterbath.
3. At ?' second intervals remove the cuvette, wipe it dry and measure the absorbance.

#mmediately replace the sample and cuvette back in the 1'G 7 waterbath. Repeat this for a total period of ? minutes.
4. After the ? minute interval, place the cuvette and its contents in the ?>G 7 waterbath. 5. 7ontinue to read the absorbance at ?' second intervals, returning the sample to the ?>G 7

waterbath between readings.

6. .hen the absorbance value stabili&es ,about * minutes-, replace the sample and cuvette

back in the 1'G 7 bath and continue to measure the absorbance at ?' second intervals.
7. <lot absorbance against time, indicating points of temperature change.

"otes Jou may monitor the kinetics of tubule formation by continuously measuring the absorbance of the solution at ?*' nm, provided a temperature controlled sample holder is available. <referably,

the sample temperature should be able to be rapidly changed from 1'G 7 to ?>G 7 and back. $his can be accomplished via a temperature4controlled kinetic spectrophotometer ,such as a %eckman C1 Binetic spectrophotometer, or e3uivalent-. Alternatively, a water "acketed sample cuvette can be engineered with some simple tubing and valves to switch the water flow through the "acket.

Exercise >.< - TEM :isuali3ation of Microtu+ules

!D=D! ## Materials

7oated grid for $DM '.1 M ammonium acetate *S ethanol saturated uranyl acetate $ransmission electron microscope

Procedure
1. At the conclusion of a ?>G 7 incubation from D/ercise O.5, remove one drop

,appro/imately 9' microliters- of the e/tract, place it on a collodion or formvar and carbon4coated DM grid, and allow it to remain for about 9'4?' seconds. 5
2. Rinse the grid carefully with '.1 M ammonium acetate, and float the grid upside down on

a drop of *S uranyl acetate for 1* minutes. Rinse the grid with water, blot all e/cess fluids from the grid, and allow it to air dry.
3. D/amine the stained grid in a transmission electron microscope. 4. Craw or photograph your observations 44 calculate all appropriate dimensions ,length,

diameter- of the formed tubules.

Exercise >.= - 6*6 Ael Electrophoresis of Tu+ulinIM$Ps

!D=D! ## Figure O.? SCS4<AAD of tubulin and associated proteins Materials

Stock Acrylamide2 ,?'S$2'.IS7o

?'S by weight of acrylamide

'.IS by weight of +,+@4bis4methylene acrylamide

Separation Ael ,Final 7oncentrationso o o

1'S acrylamide ,12? dilution of stock'.?>* M $ris4:7l ,p: I.I'.1 S SCS

Stacking Ael ,Final 7oncentrationso o o

? S acrylamide ,121' dilution of stock'.19* M $ris4:7l ,p: 5.I'.1S SCS

Dlectrode %uffer
o o o o

'.'9* M $ris '.1O9 M Alycine '.1S SCS Ad"ust p: to I.?

'.94'.? ml samples of brain e/tract, and containing

o o o o o o

9'' micrograms protein '.'59* M $ris4:7l ,p: 5.I9S SCS 1'S glycerol *S 4mercaptoethanol '.''1 S bromophenol blue

$DMDC 1'S ,w8v- Ammonium persulphate *'S and >S ,w8v- $7A ,trichloroacetic acid'.1S ,w8v- 7oomassie brilliant blue in *'S $7A

Procedure

1. <repare 1* cm glass tubes with 5 mm internal diameter. <olymeri&e a 1' cm separation

gel and a 1 cm stacking gel within the tubes by the addition of $DMDC and ammonium persulfate as directed in 7hapter Four.
2. Assemble all the e3uipment and place 9'' Ll of sample in one tube. Set up a second gel

containing 9'' Ll of protein molecular weight standards.

3. Dlectrophoresis is carried out at ? mA per gel until the bromophenol blue dye reaches the

bottom of the tube ,appro/imately > hours-.

4. Fi/ the gels overnight in *'S $7A. 5. Stain with '.1S 7oomassie brilliant blue ,fresh in *'S $7A- for 1 hour at ?>G 7. 6. Ciffusion4destain with repeated washing in >S $7A. 7. Scan the gels or photographically analy&e them. 8. Cetermine the molecular weights of the proteins ,tubulin and MA<@s-.

Exercise >.> - Isolation of $ctin and M0osin Fila#ents

!D=D! ### Materials

Rela/ing Solution
o o o o o o

'.1 M B7l '.''1 M Mg7l * mM A$< '.'15 M +a: <; +a :<; Ad"ust p: to >.?

'.'* M Sodium phosphate buffer, p: >.' '.''1 M DC$A ,Dthylene diamine tetra acetic acid%lender <reparative centrifuge Materials for $DM fi/ation, embedding and observation

Procedure >

1. ;btain fresh chicken gi&&ards I and dissect the lateral muscles free from all attachments.

<lace the muscles on crushed ice and then grind them in a standard worm4 drive meat grinder. Small samples can be pushed through a hand press, if desired.
2. .eigh the tissue and add an e3ual volume of cold '.'* M Sodium phosphate buffer, p:

>.' with '.''1 M DC$A. %lend this mi/ in a standard blender at low speed and pour the slurry into a large beaker.
3. 1pon settling, the muscle fragments will settle on top of the underlying connective tissue.

Separate the two by decanting, and concentrate by low speed centrifugation.

4. Suspend ali3uots of blended muscle in two volumes of rela/ing solution and homogeni&e

in a blender at high speed for ?' seconds.

5. 7entrifuge the homogenate at *'' /g to remove membraneous organelles and whole

cells. $he myofilaments are preferentially locali&ed in the middle, clear solution of the centrifuge tube.
6. 7ollect the middle layer and recentrifuge at )',''' /g to pellet the myofilaments. .ash

and gently resuspend. $his will rid the preparation of soluble proteins.
7. <repare a small sample of the middle layer from Step * for DM observation, following the

procedure outlined above for tubulin ,D/ercise O.>-.

Exercise >.1, - Further 6tudies

!D=D! ### Additional analysis of the interaction of tubulin and MA<s can be made following the separation of the proteins. $his can be accomplished by phophocellulose chromatography, as described in Aamodt and .illiams. O ;nce separated, the polymeri&ation can be monitored with varying ratios of tubulin to MA<, and in the presence of various polymeri&ation inhibitors ,colchicine, vincristine-. ;ther studies could be made of isolated cellular structures, such as flagella, cilia, or even isolated spindle fibers. $he latter can be isolated in 3uantity from sea urchin embryos, as outlined by B.A. Suprenant. 1' 7ollect egg and sperm by in"ection of B7!, and combine the two for fertili&ation. As the embryo@s reach metaphase ,I'4I* minutes at 1IG 7-, they are collected, washed free of salt water and homogeni&ed in a low p: <#<DS buffer containing Mg , DA$A and $riton (41''

detergent. $he spindles are pelleted at 1''' (g ,clinical centrifuge-, washed several times and monitored with polari&ation microscopy. Since the spindles are highly birefringent, they can be studied without the use of a $DM. $hey can be observed fairly readily with standard enhanced phase contrast optics or stained for bright field optics. $he sea urchin also serves as a ready source of flagella ,sperm- and cilia ,developing pluteus-. =irtually all of the early work on tubulin polymeri&ation was accomplished with this model system. Chapter >: Tu+ulesIFila#ents - Endnotes

1.

#t is convenient to use permanent photographic slides, since the microscope preparations are unstable ,flurorescence causes bleaching of the dye-. Several companies produce fluorescent antibodies to tubulin and also supply protocols for producing the slides. Refer to D/ercise 9.> for further details. Available from slaughter houses, or any animal brain may be substituted. #t is important that the brain be freshly removed from a sacrificied animal, however. From Shelanski, M.!., F. Aaskin and 7.R. 7antor. 1O>?. 0Microtubule assembly in the absence of added nucleotides.0 P6"C. 4AT5. ACA-. +C7. U+A >'2>5*4>5I. For a modified .eissenberg $echni3ue and Sephade/ <urification of tubulin cf. A.7. +a and S.+. $imasheff 0<hysical <roperties of <urified 7alf %rain $ubulin0 in Methods in Dn&ymology, =ol. I*, p. ?O?4)'I. 1OI9. A household blender should only be used as a last resort. 7rude estimation of an increase in viscosity can be had by observing the rate of escape for trapped air bubbles as the solution is swirled. =iscosmeters are available commercially at reasonable prices. Alternatively, a pipette can be utili&ed. Mi/ a reasonable sample of sucrose concentrations ,look up viscosities in handbook of physical constants- and simply calibrate the viscometer by measuring the time it takes to deliver ? ml from the filled pipette. Alternatively, the pellets containin microtubules can be collected, fi/ed, embedded and sectioned for DM e/amination. $his will re3uire several days, however, and will yield sectioned views of the tubules, rather than intact whole tubules.

2.

3.

4. 5.

6.

7.

7ooke, <. 0<reparation of #solated $hick and $hin Filaments0 in Methods in Dn&ymology, =ol. I*, pp 9>>49I)., 1OI9. #t is possible to isolate the individual fibers from various sources, but avian gi&&ards have been most often used for preparation of thick and thin filaments. $hey can be obtained from chicken slaughterhouses in mass 3uantity, and are virtually pure smooth muscle. Aamodt, D.T. and R.7. .illiams, Tr. 1OI). 0Microtubule4associated proteins connect microtubules and neurofilaments in vitro.0 %iochemistry 9?25'9?45'?1.

8.

9.

10. #n #etho&s in Cell %iology =ol. 9>2 Dchinoderm Aametes and Dmbryos ,$.D. Schroeder,

ed.-. Academic <ress, ;rlando, 1OI5. pp. 1IO4915.

Chapter 1,: Chro#oso#es - Introduction

Figure 1'.1 Ducaryotic chromosome structure $he interphase chromosomes of eucaryotic cells are comple/ molecular structures composed primarily of a C+A core and a protein matri/ comple/ed into a long thread4like structure. $his basic chromosome thread is then coiled through several layers of organi&ation and ultimately gives rise to a structure that can be visuali&ed with a light microscope. 7hemically, the interphase nucleus is composed of a substance known as 0chromatin,0 which is further subdivided into euchromatin and heterochromatin. $he distinction between these subdivisions is based on 3uantitative distribution of the basic chromosome fiber, with a higher concentration found in heterochromatin. :eterochromatin, therefore, will stain more intensely than euchromatin, since the fiber is packed tighter within a given volume. <roteins e/tracted from chromatin have been classified as either basic or acidic in nature. $he basic proteins are referred to as 0histones0 and the acidic as 0non4histone proteins0. :istones play an integral part in the structural integrity of a eucaryotic chromosome. $hey are organi&ed into specific comple/es, known as nucleosomes, and around which the C+A molecule is coiled. Acidic proteins within the nucleus compose many of the C+A replication and R+A transcription

en&ymes and regulatory molecules. $hey vary in si&e from small peptides of a few amino acids to large duplicase and replicase en&ymes ,respectively C+A and R+A polymerases-. $ranscription of C+A on the chromosome fiber results in the presence of a host of R+A species found within the nucleus of the cell. .hen the R+A is transcribed from the 0nucleolar organi&er0 region of a genome and comple/ed with ribosomal proteins, granules are formed which collectively produce a 0nucleolus,0 visible at the light microscope level of resolution. .hen transcribed from other portions of the genome, the R+A is either in the form of pre4transfer R+A, or heterogeneous nuclear R+A ,hnR+A-. $he precursor tR+A must be methylated and altered before becoming functional within the cytoplasm, and the hnR+A will also be significantly modified to form functional mR+A in the cytoplasm. $hus, a chemical analysis of chromosomes will yield C+A, R+A and both acidic and basic proteins. 1 #t is possible to e/tract these compounds from an interphase nucleus ,i.e. from chromatin- or to physically isolate metaphase chromosomes and then e/tract the components. For the former, the nuclear envelope will be a contaminating factor, as will the nucleolus. For isolated chromosomes, many of the regulatory molecules may be lost, since the chromosomes are essentially non4 functional during this condensation period.

Exercise 1,.1 - Pol0tene Chro#oso#es of *ipterans

!D=D! # Materials

<repared slides of -rosophila ,fruit fly- salivary gland chromosomes Aenetic map of polytene chromosome bands

Procedure
1. D/amine the slides for the presence of bands. Select a single chromosome spread

demonstrating all four chromosomes and draw the complete structure.

2. !abel each of the four chromosomes of the fruit fly, as well as the chromocenter of the

connected chromosomes.

3. 7ompare your drawings to the genetic map for -rosophila.

"otes $he glands of dipterans ,flies- have a useful characteristic for analysis of gene location on chromosomes. Curing their mitotic division, the normal division of the chromosomes is aborted and the replicated chromosomes remain as an integral unit. $he chromosome content thus increases geometrically and produces 0giant0 polytene chromosomes. $he chromosomes remain attached at a point where the centromeres fuse, at the chromocenter.1 $his is clearly observed in the chromosomes of the fruit fly salivary gland tissue. $he fruit fly chromosomes are ideal specimens since they are in a near constant state of prophase and are incapable of further division. %ecause they have been e/tensively analy&ed for their genetic composition, co4linear maps of genes within genetic linkage groups have been produced and correlated with the physical location of a band on the chromosomes.

Exercise 1,.. - 6ali(ar0 Aland Preparation (6;uash technigue!

!D=D! # Figure 1'.9 Fruit fly larva Materials

Fruit fly larva ,wild type and tandem duplication mutantsRingers insect saline Fine forceps and probe Aceto4orcein Cissecting and regular microscopes Slides, coverslips Small dish of melted paraffin and paintbrush 9

Procedure
1. Select a third instar larva, for which the cuticle has not yet hardened, from a wild4type

culture of-rosophila. <lace it into a drop of Ringer@s saline solution on a slide.

2. <lace the slide on the stage of a dissecting microscope and view the larva with low

power. Arasp the anterior of the larva with a fine point forceps and pin down the posterior portion with a probe. Aently pull the head off and discard the tail of the larva.
3. !ocate the salivary glands and their attached fat bodies. $he glands are semitransparent

and attached by ducts to the digestive system. $he fat bodies are white and opa3ue. $ease away the fat bodies and discard.
4. <lace a drop of aceto4orcein on the slide ne/t to the Ringer@s and move the salivary

glands into the stain. %lot away any e/cess Ringer@s.

5. <lace a coverslip over the preparation and allow it to stand for 14? minutes ,it will take a

few trials to obtain properly stained chromosomes-. Aently s3uash the gland preparation in the following manner2
o o

<lace the slide between several layers of paper toweling. <lace your thumb on the top of the towel immediately over the coverslip and gently roll your thumb while e/erting a small amount of pressure ,as though you were making a finger print-. Co not move your thumb back and forth. ;ne gentle roll is sufficient. Remove the slide from the towels, and seal the edges of the coverslip by using a paint brush dipped in melted paraffin.

6. D/amine the slide with the microscope and diagram the banding patterns that are

observed.
7. 7ompare your s3uash preparation to that of the prepared slides e/amined in D/ercise

1'.1.
8. Repeat the s3uash techni3ue using larva from a genetic variant known to be the result of a

deletion and8or tandem duplication. Cetermine the location of the deleted or duplicated bands on the chromosomes.

Exercise 1,.5 - Extraction of Chro#atin

!D=D! ## Materials

%ovine or porcine brain '.9* M Sucrose containing '.''?? M calcium acetate

9.' M Sucrose with '.''?? M calcium acetate '.'>* M +a7l with '.'9) M DC$A, p: I.' $ris4:7l buffer, p: I.' with the following molarities '.'* M, '.''9 M, '.''') M

$7A ,$richloroacetic acid$issue homogeni&er 7heesecloth Refrigerated preparative centrifuge %radford or !owry protein assay 1= spectrophotometer ,;ptional-

Procedure ?
1. :omogeni&e appro/imately ?' gms of bovine or porcine cerebellar tissue in a teflon4

glass homogeni&er in O volumes of cold '.9* M sucrose containing '.''?? M 7alcium Acetate.
2. Filter the resulting brei through several layers of cheesecloth and obtain crude nuclear

pellets by centrifuging at ?,*'' /g for 9' minutes.

3. Resuspend the nuclear pellet in I' ml of cold '.9* Sucrose containing '.''?? M 7alcium

Acetate. )
4. ;btain three cellulose nitrate centrifuge tubes and place 9* ml. ali3uots of the

resuspended nuclear pellet in each. 7arefully pipette *.' ml of 9.' M Sucrose4'.''?? M 7alcium Acetate into the bottom of each tube. #nsert a pipette with the 9.' M sucrose through the suspended nuclei and allow the viscous sucrose to layer on the bottom of the tube. 7entrifuge the tubes at )',''' /g for 5' minutes.
5. 1sing the resulting nuclear pellet is "ust above the dense sucrose layer. #t is used to

e/tract chromatin proteins. 7arefully remove the supernatant above the pellet with a pipet. $hen, insert the pipet through the nuclear layer and remove the bottom sucrose layer. $he nuclear pellet will remain in the tube. Resuspend the pellet in )' ml of '.'>* M +a7l4'.'9) M DC$A, p: I.' and centrifuge at >>'' /g for 1* minutes.

6. Remove and discard the supernatant, resuspend the pellet once again in )' ml of '.'>* M

+a7l4'.'9) M DC$A, p: I.' and centrifuge again at >>'' /g for 1* minutes. Repeat this process one more time.
7. Resuspend the nuclear pellet in )' ml of '.'* M $ris4:7l, p: I.' and centrifuge at >,>''

/g for 1' minutes.

8. Repeat step > to thoroughly wash the nuclei and then wash 9/ each in '.'1 M $ris p:

I.', '.''9 M $ris p: I.' and '.''') M $ris p: I.'.

9. Resuspend the final washed nuclear pellet in ice cold distilled water to a final volume of

1'' ml and allow to swell overnight at )G 7. Aently stir the mi/ture on the following day. $his solution is the pure chromatin to be used for subse3uent analysis.
10. Cetermine the purity of the chromatin sample within the nuclear pellet using one of the

following2
o o

Cetermine the protein concentration by !owry or %radford procedure. Measure the optical absorbance at 9?' nm ,1=-. $he absorbance of a 1 mg8ml concentration of pea bud histone at 9?' nm e3uals ?.*. ;C units. $he absorbance follows the %eer4!ambert law, and is linear with histone concentration. Since it is merely an optical reading, the sample is not destroyed in the measurement. Measure the turbidity of the solution. Add trichloroacetic acid to a final concentration of 1.1 M and wait e/actly 1* minutes. Read the ;C . A 1' ug8ml solution of pea bud histone has an ;C 6 '.'I? at )'' nm. $his techni3ue is e/cellent for readings between ' and '.1* ;C. $he $7A precipitates some proteins and thus this procedure is more specific to histones than %. #t can also be performed without a 1= spectrophotometer. Measure by non4destructive fluorometry. :istones can be detected by an e/citation wavelength of 9I' nm and a fluorescence measurement at ?'I nm. +on4histones can be detected in the same sample by e/citation at 9O' nm and measurement at ?)* nm. ;f course, this procedure re3uires the use of a fluorescence spectrophotometer.

"otes

$he e/traction of chromatin proteins starts with the isolation of a good nuclear fraction. +uclear pellets and chromatin should be e/tracted one day before the laboratory period. #f C+A, R+A, and both :istone and +on4:istone proteins are to be separated, begin the procedure appro/imately three working days prior to lab.

Exercise 1,.1 - Chro#atin Electrophoresis

!D=D! ## Materials

1) M 1rea 5 M +a7l '.'* M and '.O M Acetic acid Cialysis tubing Dlectrophoresis apparatus <repared gels 1' M urea4'.O + acetic acid4'.* M 4mercaptoethanol '.9*S 7oomasie blue

Procedure
1. $o the chromatin suspension from D/ercise 1'.?, add concentrated urea and concentrated

+a7l separately to yield a final concentration of > M urea and ? M +a7l.

2. 7entrifuge the clear solution at I*,*'' /g for )I hours at )G 7 to pellet e/tracted C+A. 3. 7ollect the supernatant and dialy&e it against '.'* M acetic acid ,three changes, 5 liters

each at )G 7-. Remove the dialy&ed protein solution and lyophili&e it to dryness.
4. Meanwhile, set up a standard polyacrylamide gel ,D/ercise ).1-, using 1*S acrylamide

,1*S$2*S7- in 9.* M urea and '.O M acetic acid. Set up the gel in the electrophoresis unit and run the gel at 9 mA8gel for 9 hours with no sample, using '.O M acetic acid for the running buffer.
5. Cissolve the lyophili&ed protein from step ? in 1' M urea4'.O + acetic acid4'.* M 4

mercaptoethanol ,to a final concentration of *'' micrograms protein per 1'' Ll of bufferand incubate at room temperature for 1941) hours prior to the ne/t step.

6. Apply 9' Ll samples of the redissolved protein e/tract to '.5 / I.' cm polyacrylamide

prepared as in step ).
7. $he gels are run against '.O M acetic acid in both upper and lower baths for

appro/imately ? hours at 1'' =.

8. Stain the gels for 1 hour in 7oomasie blue, rinse with water, destain and store in >S

acetic acid.
9. #f densitometry measurements are made, * Lg of pea bud fraction ##a protein has a

density of 1.?5' density units / mm with a O*S confidence limit of 1'S. %y comparison, the density value can be used to 3uantitate the concentration of protein fractions in Lg of your sample.

Exercise 1,.7 - Extraction and Electrophoresis of ?istones

!D=D! ## $able 1'.1 <roperties of chromatin Figure 1'.? 7hromatin electrophoresis pattern Materials

Saline 7itrate ,181' SS71.' + : S; Refrigerated preparative centrifuge Absolute ethanol 4mercaptoethanol '.'1 M Sodium phosphate buffer, p: >.' W 1S ,w8v- SCS W '.1S ,v8v- 4 mercaptoethanol 1'S acrylamide gels ,1'S$2*S7- with '.1S ,w8v- SCS >S ,w8v- Acetic acid '.9*S 7oomasie blue

Procedure *

1. Cissolve crude chromatin in cold dilute saline citrate ,'.'1* M +a7l W '.''1 M Sodium

7itrate- to a final C+A concentration of *'' Lg8ml.

2. Stir the solution on ice and slowly add 18) volume of cold 1.' + : S; . 7ontinue stirring

for ?' minutes.

3. 7entrifuge the suspension at 19,''' /g for 9' minutes at )G 7. Save the supernatant. For

ma/imum yield, break up the pellet, resuspend in fresh, cold '.) + : S; , re4e/tract, centrifuge, and add the resulting supernatant to the first.
4. Add ) volumes of cold absolute ethanol to the supernatant and store for 9) hours at 41'G

7 to precipitate the histone4sulfates.

5. 7ollect the precipitate by centrifugation at 9,''' /g for ?' minutes. 6. Cecant as much of the alcohol as possible, and resuspend the pellet in cold absolute

ethanol.
7. 7entrifuge at 1',''' /g for 1* minutes. 8. 7ollect the pellet and free&e dry for later analysis.

$o continue with the electrophoresis, carefully weigh the histone protein sample and dissolve in '.'1 M sodium phosphate buffer, with a p: >.' and containing 1S sodium dodecyl sulfate and '.1S 4mercaptoethanol final volume should contain appro/imately ?'' Lg of protein in 1'' Ll of buffer.
9. <repare the electrophoresis chamber with a 1'S acrylamide gel with '.1S SCS. 10. Add separately 9* Ll of the dissolved protein and 9* Ll of protein standards to2

*' Ll of '.1S SCS, '.1S 4mercaptoethanol in %uffer * Ll of 4mercaptoethanol 1 Ll of '.1S bromophenol blue in water
11. Mi/ thoroughly and apply the histone e/tract and protein standards to separate wells of

the electrophoresis gel.

12. Separate the proteins in the anode direction ,Anionic system-. 5
o

$he addition of SCS anions to the proteins results in negatively charged proteins which will separate according to molecular weight.

Dlectrophoresis is carried out in the standard manner following the basic steps given in 7hapter Four. $he buffer utili&ed is that of !aemmli, > '.'9* M $ris4 '.1O9 Alycine and '.1S SCS, p: I.?. <roteins are separated by a current of ?4) mA per gel until the bromophenol marker reaches the bottom of the tube ,about > hours at ? mA, and ) hours at ) mA-.

13. Stain the gels with '.9*S 7oomasie %lue for 9 hours. 14. Cestain and store in >S acetic acid. 15. Scan the gels and determine the molecular weights of each component.

"otes <reparation of a total histone fraction from nuclei is normally accomplished by e/traction with a dilute acid or a high molarity salt solution. $he acidic e/traction removes histones from C+A and non4histones immediately, while the dissociation of chromatin in salt solutions will re3uire further purification. #n either event, the histones themselves are subdivided into five ma"or types, designated as :1, :9, :?, :) and :*. :9 dissociates into two peptides, which are thus designated as :9A and :9%. $he classification of histones is based on their electrophoretic mobility. +on4histone proteins can also be e/tracted and separated by electrophoresis. .hereas histones have only * ma"or types, non4 histones are e/tremely heterogeneous and up to *'' different proteins have been identified from one cell type, while the ma"or proteins comprise less than 9' types. $he e/traction of chromatin C+A was possible with the > M urea 4 ? M +a7l e/traction performed inD/ercise 1'.). Further analysis of C+A will be undertaken as part of a later lab e/ercise ,on $ranscription-, and the C+A sample from this lab may be kept lyophili&ed and fro&en until that time. For our current needs it is sufficient to note that the genes are composed of C+A, and that various specific regions of the C+A8Aenetic information can be physically isolated to a specific locus on a chromosome. $his in turn is readily observed and correlated with banding patterns, such as those in the fruit fly poltytene chromosomes.

Exercise 1,.9 - Ear0ot0pe $nal0sis

!D=D! ## $able 1'.9 :uman chromosome dimensions 7lick here for karyotype form Materials

Fresh venous blood I :eparini&ed syringes Dagle@s spinner modified media with <:A 7ulture flasks $issue culture grade incubator at ?>G 7 1' L g8ml 7olcemid 7linical centrifuge and tubes '.'>* M B7l Absolute Methanol and glacial acetic acid ,?21 Mi/ture, prepared freshCry ice Slides, coverslips and permount Alkaline solution for A4banding Saline47itrate for A4banding Dthanol ,>'S and O*S ,v8v-Aiemsa stain

Procedure O
1. Craw * ml of venous blood into a sterile syringe containing '.* ml of sodium heparin

,1''' units8ml-. $he blood may be collected in a heparini&ed =acutainer, 1' and transferred to a syringe.
2. %end a clean, covered 1I gauge needle to a )*G angle and place on the syringe. #nvert the

syringe ,needle pointing up, plunger down-, and stand it on end for 14189 to 9 hours at room temperature. Curing this time the erythrocytes settle by gravity, leaving appro/imately ) ml of leukocyte4rich plasma on the top, and a white buffy coat of leukocytes in the middle.

3. 7arefully tip the syringe ,do not invert- and slowly e/pel the leukocyte4rich plasma and

the buffy coat into a sterile tissue culture flask containing I ml of Dagle@s spinner modified media supplemented with '.1 ml of phytohemagllutin ,<:A-. %D D($RDMD!J 7ARDF1! +;$ $; C#SR1<$ $:D RDC %!;;C 7D!!S #+ $:D %;$$;M ;F $:D SJR#+AD. R%7@S .#!! #+:#%#$ AR;.$: ;F $:D !D1B;7J$DS.
4. #ncubate the culture for 554>9 hours at ?>G 7. Aently agitate the culture once or twice

daily during the incubation period.

5. Add '.1 ml of colcemid ,1' micrograms8ml- to the culture flasks and incubate for an

additional 9 hours.11
6. $ransfer the colcemid4treated cells to a 1* ml centrifuge tube and centrifuge at 99* /g for

1' minutes.
7. Aspirate and discard all but '.* ml of the supernatant. Aently tap the bottom of the

centrifuge tube to resuspend the cells in the remaining '.* ml of culture media.
8. Add 1' ml of '.'>* M B7l, dropwise at first, and then with gentle agitation to the

centrifuge tube. Aently mi/ with each drop. S$AR$ $#M#+A $:D +D($ S$D< #MMDC#A$D!J .#$: $:D F#RS$ CR;< ;F B7!.
9. !et the cells stand E-$CTBK 5 minutes in the hypotonic B7l.

$:D :J<;$;+#7 S;!1$#;+ S:;1!C +;$ %D #+ 7;+$A7$ .#$: $:D 7D!!S #+ D(7DSS ;F 1* M#+1$DS FR;M $:D $#MD #$ #S ACCDC.
10. 7entrifuge the cells at 99* /g for 5 minutes. Aspirate the B7l and discard all but '.* ml

of the supernatant. Aently resuspend the cells in this small volume of fluid.
11. Add 1' ml freshly prepared fi/ative dropwise at first and then with gentle agitation.

Aentle and continuous agitation is important at this step to prevent clumping of the cells. #f the cells were not properly resuspended in step 1', the cells will clump beyond any further use.
12. Allow the cells to stand in fi/ative at room temperature for ?' minutes. 13. 7entrifuge at 9'' /g for * minutes and remove all but '.* ml of supernatant. Resuspend

the cells in fresh fi/ative.

14. .ash the cells twice more in 1' ml volumes of fi/ative. Add the fi/ative slowly,

recentrifuge, and aspirate the fi/ative as previously directed. $:D F#(DC, <D!!D$DC 7D!!S MAJ %D S$;RDC F;R SD=DRA! .DDBS A$ )G 7.
15. Resuspend the pellet of cells in "ust enough fi/ative to give a slightly turbid appearance. 16. <rop a piece of dry ice against the side of a styrofoam container and lace a clean slide

onto the dry ice to chill the slide. CRJ #7D .#!! 7A1SD FR;S$ %#$D. :A+C!D .#$: $;+AS ;+!J. 1se a siliconi3ed pasteur pipette to draw up a few drops of the suspended cells and drop the cells onto the surface of the chilled slide. Spreading of the chromosomes may be enhanced by dropping the cell suspension from a height of at least 19 inches. As soon as the cells strike the slide, blow hard on the slide to rapidly spread the cells. 19 F;R %DS$ RDS1!$S A!!;. ;+!J ;+D CR;< <DR S!#CD.
17. Remove the slides from the dry ice and allow them to air dry. <erform the desired

banding and8or staining procedures. <rocedure 6 <reparation of chromosomes for karyotype analysis can be performed in a number of ways and each will yield differing pieces of information. $he chromosomes may be stained with aceto4 orcein, feulgen or a basophilic dye such as toluidine blue or methylene blue if only the general morphology is desired. <rocedure 6 #f more detail is desired, the chromosomes can be treated with various en&ymes in combination with stains to yield banding patterns on each chromosome. $hese techni3ues have become common place and will yield far more diagnostic information than giemsa stain alone ,the most commonly used process-. A band is an area of a chromosome which is clearly distinct from its neighboring area, but may be lighter or darker than its neighboring region. $he standard methods of banding are the M, A, R, and 7 banding techni3ues. $hese are defined as follows2
o

M4banding
1. Muinacrine stain 2. Fluorescence microscopy

A4banding

1. Aiemsa stain 2. Additional 7onditions

o

a. :eat hydrolysis b. $rypsin treatment c. Aiemsa at p: O.'

R4banding
1. Aiemsa or acridine orange 2. +egative bands of M and A reversed 3. :eat hydrolysis in buffered salt

74banding
1. Aiemsa stain 2. <retreatment with %a;: or +a;: followed by heat and salt.

$he following directions are for a A4banding2 1?

o

$reat fi/ed and flamed slides in alkaline solution, room temperature for ?' seconds. Rinse in saline4citrate solution, ? changes for *41' minutes each. #ncubate in saline4citrate solution, 5*G 7 for 5'4>9 hours. $reat with ? changes of >'S ethanol and ? changes of O*S ethanol ,? minuteseach.

o o o

o o o o

Air dry. Stain in buffered Aiemsa for * minutes. Rinse briefly in distilled water. Air dry and mount.

18. <hotograph appropriate spreads and produce I ( 1' high contrast photographs of your

chromosome spreads.
19. 7ut each chromosome from the photograph and arrange the chromosomes according to

si&e and position of the centromere.

20. 1se $able 1'.9 to identify the specific chromosomes.

21. $ape or glue each chromosome to the form supplied for this purpose.

Exercise 1,.< - In Situ ?0+ridi3ation

!D=D! ### Figure 1'.) #n situ hybridi&ation A modern approach to the specific location of genes on chromosomes is a techni3ue for the hybridi&ation of C+A and R+A 0in situ.0 .ith this procedure, specific radioactive R+A or C+A ,known as probes- can be isolated ,or synthesi&ed 0in vitro0- and then annealed to chromosomes which have been treated in such a manner that their basic double stranded C+A has been 0melted0 or dissociated. #n theory, and fortunately in practice, when the C+A is allowed to re4anneal, the probe competes for the binding, but only where it mirrors a complimentary se3uence. $hus, R+A will attach to the location on the chromosome where the code for its production is to be found. C+A will anneal to either R+A which is still attached to a chromosome, or to the complimentary se3uence C+A strand within the chromosome. Since the probe is radioactive, it can be locali&ed via autoradiographic techni3ues. Finally, it is possible to produce an R+A probe that is synthesi&ed directly from repetitive se3uences of C+A, such as that found within the nucleolar organi&er region of the genome. $his R+A is known as cR+A ,for copied R+A- and is a convenient source of a probe for locali&ing the nucleolar organi&er gene within the nucleus, or on a specific chromosome. $he use of in situ hybridi&ation begins with good cytological preparations of the cells to be studied, and the preparation of pure radioactive probes for the analysis. $he details depend upon whether the hybridi&ation is between C+A ,probe- and C+A ,chromosome-, C+A ,probe- and R+A ,chromosome-, or between R+A ,probe- and C+A ,chromosome-. Preparation of the Probe1 1) <roduce radioactive R+A by incubating the cells to be measured in the presence of :4uracil, a specific precursor to R+A. Subse3uent to this incubation, e/tract rR+A from the sample and purify through differential centrifugation, column chromatography or electrophoresis. Cissolve the radioactive R+A probe in )( Saline47itrate containing *'S formamide to yield a sample

that has *',''' to 1'',''' counts per minute, per ?' microliter sample, as determined with a scintillation counter. Add the formamide is added to prevent the aggregation of R+A. Preparation of the +li&es1 Fi/ the materials to be studied in either O*S ethanol or in ?21 methanol2water, attach to pre4 subbed slides ,as s3uashes for chromosomes- and air dry. ybri&i!ation <lace the air dried slides into a moist chamber, usually a disposable petri dish containing filter paper and carefully place ?' microliters of R+A probe in )( SS74*'S formamide onto the sample. 7arefully add a cover slip ,as in the preparation of a wet mount-, place the top on the container and place in an incubator at ?>G 7 for 5419 hours. 8ashing1
1. <ick up the slides and dip into 9( SS7 so that the coverglass falls off. 2. <lace the slides in a coplin "ar containing 9( SS7 for 1* minutes at room temperature. 3. $ransfer the slides to a treatment with R+ase ,*' microgram8ml R+ase A, 1'' units8ml

R+ase $1 in 9( SS7- at ?>G 7 for 1 hour.

4. .ash twice in 9( SS7, 1* minutes each. 5. .ash twice in >'S ethanol, twice in O*S ethanol and air dry.

Autora&iography1 Add photographic emulsions to the slides and after a suitable e/posure period, develop the slides, counterstain and add cover slips. Analy&e the slides by determining the location of the radioactive probe on the chromosomes or within the nuclei. Chapter 1,: Chro#oso#es - Endnotes

1.

;ccasionally, lipids are found within nuclei. #t is usually associated with degeneration of the nuclear structure, or with neoplastic alterations of the nucleus. $his is a standard means of sealing wet mounts. Melted paraffin is highly flammable, however, and should never be melted with an open flame. $he author has found that the slides can be sealed "ust as well with a coat of nail polish.

2.

3.

From2 !.M.T. Shaw and R.7.7. :uang. 0A Cescription of $wo <rocedures .hich Avoid the 1se of D/treme p: 7onditions for the Resolution of 7omponents #solated from 7hromatins <repared from <ig 7erebellar and <ituitary +uclei0 %iochemistry O29? 1O>'. pp )*?'4)*)9. A sample may be taken and fi/ed for light and electron microscope observation at a later time. %onner et al. B. .eber and M. ;sborn. 1O5O. 0$he Reliability of Molecular .eight Ceterminations by Codecyl Sulfate <olyacrylamide Ael Dlectrophoresis0 9. %iol. Chem. 9))215, ))'54))19. 1.B !aemmli. 1O>'. 07leavage of Structural <roteins during the Assembly of the :ead of %acteriophage $)0 4ature =ol. 99>. pp. 5I'45I*. $he procedure is based on one performed at the 7entersfor Cisease 7ontrol, Atlanta, AA on human blood samples. =irtually any animal blood can be substituted, or if cells in culture are available, log phase cultures can be substituted and the process begun with step *. %lood can be drawn at a local clinic directly into heparani&ed tubes, and should be disposed of properly. $his procedure can be handled as a !evel # e/ercise by purchasing photographs of chromosme spreads ,7arolina %iological- and skipping to step 1O.

4.

5. 6.

7.

8.

9.

10. =acutainer Systems, ;rder R5)I1. %ecton Cickinson, Rutherford, +T '>'>' 11. #f an inverted phase microscope is available, you can check that the cultures are in a log

phase of growth before adding the colcemid.

12. $he general process involves swelling of the cells in the hypotonic B7l treatment and

dropping them like big water balloons onto the slide. $he bigger 0splash0 they make on impact, the better the chromosomes will spread out as the nuclei burst on contact with the slide. Some investigators actually drop the cells from a distance of five feet. $he author has found this to be challenging and fun, but of no real advantage. $he chilled slide aids in sticking the chromosomes onto the slide. Siliconi&ed pipettes are necessary to prevent the cells from adhering to the walls of the pipette during transfer. $his is the most common reason for not obtaining good chromosome preparations.

13. #f only morphology is to be studied, skip steps a4e and begin with step f, but increase the

staining to 1'49' minutes.

14. Summari&ed from :. Macgregor and T. =arley. 8or:ing 0ith Animal Chromosomes. Tohn

.iley U Sons, 7hichester, 1OI?. pp. 1O549'1.

Chapter 11: Cell C0cles - Introduction

$he onion root tip and the whitefish blastula remain as the standard introduction to the study of mitosis. $he onion has easily observable chromosomes, and the whitefish has one of the clearest views of the spindle apparatus. $he testis of the grasshopper and the developing &ygote of the roundworm Ascaris are the traditional materials used for viewing the various stages of meiosis. #n a single longitudinal section of a grasshopper testis one can usually find all of the stages of meiotic development. $he stages are also aligned from one pole of the testis. Few other meiotic samples are as convenient. For most material, meiosis occurs in a more randomly distributed pattern throughout the testis. Ascaris is utili&ed to observe the final stages of development in eggs ,oogenesis-. $he Ascaris egg lies dormant until fertili&ed. #t then completes meiosis forming two polar bodies while the sperm nucleus awaits fushion with the female nucleus. .hen this phenomenon is coupled to the large abundance of eggs in the Ascaris body, it makes an ideal specimen for observing the events of fertili&ation, polar body formation, fusion of pronuclei and the subse3uent division of the cell ,cytokinesis-. #nterphase A14S4A9 $he stages of mitosis were originally detailed after careful analysis of fi/ed cells. More recently, time lapse photography coupled with phase contrast microscopy has allowed us to visuali&e the process in its entirety, revealing a dynamic state of flu/. #n early work, so much emphasis was placed on the movement of the chromosomes that the cell was considered to be 0at rest0 when not in mitosis. As significant as mitotic division is, it represents only a small fraction of the life span of a cell. +one the less, you may still come across the term 0resting phase0 in some older te/ts. $his term is rarely used today, and the term interphase is sufficient for all activities between two mitotic divisions. $he cell is highly active

during interphase and most of the metabolic and genetic functions of the cell are reduced during the physical division of the nuclear and cellular materials ,mitosis-. Figure 11.1 presents our current view of a cell cycles. +ote that interphase is divided into three sub4phases, A1, S and A9. $he basis for this division is the synthesis of C+A. +ote also that while the entire cycle may be as long as 9) hours, mitosis is normally less than one hour in length. %ecause of the synthesis of C+A in interphase, the amount of C+A per nucleus is different depending on which sub phase of interphase the cell is in. C+A can be measured using the fuelgen reaction and a microscpectrophotometer. $he basic amount of C+A in a haploid nucleus is given the value 7. A diploid nucleus would be 97. A triploid and tetraploid cell would be ?7 and )7 respectivesly. :owever, when nuclei are actually measured, diploid cells in interphase can be divided into three groups some are 97, some are )7 while a few are at intermediate values between 97 and )7. $he conclusion is that the genetic material ,C+A- and presumably the chromosomes must duplicate. $he period within interphase and during which C+A is synthesi&ed is termed the S phase ,for Synthesis-. $he period of interphase preceding the S phase is the A1 phase ,for 1st Arowth <hase-, while the period subse3uent to the S phase is the A9 phase ,for the 9nd Arowth <hase-. Curing the A1 period, the cell is generally increasing in si&e and protein content. Curing S, the cell replicates the chromosomes and synthesi&es C+A. Curing A9, it continues to increase in si&e, but also begins to build a significant pool of A$< and other high energy phosphates, which are believed to be a significant part of the triggering mechanism for the subse3uent karyokinetic and cytokinetic events of mitosis. Mitosis returns the cells to the 97 state. Meiosis reduces the amount of C+A even further, to 17. Meanwhile the number of chromosomes ,designated with the letter +- is also changing. For a diploid cell, the number of chromosomes is twice that of a haploid, or 9+. Curing mitosis, a diploid cell would go from one 9+ cell to two 9+ cells. Since the daughter cells have the same chromosome number as the parent, mitosis is also referred to as e3uational division. #f a diploid ,9+- cell undergoes meiosis, it will result in four haploid cells, each 1+. $hus, meiosis is also referred to as reductional division. Refer to Figure 11.9 for comparison of 7 and + values during division.

Figure 11.1 7ell 7ycle #t is possible to visuali&e the process of C+A synthesis within either nuclei or chromosomes by the incorporation of a radioactive precursor to C+A into cells and subse3uent detection by autoradiography. #ncorporation of thymidine, a C+A precursor, will only occur during the S phase, and not during A1 nor A9. #f a pulse ,short period of e/posure- of :4thymidine is presented to cells, those that are in the S period will incorporate this radioactive substance, while all others will not. 7areful application of the pulse will allow the timing of the S phase. %y knowing the timing for the entire cycle ,from mitosis to mitosis-, one can deduce the A1 and A9 periods.

Figure 11.9 Amount of C+A present during division Meiotic division differs from mitosis in that there are two division cycles instead on one. #n the first cycle, interphase is the same as for mitosis. $hat is there is an S phase with corresponding A1 and A9. Curing the second interphase, however, no C+A synthesis occurs. 7onse3uently there is no A1 or A9 in the second interphase. $he result is that chromosomes are replicated prior to Meiosis, and do not replicate again during meiosis. For the following details of Mitosis, refer to

Figure 11.? Stages of Mitosis in ;nion <rophase $he first phase of mitosis is marked by the early condensation of the chromosomes into visible structures. At first, the chromatids are barely visible, but as they continue to coil, the chromosomes become thicker and shorter. $he nuclear envelope is still present during this stage, as are any nucleolar structures. $he centrioles are moving to the poles of the cell and spindle fibers are "ust beginning to form. Metaphase Curing the middle phase of karyokinesis, the chromosomes line up in the center of the cell, and form a metaphase plate. =iewed on edge, the chromosomes appear to be aligned across the entire cell, but viewed from O' Cegrees they appear to be spread throughout the entire cell ,visuali&e a plate from its edge or from above-. Dach chromosome has a clear primary constriction, the centromere, and attached to each is a definitive spindle fiber. $he spindle apparatus is completely formed, and the centrioles have reached their respective poles. $he nucleolus and the nuclear envelope have disappeared. Anaphase $he movement phase begins precisely as the two halves of a chromosome, the chromatids, separate and begin moving to the opposite poles. $he centromere will lead the way in this process, and the chromatids form a = with the centromeres pointing toward the respective poles. $elophase $he last phase is identified by the aggregation of the chromatids ,now known as chromosomesat the respective poles. Curing this phase, the chromosomes uncoil, the nuclear envelope is resynthesi&ed, the spindle apparatus is dismantled and the nucleolus begins to appear.

Meiosis For meiosis, the phases prophase, metaphase, anaphase and telophase are identified, but because there are two divisions, there are two sets. $hese are designated by Roman numberals thus <rophase #, Metaphase #, Anaphase #, $elophase #, #nterphase, <rophase ##, Metaphase ##, Anaphase ## and $elophase ##. #nterphase is normally not designated with a Roman numberal. %ecause of the significance of the chromosome pairing which occurs in <rophase #, it is further subdivided into stages. For the following descriptions of meiosis, refer to Figure 11.) Stages of Meiosis in Arasshopper $estis. $he phases of <rophase # are named for the appearance of a thread4like structure, known as nema. !eptonema means 0thin thread0 and leptotene is the ad"ective applied to the term stage, i.e. proper terminology is the leptotene stage of prophase #. $he word stage is often omitted. <rophase #2 !eptotene 1 $his stage is marked by the first appearance of the chromosomes when the chromosomes are in their most e/tended form ,e/cept for during interphase-. $hey appear to be a string with beads. $he beads are known as chromomeres. $he chromatids have already replicated prior to this phase, but typically, the replicated chromatids can not be observed during the leptotene stage. <rophase #2 Fygotene Fygos means 0yoked,0 and during this stage, the homologous chromosomes are seen as paired units. $he chromosomes are shorter and thicker than in leptotene, and in some cells they remain attached to the nuclear envelope at the points near the aster. $his gives rise to an image termed the 0bou3uet.0 $his attachment is rare in invertebrates and absent in plants, where the chromosomes appear to be a tangled mass. <rophase #2 <achytene .hen the pairing of &ygotene is complete, the chromosomes appear as 0thick0 strings, or pachynema. $he chromsomes are about 18) the length they were in leptotene, and there are obviously two chromosomes, each with two chromatids in each bundle. $he two chromosomes are referred to as a 0bivalent,0 while the same structure viewed as four chromatids is known as a 0tetrad.0 <rophase #2 Ciplotene

$his stage results as the gap between the two homologous chromosomes widens. $he homologs have already paired during &ygotene, recombined during pachytene and are now beginning to repel each other. Curing this stage, the chromosomes of some species uncoil somewhat, reversing the normal direction typical of prophase. As the chromosomes separate, they are observed to remain attached at points known as 0chiasmata.0 $hese are believed to be the locations where genetic recombination of the genes has taken place. <rophase #2 Ciakinesis <rophase # ends as the homologs completely repel each other. $he chromosomes will continue to coil tightly ,reversing the slight uncoiling of the diplotene- and will reach their greatest state of contraction. As diakinesis progresses, chiasmata appear to move toward the ends of the chromosomes, a process known as 0terminali&ation.0 Since this stage is the end of prophase, the nucleolus usually disappears, along with the nuclear envelope. Metaphase # $he tetrads move toward the center and line up on a metaphase plate. $he nuclear envelope completely disappears. As the tetrads align themselves in the middle of the cell, they attach to spindle fibers in a uni3ue manner. $he centromeres of a given homolog will attach to the spindles from only one pole. Anaphase # $he uni3ue event occurring at this phase is the separation of the homologs. #n contrast to mitotic anaphase, the centromeres of a given homolog do not divide, and conse3uently each homolog moves toward opposite poles. $his results in a halving of the number of chromosomes, and is the basis of the reduction division that characteri&es meiosis. $elophase #2 #nterphase $elophase in meiosis is similar to that of mitosis, e/cept that in many species, the chromosomes do not completely uncoil. #f the chromosomes do uncoil and enter a brief interphase, there is no replication of the chromatids. Remember that the chromatids have already been replicated prior to <rophase #. <rophase ## 4 $elophase ## $hese phases are essentially identical in meiotic and mitotic division2 the only distinction is that the chromosome number is half of the number the cell had prior to meiosis. Dach chromosome

,homolog- is composed of two chromatids, and during Anaphase ##, the two chromatids of each chromosome move apart and become separate chromosomes. 9 $here is a shift in the terminology applied to these units. .hile the two chromatids remain attached at the centromere, they are known as chromatids. #mmediately upon separating, each chromatid becomes known as a chromosome and is no longer referred to as a chromatid. $his is the reason that a cell can divide one chromosome ,with two chromatids- into two cells, each with a chromosome 4 the term applied to the chromatid is changed. Camage induced during division #n 1O)O, !evan developed what was to become known as the Allium test for chromosome damage. Arowing roots from onion bulbs were soaked in various agents and analy&ed for their effect on mitosis. #t was discovered that caffeine, for e/ample, caused complete inhibition of mitosis, primarily through the inhibition of cell plate formation. $his test was later used e/tensively by %.A. Bihlman and e/tended to other higher plants. Bihlman found that 1 to 9) hour treatments of cells with caffeine and related o/ypurines not only inhibited mitosis, but also induced signficant chromosome alterations ,aberrations-. Specifically, this treatment induced 0stickiness0 and 0pseudochiasmata.0 Stickiness is the clumping of chromosomes at metaphase and the formation of chromatin bridges at anaphase. <seudochiasmata is the formation of side4arm bridges during anaphase. 7affeine also causes the formation of other chromosome and chromatid breaks and e/changes. 7olchicine, a drug which inhibits spindle fiber formation during mitosis, can be added to the growing cells to halt cell division at metaphase. $his often will result in a doubling of the chromosome number, since colchicine typically inhibits cytokinesis, but not karyokinesis. $he doubled chromosomes will fuse within a single nucleus, thus increasing the ploidy value of the nucleus. Moreover, methylated o/ypurines ,caffeine, theophylline, I4etho/ycaffeine- are inhibitors of cell plate formation. $reatment with these agents for '.* 4 1 hour, with concentrations as low as '.'94 '.')S, results in the cell@s failure to undergo cytokinesis in addition, the nuclei do not fuse into a single unit. $hus, treatment with any of these agents should result in binucleate or multinucleate cells.

Some antibiotics ,a&aserine, mitomycin 7 and streptonigrin- have been known to have chromosome4breaking properties, usually associated with A1 or A9 of #nterphase. A1 inhibition would result in chromosome breaks, while A9 inhibition would result in chromatid breaks. #n addition, alkylating agents such as ,Ci,94 chloroethyl-methylamine or nitrogen mustard, Ci,9.?4epo/ypropyl- ether ,CD<D- and 4<ropiolactone ,%<!--, +itrosocompounds ,+4+itroso4 +4methylurethan ,+M1--, +4Methyl4phenyl4nitrosamine ,M<+A-, +4 hydro/ylphenylnitrosamine4ammonium ,cupferron- and 14Methyl4?4nitrosoguanidine ,M++Ahave all been indicated as potent chromosome4breaking agents. ;ther compounds have included such things as maleic hydra&ide, potassium cyanide, hydro/ylamine, and dyes such as acridine orange in visible light. $he damages involve abnormal metaphases, isochromatid breaks, chromatid e/changes and anaphase bridges to name a few. Figure 11.* illustrates some abnormalities induced in plant chromosomes by these compounds. Figure 11.* 7hemically induced chromosome abnormalities

Exercise 11.1 - Mitosis

!D=D! # Materials

<repared slide of ;nion root tip <repared slide of whitefish blastula Microscope

Procedure
1. ;btain a slide of an onion root tip and e/amine it for the basic stages of mitosis. Refer

to Figure 11.? for comparison.

2. Craw and label each of the stages. 3. ;btain a slide of a whitefish blastula for observation of the stages of mitosis in an animal

cell. Since early embryogenesis involves rapid cellular division, the whitefish blastula has long served as a model of mitotic division in animals. #t also has the advantage of demonstrating clear spindle formation in the cytoplasm.

4. 1sing Figure 11.?A as a guide, draw and label all stages of mitosis, including spindle

formation, in the whitefish blastula.

5. 7ompare the division of the cell ,cytokinesis- from the onion root tip to that of the

whitefish blastomeres.

Exercise 11.. - Arasshopper 6per#atogenesis

!D=D! #

Figure 11.5 Stages of meiosis in grasshopper testis Materials _ <repared longitudinal section of grasshopper testis _ Microscope Procedure
1. <lace the slide on the microscope and with low power identify the apical end of the testis

and the region where the testis "oins with the vas deferens. $he apical end is round and packed with cells, while the opposite end is a more open cavity lined with formed sperm.
2. #dentify the individual compartments of the testis, known as cysts. $he cysts are

separated by connective tissue walls or septa.

3. At the apical end, the cells are grouped into presumptive germ cells known as

spermatogonia. $hese cells are undergoing mitosis and are giving rise to all of the remaining germ cells in the testis. Moving from the spermatogonia in the apical end to the vas deferens at the opposite end of the testis, the cells mature as a group. $hat is, all of the cells within a given cyst will be in the same appro/imate stage of meiosis. A good longitutinal section will have nearly all of the stages of meiosis displayed on one section, but you may have to e/amine several slides to observe all of the stages.
4. 1sing Figure 11.5 as a guide, identify , draw and label each of the following stages2

Spermatogonia <rimary spermatocytes in the following phases of <rophase # leptotene &ygotene pachytene diplotene diakinesis. Secondary spermatocytes Spermatids Sperm

Exercise 11.5 - $scaris /ogenesis

!D=D! # Figure 11.> Ascaris maturation Materials

<repared slides of Ascaris megalocephala maturation Microscope

Procedure
1. <lace the slide,s- of Ascaris on the microscope and focus on the eggs within the body.

1nlike the grasshopper testis where division is continuous, the ascaris egg halts its meiotic division until fertili&ed by a sperm. Subse3uently, the final stages of meiosis are completed and nuclear fusion occurs. .ithin an average cross section of the female, it is usually possible to observe all of the stages of meiosis and early cleavage.
2. 1sing Figure 11.> as a guide, identify, draw and label the following2

Sperm penetration First polar body formation ,first maturation divisionSecond polar body formation ,second maturation division<ronuclear stage ,fusion of egg and sperm nucleiFirst cleavage division ,beginning of mitosis-

3. +ote the presence of any spindle fibers, and the shape and location of the chromosomes

during each phase of development.

Exercise 11.1 - ?-Th0#idine %pta4e +0 Cultured Cells

!D=D! ## Materials _ Fibroblast cells in log phase growth

_ 7a

, Mg

free4phosphate buffered saline ,<%SA-

_ *S ,w8v- Alutaraldehyde ,A$A_ 9S ,w8v- <erchloric Acid ,<7A_ Subbed slides ,coated with chrom alum gelatin- and <ermount _ +uclear $rack Dmulsion ,Bodak or #lford_ Carkroom and chemicals for photographic processing

Cektol developer Bodak Fi/er

_ Aiemsa stain, graded series of alcohols, /ylol Procedure

1. Arow either ! cells ,Mouse fibroblasts- or chick embryo fibroblasts on coverglasses and

then give them :4thymidine for a short period of time.

2. At the end of the labeling period, wash the coverslips in <%SA by gently grasping a

coverslip with forceps and passing it through a beaker of saline.

3. Fi/ cultures in glutaraldehyde for 1* minutes. 4. .ash in several changes of water. 5. .ash in cold 9S <7A for * minutes to remove unincorporated labeled precursors to

C+A. Repeat twice.

6. .ash in water * minutes. Repeat. 7. Cry the backs of the coverslips with filter paper, and mount 7D!! S#CD 1< on slides

with <ermount. $he slides should be very clean. 7oat the slides beforehand with chrom alum gelatin ,7AA- by dipping the slides into 7AA solution and draining until dry. $his coating, and the gelatin coating on the coverslips, help to prevent the emulsion layer ,below- from pulling away from the slide during later development.
8. Allow <ermount to dry overnight. 9. #n a darkroom, spoon out a small amount of gel into a suitable vessel, and slowly melt it

at )*G 7 in a water bath. Bodak +$%4? emulsion is stored refrigerated as a gel in a screw cap bottle inside a double light4tight bo/.
10. Cip the slide in the emulsion and drain momentarily. <lace the slides vertically on a test

tube rack in an oven set at 9IG 7 for at least 1 hour in darkness. #n general, the slides should be dried at a temperature greater than will be used for developing. $his minimi&es undesirable separation of emulsion from the slide.
11. <lace the slides in light4tight bo/es containing dessicant and store at )G 7. 12. Cevelop a sample slide in Cektol diluted 1 part developer to 9 parts distilled water at 1IG

7 for O' seconds. $he temperature of the developer will control the si&e of the silver grains. #ncreased length increases background fog of development. Cevelop the slides as indicated in 7hapter $wo. $he length of e/posure ,refrigerator storage- must be determined for each system ,a function of specific activity of label in medium, pool si&es, length of labeling, synthetic rate, etc.-. $hus e/tra control slides are always included to allow repeated sample developing until a useful number of silver grains have accumulated.
13. <ass the slides through two changes of distilled water, and into photographic fi/er at 1IG

7. Fi/ for * minutes.

14. .ash slides in two changes of distilled water, for a total of * minutes. 15. Stain the cells with Aiemsa diluted 12?' as re3uired, or dry slides slowly in a dust4free

atmosphere, and stain later.

16. .ash off e/cess stain briefly in distilled water, dip slide briefly in >'S ethanol,

dehydrate in O*S and 1''S alcohol, and clear in /ylene. Mount coverslip with <ermount.
17. D/amine the slides with a microscope at 1'( magnification and look for clusters of silver

grains over the cells. 7ount and calculate the percent of cells that are labeled.
18. D/amine the slides at )'( magnification. 7ount the number of grains per cell. 19. <repare a histogram by plotting the number of cells versus the number of silver grains.

Exercise 11.7 - Ti#ing of C0cles

!D=D! ### Figure 11.I $iming of mitotic cycle Materials

Monolayer cultures grown in >* mm culture flasks ,7ells from D/ercise 11.) may be used, or cultures of tetrahymena, yeast, or algae may be used.-

:4thymidine with at least ) Lc8ml, '.?5 c8mM <hosphate buffered saline ,<%S-, $rypsin Methanol2Acetic acid ,?21- fi/ative +uclear track emulsion and e3uipment for autoradiographic analysis Subbed slides, coverslips, permount Aiemsa stain Microscope ,<hase contrast if total cycle is to be measured7linical centrifuge

Procedure ?
1. D/pose log cultures of cells ) to :4thymidine for a period of ?' minutes. $he Mean

7ycle $ime ,hours- of the culture must be known. * 7alculate the M7$ by plotting the growth of the culture and determining the average time for the cell population to double.
2. <our off the radioactive media ,discard with radioactive wastes- and wash cells twice

with <%S. Add washings to radioactive waste.

3. Add 1.* ml of '.9*S trypsin to the flask to dislodge the cells from the flask. Add 1' ml

of <%S, mi/ and pour into a centrifuge tube.

4. 7entrifuge in a clinical centrifuge at 5'' R<M for * minutes to pellet the cells. 5. Aspirate the supernatant, leaving about '.* ml of cells packed in the bottom of the tube.

Resuspend in <%S to wash, and collect again by centrifugation at 5'' R<M for * minutes.
6. Aspirate all but '.* ml of the <%S from the tube. Aently stir the cells by tapping the

centrifuge tube and add *.' ml of freshly prepared fi/ative, drop by drop, gently mi/ing between each drop. Allow the cells to fi/ for 149 hours.
7. 7ollect the cells, rinse once with fresh fi/ative and pellet cells into a final volume of

about '.* ml of fi/ative.

8. 1se a pasteur pipette to transfer the cells onto clean, subbed slides and allow to air dry. 9. <repare slides for autoradiographic analysis as in D/ercise 11.). 7oat with nuclear track

emulsion and e/pose for one week. Cevelop autoradiograms and stain lightly with Aiemsa as directed in D/ercise 11.). <repare permanent slides by attaching coverslips with <ermount.
10. D/amine the slide and count the total number of cells in interphase and the number of

cells that are radioactively labeled. D/press this number as a decimal fraction ,i.e. if )*S of the cells are labeled, the fraction is '.)*-.
11. 1se the following formula to compute the length of the S phase.

$ime for S phase 6 Mean 7ycle $ime / Fraction of !abeled 7ells /ptional $he entire process can be repeated with e/posure to the radioactive thymidine followed by a brief period of e/posure to non4radioactive thymidine. Fi/ a series of cultures at half hour intervals after removal of the pulsed radioactive label. D/pose the cultures to nuclear track emulsion and e/amine for labeled mitotic images ,as opposed to interphase cells-. $he time between the appearance of the first mitotic cells with label and the level of *'S of the mitotic images labeled represents an appro/imation of A9.

$he length of the mitotic division can be measured directly with phase contrast microscopy ,usually less than 1 hour-. Dstimate A1 by subtracting the time for mitotic division, A9 and S from the Mean 7ycle $ime.

Exercise 11.9 - Vicia Faba and Che#ical *a#age

!evel ### Materials

Arowing root tips of the broad bean,,. faba ,9n619Solutions of chromosome4damaging agents listed in the introduction to this 7hapter Reagents for Feulgen stain ,D/ercise 9.*;ven at 5'G 7 Microscope

Procedure
1. <lace freshly germinated bean seedlings into petri plates containing serial diluted samples

of suspected chromosome damaging agents.

2. Remove a seedling, rinse with distilled water and cut off root tips. <lace the root tips into

1 + :7l, 5'G 7, for 1' minutes.

3. Rinse the root tip and place in Schiff@s reagent in dark for ?' minutes. 4. Rinse the tip again, blot it gently and very gently rub the e/treme tip of the root to

remove the root cap.

5. <lace the root tip into a drop of )*S acetic acid on a clean slide and macerate the tissue

with a ra&or blade.

6. <lace a coverslip over the macerated tissue and s3uash as in the procedure for the

Crosophila polytene chromosomes ,7hapter $en-.

7. #dentify as many types of chromosome damage as found, referring to Figure 11.*. Craw

and label representative views.

8. 1se the caffeine treated cells to count the number of abberrations appearing per 1''

anaphases e/amined and record the data in this manner ,i.e. number of abnormalities per 1'' anaphases e/amined-.

Chapter 11: Cell C0cles - Endnotes

1.

$he phases of <rophase # are named for the appearance of a thread4like structure, known as ;nema. !eptonema means 0thin thread0 and leptotene is the ad"ective applied to the term stage, i.e. proper terminology is the leptotene stage of <rohase #. $he word stage is often omitted.

2. [600 a600Z$here is a shift in the terminology applied to these units. .hile the two

chromatids remain attached at the centromere, they are known as chromatids. #mmediately upon separating, each chromatid becomes known as a chromosome and is no longer referred to as a chromatid. $his is the reason that a cell can divide one chromosome ,with two chromatids- into two cells, each with a chromosome 4 the term applied to the chromatid is changed.
3.

From T.:. <riest. #e&ical Cytogenetics an& Cell Culture, 9nd ed. !ea U Febiger, <hiladelphia, 1O>>. 7ells from D/ercise 11.) may be used, or cultures of tetrahymena, yeast, or algae may be used. $his is calculated by plotting the growth of the culture and determining the average time for the cell population to double.

4.

5.

Chapter 1.: Cell Cultures - Introduction

A ma"or advance in our knowledge of cells came about with the ability to maintain them in continous culture. <rokaryotes have been cultured for a relatively long time, but eukaryotic cultures were first accomplished in the early 1O''@s ,:arrison, 1 7arrel 9-, with ma"or advances being made only in the past two or three decades $he procedures employed during this e/ercise will e/amine several types of prokaryotic cultures as well as a eukaryotic suspension culture, and establish a culture of embryonic fibroblasts. $he prokaryotic cultures and the eukaryotic suspension culture are 0established0 while the embryonic culture will be a 0primary0 culture. 7hick embryos are used for this latter procedure because of the relative ease of culture, ease of obtaining embryos, and relative simplicity of their nutritional needs. :ighly differentiated cells would be more difficult to establish and in some cases not possible at all ,given current technology-.

<rokaryotes <rokaryotes are cells without nuclei and are generally considered to be more primitive than eukaryotes ,cells with true nuclei-. $ypically, prokaryotes are easier to culture in a laboratory because most of them have less stringent nutrient re3uirements. For this lab we will utili&e bacterial cultures grown in nutrient agar, an environment in which human pathogenic organisms are e/tremely unlikely to grow. %acteria may be e/amined by observing the living, unstained microbes in a wet mount ,phase contrast or dark field illumination-, by observing dead cells stained with dyes under bright field illumination, or by observing cells prepared for electron microscopy. ;ur procedures will use 0fi/ed0 ,i.e. dead- cells stained for standard light microscopy.

Figure 19.1 %acterial growth curve .hen cells are grown, they will have specific growth characteristics, depending on the media, the temperature and the strain of cells utili&ed. For bacteria ,and some algae, fungi and other eukaryotic tissue culture- it is possible to measure the growth of cell populations by calculating cell number or mass. .ith modern e3uipment and the proper computer software, this can be a completely automated analysis, that would include the specific morphological data as well ,si&e, shape and density of colonies or individual cells-. Since bacteria are small, they are difficult to count through direct visuali&ation, but can be counted if one makes an assumption that each bacterium is capable of forming an individual colony. $he mass of a bacterial suspension can be deduced from the optical turbidity of a suspension. Dukaryotes %y contrast to the simple broth cultures of E. coli, the nutrient re3uirements of even the simplest eukaryotic culture are comple/. Refer to $able 19.1 for a comparison of the ingredients of +utrient %roth and Minimum Dssential Media ,MDM-, a typical eukaryotic media. Dukaryotes

also re3uire supplemental sources of materials, most often in the form of blood serum. Fetal calf serum is used e/tensively for this purpose, since it is readily available, and the fetal nature of the serum limits the presence of antibodies, which might negatively effect cell growth. ;ur first procedure will involve the simple transfer of an established culture from a suspension culture. An ali3uot will be removed from a commercially available cell line and transferred to prepared transfer vessels. Dach day students will observe these transfer cultures with an inverted phase contrast microscope, and remove ali3uots for cell counting with a hemacytometer. Simultaneously, they will check on the viability of the cells through a dye e/clusion techni3ue. $he second procedure will be somewhat more complicated. Students will remove chicken embryos from the egg, trypsini&e them to disaggregate the cells, and transfer the resulting cells to culture flasks. $his procedure establishes a primary culture, or one that is a first generation growth from 0in vivo0 cells. Dstablished culture lines are the result of long term selection for cells capable of growth under 0in vitro0 conditions. As such, they are more consistent clones, but often have genetic and structural alterations that differ significantly from the starting cell lines. $able 19.1 Dukaryote and prokaryote culture media As the cells grow in culture, we can observe three distinct phases. $he first is a !ag <hase, usually no more than 149 days in length, and during which there is little or no increase in cell number. Curing this time, the cells are 0conditioning0 the media, undergoing internal cytoskeletal and en&yme changes and ad"usting to the new media. $his is followed by a !og <hase. Curing this phase the cell number increases e/ponentially. $his growth will continue as long as there is sufficient nutrient to support the increasing cell number. Dventually some critical nutrient will become limiting, however. $he final phase is the <lateau <hase. Curing this phase the number of cells remains constant ,although not necessarily viable-. Dventually, of course, the cells will die unless subcultured or fresh media is added. $he final procedure in this laboratory e/ercise involves establishment of a primary culture from a chick embryo. :ere, the cells are not established in culture, but must be disaggregated ,detached from each other- and placed into a foreign environment ,the culture media-. Cisaggregation in embryos is reasonably easy, but depends on the en&ymatic dissolution of the cells glycocaly/, and the disruption of many plasma membrane structures and chemical elements. 7onse3uently, the cells will take a longer time before they grow ,that is, there is a long

!ag <hase-, and the selection process will favor cells which grow in contact with the culture vessel surface. $he cells will continue to grow in contact with the vessel and give rise to a 0monolayer0 culture. $he cells will cease to divide when they reach confluency they are said to demonstrate contact inhibition. 7onse3uently, growth curves are not measured by removing ali3uots and determining cell concentration, but are measured by the density of cells growing on the vessel surface. $his is accomplished through the use of an ocular grid inserted into an inverted phase contrast microscope. 7ell density ,cells8cm - is then plotted on a log scale against time in culture.

Figure 19.9 Dukaryotic growth curve

Exercise 1..1 - $septic Cell Transfers

!evel #

Figure 19.? Flaming a wire loop and removing cap Materials

%unsen burner .ire loop

<etri plate or broth tube %acterial culture Microscope slides

Procedure
1. <ick up the inoculating loop and hold it pointed down into an open flame until the loop

glows red. $his process sterili&es the loop of wire and is known as 0flaming0 the loop. #t will result in a sterile loop and will not contaminate your stock culture. #f there are li3uids already on the loop, the loop should be gradually placed into the flame to dry the loop. #f the loop is rushed into the flame, the drop of li3uid will splatter and spread bacteria over your work surface.
2. <ick up a broth culture in one hand, while holding the loop in the other. .ith the last two

fingers of the hand holding the loop remove the cap from the culture and gently flame the top of the test tube ,do not overheat-. #nsert the flamed inoculating loop into the test tube until it is submersed in the broth. $he loop should be allowed to cool slightly before immersion. Retract a small 3uantity of the broth held in the loop and replace the cap on the culture.
3. ;pen the top of your transfer vessel ,tube with water or nutrient agar- and flame the open

top of the tube. #nsert the loop into the tube and into the li3uid in the tube. .ithdraw the inoculating loop slightly from the li3uid, blot gently on the inside of the tube and completely remove from the tube. Replace the top on the tranfer tube.
4. #mmediately flame the inoculating loop.

Exersise 1... - Exa#ination of Bacterial Colonies

!D=D! # Materials

<etri plate cultures of various bacteria

Procedure
1. ;btain an established culture. +ote that the bacteria grow in clearly defined groups,

known as colonies. #n most cases, each colony is the outgrowth from an individual cell, although they may overlap if e/cessive numbers of cells were plated.

2. =isually e/amine the individual colonies of bacteria and describe them according to the

following characteristics2
o

Si&e. <inpoint, small, medium, or large, based on the relative differences between the largest and smallest colonies seen. Shape and Margins. Round, regular or irregular. Dlevation. Flat, conve/ or rounded, umbonate ,flat on margins and raised in the center 4 like a fried egg-, craterlike ,with depressed center-. 7onsistency. Shiny or rough. 7olor. Cescribe the color as accurately as possible, distinguishing between different types of gray or white, yellows, and red. #f the pigment appears to diffuse into the surrounding medium, rather than coloring only the colony, it is a water4soluble pigment.

o o

o o

3. Cetermine and record the identity of your colonies.

Colon0 1 Colon0 . Colon0 5 Colon0 1 6i3e 6hape Margins Consistenc0 Color

Exercise 1..5 - Ara# 6tain (LI-!

!D=D! #

Figure 19.) $ypical bacterial shapes Materials

7olonies of bacteria from D/ercise 19.9 $oothpicks

7rystal violet Aram@s iodine O*S ethanol Safranin Microscopes with oil immersion

Procedure
1. %efore staining the individual colonies, you should first practice the techni3ue by

observation of the gram positive micro4 organisms normally found in the gum linings of your mouth.
2. 1se a clean toothpick to rub along the gingival crevices ,area between tooth surface and

gums- of your mouth. @u+ lightl0)

3. Mi/ the scrapings with a drop of water previously placed on a clean slide, spread in a thin

film over the center of the slide and allow to air dry.
4. Fi/ the smear to the slide by passing the slide ,smear side up- 3uickly through a flame

three times. #f the slide is held directly in the flame, it will heat up too rapidly and break. $he trick is to gently dry the smear without overheating the slide.
5. <lace the slide on a staining rack. Apply the stains on the fi/ed smear as follows2
o o o o o o o o o

Flood the slide with crystal violet for ?' sec. Rinse with water. Flood with Aram@s iodine for 5' sec. Rinse with water. Cecolori&e with O*S ethanol. Rinse with water. 7ounterstain with safranin for 5' sec. Rinse with water and blot dry ,no rubbingN-. D/amine under oil4immersion ob"ective lens. ?

Aram4positive bacteria retain crystal violet after washing with O*S ethanol, while gram4 negative bacteria lose the purple dye after washing with O*S ethanol. $he positive or

negative reaction is a measure of the presence or absence of specific polysaccharide components of their cell walls. Safranin is used as a pink counterstain, so that Aram 4 cells can be visuali&ed. #n practice then, the distinction is made between purple cells ,Wand pink cells,4-.
6. Cetermine the basic cell shape of the bacteria. 7. Add the information on Aram stain and cell shape to the work done in D/ercise 19.9.

Exercise 1..1 - Pro4ar0ote Cell "u#+er +0 *ilution Plating

!D=D! ##

Figure 19.* 7olony counter Materials

%roth culture of E. coli $ubes of nutrient broth ,O.O ml each+utrient agar plates Sterile transfer pipettes ,1.' mlMuebec colony counter ,;ptional-

Procedure
1. ;btain a broth culture of E. coli and carefully mi/ the contents to ensure e3ual

suspension of the bacteria.

2. ;btain four test tubes each containing O.O ml. of nutrient broth. $hese will be used to

produce a serial dilution of the stock culture.

3. 1sing sterile pipettes, remove '.1 ml of well suspended cells from the stock culture and

transfer these aseptically to one of the waiting test tubes of broth. $his tube now contains a dilution of 181'' or 1' . Aently but thoroughtly mi/ the contents and label this tube at 1' .

4. Repeat this process, but now aseptically remove '.1 ml of culture from the 1'

tube and

place it into a new broth tube, which now becomes a 181',''' or 1' contents and label as 1' .
5. Repeat this procedure twice more to produce respectively a 1'

dilution. Mi/ the

and a 1'

dilution. %e

sure to mi/ thoroughly and label each tube.

6. Jou should now have a serial dilution of the stock culture with tubes at 1' , 1' , 1' ,

and 1'

. $he original stock culture will almost invariably be too high a population for

the ne/t step, so you will only use the four dilutions that you have produced. 1sing four separate petri plates containing 1* ml. of nutrient agar each and four separate sterile pipettes, transfer 1.' ml of each dilution broth suspension onto the surface of a petri plate. 7arefully label the plates and place them in an incubator for 9) hours at ?>G 7.
7. At the conclusion of incubation, remove each of the four petri plates and count the

number of colonies formed on the plates. For proper statistical analysis, the plate containing between ?' and ?'' colonies will give the most accurate results. $he colonies can be more easily counted by using a Muebec 7olony 7ounter which allows proper illumination, a grid overlay and by slight magnfication of the plate surface.
8. Multiply the number of colonies counted by the dilution factor to obtain the population

density of the original broth culture. "otes A growth curve can be established by repeating this procedure every two hours. Since the number of bacteria can be large, it will be necessary to plate cultures serially diluted. 7ount the number of colonies for each dilution and average the results.

Exercise 1..7 - Cell Mass +0 Measure#ent of Tur+idit0

!D=D! ## Materials

$rypticase soy broth culture of E. coli $ubes of trypticase soy broth ,O.O ml each$ubes of trypticase soy broth ,*.' ml each+utrient agar plates Sterile transfer pipettes Spectrophotometer and cuvettes )

Procedure
1. ;btain a trypticase soy broth culture of E. coli. $rypticase broth is better than nutrient

broth, since it is inherently less light absorbent.

2. <repare trypticase soy broth dilutions of 1'

and 1'

of an appropriate bacteria culture,

as described in D/ercise 19.).

3. +ow, for each of the two dilutions, set up a second series of dilutions, this time diluting

by 189 each time. $hat is, transfer *.' ml of the 1'

culture to *.' ml of fresh broth and

mi/ thoroughly. 1se *.' ml of this and transfer to *.' ml of fresh broth to produce a 18) dilution. Repeat for 18I, 1815, and 18?9 dilutions. Repeat the entire 189 dilution series for the 1' dilution. and the 1' dilutions. $hese

Jou should now have twelve tubes, si/ for each of the 1' are then diluted 189, 18), 18I, 1815, and 18?9.

4. 1se 1.' ml of each of the twelve dilutions and plate on nutrient agar plates to perform a

colony count as in the preceeding section.

5. Set a spectrophotometer for 5I5 nm wavelength and be sure it is turned on and

functioning properly. Ad"ust the dark current to 'S $.

6. <lace a cuvette containing trypticase soy broth into the spectrophotometer and ad"ust the

reading to 1''S $. $his is the blank for all subse3uent measurements.

7. $ransfer the remaining contents of the 19 dilutions to spectrophotometer cuvettes and

measure the S$ for each. 7ompute the absorbance for each sample. *
8. 7ount the number of colonies formed for each sample after 9) hours. of incubation. 9. <lot the absorbance of each sample against the plate count for that sample.

;nce this has been accomplished, you will have a value for computing the cell population number directly by measuring the absorbance of a suspension. $his can be more readily measured on a continuous basis. $o do this, grow suspensions directly in cuvette tubes and measure the A at timed intervals. Record a growth curve. %y connecting a recorder

to a spectrophotometer and keeping the chamber at ?>G 7, a continuous growth curve can be automatically recorded ,assuming care is taken to ensure proper suspension of the bacteria throughout the time period involved-. !acking such sophisticated e3uipment, the tube can be removed from an incubator at intervals, gently suspended, measured for absorbance and returned immediately to the incubator. <lot cell growth against time to produce a growth curve.

Exercise 1..9 - Transfer of Eu4ar0ote 6uspension Cultures

!D=D! ## Materials _ Fibroblast suspension culture _ $issue culture laminar flow hood _ Media appropriate to culture line used _ Cisposable pipettes ,1' ml and 1.' ml_ Cisposable culture flasks Procedure
1. ;btain a culture of mouse fibroblast cells in suspension culture. $his will be a simple

culture with minimal re3uirements, and one selected for e/cellent growth characteristics. $he transfer procedure will be similar to that for prokaryotes, with a few ma"or changes. First, all transfers will be done in a tissue culture hood in order to ma/imi&e asepsis. Secondly, the cells will be transferred with siliconi&ed pipettes rather than wire loops. $he silicone prevents adherance of the cells to the glass wall of the pipette.

A tissue culture hood is a device that has air moving in layers and under positive pressure. Since the air is filtered, it contains minimal numbers of bacteria or fungal spores, and since it is under a positive pressure, those particles that are present are blown out of the hood. $he layering prevents airborne organisms from settling on the work surfaces.
2. Arrange the materials in front of you, easily accessible through the opening of the tissue

culture hood. Dnsure that any alcohols and wrapping paper are kept clear of the bunsen burner. <re4sterili&e the hood before use, and use disposable sterile gloves.
3. !oosen the cap of a tissue culture flask and the cap of a stock bottle of tissue culture

media. 5 #nsert the tip of a sterile pipette into the stock bottle and remove 1' ml of media. $ransfer the media to the tissue culture flask.
4. ;pen the top of the suspension culture and use a sterile 1.' ml transfer pipette to remove

a 1.' ml sample of the culture. $ransfer it to the fresh media in the culture flask. Secure all caps that have been loosened.
5. <lace the new cultures in an incubator at ?>G 7.

Exercise 1..< - :ia+ilit0 Cell Count

Materials

Suspension culture of cells Sterile transfer pipettes Stock '.9S ,w8v- $rypan blue :emacytometer and microscope

Procedure
1. Aently swirl a suspension culture to distribute the cells evenly. Aseptically remove a

small sample ,'.1 ml- of cells from the cultures. <lace the sample in a separate test tube ,it need not be sterile-.
2. Cilute ) parts of stock $rypan %lue with 1 part of *( saline and add '.1 ml of the diluted

dye to your sample. Mi/ gently.

3. Set up a hemocytometer and cover slip. #mmediately place a drop of the stain8culture

combination on the hemocytometer ,remember to use both sides of the hemocytometerand wait one minute.
4. ;bserve the cells with low power microscopy. 7ount the total number of cells, and the

number of stained cells.

5. 7ompute the concentration of viable cells per ml. of culture.

"otes $rypan %lue is a stain that is actively e/truded from viable cells, but which readily enters and stains dead cells. $herefore, the cells which are blue are dead. $he difference between the total number of cells and the number of dead cells would be the number of viable cells in a given ali3uot of your culture. $rypan %lue actually significantly overestimates the number of viable cells, but is sufficient for purposes of this lab. Appro/imately ?'S of the cells measured as viable with $rypan %lue will not be able to continue growth beyond a 9) hour period.

Exercise 1..= - Co#putation of Transfer $li;uots

!D=D! ## Materials

Suspension culture of cells Media appropriate to culture 7ulture flasks $ransfer pipettes :emacytometer and microscope

Procedure
1. ;btain ? transfer culture vessels. $he vessels may already contain fresh culture media, or

you may be asked to transfer your own.

2. ;btain a suspension culture and count the number of cells8ml of culture using the

procedure listed inD/ercise 19.>.

3. 7ompute the volumes of the suspension culture needed so that when added to 9* ml of

fresh media, the final concentrations will be2

1 ( 1' cells8ml * ( 1' cells8ml 1 ( 1' cells8ml For e/ample2 #f you have 9* ml of fresh media, you will need to add 9* ( 1' cells to obtain a final concentration of 1 ( 1' cells8ml #f your suspension culture contains * ( 1' cells8ml, you will need to transfer * ( 1' ,9* / 1' cells divided by * / 1' cells8ml-. 1se the formula2 Ali3uot to be transferred 6 +umber of cells to be transferred 4444444444444444444444444 7ulture concentration ml or '.* ml of culture to the fresh media

4. Seed three flasks each containing 9* ml of fresh media to a final concentration of 1'

cells8ml, * ( 1' cells8ml and 1' cells8ml.

5. !abel and place your fresh cultures in the tissue culture incubator at ?>G 7.

!ay the culture flasks on their sides in order to ma/imi&e the air e/change surface of the culture and to prevent the cells from drowning. An alternative would be to incorporate mechanical shaking of some type.

Exercise 1..> - Eu4ar0ote Aro&th *0na#ics

!D=D! ## Materials _ Suspension cultures set up from D/ercise 19.I _ Sterile transfer pipettes _ Materials for viability counting ,D/ercise 19.>Procedure

1. After 19 hours, aseptically remove '.1 ml from each of the three cultures, add '.1 ml of

trypan blue and count the total number of cells and the number of blue cells. 7ompute the number of viable cells8ml.
2. After 9) hours ,from the time of seeding-, repeat step 1. 3. 7ontinue to repeat step 1 at 9) hour periods ,i.e. daily- until there is no change in the

number of cells8ml of culture.

4. <lot cell concentration on a log scale vs time of culture. #dentify and label the !ag, !og

and <lateau phases for your culture.

5. Select a period of time during the !og <hase and compute the doubling time for your

culture. $hat is, the time re3uired during the !og <hase to e/actly double the number of cells8ml.

Exercise 1..1, - Esta+lish#ent of a Pri#ar0 Culture

!D=D! ### Materials

7hick embryo ,appro/imately I days old>'S ,v8v- ethanol for swabbing Sterile scissors, forceps and probes Sterile petri plates <hosphate buffered saline ,<%S$rypsin, cold sterili&ed in a 19* ml sterile erlenmeyer containing a magnetic stirring bar Minimum Dssential Medium Fetal 7alf Serum 7linical centrifuge with sterile capped centrifuge tubes 7ulture flasks #nverted phase contrast microscope ,;ptional-

Procedure I
1. 7andle an I day old egg to ensure that it is alive. $his is easily accomplished by holding

the egg in front of a bright light source the embryo can be seen as a shadow. 7ircle the embryo with a pencil.
2. <lace the egg in a beaker with the blunt end up, and wash the top with a mild detergent,

followed by swabbing with ethanol.

3. 7arefully puncture the top of the egg with the point of a pair of sterile scissors and cut

away a circle of shell, thus e/posing the underlying membrane ,the chorioallantois-.
4. .ith a second pair of sterile scissors, carefully cut away and remove the chorioallantoic

membrane, e/posing the embryo.

5. #dentify and carefully remove the embryo by the neck, using a sterile metal hook or a

bent glass rod, and place the embryo in a 1''mm petri dish containing phosphate buffered saline ,<%S-. .ash several times with <%S by transferring the embryo to fresh petri plates. After removal of all yolk and8or blood, move the embryo to a clean dish with <%S.

6. 1sing two sterile forceps, remove the head, limbs, and viscera. %e sure to remove the

entire limb by pulling at the pro/imal end. Move the remaining tissues of the embryo to yet another dish and wash with <%S.
7. Mince the embryo finely with scissors and transfer the minced tissue to a flask containing

<%S. Allow the tissue pieces to settle.

8. Remove the <%S with a sterile pipette and add 9* ml of trypsin, a proteolytic en&yme.

Stir the solution gently at ?>G 7 for 1*49' minutes.

9. Allow the larger, undigested tissue pieces to settle and decant the supernatant into an

e3ual volume of Minimal Dssential Medium ,MDM- W 1'S Fetal 7alf Serum ,F7S-. F7S contains protease inhibitors which will inactivate the trypsin.
10. 7entrifuge the cells in MDM at 1''' rpm for 1' minutes in a standard clinical centrifuge.

Remove the supernatant and resuspend the pellet in 9* ml of fresh MDM W 1'S F7S.
11. Remove '.1 ml of the culture and determine cell concentration and viability as directed in

the previous section.

12. Seed two 9* cm plastic culture flasks containing 9* ml of MDM W 1'S F7S to a final

concentration of 1' cells8ml.

13. !abel and place your cultures in the tissue culture incubator at ?>G 7 and e/amine daily

for cell density and morphology.

14. +ote any changes in the color of the media. $issue 7ulture media has a p: indicator

,<henol Red- added in order to check on the growth of cells. $he media initially is a cherry red ,with slight blue ha&e- and turns orange and then yellow as the cells grow, thereby reducing the media. Should this color change occur within 9) hours, the culture is most likely contaminated and should be disposed of.
15. D/amine the cultures using an inverted phase contrast microscope. $his will allow

observation of the cells without opening or disturbing the growth.

16. Make cell density determinations at 1' ( magnification using a s3uare ocular grid, as

e/plained in 7hapter ;ne for the determination of area.

17. <lot the cell density on a log scale vs. time of culture. 18. Ciagram the shape of the cells at each phase.

"otes

$he cultures will develop differently than the suspension cultures. $he viable cells will grow out of the trypsini&ed pieces of tissue and will remain in contact with the bottom of the culture flask. $hey will continue to divide and migrate until the entire bottom of the flask is covered with a single layer of cells ,contact inhibition and the formation of a monolayer-. Chapter 1.: Cell Cultures - Endnotes
1.

:arrison, R.A. ,1O'>- ;bservations on the living developing nerve fiber. <roc. Soc. D/p. %iol. Med. )21)'41)?. 7arrel, A. ,1O19- ;n the permanent life of tissues outside the organism. T. D/p. Med. 1*2*154*9I. ;ne of the continuing differences between microbiologists and microscopists is the lack of a coverslip when viewing bacteria. 7onvenience causes microbiologists to skip the process of placing mounting media and a coverslip on their slides. $his causes microscopists to cringe at the thoughtN #f the cultures are to remain aseptic, the cuvettes can be sterili&ed and plugged. Alternatively, culture flasks with cuvette side arms can be used. Absorbance may be read directly if the spectrophotometer is e3uipped with digital display. Absorbance is more difficult to interpret on an analog display. #t is assumed that the media has been pre4mi/ed, with serum and other additives put into the media. $he media should be in small aseptic containers for student use. From2 %arbara %. Mischell and Stanley M. Shiigi. 0Selected Methods in 7ellular #mmunity0. ..:.Freeman U 7o. San Francisco, 1OI', p. 1>.

2.

3.

4.

5.

6.

7.

Modified fro# Freshne0' @. Ian. Culture of Animal Cells: A Manual of Basic Chapter 15: *ifferentiation - Introduction Cifferentiation occurs when cells change in structure and function within a period of time. As such, one of the ma"or problems in research is the factor of time. 7ells must be 0competent0 or primed for a differentiating change by being properly 0induced0 by some internal influence ,gene, ion flu/, metabolic alteration- or e/ternal factor ,hormone, growth factor, cell4cell communication-. Finally, they must be in the correct association with neighboring cells ,position effect in embryos, contact inhibition or guidance- and strata for the change to occur. All of these

factors combine to make an afternoon laboratory session on differentiation difficult to coordinate. 7onse3uently, the laboratory will use prepared slides of materials that have undergone differentiation, so that the time can be compacted into convenient laboratory periods. .e will use some living cells by carefully choosing systems which can be closely monitored ,slime mold development- or which are slower to develop and thus able to be 0ready0 at the time of a lab session ,fern gametophytes-. .e will observe specific molecular alterations indicating differential gene activity through indirect observation of the changes, primarily through the use of inhibitors of the basic C+A8R+A8<rotein system. Dmbryogenesis A classical approach to the basic process of cellular differentiation has involved the primary formation of the three germ tissues of embryos, namely ectoderm, endoderm and mesoderm. $he sea urchin provides readily available egg and sperm which can be fertili&ed in laboratory with ease. Further , there is little yolk within the egg, and the yolk is evenly distributed ,i.e. an isolecithal egg-. $he embryo demonstrates 0regulative0 development ,each embryo cell or blastomere has the ability to form a complete organism-, it has several mutant strains available for genetic analysis, and it develops in a synthetic sea water environment, without the need for elaborate culture or 0in utero0 studies. $he eggs are large enough to be studied with standard light microscopes and can be conveniently micro4 manipulated for nuclear transplant and cytoplasmic in"ections. $he sea urchin embryo is thus a convenient model system for the complete analysis of early embryogenesis. For our purposes, we will limit the laboratory study to descriptions of the basic processes of cleavage, induction, migration and invagination of cells. Figure 1?.1 Adult forms and life cycle of 7. elegans Caenorhab&itis elegans is rapidly becoming a favorite organism for early embryogenesis. $his invertebrate worm ,nonparasitic nematode- reproduces as a self4fertili&ing hermaphrodite. $he genetics of the nematode is fairly simple each contains a pair of se/ chromosomes ,(( 6 Female, (; 6 Male- and five pairs of autosomes. $he male karyotype develops spontaneously in

18>'' developing embryos. Males can be mated to hermaphrodites, but hermaphrodites never mate with each other. Figure 1?.1 presents the life cycle of 7. elegans. C. elegans has an asymmetric first cleavage which clearly establishes two distinctly different cells, which in turn are the progenitors of specific parts of the final organism. Starting from the single cell &ygote, the first stage larva hatches in about 1) hours after fertili&ation with a total of *)5 somatic nuclei and four primordial gonadal nuclei. $he embryo is a classic 0mosaic0 where each cell carries the information for a piece of the whole. %locking cell division as early as the two4 cell stage will result in cells which are unable to form complete organisms, but which will demonstrate specific fates. $he cells are each given labels indicative of their ultimate fate, and limited potential.1, 9 Figure 1?.9 presents the general scheme for this cellular development. $he eggs of C. elegans are small, transparent, capable of developing outside of the mother, have relatively small genomes and have had each cell@s development characteri&ed. ? .ith the isolation of mutants, and characteri&ation of the specific gene loci ,C+A se3uencing-, powerful probes of early gene control over embryogenesis have become available. ) $he se3uence of events in the development of this nematode are observed by the formation of polar bodies and completion of meiosis following fertili&ation. At this time, we can see intensive cytoplasmic streaming which results in the observable segration of germ line specific granules to the posterior of the embryo. Subse3uently, the pronuclei fuse and the first cleavage is initiated, giving rise to a large A% blastomere and a small <41 blastomere. Antibodies can be made to the < granules and we can observe a clear mosaic pattern distribution can be observed with immunofluoresence. Figure 1?.9 Fate of 7. elegans blastomeres :ematopoietic System $he development of the blood and related cells has long been a sub"ect of intensive study. ;ne reason is that blood represents a type of differentiation which is continuous throughout the life of an organism, and demonstrates the role of stem cells in development. Stem cells are embryonic derivatives which retain the ability to form clones, yet remain relatively undifferentiated themselves. $his is accomplished by the division of a stem cell into two cells, typically one of which will continue on to a highly differentiated role, while the other remains as a stem cell. #t is

possible for stem cells to divide into two stem cells, and on occasion, a stem cell will divide and form two differentiated cells ,thus ceasing to be a stem cell-. $he hematopoietic system ,blood forming- demonstrates each of these modes. #n adults, blood cells do not divide within the bloodstream, but are produced either within the bone marrow, lymph nodes, spleen or thymus. $hus, these organs make up the hematopoietic system, or blood forming system. ;nce the cells are formed through cellular division, they must mature before attaining their final differentiated state. Smears of bone marrow ,and sometimes whole blood- will display many intermediate states of differentiation. #t is generally believed that all blood cells arise from a single type of cell ,the 0unitarian0 theory-, which is itself derived directly from embryonic mesenchyme. $he cell is known as a hemocytoblast and is characteri&ed by its large si&e ,I4?' microns-, minimal 3uantity of basophilic cytoplasm, lack of cytoplasmic granules, large nucleus and presence of 94? prominent nucleoli. $hey are fairly scarce within bone marrow ,S of cells present-, yet they are the progenitors of all other blood cells. A hemocytoblast within bond marrow will give rise to granulocytes, megakaryocytes and erythrocytes. $he same cell found in lymphoid tissues will give rise to lymphocytes. For the e/ercises used in this manual, we will limit our observations to those cells found in the bond marrow. $he lymphocytic series is more difficult to study since it has fewer distinguishing marks ,granules, basophilia-. $he erythrocyte, granulocyte and megakaryocyte series are clearly defined by observable light microscope characteristics. 7ollectively they represent a model series of cellular alterations leading toward differentiation from a stem cell population. Figure 1?.? Drythrocytic and granulocytic development <hotomorphogenesis of Ferns A/enic growth of fern prothallia germinated from spores represents a ready se3uence of cellular differentiation which has many of the standard characteristics. *, 5, > #n addition, the developing gametophyte is haploid ,simplifying the genetics-, and since the fern fronds grow ase/ually, usually all the spores from a given field are from the same plant, thus ensuring genetic homogeneity.

As the spore germinates and begins to develop a heart4shaped gametophyte, it will pass through several stages of recogni&able development. $he first is the formation and e/tension of a rhi&oid, followed almost immediately by cellular division within a single plane. $his division takes place within the 0tip0 cell and gives rise to a filamentous protonema. $he tip cell will then alter the planes of division by O'G and subse3uently, the growth will become two4dimensional. As this process continues, the typical shape of a young fern prothallus is established. $he division continues forming a structure with a single layer of cells, with differentiated areas forming rhi&oids, the body of the prothallus, antheridia ,sperm- and archegonia ,egg-. $hus, there are a small number of easily recogni&able differentiated states, within an organism that can be propagated from spores with genetic homogeneity and which can be grown easily under aseptic and a/enic conditions. $he body of the fern gametophyte can be sliced into various components and a new prothallus will develop from each. $he spores can be irradiated and will give rise to tumors, abnormal three4dimensional growths with comple/ structure and physiology. $he cultures of developing gametophytes can, of course, be treated with any number of drugs for molecular analysis of function. #n addition, the developing gametophyte demonstrates a characteristic process of photomorphogenesis. $his process is one in which light has an effect upon the structure and function of the plant, independent of photosynthesis. $here are several e/amples of this process, most of which involve red light and far red light wavelengths and the germination and development of seeds. $hese systems involve the pigment phytochrome, and re3uire somewhat difficult illumination control. $he fern gametophyte, by comparison, alters its growth when grown in various components of visible light. Specifically, the shape of the gametophyte will differ when grown in red, blue and green light. Figure 1?.) Cevelopment of fern gametophytes 1nder sufficient illumination with blue light, the gametophyte will develop as indicated above, that is, as though it were in white light. 1nder red illumination ,corrected for the same energy intensity-, the gametophytes will develop as filaments only. $hat is, the alteration in the plane of development which gives rise to the two4dimensional growth will not occur. 1nder green

illumination, the gametophyte will grow filamentous, but will also develop and differentiate massive amounts of antheridia. Refer to Figure 1?.) for details of fern gametophyte development. 7ell 7ommunication 4 Dictyostelium and cAM< $he development of the slime mold -ictyostelium &iscoi&eum has been chronicled for several decades, with the primary establishment of this important system accomplished by T.$. %onner I, O, 1' and followed by too many investigators to list here. ;ne would be remiss, however, to not acknowledge the e/tensive work of the Sussmans. 11 $here has been a more recent review of the growth characteristics in Ashworth. 19 $he basic developmental pattern of -. &iscoi&eum is given in Figure 1?.*. $his organism is a cellular slime mold that lives most of its life cycle as a free amoeboid cell. $he amoeba of -. &iscoi&eum function as simple protists, yet when nutrition becomes limited, they can aggregate and form a multicellular slug, with many of the characteristics of a true multicellular organism. .ithin its life cycle, the amoeboid cell emerges from a spore, is strictly parasitic on bacteria and divides by simple binary fission. .hen the number of amoeboid cells increases, and there is a slight drying of the environment, the amoeba will gather, form protective slime sheaths around themselves and begin differentiating. $he cells arrange themselves into positions, and the location a cell has will in turn determine its ultimate fate, whether it will become a part of the stalk ,prestalk- or will be involved in the se/ual reproduction of the species ,prespore-.

Figure 1?.* Cevelopment of -. &iscoi&eum $he aggregated amoeba first form a slug or pseudoplasmodium which travels about as though it were a multicellular organism. Dventually, it will settle down on the agar plate ,or a leaf if in a

lake- and form a base, stalk, and fruiting body ,sorocarp-. Spores are formed within the fruiting body and the process begins again. #t has long been known that the primary induction of this phenomenon is a pulsed level of the nucleotide cAM<.1? $he level of cAM< that a cell is e/posed to is fundamental to its differentiation into either stalk cell or spore cell. 1) 7ells can be isolated, which are thus prestalk or prespore, after cAM< is administered. #f agar cultures of the amoeba are supplemented with 1' M cAM<, and the amoeba are placed

in small drops onto the surface of the media ,?4I mm diameter-, the cells will form a ring around the drop as they migrate from the center. 1* .ithin 9) hours, some of the advancing cells within the ring will differentiate into separate stalk cells. %y contrast, in the absence of cAM<, the cells aggregate and form normal pseudoplasmodia.

Exercise 15.1 - 6ea %rchin E#+r0olog0

!D=D! # Materials

<repared slides of sea urchin development Microscope Male and female sea urchins 15 '.* M B7l Cepression slides

Procedure
1. <lace a commercially prepared slide of the sea urchin embryo development on the stage

of the microscope and locate representative stages in the development of the organism. Craw and label the following stages2 <rimary oocyte with Aerminal vesicle Fertili&ed egg with aster $wo, Four, Dight 7ell stages Morula ,Si/teen cell stage%lastula Aastrula

2. #ndicate on your drawings the development of micromeres, mesomeres, and macromeres.

Also identify the formation of primary mesenchyme and secondary mesenchyme during the formation of the gastrula.
3. 7ollect a living sea urchin and place it 0upside4down0 in the top of a small beaker. $he

mouth should be e/posed. .ith a clean, new needle, in"ect 1.' ml of '.* M B7l into the tissue "ust ad"acent to the oral opening. $he fluid should enter the coelomic cavity, not the gut of the animal.
4. .ithin a short period of time ,several minutes- the sea urchin will discharge its gametes.

$he gametes will appear as either a yellow collection of eggs, or a white collection of sperm. For the eggs, immediately invert the sea urchin so that the eggs are shed directly into a small amount of salt water in the bottom of a new, clean beaker. #t is important that the bottom of the sea urchin be in contact with the sea water and that the salt water be kept cool ,1*G 7 is ideal-. $he shed eggs are stable for several hours if kept cool. For sperm, the animal should be inverted over a dry beaker, that is with no salt water. Sperm are activated when diluted in sea water. #f collected in the dry state and stored in a refrigerator, they will survive for several hours. .hen diluted, they will last only a matter of minutes.
5. <lace a small sample of the eggs in sea water onto a microscope slide and place the slide

on the stage of a microscope. Focus on the eggs.

6. .ith a clean pipette, collect a small drop of the sperm and place it into about 1 ml of salt

water in a test tube. Aently stir to mi/ and activate the sperm, and then transfer a drop of the diluted sperm to the waiting eggs on the microscope stage. *o not conta#inate the re#aining eggs +0 contact &ith the sper#.
7. #mmediately place a coverslip on the suspension of sperm and eggs and observe. ;nce

fertili&ation occurs, the edges of the coverslip can be sealed with parafin to prevent drying. For long term observation, a diluted sample of eggs should be fertili&ed and kept at 'G 7. <eriodically, wet mounts can be made of the developing embryos to e/amine the progress.
8. 7ompare the development of the living embryos to the prepared slide stages. 1>

"otes $he development of the sea urchin embryo occurs rather rapidly. Fertili&ation will probably occur before you can get the coverslip on the slide. .ithin about 149 minutes the fertili&ation membrane will lift off, and the first division should occur within 94? hours, depending on the temperature. $he embryo will hatch in "ust over 9) hours and will complete its entire development within >9 hours. #f close e/amination of the fertili&ation process is desired, place the eggs on a slide with a support to keep the coverslip "ust off the eggs ,a second coverslip will do-, add a coverslip and focus on the eggs with darkfield illumination. <lace a drop of sperm "ust at the edge of the coverslip while observing the eggs through the microscope. .ith darkfield illumination the sperm will be visible and minute changes in the cortical region of the egg will be observed as a sperm penetrates the outer membrane.

Exercise 15.. - *eter#inants and Fate - C. elegans e#+r0os

!D=D! # Stages in development of C. elegans Materials

Caenorhab&itis elegans cultures on agar plates E. coli strain ;<*' +A agar plates C. elegans Ringer@s solution Microscope ,preferrably +omarski interference or phaseSlides and coverslips

Procedure
1. <lace a culture of C. elegans on the stage of a dissecting microscope and focus on the

surface of the agar plate. $he cultures contain both small males and the larger hermaphrodites. !ocate a specimen of each.
2. For embryological studies, select a large hermaphrodite and identify the various organs

within the worm. .ith a wooden applicator, select a hermaphrodite and transfer it to a slide containing a drop of Ringer@s solution. Make a wet mount. 1I

$he coverslip should hold the worm down, but will allow it to continue moving. #t will normally be very active at first, but then 3uiet down. Since the worm can only fle/ its body in a dorsal8ventral plane, it will also normally be on its side.
3. <lace the slide on a microscope 1O and locate the worm in the field of view. #dentify the

eggs and developing embryos within the ovary of the worm. 7areful ad"ustment of the light will be necessary. As the slide dries out, it will be necessary to add a drop of Ringer@s to the edge of the coverslip.
4. Craw the various stages of development and label each cell according the the scheme

in Figure 1?.5. /ptional Select several gravid worms ,hermaphrodites- and transfer them to a fresh plate of +A agar pre4 seeded with a lawn of E. coli. Allow the worms to lay eggs on the agar. After the eggs are sown, use Ringer@s solution to rinse the adult worms from the agar plate. Any remaining adults should be picked off the agar, but the eggs will remain stuck in the agar. $his process will nearly synchroni&e the eggs and their development can then be observed over time. !1 larva can be washed from the plates in about two hours and the subse3uent development of these larva monitored as a synchronous population.

Exercise 15.5 - The ?e#atopoietic 60ste#

!D=D! # Materials

<repared slide of bone marrow Microscope

Procedure
1. D/amine the bone marrow slide to identify the erythrocytic series2 proerythroblast,

basophilic erythroblast, polychromatophilic erythroblast, normoblast, reticulocyte and mature erythrocyte. Craw and label each cell type.
2. Craw and label the granulocytic series2 myeloblast, promyelocyte, myelocyte,

metamyelocyte, granulocyte with a band4 shaped nucleus, and the mature granulocyte ,neutrophil, eosinophil, basophil-.

3. Craw and label the megakaryocytic series2 megakaryoblast, megakaryocyte and

platelet. 9'

Exercise 15.1 - Photo#orphogenesis

!D=D! # Materials

Pteri&ium aquilinum spores ,%racken fernAgar plates of Bnudson media 91 7ellophane filters ,red, blue and green 99Cissecting microscope 1S ,v8v- $ween I' ,<olyo/yethylene sorbitan mono4oleate-

Procedure
1. <lace 1' ml of $ween I' into a capped test tube and add about 149 mg of dry fern spores.

7ap the tube and shake the vial to wet the spores.
2. ;btain eight petri plates containing a minimum balanced salt media. <ipette 1.' ml of the

spore suspension onto the surface of each plate and swirl the plate to evenly distribute the spores. <lace the lids on the petri plates and seal the plates with tape running completely around the edge. 9?
3. .rap the plates in pairs of red, blue or green cellophane. !eave two plates with no

wrapping. <lace the plates under a light source ,window sill or fluorescent 0Aro4lu/0 with a 19819 ,19 hrs. light, 19 dark- light regime.
4. Monitor the spores daily by observation with a dissecting microscope. .hen they begin

to germinate, monitor with the low power of a regular microscope ,or inverted, if the agar is not too thick-. $he spores will e/tend a rhi&oid in about * days and begin cell division within >4I days. $he first divisions are then crucial to the development of the gametophyte shape. 7ontinue to monitor the division planes for a period of two to three weeks.
5. +ote the number of spores that germinate and calculate the percent germination. As the

prothalli grow, note the position and direction of each cell division. Sketch each stage in the development of the fern gametophyte. /ptional

Fi/ gametophytes periodically with acid4alcohol and prepare the gametophytes for histological staining of the chromosomes. +ote the presence of mitotic figures and identify the poles of each cell division. %ased on the location of the division planes, predict the direction for spindle fiber growth and hypothesi&e a mechanism for controlling the direction of cell division ,and therefore the morphology of the gametophyte-. Cevise an e/periment to test your hypothesis. ;ne can do more advanced study of morphology by combining the growth of the gametophyte with the presence of mitotic inhibitors or chromosome damaging agents ,7hapter $en-. Finally, the spores can be sub"ected to /4ray e/posure ,)'45',''' roentgens- and the morphology studied. As the fern 0tumors0 develop, the planes of division will appear randomly, giving rise to a ?4 dimensional growth rather than the typical 94dimensional gametophyte.

Exercise 15.7 - D.discoideum Aro&th on $gar

!D=D! ## Materials

-ictyosteleum &iscoi&eum grown a/enically on agar plates <etri plates with SM agar media %roth culture of <lebsiella aerogenes or E. coli ,non4mucoid variety.ire loop for transfers Microscope, slides, coverslips

Procedure
1. Select a petri plate with SM agar media, a broth culture of and a culture of C. discoideum

containing fruiting bodies.

2. #noculate an agar plate with a thin layer of the bacteria. Select a fruiting body from the

fungal culture and place it in the center of the petri plate containing the bacterial suspension.
3. 7ontinue to monitor the growth of the fungus for the ne/t week ,make daily observations

4 new fruiting bodies will appear in about ) days-. +ote the initial germination of the spores from the fruiting bodies, the subse3uent growth and the increased number of amoeba in the culture. As the culture begins to dry, the amoeba will aggregate and form the migrating slug. $he slugs will migrate around the

plate, consuming the bacteria as food. Dventually, the slug will anchor itself to the agar and undergo a process known as culmination. Curing this stage, the cells in the anterior of the slug will begin differentiating into pre4stalk cells, while those in the rear will develop into pre4spore cells.
4. Sketch each stage in the development of the fungus and note the time of each stage

appearance within the culture. /ptional <eriodically remove a sample of the cells or slug and fi/ with 7arnoy fi/ative. <lace the cells on a slide and allow to air dry. Stain with a a basophilic dye and note any alterations in the nuclear material during each phase. #n particular, note the presence and si&e of the nucleoli, and the degree of basophilia demonstrated by the cytoplasm.

Exercise 15.9 - 6uspension cultures of D. discoideum

!D=D! ## Materials _ A/enic culture selected for suspension growth ,e.g. -. &iscoi&eum Strain A(4? 9)_ 7ultures supplemented with 1' M cAM<9*

_ Fresh flasks of growth media, with and without cAM< _ MDS4<CF containing 1' mM DC$A ,;ptionalProcedure
1. 7ollect amoeba from the normal media cultures. D/amine the culture for the presence of

the amoeba and note the stage of their development ,i.e. individual amoeba, aggregates, slugs-.
2. Select amoeba from the cAM< treated culture and compare the amoeba to those grown in

the absence of the nucleotide.

3. 1sing a pipette, transfer an ali3uot of the amoeba previously grown in the absence of

cAM< to fresh media without cAM<. Dstablish a second culture by transferring the same

amoeba to media containing cAM<. Follow the techni3ues outlined for the transfer of eukaryotic cells in 7hapter $welve.
4. <repare a growth curve for each of the amoeba subcultures ,i.e. amoeba grown in the

presence and absence of cAM<-.

5. +ote on the growth curve the times for any observed differentiation.

/ptional .hen the amoeba aggregate, the position the cells have within the slug determines its ultimate fate. #f a cell is in the anterior of the slug, it will become part of the subse3uent stalk. #f in the posterior, the same cell would become part of the spore forming body. $he pseudoplasmodia are attracted toward a strong light, and this ta/is has been used to orient slugs and conse3uently separate cells on the basis of their position. <late A/4? cells onto Millipore filters for development ,* / 1' cells8cm -. At the finger stage, shake the cells from the filters and dissociate them into single cells by vigorous pipetting in cold MDS4<CF containing 1' mM DC$A. Shake the suspension for ? hours under one of the following conditions2 +o added cAM< X9?' R<M 1 mM cAM<, with hourly additions to 1'' microM, 9?' rpm +o added cAM< X >' R<M $he fast shaking cultures will remain as predominantly single cells while those in the slowly shaking culture will aggregate. 95 :arvest the cells by centrifugation and analy&e for si&e of the aggregates as well as formation of the multicellular structures.

Exercise 15.< - Isolation of Fruiting Bodies &ithout slugs

!D=D! ### Materials

9) suspension cultures of -. &iscoi&eum 7linical centrifuge and tubes %lack Millipore filters ,AA%<)>S;7ellulose pads for Millipore filters !<S buffer

Procedure 9>
1. 7ultures grown for 9)4?' hours ,agar or suspension- are washed free of bacteria by

several centrifugations in fresh, cold water ,9 minutes X ?'' (g each-.

2. Apply the cells at a density of 5 ( 1' cells per cm to a water washed black Millipore

filter supported by two cellulose absorbent pads previously saturated with !<S %uffer.
3. Allow the cells to develop in the dark at 99G 7 in a humidity saturated chamber. 4. <eriodically withdraw a sample of the amoeba and sketch the development of the

aggregates. +ote in particular the induction of synchronous fruiting without the formation of migrating slugs.

Exercise 15.= - Prestal4 (s Prespore Effect

!D=D! ### Figure 1?.> <restalk8prespore immunofluoresence Materials

Agar cultures of -. &iscoi&eum 9' mM +a4B <hosphate buffer, p: >.' 9' mM +a4B <hosphate buffer plus 9' mM DC$A, p: >.' :ypodermic syringe with 15 gauge needle +ylon mesh >'S v8v <ercoll in 9' mM DC$A, * mM MDS, p: >.' <reparative centrifuge, rotor and tubes

Procedure 9I
1. 7ollect migrating slugs by washing them from the agar with cold 9' mM +a4B

<hosphate %uffer, p: >.'. A bent glass rod may be used to assist in dislodging the slugs.
2. .ash the slugs once with the same buffer and resuspend in 9' mM +a4B <hosphate

%uffer containing 9' mM DC$A.

3. <ipette the slugs up and down through a 15 gauge needle for about * minutes to

dissociate the cells. 9O

4. <ass the suspension of cells through a fine nylon mesh to remove clumps, wash with the

DC$A buffer and resuspend in 94? ml of fresh DC$A buffer. 7ollect the cells by centrifuging for 9 minutes at ?'' /g. Resuspend the cells to make a final concentration of ) ( 1' cells per ml of DC$A buffer.
5. Form two <ercoll gradients by placing 19 ml of <ercoll into two centrifuge tubes and

centrifuging at 91,''' /g for )' minutes at )G 7.

6. !ayer 9*' Ll of the cell suspension onto a preformed <ercoll gradient. Dither distribute

the cells between the two gradients, or use one tube as a balance in the centrifuge.
7. 7entrifuge the amoeba in the preformed <ercoll gradient at 1?,''' /g at 1*G 7 for ?

minutes.
8. 7ollect and identify the bands of cells. .ash the cells free of <ercoll with DC$A buffer.

"otes ;nce separated on <ercoll, the cells can be analy&ed for any number of activities. ;ne of the principal analyses is for the production of specific gene products which would indicate C+A control of differentiation. $he effect of differential gene activity can be studied by e/tracting R+A from the prestalk and prespore cells with subse3uent analysis of the R+A. Since the ultimate gene product is a protein, several investigators have identified and isolated proteins characteristic of prestalk and prespore differentiation. %y producing antibodies to those proteins, immunofluorescent techni3ues may be applied to the detection of early differentiation. Figure 1?.> demonstrates such an analysis applied to prestalk8prespore cells. Chapter 15: *ifferentation - Endnotes
1.

Schierenberg, D. 07ell Cetermination during Darly Dmbryogenesis of the +ematode Caenorhab&itis elegans.0 in #olecular %iology of -e*elopment1 Col& +pring arbor +ymposia on =uantitati*e %iology, =ol !, 7old Spring :arbor !aboratory, 7old Spring :arbor, 1OI*. pp. *O45I. :irsh, C. B.T. Bemphues, C.$. Stinchcomb and R. Tefferson. 0Aenes Affecting Darly Cevelopment inCaenorhab&itis elegans.0 #olecular %iology of -e*elopment1 Col& +pring arbor +ymposia on =uantitati*e %iology$ ,ol 5$ Col& +pring arbor 5aboratory$ Col& +pring arbor$ 1>3?. pp. @>;A3.

2.

3.

Sulston, T.D., D. Schierenberg, T. .hite, and +. $homson. 1OI?. 0$he Dmbryonic cell lineage of the nematode Caenorhab&itis elegans0. -e*. %iol. 1''25). Fi/sen, .., <. Sternberg, :. Dllis and R. :orvit&. 0Aenes $hat Affect 7ell Fates during the Cevelopment of Caenorhab&itis elegans0. in #olecular %iology of -e*elopment1 Col& +pring arbor +ymposia on =uantitati*e %iology, =ol !, 7old Spring :arbor !aboratory, 7old Spring :arbor, 1OI*. pp OO41'). Sobota A.D. and <artanen, 7.R. 1O55. 0$he growth and division of cells in relation to morphogenesis in fern gametophytes. #. <hotomorphogenetic studies in Pteri&ium aquilinum0. Cana&. 9our. %ot.))2)O>4*'5. Miller, T.:., and Miller, <.M. 1O51. 0$he effect of different light conditions and sucrose on the growth and development of the fern, "noclea sensibilis0 Amer. 9our. %ot. )I21*)4 1*O. Cavis, %.C. 1O5I. 0Dffect of light 3uality on the transition to two4dimensional growth by gametophytes of Pteri&ium aquilinum0. %ulletin Torrey %ot. Club O*2?14?5. %onner, T.$. 1O)>. 9. E(p. .ool. 1'5,1. %onner, T.$. 1O5>. The Cellular +lime #oul&s. ), <rinceton 1niversity <ress, <rinceton. Cifferentiation in the 7ellular Slime Molds0. 9. E(p. .ool. 1?'21??41*>. 1O**.

4.

5.

6.

7.

8. 9.

10. %onner, T.$., 7hi3uoine, A.C. and Bolderie, M.M. 0A :istochemical study of

11. R.R. Sussman and M. Sussman have the most e/tensive list of publications, much too

long to list. For reference, see Sussman, M. 0%iochemical and Aenetic Methods in the Study of 7ellular Slime Mold Cevelopment.0 #n Methods in 7ell <hysiology. C. <rescott, ed. Academic <ress, +ew Jork, 1O55.
12. Ashworth, T... 1O>). 0$he Cevelopment of the 7ellular Slime Moulds0 in %iochemistry

of Cell -ifferentiation ,T. <aul, ed.-, %iochemistry Series ;ne, =ol. O, M$< #nternational Review of Science. 1niversity <ark <ress ,%utterworths, !ondon-, %altimore, 1O>). pp. >4?).
13. For an e/cellent mathematical treatment of this topic, refer to Segel, !.A. #o&eling

&ynamic phenomena in molecular an& Cellular %iology, 7ambridge 1niversity <ress, 7ambridge, 1OI), pp. O>419O.

14. .ang, Mei and <. Schaap. 07orrelations between $ip Cominance, <restalk8<respore

<atterns and cAM< relay efficiency in Slugs of Cictyostelium discoideum.0 -ifferentiation ?'2>41). 1OI*.
15. T.$. %onner. 0#nduction of Stalk 7ell Cifferentiation by 7yclic AM< in the 7ellular

Slime Mold-ictyostelium &iscoi&eum0. Proc. 4at. Aca&. +ciences. 5*,1- Tan. 1O>'. pp. 11'411?.
16. 1nfortunately there is no e/ternal means of determining gender of the sea urchin.

.hen+trongylocentrotus purpuratus is induced to shed gametes, the eggs that are released will be a yellow color, while the sperm is white. $hus, the gender is determined via the gametes that are released.
17. For assistance in your drawings, e/cellent photographs of early embryology are found in

Mathews, .ills .. Atlas of -escripti*e Embryology, ?rd Dd. Macmillan, +ew Jork, 1OI9.
18. #f long term obsrvation is desired, a special slide needs to be made. <lace masking tape

on a slide, with a hold cut in the middle. <ut a small drop of li3uified +A agar on the slide and place a siliconi&ed coverslip immediately on the agar. .hen the agar solidifies, remove the coverslip. Tust prior to use, place a very small amount of E. coli on the agar, transfer a worm to the agar, and place a coverslip over the agar and the tape. ;nce the worm takes to eating the bacteria, it will settle down and can be readily observed for hours.
19. 1se +ormarski interference optics if available. ;therwise alternate between bright field

and dark field views.

20. For a thorough review of the hematopoietic system, refer to Tun3ueira, !.7., T. 7arneiro,

and A.+. 7ontopoulos. %asic istology. !ange Medical <ublications, !os Altos, 1O>*.
21. Although Bnop@s is more commonly used, the author has used Bnudson@s for many

years. Any minimal plant media with minor and ma"or salts will work. $he critical factor is low p:, with an ideal of about *.*. #f organic elements are added ,such as sucrose- then the media must be sterili&ed to prevent the growth of fungi. Sterility is not re3uired if the organic elements are eliminated, and the fern spores will germinate e/tremely well. #n fact they will germinate reasonably well on the surface of a clay flower pot that is kept moist with plain tap water.

22. <hotomorphogenesis depends on the 3uality of these filters. Many lightly colored filters

allow transmission of significant amounts of light other than the color they appear to be. $o check, cut a small piece of the cellophane and tape it to a spectrophotometer cuvette. Run an absorption spectrum on the cellophane to identify the wavelengths which are transmitted.
23. Alternatively, the agar media can be put in baby food "ars, the spores placed on the agar,

the tops sealed with plastic wrap and held in place by a rubber band. $his system will allow the gametophytes to develop completely over periods greater than 5 months, and will even support the early growth of the sporophyte. %y contrast, the petri plates are useful only for about two weeks.
24. Firtel, R.A. and T.$. %onner. 07haracteri&ation of the genome of the cellular slime

mold -ictyostelium &iscoi&eum.0 9. #ol. %iol. 552??O4?51. 1O>9. $his culture has the advantage of easy administration of cAM< and differentiation can be controlled to some e/tent by the speed with which the amoeba are shaken. Slow speeds allow the amoeba to form aggregates, while higher speeds impede this formation.
25. Further differentiation can be had by 0pulsing0 the cultures with cAM<. #n the normal

aggregation and development of the slime mold, the cells do not continuously secrete cAM<, but secrete it in increasing measured doses.
26. Reymond, et al. Cell ?O21OI). 27. Ratner, C. and .. %orth. 07omparison of Cifferentiating -ictyostelium &iscoi&eum 7ell

$ypes Separated by an #mproved Method of Censity Aradient 7entrifugation0. E(per. Cell 6esearch 1)? ,1OI?- p. 141?.
28. Ratner, C. and %orth .. 1OI9. 07omparison of differentiating -ictyostelium

&iscoi&eum cell types separated by an improved method of density gradient centrifugation.0 E(p. Cell 6es. 1)? 141?. 1OI?.
29. #f the slugs do not readily dissociate, 19.* mM 9,?4dimercaptopropanol and 1 mg8ml

pronase may be added.

Chapter 11: "ucleic $cids - Introduction

Figure 1).1 C+A, R+A and protein structures

$he previous e/ercise dealt with differentiation, and specifically with the events leading to differential gene activity. #n molecular terms, this process involves regulation using C+A as a primer molecule for the selective synthesis of R+A. C+A primed R+A synthesis is termed 0transcription.0 $ranscription is a comple/ series of reactions which involve the use of an R+A polymerase en&yme, known as transcriptase. #f the reaction occurs in reverse, that is with an R+A primer synthesi&ing C+A, the process is known as reverse transcription, and the en&yme is reverse transcriptase. $his latter process is important for R+A virus replication in general, and is most significant when e/amining oncornagenic virus. ;ncornagenic virus are R+A4containing virus that are also causal agents for some forms of cancer ,primarily in birds-. Reverse transcription is also important in the development of amphibians, and in the process of gene amplification.

Exercise 11.1 - Extraction of *"$ fro# Bo(ine 6pleen

!D=D! # Materials

%ovine spleen Saline 7itrate %uffer ,SS77hilled blender Refrigerated preparative centrifuge 9.5 M +a7l O*S ,v8v- ethanol

Procedure
1. .eigh out appro/imately 1* grams of fro&en bovine spleen. Record the e/act weight for

future reference. .eight of the liver KKKKKKKKKKK gm

2. Crop the pieces of spleen one at a time into a chilled blender containing 1*' ml of cold

citrate4saline buffer ,SS7-. 7ontinue to homogeni&e the tissue until it is thoroughly macerated. Co not over4homogeni&e and allow the contents to warm up. <roper procedure should take about ?'45' seconds of blending.
3. <our the homogenate into nalgene centrifuge tubes and centrifuge at ),''' /g for 1*

minutes at )G 7.

4. Cecant the supernatant and discard.

$he supernatant contains most of the materials that are soluble in physiological buffer. R+A, protein and many carbohydrates are found in this portion. $he pellet contains most of the C+A, but it is comple/ed and in the form of C+<. $he pellet also contains any cell debris and unbroken cells resulting from the homogeni&ation.
5. Resuspend the pellet in about 9' ml. of saline8citrate buffer by gently stirring with a glass

rod. #f the pellet is packed hard and will not disperse easily, you may use a vorte/ mi/er to aid in the dispersion.
6. Recentrifuge as in step ?. Ciscard the supernatant. 7. Add 9' ml. of cold 9.5 M +a7l. %reak up the pellet with a glass rod, close the centrifuge

tube with a tight fitting cap and shake vigorously. C+A is soluble in cold +a7l and will also dissociate from the protein. <our off the li3uid portion from this procedure and save for ne/t step. Add another 9' ml. of cold 9.5 M +a7l and shake vigorously. 7ontinue to do this for two more e/tractions. #t is important that the salt be kept cold ,use an ice bathand that the shaking be vigorous. %reaking the pellet with a glass rod may also help.
8. 7ombine all four e/tractions from above and centrifuge at 9',''' /g for 9' minutes. $his

will pellet the insoluble proteins.

9. <our the supernatant carefully into a liter beaker and slowly add 9.? volumes of cold O*S

ethanol allowing it to pour down the side of the beaker and layer on top of the a3ueous supernatant.
10. 7ollect the C+A by gently stirring the mi/ture together.

C+A, if it is highly polymeri&ed, will 0spool0 onto a clean glass rod as the salt solution is mi/ed with the alcohol. #t can then be removed from the solution in the beaker, washed twice with cold >'S ethanol and placed into >'S ethanol or lyophili&ed for storage. "otes C+A spooled by this procedure is impure. %efore it can be used for further analysis, care must be taken to remove contaminating protein, R+A, and carbohydrate. $here are a number of means to accomplish this, most involving either en&yme digestions ,pronase, amylase, and R+Ase- or differential salt solubility or combinations of these techni3ues ,D/ercise 1).9-.

Exercise 11.. - Purification of *"$

!D=D! # Materials

Spooled C+A from D/ercise 1).1 SS7 buffer <ancreatic Ribonuclease A ,1'' Lg8ml<ronase Sodium lauryl sulfate ,S!SSodium perchlorate 7hloroform2isoamyl alcohol ,9)21O*S and >'S ,v8v- ethanol Refrigerated centrifuge, rotor and tubes

Procedure 1
1. Cecant off the alcohol, and dissolve the e/tracted C+A in ?' ml. of diluted SS7 buffer

,'.1 ( SS7- in a 19* ml. erlenmeyer flask. $his will re3uire some time, as polymeri&ed C+A dissolves slowly. Aentle swirling of the material will help.
2. Add pancreatic ribonuclease A to a final concentration of 1'' micrograms8ml and agitate

slowly at ?>G 7 for 1 hour.

3. Add pronase to a final concentration of *' micrograms8ml and again agitate slowly at ?>G

7 for another hour.

4. Add sodium lauryl sulfate ,S!S- 9 to make a 1S concentration ,w8v- and sodium

perchlorate to a final concentration of 1 M. Agitate for ?' minutes at room temperature.

5. D/tract the solution with chloroform2isoamyl alcohol ,9)21 v8v- by adding an e3ual

volume and shaking virogously for at least 1* minutes.

6. <lace the solution into appropriate centrifuge tubes and centrifuge for * minutes at I'' /g

and )G 7.
7. Remove the upper a3ueous phase, add 9.? volumes of O*S cold ethanol and respool the

C+A from this solution onto a glass rod.

8. .ash the spooled C+A twice with cold >'S ethanol and store for future analysis.

Exercise 11.5 - Characteri3ation of dna

!D=D! # Materials

C+A sample SS7 buffer 1= spectrophotometer ? and 3uart& cuvettes

Procedure
1. Cissolve a small 3uantity of your e/tracted C+A in ?.' ml of '.1( SS7. 2. $urn on and blank a 1= spectrophotometer at 99' nm ,use '.1( SS7 as the blank-.

Cetermine the absorbance of your sample C+A at 9?' nm.

3. 7hange the wavelength to 9?' nm, reblank the spectrophotometer and measure the

absorbance of the sample at 9?' nm.

4. #ncrement the wavelength by 1' nm and repeat blanking and measuring the absorbance

until readings are taken through ?'' nm.

5. 7ompute the absorbance ratio 95' nm to 9I' nm. <ure C+A ,without protein or R+A-

will have a 95'29I' absorbance ratio of 1.I*. R+A will have a 95'29I' ratio of 9.'.
6. <lot the absorbance spectrum of your sample and indicate the 95'29I' ratio, as well as

the amount of protein contamination on the graph. Ca(elength 99' 9?' 9)' 9*' 95' 9>' 9I' 9O' ?'' $+sor+ance

Exercise 11.1 - *"$ - *ische *iphen0la#ine *eter#ination

!D=D! # Material

!yophili&ed C+A standard Sample C+A SS7 Cische diphenylamine reagent Spectrophotometer

Procedure
1. .eigh out 1*.' mg of commercial lyophili&ed C+A and prepare a stock solution of ?.'

mg8ml by dissolving the C+A in *.' ml of SS7. $his material will be used to prepare a standard curve for the diphenylamine reaction. +ote that lyophili&ed, highly polymeri&ed C+A is e/tremely slow to go into solution. #t will re3uire preparation at least one day in advance of lab with constant shaking.
2. <repare a series of known standard solutions by serially diluting the stock solution of

C+A. Set up a series of test tubes containing 9.' ml of SS7 each. <ipette 9.' ml of stock solution into tube R1, mi/ and pipette 9.' ml of the resulting mi/ture into tube R9 and so on. $his will yield a series of tubes containing 1.*, '.>* and '.?>* mg8ml of C+A. Jour original stock solution is ?.' mg8ml and SS7 should be used for the blank.
3. Remove and discard 9.' ml of the final dilution. $o each of the five tubes containing in

step 9 ,each should contain only 9.' ml-, add e/actly ).' ml of Cische diphenylamine reagent and mi/ well. This @eagent contains glacial acetic acid. It is caustic and should +e handled &ith care.
4. <lace a marble on the top of each test tube ,it should not fall into the test tube, as it will

act as a reflu/ to prevent evaporation, while allowing for pressure changes-. <lace the tubes in a boiling water bath for 1' minutes, remove from the bath and immediately immerse in an ice bath to cool.
5. $urn on a spectrophotometer and ad"ust the wavelength to 5*' nm. 1se the tube

containing no C+A from step 9 to blank the instrument and measure the absorbance of each of your standards.

<lot the absorbance against C+A concentration, perform a linear regression of the data and compute the e/tinction coefficient.
6. Cissolve your e/tracted or sample C+A in 1' ml of SS7. Make serial dilutions of 181',

181'' and 181''' with SS7. Measure the absorbance of your e/tracted or sample C+A dilutions and calculate the concentration of C+A in the sample. 1se the dilution which gives an absorbance in the '.1 to 1.* range.

Exercise 11.7 - Melting Point *eter#ination

!D=D! ## Materials

C+A SS7 1= spectrophotometer ,preferably with temperature control-

Procedure )
1. Cissolve your C+A preparation in SS7 to give a final concentration of appro/imately 9'

Lg C+A8ml.
2. <lace the dissolved C+A in an appropriate 3uart& cuvette along with a second cuvette

containing SS7 as a blank.

3. <lace both cuvettes into a dual beam temperature regulated 1= spectrophotometer and

measure the absorbance of the sample at 95' nm at temperatures ranging from 9*G 7 to I'G 7. 7ontinue to increase the temperature slowly and continue reading the absorbance until a sharp rise in absorbance is noted. Alternatively2 a. <lace the cuvettes into a waterbath at 9* G 7 and allow to temperature e3uilibrate. Remove the blank, wipe the outside dry and rapidly blank the instrument at 95' nm. $ransfer the sample to the spectrophotometer ,be sure to dry and work rapidly- and read the absorbance. b. Raise the temperature of the bath to *'G 7 and repeat step a. c. Raise the temperature se3uentially to 5'G 7, 5*G 7, >'G 7, >*G 7 and I'G 7 and repeat the absorbance measurements.

d. Slowly raise the temperature above I'G and make absorbance measurements every 9G until the absorbance begins to increase. At that point, increase the temperature, but continue to take readings at 1G 7 intervals.
4. 7orrect all of the absorbance readings for solvent e/pansion relative to 9*G 7. * !ist the

corrected values as A
5. <lot the value of A 8A vs temperature and calculate the midpoint of any increased

absorbance. $his midpoint is the melting point ,$m- for your C+A sample.
6. 7alculate the A7 content of your sample using the formula

<ercent of A W 7 6 k,$m 45O.?- / 9.)) "otes Single strand C+A absorbs more 1= light than double strands. Moreover, double strands can be separated by heat ,melted- and the temperature at which the strands separate ,$m- is related to the number of guanine4cytosine residues ,each having three hydrogen bonds as opposed to the two in adenine4thymine-. $his has led to the development of a rapid test for an appro/imation of the A78A$ ratio using melting points and the change in 1= absorbance ,known as

0hyperchromicity0 or 0hyperchromatic shift0-. ;f course, the separation is also dependent upon environmental influences, particularly the salt concentration of the C+A solution. $o standardi&e this, all $m measurements are made in SS7 buffer. C+A melts between I*G and 1''G 7 in this buffer ,as opposed to 9*G 7 in distilled water-.

Exercise 11.9 - CsCl - *ensit0 6eparation of *"$

!D=D! ## Materials

C+A 7s7l '.? + +a;: '.9 M $ris4:7l buffer, p: >.' 1ltracentrifuge and rotor 1= spectrophotometer and cuvettes

Procedure

1. Cetermine the AW7 content of the sample C+A ,D/ercise 1).*2. ;nce the AW7 content is determined, the bouyant density of the C+A can be determined

from the formula2 p 6 1.55' g8cm W '.'OI / ,AW7 fraction1sing the bouyant density and the 7s7l $able in Appendi/ F, determine the concentration of 7s7l salts to use for dissolution of the C+A.
3. Cissolve appro/imately 1'' micrograms of C+A in ).9 ml of the appropriate 7s7l

solution in '.? + +a;:.

4. !oad the dissolved C+A87s7l solution onto a centrifuge tube suitable for a ).9 ml

sample and speeds of ?'4)',''' R<M ,%eckman S.?O rotor, or e3uivalent-.

5. For the %eckman S.?O rotor, centrifuge the material at ?*,''' R<M for 5* hours at 99G

7.
6. 7ollect the fractions in '.1 ml steps. 7. Add '.9 M $ris4:7l, p: >.' to each fraction and measure the A

for each fraction. #f

available, a continuous flow system using a fraction collecting device may be used.

Exercise 11.< - Phenol Extraction of r@"$ (@at li(er!

!D=D! ## \\\ RDAC $:R;1A: A!! 7A1$#;+S %DF;RD $RJ#+A $:#S D(<DR#MD+$ \\\ Materials

Rat liver ,fasted rat!i3uid nitrogen p4Amino4salicic acid <henol mi/ture :omogeni&er or blender5 Refrigerated preparative centrifuge +a7l O*S and >'S ,v8v- ethanol

Procedure

1. ;btain a rat which has been fasted for 9) hours ,to remove glycogen from the liver-,

decaptitate, e/sanguinate and remove the liver as rapidly as possible.

2. .eigh the liver, being careful not to allow the it to dehydrate. 3. #mmediately drop the liver into a container of li3uid nitrogen.

CAUT7"41 5iqui& nitrogen 0ill cause se*ere frostbiteB

4. 1sing the weight of the liver as an indication of the volume ,1 gm of liver e3uivalent to 1

ml-, add 1* volumes of freshly prepared 5S para4amino4salicylate ,pAS- to a chilled blender or homogeni&er.
5. Add an e3ual volume ,e3ual to the pAS- of phenol mi/ture to the blender and turn on the

blender for a short burst to mi/ the pAS and phenol. CAUT7"41 Phenol is e(tremely caustic. <henol causes severe skin burns, yet it is a local anesthetic. Jou will be unaware of the burn at first, e/cept for tell4tale discoloration of the skin and blisters. Jou will become aware of the burn as the anesthetic properties wear off. <henol also readily dissolves most countertops and all rubber compounds. C5EA4 UP A55 +P755+ 7##E-7ATE5CB 4"T7/C C"U6 74+T6UCT"6 "/ A4C +P755+ A/TE6 C"U A,E T "6"U) 5C 674+E- A4- 8A+ E- A8AC A4C #ATE67A5+ 74 C"4TACT 87T C"U6 +<74.
6. Stop the blender and add the fro&en liver ,handle the liver with long forceps, or tongs-.

%lend the entire mi/ture ,pAS, phenol and liver- for ?' seconds at full speed. Co not blend for longer periods or you will sheer the R+A.
7. 7arefully transfer the homogenate to a beaker and continue to stir the mi/ture for 1'

minutes at room temperature.

8. $ransfer the homogenate to nalgene centrifuge tubes and centrifuge the mi/ture at 1*,5''

/g at )G 7 for 9' minutes.

9. Remove the centrifuge tubes and carefully separate the upper a3ueous layer from the

lower phenol layer. $ake care that none of the white interphase material is mi/ed into the a3ueous layer. $he upper layer can most efficiently be removed by using a large hypodermic e3uipped with a long, large bore, s3uare tipped needle. Should some of the interphase material be stirred into the a3ueous phase, it will be necessary to repeat step I.

10. Measure the volume of the a3ueous layer and discard the phenol layer and interphase

material.
11. Add ?.' grams of +a7l per 1'' ml. of a3ueous phase and stir until dissolved. 12. Add '.* volumes of phenol mi/ture to the a3ueous phase, place into a suitable flask and

shake vigorously for about five minutes. Recentrifuge as in step I above, but for 1' minutes.
13. Separate the a3ueous phase and add 9.? volumes of cold O*S ethanol. Allow the mi/ture

to stand in the free&er until a precipitate forms.

14. 7ollect the R+A precipitate by centrifugation, wash once in >'S ethanol and store in

>'S ethanol at '4*G 7. "otes Bnowledge of transcription is based on our ability to e/tract 0native0 or functional R+A molecules from cells, with subse3uent use of those molecules 0in vitro.0 ;ne of the earliest methods for this type of analysis is a phenol4detergent e/traction of R+A > coupled with separation of the various si&ed molecules of R+A with centrifugation in a gradient. $his basic procedure remains useful today, although there have been myriad additions and alterations to the procedure using a host of e/traction techni3ues and separation procedures ,such as electrophoresis or column chromatography-. For the purposes of introduction to the techni3ue, this e/ercise e/tracts R+A from rat liver using a phenol e/traction which yields predominantly rR+A and tR+A. $here is some mR+A present, but it is variable and should be considered as a background contaminant. $here is also a good portion of sR+A caused by sheering of the R+A during homogeni&ation, and by en&ymatic digestion by R+Aase during the e/traction.

Exercise 11.= - 6pectrophoto#etric $nal0sis of r@"$

!D=D! # Materials

R+A 1= spectrophotometer and cuvettes Alkaline distilled water

Procedure

1. Cissolve 1' mg of commercial R+A in 9*' ml. of slightly alkaline distilled water. 1se a

volumetric flask and proper analytical techni3ue. $his will give a standard solution of )' micrograms R+A8ml.
2. <repare a series of dilutions so that you have )', 9', 1', * and 9.* micrograms of R+A

per ml.
3. $urn on a 1= spectrophotometer and ad"ust the wavelength to 95' nm. 1se the alkaline

water to blank the spectrophotometer at 95' nm.

4. Read the A

of each of the standards. <lot the A

vs the concentration of R+A and

calculate the e/tinction coefficient.

5. Cissolve your isolated, precipitated R+A in 1'.' ml of alkaline water. <repare a serial

dilution for 181', 181'', 181''' and 181''''. Measure the absorbance of each at 95' nm and, using the dilution which gives a reading between .1 and 1.* absorbance units, compute the concentration of R+A in your sample.

Exercise 11.> - /rcinol *eter#ination of @"$

!D=D! # Materials

R+A Alkaline distilled water Acid4orcinol reagent %oiling water bath Spectrophotometer and cuvettes

Procedure
1. <repare a series of serially diluted R+A standards as in D/ercise 1).I, but having a range

from 1.' mg8ml down to '.19* mg8ml.

2. <repare a serial dilution of your sample R+A as in D/ercise 1).I. 3. <lace ?.' ml of each standard and ?.' ml of each serial dilution of the sample R+A into

separate test tubes. <lace ?.' ml of alkaline water in a separate tube.

4. Add ?.' ml of acid4orcinol reagent to each tube and mi/ well. 5. Add '.? ml of alcohol4orcinol reagent to each tube and mi/ well.

6. <lace the tubes in a boiling water bath for 9' minutes with marbles placed on top of each

tube to prevent evaporation. 7ool the tubes by immersion in an ice bath at the end of the 9' minute period.
7. $urn on a spectrophotometer and ad"ust the wavelength to 55' nm. %lank the

spectrophotometer with the alkaline water8orcinol tube. Measure the A remaining standards and diluted samples.

of each of the

8. <lot the absorbance of the standards against the known concentrations. 7alculate the

e/tinction coefficient, and calculate the concentration of R+A in your sample. 1se the dilution yielding an absorbance between '.1 and 1.* absorbance units.

Exercise 11.1, - 6ucrose *ensit0 Fractionation

!D=D! ##

Figure 1).9 Sucrose gradient distribution of R+A Materials

1'S and )'S ,w8v- Sucrose I '.'9 M sodium acetate, p: *.1, containing '.1 M +a7l and 1 mM DC$A R+A 1ltracentrifuge, swinging bucket rotor and tubes 7entrifuge tube fractionating device 1= spectrophotometer

Procedure

1. Refer to 7hapter $hree for details of fractionation, and form a 1'4)'S linear sucrose

gradient in a nitrocellulose tube. a. 7lose all valves on the gradient device. b. <lace e/actly 1* ml.O of )'S sucrose in the right chamber and 1* ml. of 1'S sucrose in the left chamber of the gradient device. c. ;pen the flow from the right chamber to the centrifuge tube. %e sure there is a tube in place, that the flow is directed down the inside of the tube and that the magnetic stirrer is functioning. d. #mmediately open the valve on the left chamber and insure that sucrose is flowing from left to right, thereby diluting the )'S sucrose with 1'S sucrose. e. Allow the flow to continue until all of the sucrose enters the centrifuge tube.
2. Cissolve the R+A in '.'9 M sodium acetate solution to yield a final concentration of 9*'

Lg8ml. $he low p: of the solution helps to inhibit R+Ase, while the salts will keep the R+A from forming large polymeric aggregates. 1'
3. 7arefully layer 9.' ml of the dissolved R+A onto the top of a sucrose gradient. $his

should be done by slowly allowing the solution to run down the side of the tube and onto the gradient. %e careful not to disturb the gradient.
4. !oad the ultracentrifuge with the prepared tubes and centrifuge for the e3uivalent of

1I,>'' R<M for a %eckman S.9> rotor, for 9' hours at )G 7 ,Refer to Appendi/ F-.
5. At the completion of centrifugation, remove the tubes and fractionate the contents into

1.' ml fractions. a. #nsert the nitrocellulose centrifuge tube into the ple/iglass holder, but be careful +;$ to puncture the bottom. b. :ave a series of ?'4?* test tubes ready to accept the effluent from the tubes. Dach tube should be labeled and kept in order. c. <ush down on the centrifuge tube ,AentlyN- in order to puncture the bottom of the tube and immediately begin to collect the effluent. d. 7ount the drops that fall from the device, and place )' drops into each tube.
6. 1sing a 1= spectrophotometer 11 and microcuvettes, read the A

of each fraction.

7alculate the amount of R+A in each fraction.

7. <lot the concentration of R+A in each fraction against the fraction number. %ased on the

density of sucrose in each fraction, compute the density of the R+A in that fraction. %ased on the relative si&e ,greater density- of the R+A, determine the nature of the fractions ,i.e. rR+A, tR+A-.

Exercise 11.11 - "ucleotide Co#position of @"$

!D=D! ## Materials

R+A sample 1 + and '.1 + :7l %oiling water bath .hatman R1 filter paper ,for chromatography7hromatography tank 9' Ll micropipette Acetic acid2 butanol2 water ,1*25'29*- solvent 1= light source 1= spectrophotometer

Procedure
1. <lace a portion of your R+A sample ,appro/imately )' mg, hydrated- into a heavy

walled pyre/ test tube. Add 1.' ml of 1 + :7l and seal the tube.
2. :eat the tube in a boiling water bath for 1 hour. 3. 7ool the tube, open it and place the contents into a centrifuge tube. 7entrifuge the

contents at 9,''' R<M in a clinical centrifuge to remove any insoluble residue. $he supernatant contains your hydroly&ed R+A.
4. <repare .hatman filter paper +o. 1 for standard one4 dimensional chromatography. 19 5. 1sing a micropipette, spot 9' Ll of your hydroly&ate onto the paper, being careful to keep

the spots as small as possible ,repeated small drops are better than one large drop-. Allow the spots to completely dry before proceeding.
6. <lace the paper chromatogram into your chromatography tank and add the solvent ,acetic

acid2butanol2water-. Allow the system to function for an appropriate time ,appro/imately

?5 hours for a 9' cm descending strip of .hatman R1-. Remove the paper and dry it in a circulating air oven at )'G 7 for about 9 hours.
7. !ocate the spots of nucleotides by their fluorescence under an ultra4violet light source.

D/pose the paper chromatogram to a 1= light source and outline the spots using a light pencil. $he order of migration from the point of origin is guanine ,light blue fluorescence-, adenine, cytilic acid and finally, uridylic acid. *o not loo4 directl0 at the %: light source. %se a ca+inet designed to shield fro# har#ful %: radiation.
8. After cafefully marking the spots, cut them out with scissors and place the paper cut outs

into separately labeled 1* ml conical centrifuge tubes. Add *.' ml of '.1 + :7l to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.
9. <ack down the paper with a glass rod ,centrifuge in a clinical centrifuge if necessary- and

remove an ali3uot of the li3uid for spectrophotometric assay.

10. Measure the absorbance of each of the four nucleotides at the indicated 1= wavelength

,having first blanked the instrument with '.1 + :7l-. Base Auanine Adenine 7ytidylic acid 1ridylic acid Ca(elength 9*' nm 95' nm 9I' nm 95' nm Molar Extinction Coefficient 1'.5 1?.' 1O.O* O.IO

11. 1se the molar e/tinction coefficients to determine the concentration of each base in the

sample. 7alculate the percent composition of each base, and the purine8pyrimidine ratio.

Exercise 11.1. - /ther studies

!D=D! ### ;nce C+A has been purified and analy&ed, there are many procedures that can be followed. :ydro/yapatite columns can easily separate single stranded C+A from double stranded C+A and are used e/tensively in gene transplant work. $his can be appended to the formation of C+A4R+A hybrids, whereby an mR+A is re4annealed to C+A, all single strands eliminated and

the resulted hybrid re4separated. $he remaining C+A strand is the gene for that mR+A ,although not complete, some start, stop and housekeeping codes will be missing-. #f one has a sufficient 3uantity of R+A@s, it is thus possible to create a C+A 0library0 for these genes. Modern approaches will use the mR+A to synthesi&e C+A rather than isolating it through hybridi&ion. C+A synthesi&ed from R+A is known as cC+A, which presents us with powerful 0libraries0 for analysis of cell function at the gene level. ;nce obtained, the C+A can be readily se3uenced, although the techni3ues are too lengthy for our current analysis in a general cell biology laboratory. Aene se3uencing has now been automated, and the key to success is the isolation of pure uni3ue copy C+A ,that is C+A that has only one se3uence to analy&e-. ;nce se3uenced, computer searches can be made of known libraries to compare the gene with that from other organisms. For an e/cellent series of se3uencing techni3ues cf. #etho&s of -4A an& 64A +equencing, ed. S.M. .eissman. <raeger <ublishers, +ew Jork. 1OI?, or Current Procols in #"lecular %iology edited by F.M Ausubel, R. %rent, R.D. Bingston, C.C. Moore, T.A. Smith, T.A. Seidman and B.Struhl. Areene <ublishing Associates, .iley #nterscience, %rooklyn, +J, 1OII. Further analyses of R+A are also possible. Affinity chromatography allows the separation of mR+A, which can then be analy&ed for specific base se3uence. $his mR+A could be used for translation on isolated ribosomes, or used to synthetically produced C+A copies ,cC+A-.

Chapter 11: "ucleic $cids - Endnotes

1.

Modification of techni3ue of Marmur ,1O51- as given in %iochemical #etho&s in Cell Culture an& ,irology by Robert T. Buchler. Cowden, :utchinson and Ross, #nc., Stroudsburg, 1O>>. A scanning spectrophotometer may be used, if available. $he mathematics for this correction can be found in 7. Mandel, M., and T. Marmur. 1O5I. 01se of ultraviolet absorbance temperature profile for determining the guanine plus cytosine content of C+A.0 #n#etho&s in En!ymology, =ol. S## ,!. Arossman and B. Moldave, eds.-, pp. 1O*49'5. Academic <ress, #nc., +ew Jork. Birby and <arish, %iochem 9 O5,955. 1O5*.

2. 3.

4.

5.

Cirections for for use of a %eckman S.9> rotor with a capacity of ?) ml. =olumes must be ad"usted according to the capacity of the rotor employed.

6. The Cell 4ucleus D .%usch$ e&.E$ pp 1?2;223. Aca&emic Press$ 7nc. 4e0 Cor:. 7.

There are many 0ays to accomplish this$ but the simplest is to use a &escen&ing strip of paper in a chromatography tan:. This can be constructe& from rea&ily a*ailable pyre( house0are$ glass ro&s an& a bell Far$ or by using commercial units. Alternati*ely$ a simple ascen&ing system can be ma&e from large testtubes.

Chapter 17: The Central *og#a - Introduction

$he 7entral Cogma of modern biology is the conversion of the genetic message in C+A to a functional mR+A ,transcription- and subse3uent conversion of the copied genotype to a phenotype in the form of proteins. $he process of conversion of a mR+A to a functional protein is known as translation. #t involves the attachment of a messenger R+A to the smaller subunit of a ribosome, the addition of the larger subunit, plus initiation by a host of other factors. $he entire process can be accomplished in the absence of a cell, if all of the necessary factors are present. 1 1nfortunately, studies on translation and post4translational changes in protein structure are rather comple/. $hey re3uire a heavy investment in time and e3uipment. $o some e/tent, the electrophoretic identification of proteins is part of this process, and the appearance of specific proteins can be monitored during any of the developmental processes identified in 7hapter $hirteen. 7hapter Fourteen lists several means of studying both the C+A and R+A contents of cells during changes. $o study the process of translation in any meaningful way re3uires that reasonably purified sources of mR+A, ribosomes and all of the amino acids be available. #n addition, there is a re3uirement for various factors responsible for peptide chain initiation on the ribosome. 7onse3uently this chapter will deal with only one e/ercise. #t is definitely an advanced techni3ue, and re3uires mastery of many of the techni3ues presented in previous chapters. #t must be performed on an independent basis, since the e/tensive time commitment does not lend itself to typical laboratory periods.

Exercise 17.1 - Protein 60ntheses in Cell Free 60ste#s

!D=D! ###

Materials

Suspension culture of fibroblast cells ,1 liter?* mM $ris4:7l, p: >.), 1)' mM +a7l ,$%S buffer1' mM $ris4:7l, p: >.*, 1' mM B7l, and 1.* mM magnesium acetate ,$%S4M1'( $%S4M 9'' mM $ris4:7l, p: >.*, 19'' mM B7l, *' mM magnesium acetate and >' mM 4mercaptoethanol

1'( solution of 9' amino acids $eflon homogeni&er Refrigerated preparative centrifuge Sat. ,+: - S; $%S4M plus 9'S ,v8v- glycerol 1( $%S4M buffer containing '.9* M sucrose 1( $%S4M buffer containing 1.' M sucrose Sephade/ A49* column e3uilibrated with 1( $%S4M buffer !i3uid nitrogen storage Reaction mi/ture for protein synthesis, containing the following in a total volume of *' Ll 9 $ris4:7l, p: >.* Mg acetate B7! 4Mercaptoethanol A$< A$< 7reatine phosphate 7reatine kinase Dach of 1O amino acids,4leucine1.* Lm '.1* 4 '.9' Lm ).' 4 *.' Lm '.9* Lm '.'* Lm '.''* Lm '.*' Lm I.' Lg 9.' nmol '.19* L7i

74leucine ,1*' m7i8mmolRibosome fraction 1 to 9 A units =iral mR+A or Alobin OS mR+A 9.' to *.' Lg or

<oly 1? Procedure )

1'.' Lg

1. 7hill the suspension culture ,app. 1' cells- rapidly in an ice bath. 7ollect the cells as a

pellet, by centrifugation at 5'' /g for 1' minutes at )G 7. Resuspend the cells in $%S buffer and wash them three times with cold $%S buffer.
2. Suspend the final pellet in two volumes of $%S4M for * minutes at 'G 7 and homogeni&e

the cells with 1' to 9' strokes in a tight fitting $eflon homogeni&er.
3. For each '.O ml of homogenate, add '.1 ml of concentrated 1'( $%S4M buffer.

7entrifuge the mi/ture at 1',''' /g for 1' minutes at )G 7.

4. Cecant and collect the supernatant e/tract and ad"ust the e/tract such that the following

are added to yield final concentration2 A$< to 1.' mM A$< A$< to '.1 mM A$< 7reatine phosphate to 1' mM 7reatine kinase to 15' Lg8ml Amino acids to )' Lm each
5. #ncubate the mi/ture for )* minutes at ?>G 7. 6. 7entrifuge the mi/ture at 1',''' /g for 1' minutes at room temperature. 7ool the

supernatant and pass the supernatant through a Sephade/ A49* column at )G 7.

7. $urn on a 1= spectrophotometer and ad"ust the wavelength to 95' nm. %lank the

instrument with $%S buffer.

8. 7entrifuge the filtrate e/cluded from the Sephade/ column at 15*,''' /g for O' minutes

at )G 7.
9. <recipitate the proteins within the supernatant by the addition of saturated ,+: - S; to

yield a final 5'S ,+: - S; . 7ollect the precipitate by centrifugation.

10. Cissolve the precipitate in $%S4M buffer and dialy&e it against the same buffer

containing glycerol.

11. Suspend the resulting ribosome pellet in 1( $%S4M buffer containing '.9* M sucrose.

<lace * ml of $%S buffer with 1.' M sucrose into the bottom of a centrifuge tube and layer the suspended ribosomes on top. 7entrifuge at 915,''' /g for 9.* hours at )G 7.
12. .ash the resulting pellet with $%S4M buffer, and resuspend it in the same buffer with

'.9* M sucrose.
13. Cetermine the ribosome concentration using a 1= spectrophotometer to measure the A

. $he e/tinction coefficient for ribosomes is 19 A units per mg per ml at 95' nm.
14. $he ribosomes may be fro&en and stored in li3uid nitrogen, or used for in *itro protein

synthesis. #f fro&en, they should be thawed only once prior to use. $o test for protein synthesis, prepare the reaction mi/ture for protein syntheis.
15. #ncubate the reaction mi/ture at ?>G 7 for 5' minutes. $erminate the reaction by pipetting

)' Ll of the mi/ture onto a 9.* cm disk of .hatman ?MM filter paper. Cip the disk into cold 1'S $7A for 1* minutes and then in *S $7A at O'G 7 for 1* minutes.
16. Rinse the disk twice in *S $7A for * minutes, once in alcohol2ether ,121-, and then dry

it.
17. <lace the disk into a scintillation vial and add a toluene4based fluor. Refer to Appendi/

: for details on the use of a scintillation counter.

18. Measure the amount of radioactively labeled amino acid incorporated into protein. 19. Araph the protein synthesi&ed versus time.

/ptional For advanced work, compare the activity of ribosomes isolated from the fibroblast cultures to those isolated from a prokaryote culture, a plant ,yeast or pea seedlings- and from genetic L tants known to alter the structure of either rR+A or any of the ribosome structural proteins. #f sources of mR+A are available ,or if the time is taken to e/tract R+A and electrophoretically purify it-, then comparisons of the rate of protein synthesis can be made within this system. Finally, once the proteins are synthesi&ed, they can be separated on SCS4<AAD and rates of synthesis can be determined by both time of incorporation of leucine and by increasing molecular weights. $he latter will also demonstrate the possible presence of L ltiple R+A@s with the system, whereas the radioactivity will mask the presence of heterogenous R+A. Chapter 1,: Chro#oso#es' Endnotes

1.

For an e/cellent complete laboratory procedure on the regulation of translation during the development ofTenebrio refer to2 0Regulation of Messenger R+A $ranslation in Cevelopment2 $he critical Role of $ransfer R+A0 by Toseph #lan. An Addison4.e sley Module in %iology. +o. *. Addison4.esley <ublishing 7ompany, #nc. 1O>?. Falcoff, D., R. Falcoff, %. !ebleu, and M. Revel. 1O>?. 7orrelation between the antiviral effect of interferon treatment and the inhibition of in vitro mR+A translation in noninfected ! cells. 9 ,irol. 192)914)?'.

2.

3.

.hen poly 1 is used, '.19* L7i of 74phenylalanine is used instead of the amino acids, and the magnesium acetate concentration is increase d to '.9* Lm. Buehler, Robert T. %iochemical #etho&s in Cell Culture an& ,irology. Cowden, :utchinson U Ross, #nc. Sroudsburg, 1O>>. pp. ?'*4?'>.

4.

$ppendix $: %nits and Measures

6IME 7ell biology deals with things which are relatively small. $he units of measurement typically used are the micron at the light microscope level, and the nanometer at the electron microscope level. For molecular measurements, the norm is the Angstrom. $hese units are defined within the following table2 Measure Meter Cecimeter 7entimeter Millimeter Symbol Relative !ength D/ponential +otation M dm cm mm 1 .1 .'1 .''1 .'''''1 .''''''''1 1' 1' 1' 1' 1' 1'

Micrometer or micron L +anometer nm

Angstrom From this table it is apparent that2 1' E 6 1 nm 1''' nm 6 1 mm 1' mm 6 1 cm +ot apparent are that2

.'''''''''1

1'

1 inch 6 9.*) cm 6 9*.) mm 6 9*,)'' L 6 9*,)'',''' nm 1 inch 6 9.*) cm 6 9.*) ( 1' mm 6 9.*) ( 1' L 6 9.*) ( 1' nm 1 mm 6 '.') inches

Figure A.1. Si&e and resolution :/B%ME =olumes are measured relative to a liter, with the most commonly used measurements, the milliliter and the microliter. $he following table gives the relative volumes2 Measure !iter Ceciliter Symbol Relative =olume D/ponential +otation ! dl 1 .1 .''1 .'''''1 1' 1' 1' 1'

Millimeter ml Microliter Ll

$here are 1,''' L l in 1 ml. 1 ml 6 1 cm 1 gallon 6 ?.I liters

1 3uart 6 '.O* liters 1 li3uid ounce 6 9O.5 ml CEIA?T $he most common measurements of weight at the gram, milligram and microgram. Measure Bilogram Aram Milligram Symbol Relative .eight D/ponential +otation Bg g mg 1''' 1 .'''1 .''''''1 1' 1' 1' 1'

Microgram L g C/"CE"T@$TI/"

Most concentrations used throughout cell biology are those of a solute disolved or suspended within a solvent, and in most cases the solvent is water. $here are two general methods of identifying the concentration of a solution as molarity or as a percent. Molarity is based on the number of moles of solute in the solvent, while percent is based on the number of parts , either grams ,for a solid solute- or milliliters ,for a li3uid solute-. Molarity e3uals the number of moles of solute in 1 liter of solution. A mole is e3ual to the gram molecular weight ,or formula weight- of the solute. Sodium 7hloride ,+a7l-, for e/ample has a formula weight of *I.)? ,99.OI for +a and ?*.)? for 7l-. $hus, if *I.)? grams of +a7l are dissolved in 1 liter of water, the result would e3ual a 1 molar solution of +a7l. $his is designated as 1 M +a7l, or as simple M +a7l. .e often deal with solutions of less volume than 1 liter, and the following should be noted2 1 M +a7l 6 *I.)? grams 8 liter 6 *I.)? mg8ml 6 *I.)? L 1 g8L 1 l. A '.''9 M +a7l solution contains '.''9 moles of +a7l or '.115I grams ,'.''9 / *I.)?- in one liter of solvent. +ote that molar is abbreviated as M, but that there is no abbreviation for moles.

$he '. ''9 M solution contains '.''9 moles ,or 9 millimoles- or solute in one liter. A '.''9 M solution would contain '.''1 moles of solute in a half liter. $he number of moles 6 =olume ,in liters- / Molar 7oncentration $he number of millimoles 6 =olume ,in ml- / Molar 7oncentration +ote that chemical e3uations are always balanced via moles. Moreover, note that for dilutions of known concentrations, one can use the simple formula2 Molarity / =olume 6 Molarity / =olume #f you have a '.''9 M solution of +a7l and you wish to obtain 1'' ml. of a '.''1 M solution, '.''9 M / +eeded =olume 6 '.''1 M / 1'' ml +eeded =olume 6 '.''1 M / .1 liters 8 '.''9 M 6 .'*' liters6 *' ml. Measure *' ml of the '.''9 M solution, and dilute it to 1'' ml with the solvent ,usually water, or an appropriate buffer-. Molarity is appropriate for use when chemical e3uations are to be balanced. .hen we deal with physical properties of solutions, molarity is not as valuable as a similar measurement of concentration, molality. For colligative properties of solutions ,free&ing point depression, boiling point elevation, osmotic pressure, density, viscosity-, there is a better correlation between the property and molality. Molality ,designated with a lower case m- is e3ual to the number of moles of solute in 1''' gm of solvent. At first this may not appear any different from molarity, since a ml of water e3uals 1.' gm. #ndeed, for dilute solutions in water, there is little or no practical difference between a molar solution and a molal solution. #n concentrated solutions, with temperature fluctuations and with changes in solvent, there is appreciable difference. For e/ample2 A 9 m ,9 molal- solution of sucrose contains 5I).) gm of sucrose ,twice the molecular weight or two moles of sucrose- dissolved in 1''' gm ,appro/imately 1 liter- of water. $he weight of this solution is 5I).) gm W 1''' gm or 15I).) gm. $his solution ,9 m sucrose- has a density of 1.1I gm8ml or 11I' gm8liter. Since there are 15I).) gms, division by the density ,11I' gm8liter- would indicate that there are 1.)? liters of solution. $hat is, 5I).) gm of sucrose dissolved in 1''' gm of water would yield

1.)? liters of solution. $his solution would contain 9 moles of sucrose, however and would have a molarity 6 9 moles81.)? liters or 1.)' M. So, a 9 m sucrose solution e3uals a 1.) M sucrose solution. PE@CE"T 6/B%TI/"6 #n the e/ample above of 9 m sucrose, there were 5I).) gm of sucrose in the final solution which weighed 15I).) gm ,5I).) gm sucrose W 1''' gm water-. $he percent of sucrose on the basis of weight is therefore 5I).)815I).) / 1'', or )'.5S. $here are three means of e/pressing concentration in the form of a percent figure2
1. <ercent by weight ,w8w- gm solute 8 1'' gm solvent 2. <ercent weight by volume ,w8v- gm solute 81'' ml solvent 3. <ercent by volume ,v8v- ml solute 8 1'' ml solution

For dilute solutions, these differences are not significant, but at higher concentrations, they are. 7hemists ,when they use <ercent designations- usually use w8w. %iochemists and physiologists more often use w8v. %oth use v8v if the solute is a li3uid. #t is important to distinguish among these alternatives. 1sing ethanol as an e/ample, consider a 9'S solution of ethanol in water, mi/ed according to the three designations of w8w, w8v and v8v.
1. w8w would contain 9' g of absolute ethanol mi/ed with I' gm of water to yield a 9'S

,w8w- solution.
2. w8v would contain 9' g of absolute ethanol mi/ed with water to form a final volume of

1'' ml.
3. v8v would contain 9' ml of absolute ethanol diluted to 1'' ml with water.

$he three solutions are not the same. First, the density of alcohol is not e3ual to that of water, and thus conversion of g to ml is not e3uivalent. A 9'S ,w8w- solution of ethanol, for e/ample, has a density of '.O> g8ml and 9' gm of ethanol plus I' gm of water would have a volume of 1'? ml. $he S ,w8v- for this solution would be 9' gm ethanol 8 1'? ml, or 1O.)S ,w8v-. Similarly, absolute ethanol has a density of '.>O gm8ml and thus 9' ml of ethanol would weigh 1*.I gm. A 9'S ,v8v- solution would contain 1*.I gm of ethanol in 1'' ml and be a 1*.IS ,w8v- solution. So, for ethanol2

9'S ,w8w- 6 1O.) S ,w8v9'S ,w8v- 6 9'.' S ,w8v9'S ,v8v- 6 1*.I S ,w8v#n cell biology, the most common use of <ercent solution is as ,w8v-. #n practice, these are simple solutions to mi/. For a 9'S ,w8v- sucrose solution, for e/ample, simply weigh 9' gm of sucrose and dissolve to 1'' ml with water. 1nless specifically stated otherwise, solutions lacking the appropriate designation should be assumed to be ,w8v-.

$ppendix B: 6tatistics
Statistical manipulation is often necessary to order, define and8or organi&e raw data. A full analysis of statistics is beyond the scope of this work, but there are some standard analyses that anyone working in a cell biology laboratory should be aware of, and know how to perform. After data is collected, it must be ordered, or grouped according to the information which is to be sought. Cata is collected in the forms2 $ype of Cata $ype of Dntry +ominal ;rdinate +umerical yes or no W, WW, WWW ', 1, 1.?, etc.

.hen collected, the data may appear to be a mere collection of numbers, with little apparent trends. #t is first necessary to order those numbers. ;ne method is to count the times a number falls within a range increment. For e/ample, in tossing a coin, one would count the number of :eads and $ails ,eliminating the possibility of it landing on its edge-. 7oin flipping is nominal data, and thus would only have two alternatives. Should we flip the coin 1'' times, we could count the number of times it lands :eads and the number of $ails. .e would thus accumulate data relative to the categories available. A simple table of the grouping would be known as a fre3uency distribution , for e/ample2 7oin Face Fre3uency

:eads $ails $otal

)* ** 1''

Similarly, if we e/amine the following numbers ?,*,),9,*,5,9,),), several things are apparent. First, the data needs to be grouped and the first task is to establish an increment for the categories. !et us group the data according to integers, with no rounding of decimals. .e can construct a table which groups the data. #nteger Fre3uency 1 9 ? ) * 5 $otals $his data can be plotted as follows2 Figure %.1. <lot of fre3uency distribution ME$"' ME*I$" $"* M/*E From the data, we can now define and compute three important parameters of statistics. Cefinition ' 9 1 ? 9 1 O $otal ,#nteger / Fre3uency' ) ? 19 1' 5 ?1

$he mean2 $he average of all the values obtained. #t is computed by the sum of all of the values , /-, divided by the number of values ,n-. $he sum of all numbers is ?1, while there are O values, thus, the mean is ?.)). K M 6 44444444 n Cefinition $he median2 $he mid point in an arrangement of the categories by magnitude. $hus, the low for our data is 9, while the high is 5. $he middle of this range is ). $he median is ). #t represents the middle of the possible range of categories. Cefinition $he mode2 $he category that occurs with the highest fre3uency. For our data, the mode is e3ual to ), since it occurs more often than any other value. $hese values can now be used to characteri&e distribution patterns of data. For our coin flipping, the likelihood of a :ead or a $ail is e3ual. Another way of saying this is that there is e3ual probability of obtaining a :ead or not obtaining a :ead with each flip of the coin. .hen the situation e/ists that there is e3ual probability for an event as for the opposite event, the data will be graphed as a binomial distrution, and a +ormal curve will result. #f the coin is flipped ten times, the probability of one head and nine tails e3uals the probability of nine heads and one tail. $he probability of two heads and eight tails e3uals the probability of eight heads and two tails and so on. :owever, the probability of the latter ,two heads- is greater than the probability of the former ,one head-. $he most likely arrangement is five heads and five tails. .hen random data is arranged and displays a binomial distribution, a plot of fre3uency vs. occurency will result in a normal distribution curve . For an ideal set of data ,i.e. no tricks, such as two headed coin, or gum on the edge of the coin-, the data will be distributed in a bell shaped curve, where the median, mode and mean are e3ual. /

$his does not give an accurate indication of the deviation of the data, and in particular does not inform us of the degree of dispersion of the data about the mean. $he measure of the dispersion of data is known as the Standard Ceviation . #t is given mathematically by the formula2 K ,M 4 (S 6 s3rt,4444444444n41 $his value gives a measure of the variability of the data, and in particular, how it varies from an ideal set of data generated by a random binomial distribution. #n other words, how different is it from an ideal +ormal Cistribution. $he more variable the data, the higher the value of the standard deviation. ;ther measures of variability are the range ,difference between minimum and ma/imum values-, the 7oefficient of =ariation ,Standard Ceviation divided by the Mean and e/pressed as a <ercent- and the =ariance. $he variance is the deviation of several or all values from the mean and must be calculated relative to the total number of values. =ariance can be calculated from the formula2 K ,M 4 (= 6 4444444444 n41 All of these calculated parameters are for a single set of data that conforms to a normal distribution. 1nfortunately, biological data does not always conform in this way, and often sets of data must be compared. #f the data does not fit a binomial distribution, often it fits a skewed plot known as a <oisson distribution . $his distribution occurs when the probability of an event is so low, that the probability of its not occurring approaches 1. .hile this is a signficant statistical event in biology, details of the <oisson Cistribution are left to te/ts on biological statistics. !ikewise, the proper handling of comparisons of multiple sets of data. Suffice it indicate that all statistics comparing multiple sets begin with calculation of the parameters detailed here, and for each set of data. For e/ample, the standard error of the mean ,also known simply as the standard error- is often used to measure distinctions among populations. #t is defined as the standard

deviation of a distribution of means. $hus, the mean for each population is computed and the collection of means are then used to calculate a standard deviation of those means. ;nce all of these parameters are calculated, the general aim of statistical analysis is to estimate the significance of the data, and in particular the probability that the data represents effects of e/perimental treatment, or conversely, pure random distribution. $ests of significance ,Student@s t $est, Analysis of =ariance and 7onfidence !imits- will also be left to more e/tensive treatment in other volumes.

$ppendix C: Araphs
<resentation of data in an orderly manner often calls for a graphic display. $his is made somewhat easier today, with the advent of graphics programs for the personal computer, but still re3uires the application of some basic techni3ues. $he first consideration for a graph, is whether the graph is needed, and if so, the type of graph to be used. For accuracy, a well constructed table of data usually gives more information than a graph. $he values obtained and their variability are readily apparent in a table, and interpolation ,reading the graph- is unnecessary. For visual impact, however, nothing is better than a graphic display. $here are a variety of graph types to be chosen from e.g. line graphs, bar graphs and pie graphs. Dach of these has its own characteristics and subdivisions. ;ne also has to decide upon singular or multiple graphs, two4dimensional or three dimensional displays, presence or absence of error bars, and the aesthetics of the display. $he latter include such details as legend bars, a/es labels, titles and selection of the symbols to represent data, and patterns for bar graphs. Selection of a good Araphic <rogram for computer use will make most of these choices available. Refer toAppendi/ C for a 3uick review of such programs. T?E B$6IC6: <erhaps the number one rule for graphic display has to do with the a/es. Aiven a two4 dimensional graph, with two values ,/ and y-, which value is / and which is yP $he answer is always the same 4 the B+;.+ value is always the ordinate ,/- value. $he value that is MDAS1RDC is the abscissa ,y- value. For a standard curve of absorption in spectrophotometry, the known concentrations of the standards are placed on the / a/is, while the measured

absorbance would be on the y. For measurements of the diameter of cells, the / a/is would be a micron scale, while the y a/is would be the number of cells having a given diameter. 1nless you are specifically attempting to demonstrate an inverted function ,which is confusing at best-, the scales should always be arranged with the lowest value on the left of the / a/is, and the lowest value at the bottom of the y value. $he range of each scale should be determined by the lowest and higest value of your data, with the scale rounded to the nearest tenth, hundredth, thousandth, etc. $hat is, if the data ranges from 19 to O?, the scale should be from 1' to 1''. #t is not necessary to always range from ', unless you wish to demonstrate the relationship of the data to this value ,as for e/ample in a !ineweaver4%urke plot of en&yme kinetics, or a Spectrophotometric standard curve-. $he number of integrals placed on the graph will be determined by the point you wish to make, but in general, one should use about ten divisions of the scale. For our range of 19 to O?, an appropriate scale would be from ' to 1'', with an integral of 1'. <lacing smaller integrals on the scale does not convey more information, but merely adds a lot of confusing marks to the graph. $he user can estimate the values of 19 and O? from such a scale without having every possible value ticked off on a scale. An important rule of scale deals with multiple graphs drawn separately. #f the graphs are to be compared, the scales M1S$ RDMA#+ 7;+S$A+$. +othing is more disconcerting than to be shown two graphs with varying a/is values and being asked to compare the two. #t would be better to merely tabulate the data than to graphically present it. BI"E A@$P? :6. B$@ A@$P? /@ PIE A@$P? #f the presentation is to highlight various data as a percent of the total data, then a pie graph is ideal. <ie graphs might be used for e/ample to demonstrate the composition of the white cell differential count. $hey are the most often used graph type for business use, particularly in displaying budget details. <ie Araphs are circular presentations which are drawn by summing your data and computing the percent of the total for each data entry. $hese percent values are then converted to portions of a circle ,by multiplying the percent by ?5' G - and drawing the appropriate arc of a circle to represent the percent. %y connecting the arc to the center point of the circle, the pie is divided into wedges, the si&e of which demonstrate the relative si&e of the data to the total. #f one or

more wedges are to be highlighted, that wedge can be drawn slightly out of the perimeter of the circle for what is referred to as an e/ploded view. More typical of data presented in cell biology, however, are the line graph and the bar graph. $here is no hard and fast rule for choosing between these graph types, e/cept where the data is non4continous. $hen, a bar graph must be used. #n general, line graphs are used to demonstrate data which is related on a continous scale, whereas bar graphs are used to demonstrate discontinous or interval data. Suppose, for e/ample, that you decide to count the number of $4lymphocytes in four slices of tissue, one each from the thymus, <ayer@s patches, a lymph node and a healing wound on the skin. !et@s label each of these as $, <, ! and S respectively. $he numbers obtained per cubic centimeter of each tissue are $69'', <61*', !61'' and S6*'. +ote that there is a rather nice linear decrease in the numbers if $ is placed on the left of an / a/is, and S to the right. A linear graph of this data would give a nice straight line, with a statistical regression fit and slope. %ut look at the dataN $here is no reason to place $ ,or <,! or S- to the right or left of any other point on the graph 4 the placement is totally arbitrary. A line graph for this data would be completely misleading since it would imply that there is a linear decrease from the thymus to a skin in"ury A+C that there was some sort of 3uantitative relationship among the tissues. $here is certainly a decrease, and a bar graph could demonstrate that fact, by arranging the tissue type on the / a/is in such a way to demonstrate that relationship 4 but there is no inherent 3uantitative relationship between the tissue types which would force one and only one graphic display. 7ertainly, the thymus is not four times some value of skin ,although the numbers are-.N :owever, were you to plot the number of lymphocytes with increasing distance from the point of a wound in the skin, an entirely different presentation would be called for. Cistance is a continous variable. .e may choose to collect the data in 1 mm intervals, or 1 cm. $he range is continous from ' to the limit of our measurements. $hat is we may wish to measure the value at 1 mm, or 1.9 mm or 1.9? mm or 1.9?))* mm. $he important point is that the 9 mm position is 9/ the point at 1 mm. $here is a linear relationship between the values to be placed on the / a/is. $herefore a linear graph would be appropriate, with the dots connected by a single line. #f we choose to ignore the 1.9 and 1.9? and round these down to a value of 1, then a bar graph would be more appropriate. $his latter techni3ue ,dividing the data in appropriate intervals and plotting

as a bar graph- is known as a :istogram. $his graph is very familiar to students since it is the graph used for the display of grade distributions. :aving decided that the data has been collected as a continous series, and that the data will be plotted on a linear graph, there are still decisions to be made. Should the data be placed on the graph as individual points with no lines connecting them ,a Scattergram-P Should a line be drawn between the points ,known as a Cot4to4Cot-P Should the points be plotted, but curve smoothing be appliedP #f the latter, what type of smoothingP $here are many algorhythms for curve fitting, with the two most commonly used being linear regression and polynomial regression. #t is important to decide %DF;RD graphing the data, which of these is appropriate. !inear regression is used when there is good reason to suspect a linear relationship within the data ,as for e/ample in a spectrophotometric standard following the %eer4!ambert law-. #n general, the y value can be calculated from the e3uation for a straight line, y 6 m/ W b, where m is the slope and b is the y4 intercept. 7omputer programs for this can be very misleading. Any set of data can be entered into a program to calculate and plot linear regression. #t is important that there be a valid reason for supposing linearity before using this function, however. $his is also true when using polynomial regressions. $his type of regression calculates an ideal curve based on 3uadratic e3uations with increasing e/ponential values, that is y 6 ,m/Wb- , where n is greater than 1. $he mathematics of this can become 3uite comple/, but often the graphic displays begin to look better to the beginning student. #t is important to note that use of polynomial regression must be warranted by the relationship within the data, not by the individual drawing the graph. For single sets of data, that is the e/tent of the available options. For multiple sets the options increase. #f the multiple sets are data collected pertaining to identical ordinate values, then error bars ,standard deviation or standard error of the means- can be added to the graphics. <lots can be made where two lines are drawn, connecting the highest y values for each /, and a second connecting the lowest values ,the :i4!o Araph-. $he area between the two lines presents a graphic depiction of variability at each ordinate value. #f the data collected involves two or more sets of data having a common / a/is, but varying y a/es ,or values-, then a multiple graph may be used. $he rules for graphing apply to each set of

data, with the following provision Beep the number of data sets on any single graph to an absolute minimum. #t is far better to have three graphs, each with ? lines ,or bars- than to have a single graph with O lines. A graph that contains an e/cess of information ,such as O lines- is usually ignored by the viewer ,as are tables with e/tensive lists of data-. For this same reason, all unnecessary clutter should be removed from the graph e.g. grid marks on the graph are rarely useful. Finally, it is possible to plot two variables, y and &, against a common value, /. $his is done with a ?C graphic program. $he rules for designing a graph follow for this type of graph, and the use of these should clearly be left to computer graphics program. $hese graphs often look appealing with their hills and valleys, but rarely impart any more information than two separate 9C graphs. <erhaps the main reason is that people are familiar with two dimensional graphics, but have a more difficult time visually interpreting three dimensional graphs.

$ppendix *: Co#puters
$he advent of the personal computer has been one of the most significant factors impacting laboratory work in the past decades. $hese tools are now readily available and can be used at every level of work. #n the 1O5'@s computer use was limited to larger institutions and invariably centered on the use of large main frame computers, primarily for data analysis. $heir use was restricted to those with the willingness to learn comple/ high level languages, and the time and energies to write their own programs. 7omputers were somewhat unfriendly and the author can recall the most dreaded, yet common lament $he computer is down, againN 7entral main frames and their later mini computer counterparts all too often went down in the midst of your work. $he adage to save and save often was learned very 3uickly. Although there were other pioneers, it was the introduction of the Radio Shack $RS4I' and the Apple computer systems which began a revolution. $hose systems were closely followed by the introduction of the 7ommodore <D$, and Baypro computers. $hese early machines were marvels for their time, but were generally lacking in random access memory ,RAM- and speed. Manufacturers also attempted to corner markets by intentionally designing hardware and software that was incompatable with other manufacturers ,such as the lack of 7ommodore@s use of AS7## ]American Standard 7ode for #nformation #nterchange^ in its <D$ system and the substitution of its own standard, affectionately known among users as the :alf4AS7##-. Much of this changed in 1OI' with the introduction of the #%M4<7. $his machine, backed by the industry

giant, set the standard that is still in vogue. Apple was well entrenched in academic circles by this time and has not given in to the pressure for an #%M standard. Apple responded with the Mc#ntosh line, and the battle lines have been drawn since. .hich is a way of introducing the first decision that one faces when considering the use of a personal computer in cell biology. .hich system should one invest inP For many institutions, this is not a consideration since one or another system has already been purchased by the institition. For others it remains, because either there is no computer available or there are many types available within the institution. !et me settle the author@s pre"udice up front. #t doesn@t make any difference which hardware system you use. $he important point is that the computer is used as a tool to enhance learning in the laboratory. An installed Radio Shack $RS4I' can be as useful as a new Mc#ntosh ##, 7ompa3 ?I58??, or a +e($. $o those still needing to ac3uire a computer system, talk with the institutional academic computing center and follow their advice. #f you do not have such a center, contact one at the nearest university and follow their advice. ;f course availability of software programs should be considered, but in general, you can find what you need. +ewer, faster and bigger machines make some pro"ects more pleasant ,funP- but are rarely necessary. For most undergraduate instructional use of computers in the laboratory, any basic personal computer should suffice. #t is a rare event in the cell biology laboratory where multi4tasking ,having more than one program running simultaneously-, networking ,connection to other computers-, or e/tensive real4time data analysis is re3uired. #n many instances, these activities actually detract from the student@s ability to grasp the principle of the e/ercise. $he computer may act like the Siren@s of the ;dyssey ,which you may recall ended in a shipwreck-. :aving said this, it is now appropriate to indicate that most of the hardware and software discussed in this section is that appropriate to the #%M standard ,generally referred to as #%M compatable, and running under <74C;S or its counterpart, MS4 C;S-. $his is purely due to the use of this standard at the author@s institution and is not meant as an endorsement of this standard over any other ,such as Apple-. !A+A1AADS %efore discussing commercial programs, a brief note should be inserted about programming a computer. %etter than O'S of all the uses of a computer in lab can be performed using commercially available programs. For many this number can easily approach the 1''S mark. #n other words, you may never have to actually write your own program.

:owever, as often happens, you may become 3uite dissatisfied with the program established by another and wish to modify it to your own purpose. #t is at this point in time that you will need to write a program. $he list of available computer languages for writing such a program is as long as the list of computers. Dveryone has their favorite, and nothing can provoke a heated discussion among computer users faster than the merits ,or demerits- of any given program language. 7omputer hackers love to code programs in what is known as assembly language. $his is nearly an unreadable code that takes immediate command of the machine and is the closest to machine code , the only code which the computer actually uses. 7ommercial programs are usually written in assembly code or something close, in order to increase performance speed. #t is rarely used by cell biology laboratories. All beginners start with a language which needs to be interpreted before assembly and conversion into machine language. Since all of this must take place, these languages tend to slow down performance, unless compiled. 1n3uestionably, the most common language used is some version of %AS#7 ,%eginners All4 purpose Symbolic #nstructional 7ode-. $here are several reasons for this. First, it is usually bundled with the e3uipment ,the hardware- and thus is readily available. Second, it follows a straight4forward algebraic logic in its construction. #t has simple commands which are almost intuitive in their use. And finally, while it is not re3uired that programs be unstructured, it is probably the most forgiving language available for sloppy programming. +ewer enhanced versions of %AS#7 ,$rue4%asic, $urbo %asic or Muick %asic ).*- have taken this language and elevated it to a point where it can compete with virtually any language for speed and fle/ibility. %AS#7 has the distinct advantage of being the most portable language between differing systems. $he author@s favorite is Microsoft@s Muick%asic. For other reasons, Math and 7omputer Cepartments have taken to instructing <AS7A!, F;R$RA+, F;R$:, !#S<, or 7, to mention only a few. $hese are said to be more structured. %e wary, however. #t is not necessary to write unstructured code in %AS#7, and code that is written with these other languages, may not transfer easily from one machine to another, without e/tensive reworking. #n practice, once again, it hardly matters in an undergraduate cell biology laboratory. For programming your own code, use the language you are most familiar with.

<rograms given in this manual are in the more familiar %AS#7 format ,#%M %AS#7A-. Since even the core %AS#7 instructions vary slightly between computer systems, some conversion may be necessary prior to their use. 7;MMDR7#A! S;F$.ARD $here are a number of types of programs available and useful to the cell biology laboratory

Spread Sheets Araph <rograms D3uation Solvers Cata %ase <rograms .ord <rocessors ;utline <rograms <aint8Craw <rograms 7omputer Assisted #nstruction ,7A#Single <urpose <rograms

S<RDAC S:DD$S .ithout 3uestion, the single most useful program available for data collection and analysis is the spread sheet program. $here are several available, each with its own merits. $he leader ,in sales and business use- in the <7 dominated field is !otus 14 94?. ;thers of e3ual or superior value are Supercalc*, Muattro, or D/cel. #ntegrated packages ,;pen Access, Symphony, Dnable, Framework- nearly always contain powerful spreadsheets. Several of these offer educational discounts of their purchase price. A spreadsheet program is an electronic balance sheet divided in rows and columns. <ioneered by =isicalc, these programs may have had as much impact on computer use as the actual design of the hardware. Any data that can be tabulated in columns and rows can be added to this type of program. Functions are readily available for totals ,sums-, averages, means, ma/imum and minimum values, and full trigonmetric functions. $he programs listed above also include capabilities of sorting data, searching through the data, and for automatic graphing of the data. Dach allows the construction of blank masks that contain the instructions for coding input and output while allowing students ease of data entry. Spreadsheet programs have become so powerful in their latest versions, that they can be used as word processors and for data base manipulations, although they are not very sophisticated in

these functions. Many of their graph routines are as powerful as stand alone programs for graphing, and are usually easier to use. $he data entered can readily be transferred between other programs, from %AS#7 through direct analog conversions of integrated e3uipment. #f you were to purchase only one software package for the cell biology laboratory, it should be a spreadsheet. $he use of a spread sheet can be demonstrated with the calculation of o/ygen uptake by isolated mitochondria , D/ercise I.>-. $he data are collected for fourteen readings, every 1' minutes and must then be corrected for temperature and barometric fluctuations, as well as residual activity within the isolated mitochondria. $he corrected data is then graphed. 1se of a computer spreadsheet in the laboratory makes this a simple task, and allows visuali&ation of the progress of the e/ercise as it is happening. 6uperCalc 1 listing of data fro# #itochondrial respiration Q A QQ % QQ 7 QQ C QQ D QQ F QQ A QQ : QQ # QQ T QQ B QQ ! QQ M QQ + Q ; Q 1 9 ? ) 5 1 9 ? ) * Flask +umber 5 > I O 1' 11 19 1? 1) *44444444444444444444444444444444444444444444444444444444444444444444444444444 > $ime Manometer Readings Mitochondrial Respiration 7ell %iology D/ercise I.>

I44444444444444444444444444444444444444444444444444444444444444444444444444444 O 1' *'5 *'' )O' )O' ))* )OO )O5 *'? *I* )O> )OO *1' )O? )O? 1' 9' *1' )O' )I> )I> )15 )O9 )O? *19 )O' *') *'1 *15 *'' *'' 11 ?' *99 )IO )>O )>O ?O) )>I )O' *11 )O' *'5 *'* *1I )O> )O> 19 )' *?5 )I5 )>5 )>5 ?>' )>* *'1 *9* )OO *1? *1? *9> *1* *1* 1? *' *** )I5 )>? )>? ?)* )*I *'* *95 )OO *1) *1* *9> *1* *1* 1) 5' *>* )I* )5O )5O ?9> )*5 *'I *95 *'9 *15 *1> *9O *1? *1? 1* >' *O' )I9 )5) )5) ?'* ))I *1' *9O *'5 *9* *9) *?9 *1I *1I 15 1> 1I Minutes Microliters ;/ygen 7onsumed ;/ygen 1tili&ation by Mitochondria

1O 9' 91 7hange in gas volume ,Average of replicates99 Flask +umbers ,in pairs ?8) 1'.' 1?.' 91.' 9).' 9>.' ?1.' ?5.' *85 9I.' )5.' 5).' >8I O81' 11819 4).* 4I.* 411.* 49'.' 491.' 49?.' 49I.' 1?81) >.' .' ?.' 41*.' 41*.' 41?.' 41I.' 9? $ime 189 9* 1' 95 9' 9> ?' 4?.' .' 4*.*

9) 4444444444444444444444444444444444444444444444444444444444444444444444444 .* 4)1.' 49.* 4.* ?.' 9.' 45.' 45.* 4O.' 41*.*

9I )' 411.' 9O *' 49'.* ?' 5' 4?'.' ?1 >' 4?5.' ?9 ?? ?* ?5 ?> ?I 1' ?O 9' )' ?' )1 )' )9 *' )? 5' )) >'

>>.* 41?.' OI.* 41*.* 1'I.* 41>.' 19?.* 41O.*

<aired readings, averaged, and subtracted from original =alues corrected for $emp8<ressure8Dndogenous .* Alu.I AluA&ide C+< Malona7ontrol 7orrected for background ?8) 5.' 1?.' 9?.* *'.' 59.* >).' O'.' *85 9).' )5.' 55.* 1'?.* 1?).' 1*1.* 1>>.* >8I 4?.* 49.* 9.' 1?.' 9'.' 95.' ?).* O81' 4)*.' ?.' ).* 9'.' 9O.' ?).' ?I.* 11819 4I.* 4I.* 4O.' 5.' 1).* 9'.' 95.'

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7orrected values used to plot Figure C.1 Figure C.1. Super7alc ) graph of mitochondria data

A@$P?IC6 P@/A@$M6 Separate programs for graphing data are useful in that they contain more options than those found within spreadsheets, and allow for more comple/ graphs. $hey are also designed for direct presentation of results and are capable of more polish. $hat is, there are selections of such things as fonts, error bars and statistical analyses and three dimensional presentations. $he better programs will also give regressional analyses, either linear or polynomial. +early all allow for data input from a spreadsheet or data base manager, in addition to direct entry. Among the best in this area, are Sigma <lot, Dnergraphics, :arvard Araphics and 7ricket Araph. Most of these programs ,e/cept Sigma <lot- are designed for business graphics, but can be used in the cell laboratory. EH%$TI/" 6/B:E@6 #n this category, there is not a lot of choice of programs. $here are only two that come readily to the fore, $B4Solver and %orland@s Dureka. $hese programs allow for rapid entry of data for variables and simultaneous calculation of any other parameter within an e3uation. $B4Solver is ideal for insertion of such things as the +ernst e3uation, or the :odgkin4:u/ley e3uation, with subse3uent 3uery of .hat4if 3uestions. #f you wish to attempt mathematical modeling of cAM< pulsing in C. &iscoi&eum , $B4Solver or Dureka are well worth the investment. *$T$ B$6E M$"$AE@6 $hese programs are powerful for the long term storage and manipulation of data. $hey are more useful for research storage of data than for direct use in the undergraduate laboratory. $heir strength lies in the ability to do with words ,alphanumeric- what spreadsheets can do with numbers. $he leader in the business field is C% ## ,### and #=-, but there are e3ual programs available for a lower cost. Among those to be considered are MUA, Refle/, R4base and <7 File. For ease of use, while retaining powerful options of data manipulation, MUA e/cells. For tabular presentations of data, Refle/ is the choice. For relational needs, R4base or the C% series are the programs of choice. <7 File represents a good, less e/pensive alternative.

Cata base managers are e/cellent means of filing references, sources and such things as e3uipment lists, chemical inventory, etc. $hey can also be used effectively for filing of nucleic acid se3uences, but the data base must be created before it can be used. .hether or not to use a data base manager for this purpose depends on the availability of the data in an appropriate form for use within your program. #f data base managers are used, however, a hard drive becomes nearly mandatory. C/@* P@/CE66/@6 $here are perhaps as many word processors available as pebbles on the shore. For many computer users, this function is synonymous with computers. For purposes of the cell biology laboratory, any one is sufficient. $he programs are useful for writing lab reports, and marginally useful as alternatives to data base managers for such things as searching for nucleic acid codes. =irtually all word processors have a Search function, and this function can be used to search a code several thousand letters long for the presence say of a 1' base code. $his is not recommended, however, since the process is usually slow from within a word processor, and most word processing programs have severe limitations on the length of the words searched. Several word processor packages are available for scientific presentation, and these will make inclusion of scientific formulas and symbols easier. Several are e3uipped with the ability to perform red4 lining and can compare two files for any changes. $his is useful if you ask for rewriting of lab reports. $he computer will highlight those portions of the report which have been changed in the second version, eliminating the need to review the other sections. $his feature is also useful for group reports, where several individuals may make changes in the presentation. %y far the most popular word processor is .ord <erfect. $he author@s personal choice is (y.rite ###, which was used for this publication. 7losely allied with word processors are Cesk $op <ublishers . $hese programs can format both te/t and graphics for typesetter output, or for printing on laser printers. $hese programs are rarely directly useful in the undergraduate lab, but are e/cellent when it comes time to communicate your information to others. $he use of (ero/ =entura <ublisher or Aldus <agemaker ,the two representing the top of the line- is left for final publication layout. =entura <ublisher, for e/ample, was used for the final draft of this manual.

Cesk $op <ublishers are mentioned in this section merely as an option. Many software updates of familiar word processors are including the capabilities of desk top publishing into their programs. For the most part, this adds needless e/pense to the update, and increases the learning curve significantly. More importantly, it increases the need for larger 3uantities of RAM and faster hardware. $his adds to the cost of the system and although it may add impressive screen graphics, it rarely adds to any improved laboratory outcome. $here is a good deal of hype circling around which word processor is the best . $he answer is simple. #f you have a word processor, you know how to use it, and it works for you, keep it. /%TBI"E P@/A@$M6 $hese programs are also integrated within some word processors, or can be purchased separately. $he separate packages are primarily Ma/$hink or $hink$ank. $hese programs are an e/cellent means for groups or individuals to organi&e a presentation ,lab report, seminar- or even to design a protocol. $he programs allow the user to wander over the possibilities for presentation, and the program takes over the organi&ation of those thoughts into an outline form ,not without some human intervention, of course-. $he author has found these programs to be of value to students in formulating the purpose of individual laboratory e/ercises, when linked to either reports or individual pro"ects. $he entire outline for this manual was first conceived and organi&ed using Ma/$hink. #f you are heavily into writing reports and8or student papers, you may want to tie together a good outlining program with a wordprocessor and top it off with Right.riter. $he latter is a grammar and conte/t checker that is 3uite enlightening when applied to literary creations. P$I"T 8 *@$C P@/A@$M6 A useful ad"unct to various graph programs are those listed as paint or draw . <aint programs are those which plot pi/els on a graphic screen and allow simple drawing routines. Representative of these are <74<aintbrush, <74<aint, and <aintShow. $he latter was used for most of the simple computer graphics in this manual. Craw programs use mathematical formulas for their graphics ,as opposed to pi/el maps-. $hey give e/cellent resolution, but generally are more e/pensive ,the top of the line Auto7ad is over H9,'''-, and re3uire significantly more planning than the paint programs. #f you need good graphics, however, use a draw program. Mc#ntosh simply e/cels in this area. #f integrated graphics are to be used e/tensively, the Mc#ntosh is without 3uestion the

superior environment. ;n the #%M side, any of the programs running under Microsoft@s ADM Craw are good, and a batch of new programs running in the Microsoft .indows environment are approaching the 3uality and ease of the Mc#ntosh formula. $he easiest to use ,learn- is perhaps 7orel CrawN. Much of the line art for this manual was scanned or drawn using this program, with subse3uent printing by an :< ##< laser printer at ?'' C<#. C$I 7omputer Aided #nstruction is a large field, and the best advice is let the buyer beware . $here are some e/cellent programs available for instruction. Most are rather simplistic in their approach, graphics and design. Many are nothing more than childish diagrams of organs and cells which could be better presented with a real photomicrograph ,and not tie the student to the monitor-. At their worst, these programs become mere page flippers where instead of turning the page on a book, the computer regenerates a new screen crammed with information to be read. $heir only value is for the student ,now rare- who is awed by the computer and thus might turn the page. At their best, these programs can be completely interactive and can readily and accurately simulate laboratory conditions that are time or cost prohibitive. An e/cellent e/ample of this is the use of computer simulation for en&yme kinetics. $hese programs are so numerous and variable, that use of them is left to the instructors. $he best means of e/amining them is attendance at national meetings where they can often be reviewed. $here are also user@s groups which will review programs. $he only means of deciding on the purchase and use of 7A# programs is to talk with those who have used them ,not the people who wrote them or are selling them- and then try them yourself. 6I"ABE P%@P/6E P@/A@$M6. $hese programs are sometimes available through commercial channels, but more often are written for specific purposes. #t would be impossible to list all of the areas where these could be used in a cell biology laboratory, but a few should be mentioned.

Resolution 7alculations for !ight and DM Microscopy 7ell Morphometry 4 Area, =olume, +umbers 7entrifugation, 7onversion among rotors

7entrifugation, Sedimentation 7oefficients, Molecular .eights %eer4!ambert law 7alculation of Molecular .eights from Dlectrophoresis Aels #on Flu/ across Membranes Manometric 7alculations : flu/ and 7hemiosmosis Aene Mapping, Recombination Arowth 7urves Simulation of cAM< effects on Cictyostelium Radiation Cose Response

Dssentially, anything which re3uires repetitive calculations or data sorting is material for this section. #ncluded in this appendi/ are two programs. $he first ,7D!!M- allows the student to play the role of a cell membrane. $he second, ,%DDR- allows rapid calculations of e/tinction coefficients using the %eer4!ambert law. ;ther programs are available from the author, but space prohibits e/tensive listings. For e/ample, a Muick%asic program for interconversion of centrifuge rotors and calculations of viscosity, sedimentation coefficients and clearing constants would re3uire 194 1* pages if typed out. <rograms can become very comple/ over time. An e/ample of a page flipper program ,for review of photosynthesis- originally written in <7 %asic and converted to Muick%asic ).* is longer yet. $his type of program can be fun, since graphics can be included to demonstrate chlorophyll accepting light ,as lightning bolts- and transferring electrons to other molecular orbits. $here are several commercially available programs of this type. CEBBM progra# (Me#+rane control of #eta+olites! 1 1' RDM %J BD+ .#$$ $RA7J :#A: S7:;;! 9' RDM M;C#F#DC F;R %AS#74= 1? T1+D 1O>O )' RDM 5' RDM Austavus Adolphus 7ollege B. A+CDRS;+ MAR7: ?, 1OI5 ?' RDM M;C#F#DC F;R #%M %J S7;$$ S:DBD!S

*' RDM \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

>' RDM I' RDAC :,S,B,+,7,.,< O' CA$A 5>'',?',*?,',1,',' 1'' .#C$: )'27;!;R 1*,1,12BDJ ;FF27!S 11' !;7A$D 1,1*2<R#+$0.D!7;MDN0 19' !;7A$D *,1I2<R#+$0$;0 1?' !;7A$D O,152<R#+$07D!!M0 1)' !;7A$D 11,52<R#+$0A 7D!! MDM%RA+D S#M1!A$#;+.0 1*' F;R S7;$$61 $; 9'''2+D($27!S 15' <R#+$ 0Co you wish to see an e/planation0 1>' <R#+$ 0of this program0 1I' #+<1$ AH2AH6!DF$H,AH,11O' #F AH60J0 ;R AH60y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

?>' <R#+$ 0

.AS$D %D$.DD+ ' A+C 1' M;!D71!DS0

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

5*' <R#+$ 0\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\0 55' <R#+$ 0$:D 7D!! %DA#+S $; %1R+ S1AAR F;R D+DRAJ. C#F1S#;+ A+C0 5>' <R#+$ 0;SM;$#7 <;$D+$#A!S A; #+$; DFFD7$. J;1 ARD +;. RDACJ $;0

5I' <R#+$ 0A$$DM<$ $; BDD< $:#S 7D!! A!#=DN0 5O' <R#+$ 06666666666666666666666666666666660 >'' <R#+$2<R#+$0<RDSS RD$1R+ $; 7;+$#+1D0 >1' AH6#+BDJH2#F AH600 $:D+ >1' >11 .#C$: )'27!S27;!;R 1*,1,1 >9' T6' >?' !D$ <6<W1 >)' #F <61? $:D+ 1I1' >*' <R#+$2<R#+$ >5' <R#+$0 1. water0 >>' <R#+$0 9. sugar0 >I' <R#+$0 ?. potassium ions0 >O' <R#+$0 ). sodium ions0 I'' <R#+$0 *. chloride ions0 I1' <R#+$0 5. waste0 I9' <R#+$0 ,primarily ammonia compounds-0 I?' <R#+$ 07hoose the number of the item 0 I)' <R#+$0which you wish to change.0 I*' #+<1$ ( I5' !D$ :6:W)'' I>' !D$ S6S4) II' !D$ B6B49 IO' !D$ +6+W9 O'' !D$ .6.W) O1' #F (61 $:D+ O>' O9' #F (69 $:D+ 191' O?' #F (6? $:D+ 1?1' O)' #F (6) $:D+ 1?I' O*' #F (6* $:D+ 1)I' O5' #F (65 $:D+ 1*1' O>' <R#+$ 0.hat is the number of water molecules0

OI' <R#+$ 0that you want removed0 OO' #+<1$ A 1''' #F A: $:D+ 1'?' 1'1' <R#+$ 0J;1 C;+$ :A=D $:A$ MA+JN0 1'9' A;$; O>' 1'?' !D$ :6:4A 1')' <R#+$ 0Dnergy is used here to get rid of water0 1'*' !D$ S6S4) 1'5' <R#+$ 0 0 1'>' <R#+$ 0<RDSD+$ 7;+C#$#;+S ARD AS F;!!;.S20 1'I' <R#+$ 1'O' <R#+$ 0<;$ASS#1M #;+S440 B,0.AS$D M;!D71!DS440 . 11'' <R#+$ 0S;C#1M #+S440 +,0.A$DR M;!D71!DS440 : 111' <R#+$ 07:!;R#CD #;+S440 7,0S1AAR M;!D71!DS440 S 119' <R#+$ 0 0 11?' #F :p $:D+ 151' 11)' #F :I''' $:D+ 151' 11*' #F S $:D+ 15)' 115' #F S?* $:D+ 15)' 11>' #F B, $:D+ 15O' 11I' #F B5' $:D+ 15O' 11O' #F .1' $:D+ 1>?' 19'' A;$; >9' 191' <R#+$ 0:ow many sugar molecules do you want0 199' #+<1$ % 19?' #F % $:D+ 19>' 19)' <R#+$ 0$he solution cannot offer you that many0 19*' <R#+$ 0at onceN0 195' A;$; 191' 19>' !D$ S6SW% 19I' <R#+$ 0Dnergy is needed for pinocytosisN0

19O' !D$ S6S4? 1?'' A;$; 1'5' 1?1' <R#+$0<otassium ions are regulated by active0 1?9' <R#+$0transport. More specifically they are0 1??' <R#+$0regulated by the sodium pump, which 0 1?)' <R#+$0is for every sodium ion pumped out, one0 1?*' <R#+$0potassium ion is gained on the inside0 1?5' <R#+$0of the membrane.0 1?>' A;$; >?' 1?I' <R#+$ 0:ow many sodium ions do you want removed0 1?O' #+<1$ C 1)'' #F C 9' $:D+ 1)?' 1)1' <R#+$ 0J;1 C; +;$ :A=D $:A$ MA+JNNN0 1)9' A;$; 1?I' 1)?' !D$ +6+4C 1))' !D$ B6BWC 1)*' <R#+$ 0Dnergy is needed for active transportNN0 1)5' !D$ S6S49 1)>' A;$; 1'5' 1)I' <R#+$ 0J;1 :A=D !#$$!D ;R +; 7;+$R;! ;R +DDC $;0 1)O' <R#+$ 07:A+AD $:D 7:!;R#CD #;+ 7;+7D+$RA$#;+NN0 1*'' A;$; >?' 1*1' <R#+$ 0:ow many waste molecules do you want to0 1*9' <R#+$ 0get rid of0 1*?' #+<1$ D 1*)' #F D.4? $:D+ 1*>' 1**' <R#+$ 0J;1 C; +;$ :A=D $:A$ MA+J $; AD$ R#C ;FN0 1*5' A;$; 1*1' 1*>' !D$ .6.4D 1*I' <R#+$ 0Dnergy is needed to get rid of wasteN0 1*O' !D$ S6S4)

15'' A;$; 1'5' 151' <R#+$ 0\\\7R#S#S\\\0 151* <R#+$ 0ACT1S$ .A$DR 7;+7D+$RA$#;+ #MMDC#A$D!JNN0 159' !D$ T6TW1 15?' A;$; 1>5' 15)' <R#+$ 0\\\7R#S#S\\\0 15)* <R#+$ 0ACT1S$ S1AAR 7;+7D+$RA$#;+ #MMDC#A$D!JNN0 15*' #F S' $:D+ 15>' 155' <R#+$ 0J;1 ARD %1R+#+A <R;$;<!ASMN0 15>' !D$ T6TW1 15I' A;$; 1>5' 15O' <R#+$ 0\\\7R#S#S\\\0 15O* <R#+$ 0ACT1S$ <;$ASS#1M #;+ 7;+7D+$RA$#;+ 0 1>'' <R#+$ 0 1>1' !D$ T6TW1 1>9' A;$; 1>5' 1>?' <R#+$ 0\\\7R#S#S\\\0 1>?* <R#+$ 0ACT1S$ .AS$D 7;+7D+$RA$#;+ #MMDC#A$D!JNN0 1>)' !D$ T6TW1 1>*' <R#+$ 1>5' #F T $:D+ >?' 1>>' <R#+$ 0J;1 C#C +;$ F1+7$#;+ .D!! AS A0 1>I' <R#+$07D!! MDM%RA+D.0 1>O' <R#+$ 0\\\J;1R 7D!! #S +;. CDAC\\\\\\\\0 1I'' A;$; 1IO' 1I1' .#C$: I'27!S2<R#+$ 0\\\=DRJ A;;C\\\ 0 1I1* <R#+$ 0J;1 :A=D MA#+$A#+DC $:D 7D!! !;+A0 1I9' <R#+$ 0D+;1A: $; RDA7: MA$1R#$J. $:D 7D!! #S +;. A%;1$ $;0 1I?' <R#+$ 0RD<R;C17D. C; J;1 .#S: $; $RJ $; BDD< ;+D ;F $:D 0 1I)' <R#+$ 0CA1A:$DR 7D!!S A!#=D 0 1I*' #+<1$ AH2AH6!DF$H,AH,1-2#F AH60y0 ;R AH60J0 $:D+ 1O>' #MMDC#A$D!JNN0

1I5' !D$ <6' 1I>' RDM 1II' RDM 1IO' D+C 1O'' <R#+$ 0C; J;1 .#S: $; SDD A+ D(<!A+A$#;+ ;F 7;+7D+$RA$#;+S0 1O1' <R#+$ 0A+C R1!DS0 1O9' #+<1$ AH2AH6!DF$H,AH,1-2#F AH60J0 ;R AH60y0 $:D+ )O' 1O?' #F AH60n0 ;R AH60+0 $:D+ 1O*' 1O)' D+C 1O*' .#C$: )'27;!;R 1*,1,127!S 1O5' A;$; >9' 1O>' .#C$: )'27!S2<6'2A;$; >9' %DDR ,%AS#7 program for %eer4!ambert !aw1' RDM\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 9' RDM F#!D +AMD2 )' RDM *' RDM \\ R1H4R)H6RDS<;+SDS $; #+<1$ <ARAMD$DRS 5' RDM \\ M6S!;<D ;F RDARDSS#;+ 6 A%S;R<$#=#$J %6J #+$DR7D<$ >' RDM \\ + 6 +1M%DR ;F CA$A <;#+$S #+ S$A+CARC 71R=D I' RDM \\ S(,SJ,S(9,S(J ARD S1MS ;F (J ,(`9,(\J RDS<D7$#=D!J O' RDM \\ C#MD+S#;+DC =AR#A%!DS ( 6 7;+7D+$RA$#;+, J 6 A%S;R%A+7D 1'' RDM\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 11' RDM 19' C#M (,9'-, J,9'-, J$,9'-, (9,9'-, (J,9'-, J9,9'1?' RDM 1)' BDJ ;FF2 S7RDD+ '2.#C$: I'27;!;R 1),1,127!S2!;7A$D 1,1 1*' <R#+$ 0$:#S <R;ARAM .#!! 7;M<1$D $:D D($#+7$#;#+ 7;DFF#7#D+$0 15' <R#+$ 0A+C 1SD $:#S R $; 7;M<1$D $:D 7;+7D+$RA$#;+S ;F 1+B+;.+S0 1>' <R#+$2<R#+$ %DDR.%AS ..:eidcamp ?' RDM\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ S1%R;1$#+DS

1I' <R#+$ 07A!71!A$#;+S ARD %ASDC ;+ %DDR4!AM%DR$ !A. A6ecl0 1O' <R#+$ 0 9'' <R#+$ 0 91' <R#+$ 0 99' <R#+$ 0 .:DRD A 6 A%S;R%A+7D0 e 6 A%S;R%$#=#$J ,D($#+7$#;+ 7;DFF#7#D+$c 6 7;+7D+$RA$#;+ l 6 !D+A$: ;F ;<$#7A! <A$: ,1 cm F;R S<D7 9'-

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`9 S(96S(9W(9,#J9,#-6J,#-`9 SJ96SJ9 W J9,#(J,#-6(,#-\J,#S(J6S(JW(J,#-

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>O'

77 6 M\SC(8SCJ

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$ppendix E: I#age $nal0sis

Figure D.1. Schematic of image analysis system

Figure D.9. 7apabilities of ;lympus 71D 9 system 7omputers and video recorders have been combined in a powerful combination for advanced histochemical work. A video camera can be attached to a microscope and coupled to a frame4 grabber board within a personal computer. $he image can then be captured and displayed on a video monitor in either black U white, color, or pseudocolor. $he latter is an enhancement feature of the system which allows assignment of a color to a chosen gray scale of a black U white image. $hat is, the color is not real, but an assigned value, determined by the operator or the computer system. Since the eye can detect more colors than tones of gray, it increases the ability to visually distinguish subtle tone differences. A sample system suitable for the undergraduate laboratory is represented by the 71D 9 system manufactured by ;lympus. $he system consists of2 77C video camera and power supply Frame grabber board 7omputer e3uipped with math coprocessor

7omputer mouse or pointing device Software 7ables ;ptional dot matri/ printer #mage analysis systems used to be too e/pensive for routine work, but the price is steadily decreasing. A generic system can actually be pieced together from commercially available components for under H1','''. A fully integrated package will cost slightly more, and a real time system ,one which captures and analy&es the image as it is generated- can increase the price by a factor of 1'. Jou get a lot of power for the investment, however. #mage analysis begins with a digital capturing of the image as data. #n an intermediate system suitable for cell work, the image is detected with 9*5 gray levels ,the human eye can detect only O- and displayed with a resolution of either 9*5 / 9*5 or *19 / *19 pi/els. A pi/el is an abbreviation for picture elements, and represents the dots on a screen, or more significantly, the number of information points in the image. A *19 / *19 pi/el image thus contains 959,1)) pieces of information. Dach piece of information will in turn be stored as I, 15 or ?9 bits in computer memory, depending on the computer system attached. An I bit machine ,#%M <7 or e3uivalent- will satisfy the low end of this scale, a 15 bit ,#%M A$ or 9I5 clone- the intermediate, while the upper end will re3uire a significantly faster computer ,?I5 based, or minicomputer-. For an undergraduate laboratory ,and most research work-, the information stored in the low end system ,9*5 / 9*5, I bit- is sufficient, with the high end reserved for analysis of true color and8or real time images. A display resolution of *19 / *19 pi/els makes the image sharper appearing and easier to work with. ;nce the image is ac3uired in digital form, it becomes data which can be manipulated for changes in the display image, or for statistical purposes. $he operational capabilities of the 71D 9 system, as listed in Figure D49, demonstrate many of these features. Software options for image analysis include packages for densitometry ,replacing microspectrophotometers-, ?4C construction of serial sections, production of stereo images, autoradiographic analysis, planar morphometry including linear analysis ,line length, width and angles- in addition to the features listed for the basic system.

$ppendix F: Centrifugation

$he single most important advance in the use of centrifugal force to separate biologically important substances. was the coupling of mechanics, optics and mathematics by $. Svedberg and T... .illiams in the 1O9'@s. $hey initiated the mathematics and advanced the instrumentation. 1 to a point where it was possible to prove that proteins were large molecules that could be weighed in a centrifuge. 9. #n honor of that work, the value for a molecule@s ,or organelle@s- sedimentation velocity in a centrifugal field is known as its Svedberg constant or S value for short. $he instrumentation has progressed 3uite far since the early work of Svedberg and .illiams. $oday, any techni3ue employing the 3uantitative application of centrifugal force is known as ultracentrifugation. $he design of the basic instruments, the rotors and the optical systems for measurement are too e/tensive to cover in this volume. For our purposes, we will concentrate on two types of rotor, and a few selected parameters to be measured. @/T/@6 $able F.1. Rotor 7haracteristics

Figure F.1. 7ross section of Sorvall SS4?) rotor. Rotors for a centrifuge are either fi/ed angles , swinging buckets , continuous flow, or &onal, depending upon whether the sample is held at a given angle to the rotation plane, is allowed to swing out on a pivot and into the plane of rotation, designed with inlet and outlet ports for separation of large volumes, or a combination of these. Figure F.1 demonstrates the characteristics of each of these. Fi/ed angles generally work faster substances precipitate faster in a given rotational environment, or they have an increased relative centrifugal force for a given rotor speed and radius. $hey also have few ,or no- moving parts on the rotor itself and thus have virtually no ma"or mechanical failures, other than potential metal stress, which all rotors undergo. $hese

rotors are the work4horse elements of a cell laboratory, and the most common is a rotor holding I centrifuge tubes at an angle of ?) G 7 from the vertical ,such as the Sorvall SS4?) rotor or the %eckman TA49'-. Figure F.1 presents a cross4 sectional diagram of the Sorvall SS4?). Swinging bucket rotors ,also known as hori&ontal rotors- have the advantage that there is usually a clean meniscus of minimum area. #n a fi/ed angle rotor, the materials are forced against the side of the centrifuge tube, and then slide down the wall of the tube. $his action is the primary reason for their apparent faster separation, but also leads to abrasion of the particles along the wall of the centrifuge tube. For a swinging bucket, the materials must travel down the entire length of the centrifuge tube and always through the media within the tube. Since the media is usually a viscous substance, the swinging bucket appears to have a lower relative centrifugal force, that is it takes longer to precipitate anything contained within. #f, however, the point of centrifugation is to separate molecules or organelles on the basis of their movements through a viscous field, then the swinging bucket is the rotor of choice. Moreover, if there is a danger or scraping off an outer shell of a particle ,such as the outer membrane of a chloroplast-, then the swinging bucket is the rotor of choice. Most common clinical centrifuges 1 have swinging buckets. Since the buckets are easy to interchange, this type of rotor is e/tremely versatile. #ts ma"or drawback is the number of moving parts which are prone to failure with e/tended use. +early all cell biology laboratories will have several e/amples of fi/ed angle and hori&ontal rotors. .hile the sample volumes of these rotors can be significant, they are limiting. $o overcome this limitation, a continuous flow centrifuge can be used. !imnologists often employ such a device to separate plankton from gallons of lake water. 7ell biologists employ &onal rotors for the large scale separation of particles on density gradients. Fonal rotors can contain up to 9 liters of solution and can work with tissue samples measured in ounces ,or even pounds-. $he rotors are brought up to about ?''' R<M while empty, and the density media and tissues are added through speciali&ed ports. $his type of rotor has a distinct preparative advantage over the gradient capacity of more typical rotors. @/T/@ T%BE6 #n using either a fi/ed angle or swinging bucket rotor, it is necessary to contain the sample in some type of holder. 7ontinuous and &onal rotors are designed to be used without e/ternal tubes.

For biological work, the tubes are divided into functional groups, made of regular glass, 7ore/ glass, nitrocellulose, or polyallomer. Regular glass centrifuge tubes can be used at speeds below ?,''' R<M, that is in a standard clinical centrifuge. Above this speed, the /g forces will shatter the glass. A special high speed glass with the tradename of 7ore/ ,7orning Alass .orks- has been developed to handle speeds up to 1*41I,''' R<M. $hese tubes can be used in most routine cell organelle preparations, if, and only if, the proper adapters are also used within the centrifuge rotors. $hese tubes are relatively e/pensive ,about H?.*' each- and should never be used for any purpose other than the centrifuge. Any tubes with scratches or chips should be disposed of immediately. $hese high4 speed glass tubes will shatter above 1I,''' R<M. For work in the higher speed ranges, centrifuge tubes are made of plastic or nitrocellulose. <reparative centrifuge tubes are made of polypropylene ,sometimes polyethylene- and can withstand speeds up to 9',''' R<M. $hese tubes should be carefully e/amined for stress fractures before use. A tube with a fracture will hold fluids before centrifugation, but the cracks will open under centrifugal force. +itrocellulose are ine/pensive and used for most ultracentrifugation. $hey are meant to be used only once and then discarded. Repeated use increases the chance of tube collapse due to internal molecular stress within the tube walls. $here is no way to pre4determine this, so it is best to always use a new tube for ultracentrifugation. +itrocellulose also becomes less fle/ible with age, and the purchase date for all tubes should be noted. $ubes older than 1 year should be discarded. A centrifuge tube is ine/pensive when compared to the loss of time and materials for a typical ultracentrifuge run. <olyallomer tubes are re4usable, more e/pensive, and slippery. Molecules will slide down the walls of these tubes more easily, and thus are the tubes of choice for precipitation centrifugations. $hey are also more chemically inert.

@EB$TI:E CE"T@IF%A$B F/@CE

Figure F.9. +omogram for R.7.F. Modern day ultracentrifuges can generate forces in e/cess of ?'',''' times that of gravity, forces sufficient to overcome the very cohesion of most molecules ,including the metal of the rotor-. $he force is usually given as some value times that of gravity. $he centrifugal force is dependent upon the radius of the rotation of the rotor, the speed at which it rotates, and the design of the rotor itself ,fi/ed angle, vs swinging bucket-. Rotor speed and design can be held constant, but the radius will vary from the top of a centrifuge tube to the bottom. #f a measurement for the radius is taken as the mid4point, or as an average radius, and all forces are mathematically related to gravity, then one obtains a relative centrifugal force, labeled as /g. 7entrifugation procedures are given as /g measures, since R<M and other parameters will vary with the particular instrument and rotor used. Relative 7entrifugal Force is a constant that is independent of the apparatus used. Figure F.9 presents a +omogram for calculation of R.7.F. for a given radius and R<M. A simple formula for calculating this value is2 R7F 6 1.19r ,R<M81'''where r 6 radius in millimeters R<M 6 revolutions per minute $he difficulty with using the formula is establishing the value for r. $ypically, there are three r values given ,by the manufacturer- for a rotor2 the ma/imum, minimum and average r. $hese correspond to the distances from the center of rotation to the bottom, top and middle of the sample tube.

#f the density and viscosity of the medium are known, as well as the density of a given particle, then the time needed to completely sediement a particle can be determined by the formula2 $ 6 ,,C4!-8,CW!--\,+8,d ,g4p-S -where $ 6 time in minutes C 6 radial distance in cm for r ! 6 radial distance to meniscus + 6 viscosity of the fluid medium g 6 density of the fluid medium p 6 density of the particle to sediment d 6 diameter of the particle in cms. S 6 rotational velocity in R<M @/T/@ C/MP$@I6/"6 .hen separating a particle, it is convenient to be able to compare one rotor@s design characteristics with those of a differing rotor. A procedure may be given as centrifuging for 9' minutes at 1*,''' R<M in a Sorvall SS?) rotor. $he problem then becomes one of how do you e3uate that to a %eckman TA49' rotorP .orking with the ma/imum R7F for each rotor, the conversion can be made with the following e3uation2 t 6 ,t / R7F - 8 R7F where t 6 run time needed for the %eckman rotor t 6 run time specified in the procedure R7F 6 R7F of TA49' rotor at ma/imum speed R7F 6 R7F specified in the procedure ,calculated if procedure in R<M$his e3uation can be altered somewhat to be even more useful. $he efficiency of rotors can be compared for any given task. Dach rotor is given a value for k ,the clearing constant, or clearing factor-, which is an estimate of the time ,in hours- re3uired to pellet a particle of known sedimentation coefficient at the ma/imum speed of the rotor. ) $he lower the value of k, the shorter the time re3uired to sediment a given particle. $he relationship is given by

t6k8s a k 6 clearing factor e/pressed in hour4Svedbergs s a 6 Sedimentation 7oefficient in water at 9' G 7

e/pressed in Svedbergs #t is possible to compute k, but it is easiest to use the manufacturers values, which can be looked up in tables such as those in $able F.1. k 6,9*?,?''-]ln,r 8r -^8,Rotor angular velocity81'''-

1sing tabulated k values, however, the comparison of rotors is even easier, as the e3uation above becomes t 6kt 8k where the t and k values are the time and k factor for each rotor. %y substituting this e3uation, you can also determine the time of centrifugation for any change in rotor speed. * For a Sorvall SS4?), for e/ample, at 9',''' R<M ,k6)'9-, the time re3uired to sediment a particle of1'' S, in water at 9' G 7 is calculated by2 t 6 )'981''or ).'9 hours ,) hrs, 1 min$able F.9 presents the k values for the Sorvall SS4?) rotor and for the Sorvall ASA rotor. Since these two rotors will be used throughout the remainder of the course, reference should be made to this table in any future centrifugation work, when another rotor is substituted. @/T/@ 6$FETK $here are a number of safety precautions that must be adhered to when using any centrifuge and rotor. Rotor Failure2 All rotors are sub"ect to stress and with time will undergo metal fatigue. $his is a given, and conse3uently, a detailed history of the rotor use should be kept. $his is usually not done with clinical centrifuges, but is an absolute for an ultracentrifuge rotor.

E:E@K %6E /F $" %BT@$CE"T@IF%AE @/T/@ M%6T BE @EC/@*E* I" T?E CE"T@IF%AE B/A. $B6/6B%TEBK "/ E-CEPTI/"6) After a period of use, each rotor will in turn be derated , that is its ma/imum R<M will be lowered. $he %eckman rotors may contain optical speed control rings at their base 4 be sure they are present, and clean before use. $hese devices will strobiscopically monitor the ma/imum speed that a rotor can be used at. $hey are replaced as the rotor is derated. %y far the most common cause of rotor failure is corrosion stress. Salts, highly alkaline detergents and of course corrosive acids and alkali@s will cause decomposition of the coatings on aluminum rotors, which in turn will concentrate stress and eventually result in cracks and total rotor failure. $itanium rotors are more corrosion resistant, but more e/pensive. 1ltracentrifuge rotors are e/pensive ,in e/cess of H5,''' each on average- and can be potentially ha&ardous. At the forces generated in an ultracentrifuge, a rotor failure is the e3uivalent of a small bomb. T?E@EF/@E T?E F/BB/CI"A @%BE6 M%6T BE /B6E@:E*.
1. %efore running a centrifuge, check the classification decal on the centrifuge to ensure that

the rotor is safe to use in the centrifuge at hand.

2. +ever use an alkali detergent on a rotor ,most are highly alkaline 4 be sure to check

before use-.
3. Always clean and completely dry the rotor after every use. Any spilled materials,

especially salts and corrosive solvents must be removed immediately with running water. Fi/ed angle rotors are stored upside down, to drain after thorough cleaning and rinsing. Swinging buckets have only the buckets cleaned and dried, and stored inverted and with the caps removed. +D=DR immerse the rotor portion of a swinging bucket rotor. #nevitably the linkage pins will rust, as it is virtually impossible to remove all fluids from them.
4. %e especially careful not to scratch the surface of a rotor or bucket. 1se plastic brushes

only. +ormal wire brushes will scratch the anodi&ed surface of aluminum rotors which will increase the likelihood of corrosion. $he anodi&ed layer is e/tremely thin and is the main defense against corrosion of an aluminum rotor.
5. Always use the proper centrifuge tube. Alass tubes are used in clinical centrifuges only.

:igh Speed 7ore/ tubes can be used up to 1*,''' R<M ,in SS?) rotor- #F there are no

scratches or imperfections in the glass, and if the proper rubber or plastic adapter is employed. All ultracentrifugation use employs nitrocellulose or polyallomer tubes. +itrocellulose ages and will collapse in a strong centrifugal field if old.
6. Always fill the centrifuge tubes to the proper level. ,usually full to within 189 inch of the

top-. $he tubes are thin walled and will collapse if improperly filled.
7. Always balance the rotor properly. 1se a precision scale for most work. Always balance

the tube with a medium that is identical to that being centrifuged, i.e. do not balance an alcohol solution with water, or a dense sucrose solution with water only 44 the distribution of the densities will be incorrect. For swinging buckets, be sure the buckets are weighed with their caps in place, that the seals are intact and that the caps are secure. %e careful in the placement of tubes within a rotor to ensure proper balance 4 check the manufacturers guides for comple/ rotors that hold multiple tubes.
8. Dnsure that the rotor is properly seated within the centrifuge. For swinging buckets,

ensure that they are hanging properly 4 Couble or $riple checkN For preparative rotors, be sure the rotor cover is in place and properly screwed down, where appropriate. +D=DR use a rotor without its lid, when one is supplied 4 the screw actually holds the rotor to the motor shaft.
9. 7heck that the centrifuge chamber is clean, defrosted and that all membranes or

measuring devices are intact and functional ,%eckman speed and temperature controlsand that the lid is securely closed.
10. Ad"ust acceleration rates, deceleration rates, temperature and R<M controls as

appropriate. Set brake on or off as appropriate and check vacuum level where appropriate.
11. Start the centrifuge and set the timer. Co not attempt to open the centrifuge until the rotor

has come to a complete stop.

12. %efore opening the centrifuge, record the appropriate information in the centrifuge log.

+ote2 #f properly balanced and used, the rotor should accelerate smoothly and with a constant change in the pitch of the motor sound. Any vibrations, or unusual sounds should cause the cessation of operation #MMDC#A$D!J by the operator. "E:E@ leave the centrifuge until you are certain that it has reached its operating speed and is functioning properly. All rotors go through a minor vibration phase when they first start. $here will be a minor flutter when the rotor

reaches this vibration point 4 do not confuse this with a serious vibration caused by imbalance. #f in doubt, halt the centrifuge and get assistance.

$ppendix A: 6pectrophoto#etr0
D/ercise A.1 %radford <rotein Assay D/ercise A.9 !owry <rotein Assay D/ercise A.? %iuret Assay

Figure A.1 Dlectromagnetic Radiation Spectrum

Figure A.9. Schematic light transmission

Figure A.?. 1se of the Spec 9' A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution. A spectrophtometer differs from a

colorimeter in the manner in which light is separated into its component wavelengths. A spectrophotometer uses a prism to separate light and a colorimeter uses filters. %oth are based on a simple design of passing light of a known wavelength through a sample and measuring the amount of light energy that is transmitted. $his is accomplished by placing a photocell on the other side of the sample. All molecules absorb radiant energy at one wavelength of another. $hose that absorb energy from within the visible spectrum are known as pigments. <roteins and nucleic acids absorb light in the ultraviolet range. $he following figure demonstrates the radiant energy spectrum with an indication of molecules which absorb in various regions of that spectrum. $he design of the single beam spectrophotometer involves a light source, a prism, a sample holder and a photocell. 7onnected to each are the appropriate electrical or mechanical systems to control the illuminating intensity, the wavelength, and for conversion of energy received at the photocell into a voltage fluctuation. $he voltage fluctuation is then displayed on a meter scale, is displayed digitally, or is recorded via connection to a computer for later investigation. Spectrophotometers are useful because of the relation of intensity of color in a sample and its relation to the amount of solute within the sample. For e/ample, if you use a solution of red food coloring in water, and measure the amount of blue light absorbed when it passes through the solution, a measureable voltage fluctuation can be induced in a photocell on the opposite side. #f now the solution of red dye is diluted in half by the addition of water, the color will be appro/imately 189 as intense and the voltage generated on the photocell will be appro/imately half as great. $hus, there is a relationship between the voltage and the amount of dye in the sample. Aiven the geometry of a spectrophotometer, what is actually measured at the photocell is the amount of light energy which arrives at the cell. $he voltage meter is reading the amount of light $RA+SM#$$DC to the photocell. !ight transmission is not a linear function, but is rather an e/ponential function. $hat is why the solution was A<<R;(#MA$D!J half as intense when viewed in its diluted form. .e can however monitor the transmission level and convert it to a percentage of the amount transmitted when no dye is present. $hus, if 189 the light is transmitted, we can say that the solution has a *'S $ransmittance. +ote that it is always relative to a solution containing no dye. $ransmittance is the relative percent of light passed through the sample.

.hat makes all of this easy to use, however, is the conversion of that information from a percent transmittance to an inverse log function known as the Absorbance ,or ;ptical Censity-. The Beer-Ba#+ert Ba& Cefiniton Absorbance2 $he negative log of the transmittance. A 6 4 log $ DM1A$#;+ A.1

$his value is more useful in spectrophotometry than transmittance, because of plot of absorbance vs concentration yields a straight line. A plot of transmittance vs concentration is an e/ponential. $he 4 log calculates the inverse of transmittance, so that absorbance increases with increasing concentration. $ransmittance would decrease as we increased the amount of red dye in our e/ample. $he relationship of Absorbance to concentration was shown by two biochemists to follow the e3uation for a straight line, y 6 m/ Wb, where m is the slope of the line and b is the y intercept. #f the measurement is made in such a way that b 6 ' ,that is, a solution containing no dye has no absorbance-, and if we substitute Absorbance for y, concentration for /, and variant for m, we arrive at the formulation of the %eer4!ambert !aw2 A6 7 where A 6 absorbance 7 6 concentration 6 the e/tinction coefficient +ote that variant is e3ual to the slope of the straight line which will result from a plot of absorbance ,y a/is- vs concentration ,/ a/is-. $o use a spectrophotometer it is necessary to establish a known series of dilutions containing known 3uantities of a solute. ;ne of these will contain no solute and is known as the blank . #t is used to ad"ust the instrument to read 1''S transmittance or ' absorbance. #n use, a 'S transmittance value ,infinite absorbance- is established by placing a curtain between the light source and the photocell. Dlectronic control is then e/erted so that the meter will read 'S $ransmittance on its scale. $he blank sample ,containing no solute or dye- is inserted, the curtain opened and the meter read"usted to read 1''S transmittance. All other measures are then made

by merely inserting the samples into the light path and measuring the S transmittance. Most spectrophotometers have a built in means of direct conversion of this reading to absorbance. After recording the absorbance for a series of standards, a plot is made of the absorbance value ,y a/is- vs the concentration ,/ a/is-. $he slope of the line is the e/tinction coefficient. +ote that this may be computed directly by rearrangement of the %eer4!ambert law to 6 A87 DM1A$#;+ A.9

$his value can be calculated for each reading and the average taken as the value of variant. Remember that this value is a constant. $hus, once calculated, it can subse3uently be used to determine an unknown concentration by one more rearrangement of the %eer4!ambert law 7 6 A8 DM1A$#;+ A.?

Any measured value of A can be readily converted to a corresponding concentration merely by dividing the absorbance by . $he use of the %eer4!ambert law is easy to visuali&e with red food coloring. #t is not as easy to visuali&e, but none the less, "ust as accurate to measure wavelengths of light which are not visible. Dither infra red or ultraviolet can be used. 1= is more useful to biologists since many molecules ,all proteins and nucleic acids- absorb ultraviolet light. $he only changes that need to be made is the use of 3uart& cuvettes instead of glass tubes. Alass absorbs 1= light and thus is inappropriate for use in a 1= spectrophotometer. An instrument capable of using visible light ,usually with a tungsten or halogen lamp source- and 1= light is known as a 1=8=is Spectrophotometer. /PE@$TI/" /F BJB 6PEC. ., $he most commonly encountered spectrophotometer is one manufactured by %ausch and !omb and known as the Spec 9'. $he 9' refers to the band si&e of light that it is capable of producing. #f the instrument is ad"ust to a wavelength of >?', for e/ample, it actually transmits light from >9' nm to >)' nm. $hus, it is not as precise or refined as instruments designed for research purposes where the wavelength may be controlled to a fraction of a nanometer. #t is, however, the standard workhorse instrument found in nearly every lab.
1. $urn on the spectrophotometer and allow 1' minutes for warm up of the instrument

before use.

2. Ad"ust the wavelength to that specificied for the procedure you are using. 3. %e sure the cover is closed on the cuvette holder and use the left knob on the front panel

to ad"ust the dark current such that the meter is reading ' transmittance. At this point, you are simply ad"usting the internal electronics of the instrument to blank out any residual currents. $his ad"usts the lower limit of measurements. #t establishes that no light is e3uivalent to ' transmittance or infinite absorbance.
4. #nsert a clean cuvette containing the blank into the holder. %e sure that the tube is clean,

free of finger prints and that the painted line marker on the tube is aligned with the mark on the tube holder. 7lose the top of the tube holder. $he blank for this e/ercise is the solution containing no dopachrome, but all other chemicals. $he amount of solution placed in the cuvette is not important, but is usually about * ml. #t should appro/imately reach the bottom of the logo printed on the side of the cuvette.
5. Ad"ust the meter to read 1''S transmittance, using the right knob on the front of the

instrument. $his ad"usts the instrument to read the upper limit of the measurements and establishes that your blank will give a reading of 1''S transmittance ,' absorbance-.
6. Remove the blank from the instrument and recheck that your ' transmittance value has

not changed. #f it does, wait a few minutes for the instrument to stabili&e and redo steps 14*. <eriodically throughout the e/ercise, check that calibration of the instrument is stable by re4inserting the blank and checking that the ' and 1''S $ values are maintained.
7. $o read a sample, simply insert a cuvette holding your test solution and close the cover.

Read the transmittance value directly on the scale.

8. Record the S transmittance of your solution, remove the test tube cuvette and continue to

read and record any other solutions you may have. #t is possible to read the absorbance directly, but with an analog meter ,as opposed to a digital read out-, absorbance estimations are less accurate and more difficult than reading transmittance. Absorbance can be easily calculated from the transmittance value. %e sure that you note which value you measureN $B6/@PTI/" 6PECT@%M: Analysis of pigments often re3uires a slightly different use of a spectrophotometer. #n the use of the instrument for determination of concentration ,%eer4!ambert !aw-, the wavelength was pre4

set and left at a single value throughout the use of the instrument. $his value is often given by the procedure being employed, but can be determined by an analysis of the absorption of a solution as the wavelength is varied. $he easiest means of accomplishing this is to use either a dual beam spectrophotometer or a computer controlled instrument. #n either event, the baseline must be continously re4read as the wavelength is altered. $o use a single beam spectrophotometer ,such as the Spec 9'-, the machine is &eroed first, the wavelength is set, the blank is ad"usted and then the sample is inserted and read. $he wavelength is then ad"usted up or down by some determined interval, the &ero is checked, the blank re4 inserted and ad"usted, and the sample re4inserted and read. $his procedure continues until all wavelengths to be scanned have been read. #n this procedure, the sample remains the same, but the wavelength is ad"usted. 7ompounds have differing absorbtion coefficients for each wavelength. $hus, each time the wavelength is altered, the instrument must be recalibrated. A dual beam spectrophotometer divides the light into two paths. ;ne beam is used to pass through a blank, while the remaining beam passes through the sample. $hus, the machine can monitor the difference between the two as the wavelength is altered. $hese instruments usually come with a motor driven mechanism for altering the wavelength, or scanning the sample. $he newer version of this procedure is the use of an instrument which scans a blank, and places the digitali&ed information in its computer memory. #t then rescans a sample and compares the information from the sample scan to the information obtained from the blank scan. Since the information is digitali&ed ,as opposed to an analog meter reading-, manipulation of the data is possible. $hese instruments usually have direct ports for connection to personal computers, and often have built in temperature controls as well. $his latter option would allow measurement of hanges in absorbtion due to temperature changes ,known as hyperchromicity-. $hese in turn can be used to monitor viscosity changes, which is related to the degree of molecular polymeri&ation with the sample. For instruments with this capability, the voltage meter scale has given way to a 7R$ display, complete with graphics and built in functions for statistical analysis.

A temperature controlled 1= spectrophotometer capable of reading several samples at pre4 programmed time intervals is invaluable for en&yme kinetic analysis. An e/ample of this type of instrument is the %eckman C14>'. 6PECIFIC P@/CE*%@E6: For routine use, substances to be monitored by spectrophotometry are often reacted with dyes to form a comple/ that is of another color, usually one easily read within the visible light range, and with precision by an instrument such as the Spec 9'.

E-E@CI6E A.1 B@$*F/@* P@/TEI" $66$K

MA$DR#A!S

!yophili&ed bovine plasma gamma globulin or bovine serum albumin ,%SA7oomasie %rilliant %lue 1 '.1* M +a7l Spectrophotometer and tubes Micropipettes

<R;7DC1RD ,S$A+CARC ASSAJ, 9'41*' L g protein 9''41*'' L g8ml1. <repare a series of protein standards using %SA diluted with '.1* M +a7l to final

concentrations of ' ,blank 6 +a7l only-, 9*', *'', >*' and 1*'' L g %SA8ml. Also prepare serial dilutions of the unknown sample to me measured.
2. Add 1'' L l of each of the above to a separate test tube ,or spectrophotometer tube if

using a Spec 9'-.

3. Add *.' ml of 7oomasie %lue to each tube and mi/ by vorte/, or inversion. 4. Ad"ust the spectrophotometer to a wavelength of *O* nm, and blank using the tube from

step ? which contains ' %SA.

5. .ait * minutes and read each of the standards and each of the samples at *O* nm

wavelength.
6. <lot the absorbance of the standards vs their concentration. 7ompute the e/tinction

coefficient and calculate the concentrations of the unknown samples. <R;7DC1RD ,M#7R; ASSAJ, 141' L g protein

1. <repare standard concentrations of %SA of 1, *, >.* and 1' L g8ml. <repare a blank of

+a7l only. <repare a series of sample dilutions.

2. Add 1'' L l of each of the above to separate tubes ,use microcentrifuge tubes- and add

1.' ml of 7oomasie %lue to each tube.

3. $urn on and ad"ust a spectrophotometer to a wavelength of *O* nm, and blank the

spectrophotometer using 1.* ml cuvettes.

4. .ait 9 minutes and read the absorbance of each standard and sample at *O* nm. 5. <lot the absorbance of the standards vs their concentration. 7ompute the e/tinction

coefficient and calculate the concentrations of the unknown samples.

E-E@CI6E A.. B/C@K P@/TEI" $66$K

MA$DR#A!S

'.1*S ,w8v- sodium deo/ycholate >9S ,w8v- trichloroacetic acid ,$7A7opper tartrate8carbonate ,7$79'S ,v8v- Folin47iocalteu reagent %ovine Serum Albumin ,%SASpectrophotometer and tubes Micropipettes

<R;7DC1RD
1. <repare standard dilutions of %SA of 9*, *', >* and 1'' L g8ml. <repare appropriate

serial dilutions of the sample to be measured.

2. <lace 1.' ml of each of the above into separate tubes. Add 1'' L l of sodium

deo/ycholate to each tube.

3. .ait 1' minutes and add 1'' L l of $7A to each tube. 4. 7entrifuge each tube for 1* minutes at ?,''' /g and discard the supernatant. 5. Add 1.' ml of water to each tube to dissolve the pellet. Add 1.' ml of water to a new tube

to be used as a blank.
6. Add 1.' ml of 7$7 to each tube ,including the blank-, vorte/ and allow to set for 1'

minutes.

7. Add *'' L l Folin47iocalteu to each tube, vorte/ and allow to set for ?' minutes. 8. $urn on and &ero a spectrophotometer to a wavelength of >*' nm. 1se the blank from

Step > to ad"ust for 1''S $.

9. Read each of the standards and samples at >*' nm. 10. <lot the absorbance of the standards vs their concentration. 7ompute the e/tinction

coefficient and calculate the concentrations of the unknown samples. +;$DS $he !owry method depends on the presence of tyrosine within the protein to be measured. $he standard protein must contain appro(imately the same number of tyrosine residues as the sample, or the procedure will be inaccurate. #f there are no tyrosine residues in the sample to be measured, the !owry method of protein determination is useless, and use should be made of the %radford assay. #n general, the %radford assay is the method of choice for protein determinations.

E-E@CI6E A.5 BI%@ET P@/TEI" $66$K

MA$DR#A!S

%iuret Reagent %ovine serum albumin ,%SASpectrophotometer and tubes

<R;7DC1RD
1. <repare standard dilutions of %SA containing 1, 9.*, *.', >.* and 1' mg8ml protein.

<repare serial dilutions of the unknown samples.

2. Add 1.' ml of each of the standards, each sample, and 1.' ml of distilled water to

separate tubes. Add ).' ml of %iuret reagent to each tube. Mi/ by vorte/.
3. #ncubate all of the tubes at ?> G 7 for 9' minutes. 4. $urn on and ad"ust a spectrophotometer to read at a wavelength of *)' nm. 5. 7ool the tubes from Step ?, blank the spectrophotometer and read all of the standards and

samples at *)' nm.

6. <lot the absorbance of the standards vs their concentration. 7ompute the e/tinction

coefficient and calculate the concentrations of the unknown samples.

+;$DS $he %iuret reaction was one of the first for the determination of protein concentration. #t remains as a rapid determination, but is not very accurate. #t is useful during protein separation procedures since there are fewer salt interference reactions than with the %radford or !owry techni3ues. $he color formed is stable for about 149 hours and conse3uently all spectrophotometer readings must be made as soon as possible after the incubation step.

$ppendix ?: @adioacti(e tracers

$he use of radioactive tracers in cell research is an effective and safe means of monitoring molecular interactions. $here is simply no other techni3ue which allows the precision and specificity of radioactive tracers. Radiation is to be taken seriously. At a minimum, its misuse can lead to increased environmental pollution, and at worst can lead to serious long term in"ury. #t can be handled safely, however. Radioactivity is caused by the spontaneous release of either particulate and8or electromagnetic energy from the nucleus of an atom. Atoms are composed of a positively charge nucleus, surrounded by the negatively charged electrons. #n an uncharged atom, the number of orbital electrons e3uals the number of positively charged protons in the nucleus. #n addition, the nucleus contains uncharged neutrons. A proton has a mass of 1.''>5 amu ,Atomic Mass 1nits-, while a neutron has a mass of 1.''IO amu. #f the mass of a helium nucleus is e/amined, there is a difference between the e/pected mass based on its proton and neutron composition, and the actual measured mass. :elium contains two protons and two neutrons in its nucleus, and should have a corresponding mass of ).'??' amu. #t has an actual mass, however, of *.''9I amu. $he difference ,'.'?'9 amu- is the e3uivalent energy of 9I.9 Mev and is known as the binding energy . #t would re3uire 9I.9 Mev to fuse two protons and two neutrons into a helium nucleus, and the fission of the helium nucleus would yield the same energy. #n addition, the electrons orbit the nucleus with precise energy levels. .hen the electrons are in their stable orbits, they are said to be in their ground state. #f the electrons absorb energy ,e.g. from photons-, they "ump to different, yet characteristic orbits and enter the e/cited state. $he energy difference between a ground state and an e/cited state can take the form of an electromagnetic radiation.

$he number of protons in the nucleus of an atom is called the atomic number, while the number of protons plus neutrons is the mass number. $he mass number is appro/imately e3ual to the atomic weight. #n the representation of an atom used in the periodic table of elements, the atomic number is a subscript written to the left of the letter,s- designating the element, while the mass number is written as a superscript to the left. $he chemical identity of an element is determined by the number of protons in the nucleus of the atom. $he number of neutrons may vary, however. Dlements sharing the same number of protons, but having different numbers of neutrons are known as isotopes. :ydrogen, for e/ample, has one proton. All nuclei containing one proton are hydrogen nuclei. #t may have one, two, or three neutrons. $he isotopes of hydrogen would be written as : : : ,in all further references, the atomic number subscript 1 is left off for clarity-. : is the most stable form of hydrogen and is therefore the most abundant ,OO.OI*S of all forms-. : is also a stable form of hydrogen, but less stable than :, and constitutes about '.'1*S of the total hydrogen found. #t is known as deuterium. : is unstable and constitues a very small fraction of the amount of hydrogen available. $ermed tritium , this element readily reorgani&es its nucleus, and is said to decay. $he emission of its sub4atomic particles and energy is therefore known as radioactive decay, or simply radioactivity. Ceuterium is a stable, but heavy isotope of hydrogen, tritium is a radioactive isotope of hydrogen. +ote that each of the three will chemically react as hydrogen. $his is important for tracer work in cell biology. $he substitution of either deuterium or tritium for hydrogen in a molecule will not effect any chemical or physiological changes in the activity of the molecule. $ritium will, however, tag the molecule by making it radioactive. Radiation emissions have several forms. .hen an atom reorgani&es its sub4atomic structure to a more stable form, it may emit neutrons, protons, electrons, and8or electromagnetic waves ,energy-. An alpha particle is 9 protons plus 9 neutrons ,essentially a helium nucleus-. A beta particle is an electron. Aamma rays are electromagnetic energy waves similar to /4rays. $he release of sub4atomic particles and energy, resulting in the change of one element to another is known as radioactivity.

Radioactive elements thus, by their very nature, self destruct. $he loss of their sub4atomic particles is a spontaneous process, and once it has occurred, the element is no longer radioactive. .ith time a percentage of all radioactive elements in a solution will decay. Statistically, it is nearly impossible to predict which individual element will radioactively decay, but we can make a prediction about large numbers of the elements. $hat is, we can say that if we wait 1),''' years, half of the radioactivity in a sample of 7 ,a radioactive isotope of 7- will be lost ,189 remains-. .e then say that 7 has a half4 life of 1),''' years. After a second 1),''' years, half of the remaining half would have been lost, or ?8) of the original amount. %ased on this information, could you predict how long it would take for all radioactivity to have disappeared from the sampleP .ith a half4life of 1),''' years, radioactive carbon will be around for a very long time. $his is why it is used for dating rocks and fossils. #f one makes some assumptions about the activity of the carbon when the fossil was formed, and measures the current level, the age of the fossil may be determined. $he amount of radioactive material is measured by how many nuclei decay each second, and this value is known as the activity. #t is measured in curies Dach radioisotope has three important properties the type of particles emitted, the particle energy, and the half4life. $he energy and kind of decay particle will determine the penetration of the radiation, and therefore determine the degree of shielding necessary to protect the user. $he half4life determines both the remaining activity after storage or use, and the time that the isotope must be stored before disposal. #n cell biology, only a few of the many radioactive elements are used routinely. $he primary elements used are : ,$ritium-, 7 ,7arbon41)-, < 9',<hosphorus4?9-, and # 9',#odine419*-

# 9',#odine41?>-. $he characteristics of each of these are given within the following table2 +ot available at this time $A%!D :.1 Radioactive Sources and Dmission $ypes

ME$6%@EME"T /F */6E .hen alpha or beta particles, or gamma radiation pass through matter, they form ions. $hey accomplish this by knocking electrons from the orbits of the molecules they pass through. .e can monitor the ioni&ation effect by allowing the radiation to pass through dry air and measuring

the numbers of ions formed. $his is most often done by designing a chamber with an electrical charge capacitance, allowing the radiation to pass through the chamber and monitoring the amount of capacitance discharge caused by the formation of ions. $he device is a Aeiger4Mueller 7ounter and has many variations. $he ioni&ing ability is measured in roentgens, and a roentgen is the number of ioni&ations necessary to form one electrostatic unit ,e.s.u.- in 1 cc of dry air. Since the roentgen is a large unit, dosage for cell research use are normally divided into milliroentgens ,mR-. 7uries measure the amount of radioactive decay, roentgens measure the amount of radiation transmitted through matter, over distance. +either unit is useful in determining biological effect, since biological effect implies that the radiation is absorbed by the tissues that are irradiated. $he rad ,radiation absorbed dose- is a unit of absorbed dose and e3uals 1'' ergs absorbed in one gram of matter. $he roentgen is the amount of radiation e/posure in air, while the rad represents the amount of radiation e/posure in tissue. $he two are usually very close in magnitude, however, since for most biological tissues, 1 roentgen produces '.O5 rad. +ot all radioactive emissions have the same penetrating power, however. #f radiation safety ,monitoring of dose- is considered, then the rad is insufficient. A linear4energy4 transfer dependent factor must be defined for each type of emission. An alpha particle, for e/ample, would not travel very far through tissue, but it is 1' times more likely to be absorbed than a gamma wave of the same energy dose. $his factor is known as the Muality Factor ,MF- or Relative %iological Dffectiveness ,R%D-. $he R%D is limited to work in radiobiology, the MF is used in other e/posure monitor schemes. $he use of the MF results in a new parameter, the rem. $he rem is a unit of dose e3uivalent and is e3ual to the product of the MF / rad. *ETECTI/" /F @$*I/$CTI:ITK 7"47.AT7"4 C A#%E6+ $he most common method of measuring radiation e/posure is the use of an ioni&ation chamber. Among the more common forms of ioni&ation chambers are the Aeiger counter and the pocket dosimeter. $he chambers are systems that comprise two electrical plates, with a potential established between them by a battery or other electrical source. #n effect, they function as capacitors. $he plates are separated by an inert gas, which will prevent any current flow between the plates.

.hen an ioni&ing radiation enters the chamber, it induces the formation of an ion, which in turn is drawn to one of the electrical plates. $he negative ions are drawn the the anode ,W plate- while the positive ions are drawn to the cathode ,4 plate-. As the ions reach the plates, they induce an electric current to flow through the system attached to the plates. $his is then e/pressed as a calibrated output, either through the use of a digital or analog meter, or as a series of clicks , by conversion of the current through a speaker. $he sensitivity of the system depends on the voltage applied between the electric plates. Since alpha particles are significantly easier to detect than beta particles, it re3uires lower voltage to detect the high energy alpha particles. #n addition, alpha particles will penetrate through the metal casing of the counter tube, whereas beta particles can only pass through a 3uart& window on the tube. 7onse3uently, ioni&ation chambers are most useful for measuring alpha emmissions. :igh energy beta emissions are able to be measured if the tube is e3uipped with a thin 3uart& window and if the distance between the source of emission and the tube is minimal. A modification of the basic ioni&ation chamber can be made by engineering the tube such that it is miniaturi&ed and such that the tube can be charged to hold a voltage without constantly rebuilding the voltage via a battery. $his gives rise to the pocket dosimeter . $his device is a capacitor, which is charged by a base unit and which can then be carried as a portable unit. $hey are often the si&e and shape of a pen and can be thus carried in the pocket of a lab coat. .hen e/posed to an ioni&ing radiation source, the capacitor discharges slightly. ;ver a period of time, the charge remaining on the dosimeter can be monitored and used as a measure of radiation e/posure. $he dosimeters are usually inserted into a reading device which is calibrated to convert the average e/posure the dosimeter has had directly into roentgens or rems. 1 Since the instrument works by discharging the built up charge, and the charge is upon a thin wire in the center of the dosimeter, it can be completely discharged by the fle/ing of that wire, as it touches the outer shell upon impact. .hen later read for e/posure, the investigor will be informed that they have been e/posed to dangerously high levels of radiation as there will be no charge left in the dosimeter. %esides causing great consternation with the Radiation Safety ;fficer, and a good deal of paper work, it also causes some unrest with the investigator. $he dosimeters should be worn in a location where they can not impact any other ob"ects.Z Since the dosimeters normally lack the fragile and vulnerable 3uart& windows of a Aeiger tube, and carry lower voltage

potentials, they are used for the measurement of /4 ray and high energy gamma radiation, and will not detect beta emmissions. P "T")6AP 7C /75# !ow energy emissions are detected more conveniently through the use of a film badge . $his is simply a piece of photographic film sandwhiched between cardboard and made into a badge which can be pinned or clipped onto the outer clothing of the investigator. $hey can be worn routinely and collected on a regular basis for analysis. .hen the film is e/posed to radiation, it causes the conversion of the silver halide salts to reduced silver ,e/actly as e/posure of the film to light-. .hen the film is developed, the amount of reduced silver ,black- can be measured and calibrated for average e/posure to radiation. $his is normally done by a lab speciali&ing in the monitoring. %ecause of the simplicity of the system, its relative low cost and its sensitivity to nearly all forms of radiation, it is the primary means of radiation e/posure monitoring of personnel. +C74T755AT7"4 C"U4TE6+ For accurate 3uantitative measurement of low energy beta emissions and for rapid measurement of gamma emissions, nothing surpasses the use of scintillation counters. Since they can range from low to high energy detection, they are also useful for the alpha emissions. Scintillation counters are based on the use of light emitting substances, either in solution, or within a crystal. .hen a scintillant is placed in solution with a radioactive source ,li3uid scintillation counter-, the radiation strikes the scintillant molecule, which will then fluoresce as it re4emits the energy. $hus the scintillant gives a flash of light for each radiation particle it encounters. $he counter than converts light energy ,either as counts of flashes, or as an integrated light intensity- to an electrical measure calibrated as either direct counts or counts per minute ,7<M-. #f the efficiency of the system is known ,the S of actual radiatioactive decays that result in a collision with a scintillant-, then disintegrations per minute ,C<M- can readily be calculated. C<M is an absolute value, whereas 7<M is a function of the specific instrument used. !ow energy beta emissions can be detected with efficiencies of )'S or better with the inclusion of the scintillant directly into a cocktail solution. Alpha emissions can be detected with efficiencies in e/cess of O'S. $hus, with a li3uid scintillation counter, very low doses of radiation can be detected. $his makes it ideal for both sensitivity of detection and for safety. #f the system is modified such that the scintillant is a crystal placed outside of the sample chamber ,vial- then the instrument becomes a gamma counter. Aamma emissions are capable of

e/iting the sample vial and entering into a fluorescent crystal. $he light emitted from the crystal is then measured. Aamma counters are usually smaller than li3uid scintillation counters, but are limited to use with gamma emittors. Modern scintillation counters usually combine the functional capabilities of both li3uid scintillation and direct gamma counting. Since all use of radioactive materials, and particularly the e/pensive counting devices are sub"ect to local radiation safety regulations, the specific details for use must be left to the institutional discretion. 1nder no circumstances should radioactive materials be used without the e/press supervision of the radiation safety officer of the institution, following all specific institutional guidelines and manufacturer directions for the instrument used. $%T/@$*I/A@$P?K $he process of locali&ing radioactive materials onto a cell is known as autoradiography. : ,tritium- is used in cell analysis because it is a relatively weak %eta emitter ,thus making it safer to handle- and more significantly, can be locali&ed within cell organelles. 7 and < are also used, but are more radioactive, re3uire significantly more precautions in handling and are inherently less capable of resolving intracellular details. $hey are used at the tissue or organ level of analysis, however. Radioactive isotopes can be incorporated into cellular molecules. After the cell is labeled with radioactive molecules, it can be placed in contact with photographic film. #oni&ing radiations are emitted during radioactive decay and silver ions in the photographic emulsion become reduced to metallic silver grains. $he silver grains not only serve as a means of detecting radioactivity but, because of their number and distribution, provide information regarding the amount and cellular distribution of radioactive label. $he process of producing this picture is therefore called autoradiography and the picture is called an autoradiogram. $he number of silver grains produced depends on the type of photographic emulsion and the kind of ioni&ing particles emitted from the cell. Alpha particles produce straight, dense tracks a few micrometers in length. Aamma rays produce long random tracks of grains and are useless for autoradiograms. %eta particles or electrons produce single grains or tracks of grains. :igh energy %eta particles ,such as those produced by ?9<- may travel more than a millimeter before

producing a grain. !ow energy %eta particles , : and 1) G 7- produce silver grains within a few micrometers of the radioactive disintegration site and so provide very satisfactory resolution for autoradiography. $he site of synthesis of cellular molecules may be detected by feeding cells a radioactive precursor for a short period and then fi/ing the cells. Curing this pulse labeling, radioactivity is incorporated at the site of synthesis but does not have time to move from this site. $he site of utili&ation of a particular molecule may be detected be chase labeling. 7ells are e/posed to a radioactive precursor, radioactivity is then washed or diluted away and the cells allowed to grow for a period of time. #n this case, radioactivity is incorporated at the site of synthesis but then has time to move to a site of utili&ation in the cell. :4thymidine can be used to locate sites of synthesis and utili&ation of C+A. $hymidine, the deo/yribose nucleoside of thymine, can be purchased with the tritium label attached to the methyl group of thymine. $hymidine is specifically incorporated into C+A in $etrahymena. Some organisms can remove the methyl group from thymine, and incorporate the uracil product into R+A. Dven in this case R+A would not be labeled because the tritium label would be removed with the methyl group. Methyl labeled thymidine, therefore, serves as a very specific label for C+A. $his is known as pulse labeling, after which the cells are washed free of the radioactive media. All remaining radioactivity would be due to the incorporation of the thymidine into the macromolecular structure of C+A. $he cells will be fi/ed, covered with a photographic emulsion and allowed to develop . Curing this time, the activity emanating from the : will e/pose the photographic emulsion, causing the presence of reduced silver grains immediately above the location of the radioactive source ,C+A-. $hus, it will be possible to locali&e the newly synthesi&ed C+A, or that which was in the S phase of mitosis during the time period of the pulse labeling. @%BE6 F/@ 6$FE ?$"*BI"A /F @$*I/$CTI:E I6/T/PE6
1. All work with radioactive material must be done in a tray lined with absorbant paper. 2. All glassware and e3uipment contacting radioactive material must be appropriately

labeled and kept inside the tray. $he only e/ception is that microscope slides of labeled

cells may be removed from the tray after the drop of labeled cells has been applied to the slide and allowed to dry.
3. <lastic gloves should be worn when handling radioactive material. 4. All waste solutions containing radioisotopes, all contaminated gloves, paper, etc., must be

placed in appropriate li3uid or dry radioactive waste containers.

A!! #+S$#$1$#;+A! RDA1!A$#;+S M1S$ %D F;!!;.DC A$ A!! $#MDS. 1SD ;F A+J RAC#;A7$#=D D!DMD+$ #S $:D F1!! RDS<;+S#%#!#$J ;F $:D #+S$#$1$#;+ RAC#A$#;+ SAFD$J ;FF#7D A+C #$S CDS#A+A$DC ;FF#7DRS.

$ppendix I: Photograph0
$he use of photography within a cell biology laboratory allows for the capture of data and images for processing at a later time. #t also is an e/cellent means of preparing materials for presentation, either through pro"ection slides, or through illustrations. P?/T/MIC@/A@$P?K <hotographically recording visual images observed through a light microscope is a useful means of obtaining a permanent record of activities. 1sing photomicrographs is the main means of recording electron microscope images. <hotomicrography begins with proper microscope use. #t is important that the microscope be in good operation, clean and centered. #deally, the microscope will be e3uipped with either a trinocular head, or a built in camera port. D/cellent results can be had, however, by attaching a ?* mm single lens refle/ camera to an eyepiece by way of an ine/pensive adapter and a tripod or copy stand. $he adapters are designed to fit in place of the lens, and the tripod is to take the weight of the camera off of the lens tube. $he refle/ camera is useful since it has its own focusing screen. Although you will not get a sharp image in the camera ,because of the ground glass image plate-, the negative will be sharp. Special camera attachments are available from the microscope manufacturers which incorporate e/cellent optical focusing devices, but these are costly. #f a lot of photomicrography is to be performed, however, they are well worth the cost. 1se of a camera on the microscope is straightforward. Merely center the ob"ect to be photographed, focus using the camera viewer ,that is, do not focus using the microscope eye

piece- and depress the camera shutter button. D3uipping the shutter with a shutter release cable will help prevent vibrations. $his assumes that you have the proper e/posure. D/posure and film type are the ma"or problems of photomicrography. For most microscopes using a tungsten lamp source, there is very little light reaching the camera. Film that has a high enough e/posure inde/ ,ASA speed- are too grainy to be used for effective work. #n general, the faster the film the less inherent resolution the film will have. As in all things in photography, a compromise is called for. Moreover, most film sold for general use has a thicker film emulsion than is desirable. $he microscope pro"ects an image of very low contrast, with low light intensity. A thick emulsion tends to lower the contrast even more. $his results in photographs that are all gray, with no highlights ,black and white-. $he tonal range is reduced significantly using general film for photomicrography. 1se Bodak $echnical <an Film at an ASA of 1'' for photomicrography. $his is a thin emulsion film with e/tremely high contrast. $he contrast can even be controlled through the developing process and ranges from :igh ,used for photographing chromosomes-, to moderate ,used for general use- and low ,not used in photomicrography-. $his same film can be used for copy work, since it reproduces images which are black and white. #n general, you can not have too much contrast in a photomicrograph, but it is possible with this film. Another means of increasing contrast is the use of colored filters within the microscope light path. 1se a contrasting color to the ob"ect you wish to photograph. For e/ample, chromosomes stained with aceto4orcein ,dark red- can be contrast enhanced by the use of a green filter. :uman chromosome spreads stained with Aiemsa ,blue- can be enhanced by the use of a red filter. $his trick is also useful for routine viewing as well as photography. $he use of filters will increase the necessary e/posure time. $echnical <an Film is also a relatively slow film. $o establish the proper e/posure, use the light meter built into the camera. #f no light meter is available, you will have to shoot a roll of film and bracket several e/posures to determine which is best. .hen using the built in meter, remember that all light meters are designed to give an image which is a medium gray. #f you have a spot meter, be sure the spot is placed over an ob"ect which should be gray in the final image. #f you have an averaging meter, be sure there is sufficient material in the viewfinder to give a proper average e/posure. #f you do not know whether you have a spot or averaging meter, find out. $his is not trivial. Suppose you

wish to photograph a chromosome spread. $he chromosomes are typically less than 149S of the field of view. $he meter will ad"ust the e/posure such that the white field of light is e/posed as gray, and your chromosomes will appear as darker gray on a gray field 4 in other words, e/tremely murky looking. <erforming karyotype analysis on this type of image is difficult or impossible. %racket all e/posures. ;nce you have determined the appropriate e/posure, be sure to take several photographs. #f, for e/ample, the meter says 185' sec e/posure, take another at 18?' and one at 181''. $his process is known as bracketing the e/posure to ensure that one is correct. Bodak $echnical <an Film is somewhat forgiving for poor e/posure, but only somewhat. For black and white film, if you err on e/posure, overe/pose. $his is the e/act opposite for color positive film ,slides-. For ?* mm cameras, be sure to rewind the film when all e/posures have been completed. P@/CE66I"A After e/posure of the film, the film needs to be processed. <rocessing of black and white film has three steps. Cevelop the film, Stop it from developing, and Fi/ the emulsion so that it is no longer light sensitive. Jou can send your film out for processing, but it will take longer, cost more, and in general you will be more pleased with your own work. $he procedure re3uires about ?'4)' minutes and can be done while cleaning up the lab. PE@F/@M I" T/T$B *$@E"E66: Remove the film from the its cannister and roll it onto the developing tank reel. <lace the film and reel into the developing tank and place the light prrof lid onto the tank. Jou may then turn on the lights. 1 For processing, follow the following steps2
1. Select the proper developer based on the film manufacter@s recommendations. 9 Cilute

the developer as recommended and measure the temperature. For $echnical <an film e/posed at 1'' ASA, use Bodak C41O developer dilution C at a temperature of 91 G 7.

2. <our the diluted developer into the tank and allow the solution to remain for the

recommended time of development , 5 9'minutes for $< film-. $he development time is time and temperature dependent. %e sure to use the correct combination for the film used. Curing the development stage, gently swirl the solution in the tank once every ?' seconds. #t also helps to invert the tank during this stage if the tank is e3uipped with a lid. #f inverted, give the tank a mild rap on the bench top as you set it down. $his will dislodge any trapped air bubbles from the film reel. Co not slam the tank down, it takes only a mild tapN
3. Stop the developing by pouring the developer from the tank and replacing it with Bodak

#ndicator Stop for ?' seconds.

4. <our off the stop bath and replace it with Bodak Fi/er for a period of 54I minutes. Bodak

Rapid Fi/ may be substituted for a period of 9 minutes.

5. <our off the fi/er and replace with a 12) dilution of Bodak :ypo 7learing Agent for 9

minutes.
6. <our off the clearing agent, open the tank and wash the film in running water for a period

of no less than * minutes. After washing, rinse the film in distilled water ,you may use <hotoflo if available- and air dry the film. P@I"TI"A <rocessing a roll of film results in a strip of negatives. For much of the photographic work in cell biology, it is not necessary to do anything with these negatives, e/cept to store them, or use them directly for observation. :olding the negatives up to a light source, and viewing with a hand held magnifying lens is often sufficient. :owever, for presentation work, or for karyotype analysis, where you wish to cut out the chromosomes and rearrange them in some manner, a print needs to be made. <rinting the image is a more time consuming process than developing the film, re3uires a darkroom and is more e/pensive. As a minimum, there is a need for a photographic enlarger, developing trays and a good timer. <hotographic paper is more e/pensive than film. $he basic process involves inserting the negative into the carrier of an enlarger, e/posing a sheet of photographic paper to the pro"ected negative and then processing the paper film in a manner similar to that for the film. $he e/ception is that the Ceveloper is switched to Cektol and the film

is processed through trays rather than on a reel and within a tank. $here are, however, many variations. #f many prints are to be made, a stabili&ation processor can be used. $his works by virtue of the fact that the paper comes with the developer incorporated into the emulsion. #t is activated by passing the paper through a bath of strong alkali, and halted by passing it through a strong acid. $his is done automatically by a machine that resembles an old thermofa/ machine. $he paper is fed into the front and rolls out as a photograph from the back. #t is stable for about 5 months, unless fi/ed with a bath of Bodak Fi/er, which will make it as permanent as any photograph. Dlectron microscopy labs will often have one of these instruments available. For details on printing ,which has many aspects of an art form-, refer to a te/t on photographic processing. M$C@/P?/T/A@$P?K Macrophotography is used to record things too large to be viewed in the microscope. $his is an e/cellent means of making permanent records of electrophoresis gels, bands observed during ultracentrifugation, and whatever else you wish to capture on film. $wo changes are re3uired from the use of photography through a microscope. $he camera must be removed from the microscope and e3uipped with a lens, and secondly, the type of film used must be changed. =ery briefly, there are two means of adapting a ?* mm camera for macrophotography. $he simplest is to purchase a macro lens. #t is preferrable to use a real macro lens rather than a &oom with macro capability. Foom lenses do not have the inherent resolution suitable for macro work. $rue macro lenses are, on the other hand, relatively e/pensive ,bH?''-. An alternative solution is to purchase a lens reversal ring or a set of e/tension tubes for your camera. $hese are both ine/pensive options. $he former, use of a reversal ring, is an e/cellent means of optaining macro capability by simply turning around the normal *' mm lens ,cost about H1'-. $he use of e/tension rings allows some variation in the magnification capabilities ,bH?'-. For occasional photos of electrophoresis gels, it is probably worth investing in a camera specifically designed for that purpose. Fotodyne markets a <olaroid camera gun for this purpose ,bH5''-. At the other e/treme is the use of an M<) <olaroid copy stand ,H9,'''- for macrophotography. $he most economical means of performing routine photography is to

purchase a used 1O5'@s <enta/ Spotmatic ,or e3uivalent- with lens reversal ring and a microscope adapter. 7ombined with a bulk film loader and a changing bag, nearly all the re3uired aspects of film recording of data can be readily accomplished at bargain prices.

$ppendix N: Che#ical Preparations:

$he following is a list of the solutions and chemicals re3uired throughout the laboratory manual. #t is organi&ed alphabetically, and individual e/ercises list the materials needed for that e/ercise. For many solutions, directions are given for a molar solution and the user is left to dilute to the appropriat e concentration for their needs. $here are many vendors of the chemicals listed, and many of the solutions can also be purchased pre4mi/ed. Acetic acid ,M. 5'.'*Alacial acetic acid is OO.5S ,w8v- acetic acid, and is 1>.) M. 1M '.'* + '.O M >S ,w8v)*S ,w8vAdd *>.* ml of glacial acetic acid to I'' ml of water and then make to 1 liter with water. Add 9.I> ml of glacial acetic acid to I'' ml of water and then make to 1 liter with water. Add *) ml of glacial acetic acid to I'' ml of water and then make to 1 liter with water. Add >' ml of glacial acetic acid to I'' ml of water and then make to 1 liter with water. Add )*' ml of glacial acetic acid to *'' ml of water and then make to 1 liter with water.

Acetic Acid8%utanol8.ater ,1*25'29*7ombine 1*' ml of glacial acetic acid, 5'' ml of nbutanol and 9*' ml of water. Aceto4orcein Add 9.' grams of orcein to )* ml of glacial acetic acid. %ring to a boil and continue to heat until completely dissolved. 7ool and add ** ml of distilled water. Filter prior to use. +ote2 Some early investigators added a of an iron salt ,such as ferric citrate- as a mordant. #t tends to increase the intensity of the aceto4orcein stain. $he same reaction can be had by chopping plant material with an older steel ,not stainless- ra&or blade. Acid alcohol

Add 1.' ml of concentrated :7l to 1'' ml of >'S ,v8v- ethyl alcohol. Acid ;rcinol Reagent ,'.1S Fe7l in 1'S :7lAdd '.1 grams of Fe7l to *' ml of 1'S ,v8v- :7l and make to 1'' ml with 1'S :7l. Acrylamide Solutions Acrylamide solutions for <AAD are given as total concentration of acrylamide ,acrylamide W bisacrylamide- and the amount of cross linker ,bisacrylamide-. $his is listed as the $27 ratio. For e/ample, a 1'S gel ,1'S$2*S7- would contain a total of 1' g rams of acrylamide per 1'' ml, and would be composed of * grams of acrylamide and * grams of bisacrylamide. 1sually, a stock solution of ?'S acrylamide is produced containing '.IS bis4acrylamide. Many investigators use ?' grams of acrylamide plus '.I gram s of bis4acrylamide per 1'' ml of water, but 9O.9 grams of acrylamide plus '.I grams of bis would be technically correct. #n practice, it makes little difference since the gels are diluted to 1'S or less. $he ?'S stock solution is filtered through a '.)* L filter and stored at ) G 7 in the dark. For use, the stock solution is diluted with an appropriate buffer ,usually a 9( $ris4:7l-. $he stock solution is stable for about one month. Ciscard after this period. $cr0la#ides in their #ono#eric for# are neurotoxic. Pol0#eri3e all acr0la#ide solutions prior to disposal. SCS, 4mercaptoethanol and a tracker dye ,bromophenol blue- are added at various points. Refer to 7hapter ) for more complete details. Alcian blue ,M. 1?''-

'.''1 M Cissolve '.1? grams of Alcian %lue IA( ,Sigma R A49IOO- in 1'' ml of water. Alcohol ;rcinol Reagent ,1'S orcinol in O*S ethanolCissolve 1.' gram of orcinol in O*S ethanol to a final volume of 1' ml . Alkaline Cistilled .ater Add one pellet of +a;: to 1 liter of distilled water. Alkaline Solution for A4banding Cissolve 9.I grams of +a;: and 5.9 grams of +a7l to a final volume of 1 liter with water.

4Aminosalicic Acid ,<AS M. 1>*.15S ,w8v- Cissolve 5.' grams of <AS to a final volume of 1'' ml with water or buffer. Ammonium acetate ,M. >>.'I'.1 M Add >.>'I grams of Ammonium acetate to a final volume of 1 liter of water. Ammonium persulfate ,M. 99I.91'S ,w8vCissolve 1.' grams of ammonium persulfate to a final volume of 1' ml with water. Mi/ fresh, prior to use as a catalyst for <AAD. +ormally, about *' L l of ammonium persulfate is added to each 1* of gel solution for polymeri&ation.Cissolve '.1? grams o f Alcian %lue IA( ,Sigma R A49IOO- in 1'' ml of water. Ammonium sulfate ,M. 1?9.1)9S ,w8vAdd 9 grams of ammonium sulfate to a final volume of 1'' ml water.

).1M,sat.- '.''1 M Cissolve *)9 grams of ammonium sulfate to a final volume of 1 liter. n4Amyl alcohol ,<entanol 7 : ;: M. II.1*Censity 6 '.I1)) grams8ml '.?I M $he amyl alcohol can be weighed ,??.* grams- or measured volumetrically by using the density. $hat is, ??.* grams liter with water. Amylase, buffered p: >.' 1S ,w8vCissolve '.* grams of amylase to a final volume of *' ml with '.'1 M sodium phosphate buffer, p: >.'. '.I1)) grams8ml or )1.1 ml of n4amyl

alcohol. .eigh or measure the appropriate amount and dilute to a final volume of 1

Ascorbic acid ,M. 1>5.199 mM Cissolve ?*.9 mg of ascorbic acid to a final volume of 1'' ml with water. A$< ,Adenosine triphosphate, M. *'>.91-

* mM Cissolve 9*) mg of A$< to a final volume of 1'' ml with water or buffer.Cissolve ?*.9 mg of ascorbic acid to a final volume of 1'' ml with water. %aker@s Formalin Add 1.' gram of calcium chloride, 1.' gram of cadmium chloride and 1' ml of concentrated formalin to >* ml of water. Make to a final volume of 1'' ml with water. %en&oic acid ,M. 199.19I mM Cissolve OI mg of ben&oic acid to a final volume of 1'' ml with water or buffer. %is4acrylamide ,+,+@4Methylene4bis4acrylamide7ross linker for acrylamide gels. Refer to Acrylamide solutions or 7hapter Four for more details. %iuret Reagent Add 1.*' grams of 7uS; c : ; and 5.' grams of sodium potassium tartrate to *'' ml of water. Separately make ?'' ml of 1'S ,w8v- +a; : by dissolving ?'' grams of +a;: to a final volume of ?'' ml with water. 7ombine the two solutions in a 1 liter volumetric, swirl to mi/ and make up to 1 liter with water. Store the final solution in a dark, plastic bottle. Ciscard if black or red precip itate forms. %ovine Serum Albumin ,%SA$here are many grades of %SA available and care should be taken when using this protein. For routine protein concentration standards, a O54OOS pure fraction ,Sigma R A 91*?- may be used. For tissue culture, R#A, or molecular weight standardi&ation, %SA sh ould be optained which is e/tracted and purified specifically for that purpose. 1S ,w8v- Cissolve '.* grams of %SA to a final volume of *' ml in water or buffer. %radford <rotein Assay $his procedure uses an absorbance shift in an acidic 7oomasie %lue solution. #t is commercially available from <ierce 7hemical 7ompany, Rockford, #llinois as <rotein Assay Reagent, 7at. R 9?9''. #t contains methanol and solubili&ing agents and is very rel iable. #f you wish to make your own, dissolve 1'' mg of 7oomasie %rilliant %lue A49*' in *'

ml of O*S ethanol. Add 1'' ml of I*S phosphoric acid, and bring to a final volume of 1 liter with distilled water. Phosphoric acid is extre#el0 corrosi(e. ?andle &ith care. %romophenol blue ,Sodium salt, M. 5O9.''.''1 ,w8vCissolve 1 mg of %romophenol blue, sodium salt ,Sigma R %>'91- to a final volume of 1'' ml with either water or buffer.

n4%utanol ,7 : ;: M. >).19Censity 6 '.I'OI grams8ml 1.1 M $he butanol can be weighed ,I1.* grams- or measured volumetrically by using the density. $hat is, I1.* grams '.I'OI grams8ml or 1''.> ml of n4butanol. .eigh or

measure the appropriate amount and dilute to a final volume of 1 liter with water. C. elegans Ringers $his is a basic saline solution for nematodes. Cissolve 11.?5 grams of +a :<; c >: ;, ?.' grams of B: <; , '.* grams of +a7l and 1.' gram of +: 7l to a final volume of 1 liter. Ad"ust the p: to >.'. May be autoclaved for sterili&ation. 7alcium chloride ,M. 11'.OO'.''?? M Cissolve '.*99 grams of calcium acetate to a final volume of 1 liter with water or buffer.

7alcium chloride ,M. 11'.OO'.''1 M '.'I M 9S ,w8vCissolve '.111 grams of anhydrous calcium chloride to a final volume of 1 liter with water or buffer. Cissolve I.I>O grams of anhydrous calcium chloride to a final volume of 1 liter with water or buffer. Cissolve 9 grams of anhydrous calcium chloride to a final volume of 1'' ml with water or buffer.

cAM< ,Adenosine monophosphate, cyclic M. ?9O.99'.''1 M Cissolve ?? mg of cAM< to a final volume of 1'' ml with water, buffer

,1mM7arnoy fi/ative

or media.

7ombine 1'.' ml of glacial acetic acid with 5'.' ml of absolute ethyl alcohol and ?'.' ml of chloroform. 7hloroplast homogeni&ation buffer $o )'' ml of distilled water, add ?'.'*I grams of sorbitol, 9.9? grams of sodium pyrophosphate, '.)'> grams of magnesium chloride, and '.1>5 grams of ascorbic acid. Ad"ust the p: to 5.* with :7l and dilute to a final volume of *'' ml. 7hloroplast suspension buffer $o )'' ml of distilled water, add ?'.'*I grams of sorbitol, '.?>9 grams of DC$A, '.1'9 grams of magnesium chloride and *.O*I grams of :D<DS buffer. Ad"ust the p: to >.5 with +a;: and dilute to a final volume of *'' ml. 7hrom alum gelatin ,Subbing solutionCissolve *.' gram of gelatin in 1 liter of boiling water. 7ool and add '.* grams of potassium chrome alum ,7rB,S; - c 19: ;-. Store in refrigerator. $o use, dip clean slides into the solution and dry in vertical position in a dust free location. 7itric acid ,: 7 : ; c : ; M. 91'.1)'.1 M Cissolve 91.'1 grams of citric acid to a final volume of 1 liter. 7itrate buffer ,Sodium phosphate47itrate buffer'.''1 M <: ).I <: ?.5 <: ).9 <: *.) <: 5.' <: 5.5 Add )O? ml of '.9 M +a :<; to *'> ml of '.1 M citric acid. Add ?99 ml of '.9 M +a :<; to 5>I ml of '.1 M citric acid. Add )1) ml of '.9 M +a :<; to *I5 ml of '.1 M citric acid. Add **>.* ml of '.9 M +a :<; to ))9.5 ml of '.1 M citric acid. Add 5?1.* ml of '.9 M +a :<; to ?5I.* ml of '.1 M citric acid. Add >9>.* ml of '.9 M +a :<; to 9>9.* ml of '.1 M citric acid.

<: >.9 <: >.I

Add I5O.* ml of '.9 M +a :<; to 1?'.* ml of '.1 M citric acid. Add O*>.* ml of '.9 M +a :<; to )9.* ml of '.1 M citric acid.

7obaltous +itrate ,M. 1I9.O59S ,w8v7olcemid 1' L g8ml Cissolve 1' g of colcemid per ml of saline or culture medium. 7oomasie blue ,7oomasie %rilliant %lue R9*'Cissolve 9.*' grams of 7oomasie %rilliant %lue R9*' to a final volume of '.9*S 1 liter with 9'S ,w8v- trichloroacetic acid ,$7A-. Some investigators use a *41-. 7opper sulfate ,7uS; *: ; M. 9)O.5I'.*S ,w8v'.*S ,w8vCissolve '.* grams of copper sulfate to a final volume of 1'' ml with water. 7opper tartrate8carbonate ,7$7Cissolve '.* grams of copper sulfate and 1.' gram of potassium sodium tartrate to a final volume of 1'' ml with water. 7ombine 1.' ml of this solution with *' ml of 9S +a 7; in '.1 + +a;:. Must be made fresh, prior to use. Stock solutions are stable. 7rystal =iolet Cissolve '.1 grams of crystal violet and '.9* ml of glacial acetic acid to a final volume of 1'' ml with water. C7M1 ,?4,?,)4Cichlorophenyl-41,14Cimethylurea M. 9??.11 / 1' M '.''1 M Cissolve 9.? mg C7M1 to a final volume of 1'' ml with water or Cissolve '.1? grams of Alcian %lue IA( ,Sigma R A49IOO- in 1'' ml of water. ,w8v-'.''1 M '.9*S solution of 7oomasie %lue in methanol4water4glacial acetic acid ,*4 Cissolve 9.' grams of cobaltous nitrate ,he/ahydrate is very soluble- to a final volume of 1'' ml with water. Beep well closed in a cool place.

buffer. * / 1' M'.''1 M Cilute the 1 / 1'4 M solution 189'' prior to use.

Cichlorophenolindophenol ,C7<#< M. 9O'.1'.''9* M Cissolve >? mg of C7<#< to a final volume of 1'' ml with water or buffer. '.'''1 M Cissolve 9.O mg of C7<#< to a final volume of 1'' ml with water or buffer. Cinitrophenol ,C+< M. 1I).111I.) mgS Cissolve 1I.) mg of 9,)4dinitrophenol to a final volume of 1'' ml with water or buffer.

Cische diphenylamine reagent Cissolve *'' mg of diphenylamine in )O ml of glacial acetic acid. Add 1.' ml of concentrated :7l. Cithiothreitol ,7leland@s Reagent M. 1*).?'.'1 M Cissolve 1*) mg of dithiothreitol to a final volume of 1'' ml with water or buffer. Cithiothreitol is available from Sigma 7hemical 7o., St. !ouis, 7at R C'5?9. Cithioerythritol may be substituted.

C;<A ,?4,?,)4Cihydro/yphenyl-4!4alanine M. 1O>.1OI mM Cissolve 1*I mg of !4C;<A to a final volume of 1'' ml with water or buffer. +ote that the ma/imum solubility of C;<A in water is 15* mg81'' ml ,I.? mM-.

DC$A ,Dthylenediaminetetraacetic acid M. 9O9.9)1 Cissolve 9O9.9) grams of DC$A, free acid to a final volume of 1 liter. #f the more ad"usted with acetic acid or +a;:. For corresponding concentration dilutions, multiply the weight in grams by the desired molarity. For e/am ple, for 1' mM DC$A, multiply 9O9.9) ( '.'1' to obtain 9.O9 grams of DC$A per liter. DA$A ,Dthylene Alycol4bis, aminoethyl Dther- +,+,+@,+@4$etraacetic Acid M. ?I'.)1 mM issolve ?I' mg of DA$A to a final volume of 1 liter with water or buffer.

M soluble disodium salt of DC$A is used, ad"ust the weight accordingly. $he p: can be

Dosin '.*S ,w8v- Cissolve '.* grams of Dosin J in 1'' ml of water. Dthanol ,7 : ;: M. )5.'>Censity 6 '.>IO? gm8ml *'4 O*S ,v8vSince O*S ethyl alcohol is less e/pensive and easier to store than absolute, these dilutions should be made with O*S ethyl alcohol. 1nless otherwise stated, denatured alcohol works as well as the more e/pensive non4denatured. A simple way to make the S so lution is to use the appropriate amount of O*S ethanol and dilute to O*' ml instead of 1 liter. For e/ample, to make a *'S ,v8v- solution, measure out *'' ml of O*S ethyl alcohol and dilute to a final volume of O*' ml with water. For a >'S solution, meas ure >'' ml of ethyl alcohol and dilute to O*' ml with water. Absolute ethanol should be used directly as 1''S ethanol. #t is important for histology that this be truly 1''S. Since it is hydroscopic ,it absorbs water from the air-, do not assume it is abso lute unless it is sealed or treated to ensure no water. $o test, add a drop to a sample of /ylol. #f any cloudiness occurs, the alcohol is not absolute. I.* M $he ethanol can be weighed ,?O1.5 grams of absolute, )19.9 grams of O*S ,v8vor measured volumetrically by using the density. $hat is, ?O1.5 grams grams8ml or )O5.1 ml of absolute ethanol. 1sing O*S, )19.9 grams final volume of 1 liter with water. Dthanol4acetic acid fi/ative for histology ,?21$o >* ml of absolute alcohol, add 9* ml of glacial acetic acid. Must be made fresh, "ust prior to use. Fetal 7alf Serum ,F7S.hile it is possible to prepare your own serum from whole blood, it is easier ,and saferto purchase F7S from a reputable supplier. 7ommercial sources are free of mycoplasma, pre4sterili&ed and controlled for the presence of antibodies. $here are a n umber of serum substitutes available on the market and these may be less e/pensive when storage is '.>IO? '.>IO?

grams 8ml or *99.9 ml. .eigh or measure the appropriate amount and dilute to a

considered. Suppliers include Aibco, Flow !aboratories, B7 %iological and Sigma 7hemical 7o. Folin47iocalteu Reagent $his is usually purchased premi/ed, since it is difficult to make. Also known as 9+ Folin4 phenol reagent. Aiemsa stain <repare a stock solution by dissolving ?.I grams of giemsa powder in 9* ml of glycerin. :eat gently with stirring for about 9 hours at 5' G 7. 7ool and add >* ml of methanol ,neutral, acetone free-. For a working solution, dilute the stock solution 181' with water before use. For chromosome banding, combine *.' ml of stock Aiemsa, ?.' ml of absolute methanol, ?.' ml of '.1 M citric acid and IO ml of distilled water. Ad"ust the p: of the solution to 5.5 with +a :<; . Alucose ,M. 1I'.151'S ,v8vCissolve 1' grams of C4glucose ,de/trose- in a final volume of 1'' ml with water or buffer.

Alutaraldehyde ,A$A* S A$A is usually supplied as a 9*S or *'S ,w8v- solution. #t is used for electron microscope fi/ation as a *S solution in a buffer. For routine use, add 9' ml of 9*S A$A to I' ml of '.9 M sodium cacodylate buffer, p: >.). Alycerol ,M. O9.'O1'S ,v8vIM $o 1' ml of glycerol ,glycerine- add enough water to make a final volume of 1'' ml. .eigh >?.5> grams of glycerol and add to a final volume of 1'' ml. Alternatively, measure )OO.1 ml of glycerol and make to a final volume of 1 liter ,the density of glycerol at room temperature is 1.)>5- with water or buffer. For I M glycerol in M$ buffer, make a 9( M$ buffer for use as the diluent. Alycine ,M. >*.'>-

'.1O9 M Cissolve 1.)) grams of Alycine to a final volume of 1'' ml with water or buffer. Aram@s iodine Cissolve '.?? grams of iodine and '.5> grams of potassium iodide to a final volume of 1'' ml with water. :D<DS ,+4]94:ydro/yethyl^pipera&ine4+@4]94ethanesulfonic acid^ M. 9?I.?*' mM Cissolve 11.O9 grams of :D<DS, free acid to a final volume of 1 liter. #f hemisodium salt is used, ad"ust weight accordingly. Co not use sodium salts unless specified. :emisodium salt contains '.* moles of sodium for each mole of :D<DS. 1' mM Cissolve 9.?I grams of :D<DS, free acid in O'' ml of water. Ad"ust the p: with p:>.5 +a;: or :7l to >.5. Ad"ust the final volume to 1 liter with distilled water. :ydrochloric Acid ,:7l M. ?5.)57oncentrated :7l has a molarity of appro/imately 11.5. :7l is a gas, which is soluble in water and which comes in the form of concentrated reagent grade :7l. $his solution is appro/imately ?54?IS ,w8v- :7l. $o make a 1 + solution, add I5 ml of concentrated :7l to I'' ml of water and dilute to a final volume of 1 liter. For '.1 +, dilute the 1 + by a factor of 1'. For S solutions, note that li3uid :7l is only ?IS :7l, thus a 1S solution would re3uire 9.5 ml of concentrated :7l ,18.?I- per final volume of 1'' ml. Tanus Areen % '.'1S,w8v- Cissolve 1' mg of Tanus Areen % in 94? ml of absolute ethanol. Cilute to a final volume of 1'' ml with water. Bnudson Media Bnudson () Stock2 7a,+; - c ): ; ,+: -S; MgS; c >: ; Cistilled : ; ).' grams 9.' grams 1.' gram 1.' liter

%* Minor Dlements2 : S; Mn7l c ): ; : %; FnS; c >: ; 7o7l c 5: ; 7u7l c 9: ; +a Mo; c 9: ; Cistilled : ; '.* ml 9.* grams 9.' grams *' mg ?' mg 1* mg 9* mg 1.' liter

Ferric 7itrate2 Fe7 : ; c *: ; Cistilled : ; 9.* grams 1'' ml

Stock <hosphate2 B :<; 9* grams

Cistilled : ; 1'' ml 1( Media Add 9*' ml of Bnudson () to >*' ml of distilled water. Add '.* ml of %* Minor Dlements, '.* ml of Stock <hosphate, and '.) ml of Ferric citrate. Ad"ust the p: to *.* with :7l, add 1* grams of agar and heat to dissolve. Autoclave and pour into plates.[ brZ +ote2 9.*' grams of sucrose may be added prior to ad"ustment of the p:, if desired. #t is not necessary for germination of spores, but adds an organic source for mutants and abnormal fern growths. #t also increases the need for subse3uent aseptic techni3ue . Brebs <hosphate Ringers ,B<R<repare each of the following separately2

'.O'S ,w8v- +a7l 1.1*S ,w8v- B7l 1.99S ,w8v- 7a7l ?.I9S ,w8v- MgS; c : ; '.1 M phosphate buffer, p: >.) ,1>.I grams of +a :<; c : ; W 9' ml of 1 + :7l, diluted to 1 liter$o mi/, combine 9'' ml of +a7l, I ml of B7l, 5 ml of 7a7l and 9 ml of MgS; . 7arefully, and with constant stirring, add )' ml of phospha te buffer. !<S buffer ,!ower <ad Solution bufferCissolve 1.* grams of B7l, '.* grams of Mg7l , and '.* grams of steptomycin sulfate in *'' ml of water. Add )' ml of 1 M phosphate buffer, p: 5.* and dilute to 1 liter with water. Magnesium chloride ,Mg7l M. O*.9?1 mM Cissolve O*.9 mg of magnesium chloride per final volume of 1 liter. ) mM Cissolve '.?I1 grams of magnesium chloride per final volume of 1 liter. 1' mM +ote2 Cissolve '.O*9 grams of magnesium chloride per final volume of 1 liter.

'.1 M Cissolve O.*9? grams of magnesium chloride per final volume of 1 liter. A single stock solution of 1 M Mg7l can be mi/ed by dissolving O*.9? grams of magnesium chloride to a final concentration of 1 liter with water, and all dilutions made appropriately from this stock solution. Magnesium sulfate ,MgS; M. 19'.?O*S ,w8v- Cissolve *.' grams of magnesium sulfate to a final volume of 1'' ml with water or buffer. Mayer@s hemato/ylin <urchase commercially or mi/ with either of the following procedures2

A. Cissolve 1.' gram of hemato/ylin in 1' ml of absolute ethanol. Cissolve 9' grams of potassium alum ,BAl,S; - c 19: ;- in 9'' ml of water. #n a chemical hood, with protection against e/plosion, bring the potassium alum solution to a boil and add hemato/ylin8ethanol mi/ture. 7ontinue to boil for appro/imately 1 minute. Add '. * grams of mercuric o/ide and cool rapidly. Add '.* ml of glacial acetic acid. Filter before use. $his mi/ture is stable for about two months. %. Alternatively2 Cissolve *.' grams of hemato/ylin in *' ml of absolute ethanol and add to 5*' ml of warm water. :eat gently until the hemato/ylin dissolves and then add ?'' ml of glycerin, '.? grams of sodium iodate and 9' ml. of glacial acetic acid. 7o ol and make volume up to 1 liter with distilled water. Filter before use. [dt Z4Mercaptoethanol ,M. >I.1?-[8dt '.* M *S ,w8v1se *.' grams or ).15> ml in a final volume of 1'' ml of water or buffer. Censity 6 1.9 grams8ml. 1se either ?.O1 grams ;R ?.95 ml of mercaptoethanol in a final volume of 1'' ml of water or buffer.

MDS ,94,+4Morphilino-ethanesulfonic acid M. 1O*.9'.1 M Cissolve 1.O*9 grams of MDS to a final volume of 1'' ml with water or buffer. Methanol ,7: ;: M. ?9.')Censity 6 '.>O1) grams8ml 99 M $he methanol can be weighed ,>').O grams- or measured volumetrically by using the density. $hat is, >').O grams water. Methanol8Acetic Acid ,for fi/ing proteins in acrylamide gels)*S219S Add 19' ml of glacial acetic acid to )*' ml of methanol and dilute to a final volume of 1 liter with water. Methanol8Acetic Acid ,for destaining or fi/ing proteins in acrylamide gels'.>O1) grams8ml or IO'.> ml of methyl alcohol.

.eigh or measure the appropriate amount and dilute to a final volume of 1 liter with

*S2>S Add >' ml of glacial acetic acid to *' ml of methanol and dilute to a final volume of 1 liter with water. Methyl green '.9S ,w8vCissolve '.9 grams of methyl green to a final volume of 1'' ml with '.1 M acetate buffer, p: ).9. Acetate buffer ,'.1 M p: ).9- is prepared by dissolving .?51 grams of sodium acetate ,trihydrate- in appro/imately I' ml of water. Add .)9 ml of glacial acetic acid and ad"ust the volume to 1'' ml with water. Microtubule buffer ,M$ bufferCissolve 1O.*9 grams of MDS in I'' ml of distilled water. Add '.?I' grams of DA$A and )>.59 grams of Mg7l . Ad"ust the p: to 5.) with :7l or +a;: and dilute to a final volume of 1 liter with disti lled water. Minimum essential medium ,MDMFor all purposes of this manual, MDM refers to Dagle@s MDM. .hile it is possible to mi/ this medium, it is infinitely easier ,and less e/pensive- to purchase the media pre4mi/ed from any number of commercial sources ,Aibco, Flow, B7 %iological, Sigma 7hem ical-. $he ingredients are listed in $able 19.1. #t is essential that chemicals of the highest purity are used throughout. +A agar ,+ematode Arowth agarCissolve ?.' grams of +a7l, 9.* grams of peptone, and 1> grams of agar in a final volume of 1 liter. %oil to dissolve the agar, autoclave to sterili&e. Meanwhile, prepare separate sterile solutions of2 1 M 7a7l 9 mg8ml uracil 1' mg8ml cholesterol in ethanol 1 M <otassium phosphate buffer, p: 5.' 1 M MgS;

1sing proper sterile techni3ue, cool the agar solution slightly and add 1 ml of 7a7l , 1 ml of uracil, '.* ml of cholesterol, 9* ml of phosphate buffer, and 1 ml of MgS; . Swirl to mi/ all ingredients and pour plates. 4+itrophenyl phosphate ,M. 95?.1'.'* M '.IS ,w8v+ote2 Cissolve 1.?9 grams of 4nitrophenyl phosphate to a final volume of 1'' ml with water or buffer. Cissolve '.I grams of 4nitrophenyl phosphate to a final volume of 1'' ml of water or buffer. Sigma 7hemical 7o., St. !ouis, supplies this compound as Sigma 1'), <hosphatase Substrate, 7at. R 1')4'. ;smium tetro/ide ,;S; M. 9*).91S ;smium tetro/ide is a gas which is used in solution for DM preservation. #t is best purchased in sealed vials of 9 ml of )S ;s;8)8. For use, add 5.' ml of water or buffer to the 9.' ml of )S osmium tetro/ide. Seal in a tightly sealed container, wrapped with aluminium foil and keep in the refrigerator. 1se of a fume hood is mandatory when using ;s;8)8. ;smium tetro/ide will rapidly fi/ the nasal passages and e/posed cornea if not properly vented. #t should be handled with e/treme care. <erchloric acid ,<7A M. 1''.)>9S ,w8vCissolve 9.' grams of <7A to a final volume of 1'' ml with water or buffer. <ercoll 7olloidal <=< coated silica. Available from Sigma 7hemical 7o., St. !ouis. 7at. R< 15)). <eriodic Acid ,<eriodate 1sed for <AS reactionCissolve '.5 grams of periodic acid in 1'' ml of water and add '.? ml of concentrated nitric acid. <hena&ine methosulfate ,<MS M. ?'5.?)Mutagen and irritant.

'.'??S ,v8vAdd ?? L l of phena&ine methosulfate to O' ml of water or buffer and make up to 1'' ml final volume. Must be made immediately prior to use. <henol mi/ture 7ombine *** ml of a3ueous phenol ,or *'' grams of phenol crystals plus ** ml of waterwith >' ml of mcresol. Add '.* grams of I4hydro/y3uinoline. Phenol &ill cause se(ere +urns and readil0 dissol(es all plastic and ru++er co#pounds. %se extre#e caution &hen handling this co#pound. 4<henylenediamene o/alate ,<<C; M. 1OI.1I'.'9S ,w8v- Cissolve 9' mg of <<C; to a final volume of 1'' ml with water or buffer. <hosphate buffered saline ,<%SMi/ 1'' ml 7a 8Mg fr ee 1'( <%SA with I'' ml of distilled water. Separately,

dissolve '.1 gram of magnesium chloride and '.1 gram of anhydrous calcium chloride to a final volume of 1'' ml with water. .ith constant stirring, slowly add the magnesium8calcium chloride solution t o the diluted <%SA. #f a precipitate forms, start over, and add slower with continuous stirring. 7a 8Mg free <hos phate buffered saline 4 1'( ,1'( <%SA-

Cissolve I' grams of +a7l, 9.' grams of B7l, 1*.' grams of Cibasic sodium phosphate and 9.' grams of Monobasic potassium phosphate in 1 liter of distilled water. $his makes a 1'( solution of 7a '#g +tore in a refrige rator. Phosphate buffere& saline;T0een 22 D4.3E #i( P%+ an& a&& 2.1G D*'*E T0een 22. Phytohemaglutinin DP AE A*ailable as :i&ney bean lectin. 7t is typically use& as a stoc: solution of 12;22 g'ml in balance& salt solution. /or tissue culture it must be col& sterili!e& prior to use. Potassium chlori&e D<Cl #8 A4.??E 1 Cissolve >).** grams of B7l to a final volume of 1 liter with water or buffer. For free phosphate buffere& saline. -ilute 1112 prior to use.

M other concentrations, multiply the weight by the re3uired molarity. For e/ample, for '.1*' M ,1*' mM-, use '.1*' ( >).**, or 11.1I? grams of B7l in 1 liter of water or buffer. 1se half as much to obtain '.'>* M for karyotyping. Potassium cyani&e D<C4 #8 @?.11E I mM Cissolve *9 mg B7+ to a final volume of 1'' ml with water or buffer. Potassium phosphate$ monobasic D< P" #8 13@.2>E

'.'1M Cissolve 1.?5 grams of monobasic potassium phosphate to a final volume of 1 liter with water. Potassium phosphate$ &ibasic D< P" #8 1A4E

'.'1 M Cissolve 1.>) grams of dibasic potassium phosphate to a final volume of 1 liter with water. Potassium phosphate buffer '.'1M <repare *'' ml of '.'1 M B :<; and *'' ml of '.'1 M B: <; <lace the p: >.) B9:<; onto a magnetic stirrer and insert a p: electrode. Ad d the B: <; slowly to ad"ust the p: to >.). Potassium hy&ro(i&e D<" #8 ?@.12E '.* + 1'S ,w8vCissolve 9I.'* grams of B;: to a final volume of 1 liter with water. Cissolve 1' grams of B;: to a final volume of 1'' ml with water. Store in a plastic container. " H4 " #8 232.23E

Potassium so&ium tartrate D6ochelle salt <4aC n;Propanol DC " #8 @2.11E

Censity 6 '.I'?* grams8ml ?M $he n4propanol can be weighed ,1I'.? grams- or measured volumetrically by using the density. $hat is, 1I'.? grams '.I'?* grams8ml or 99).) ml

of n4propanol. .eigh or measure the appropriate amount and dilute to a final volume of 1 liter with water. Protein %uffer -issol*e 1.4@ grams of < 0ater. Pyronin C DacetoneE '.5S ,w8v6ibonuclease '.1S ,w8vCissolve 1' mg of pancreatic ribonuclease type A in 1' ml of water or buffer. 1se for en&yme treatment of histological sections by floating '.*41.' ml of this solution onto the section, with the slide set into a covered petri plate. +afranin -issol*e 2.? grams of +afranin " in 12 ml of >?G ethanol an& &ilute to 122 ml 0ith 0ater. +aline D4aClE '.I*S ,w8vSaline refers to a solution of +a7l, with the most common usage for that which is isotonic to mammalian blood cells, notable a '.I*S or '.OS solution. $o mi/, dissolve I.* grams of +a7l to a final volume of 1 liter with water. +aline citrate D1'12 &ilution of ++CE -issol*e 2.3A3 grams of 4aCl an& 2.2>4 grams of so&ium citrate to a final *olume of 1 liter 0ith 0ater. +aline citrate buffer D++CE 9'( #t is common to prepare this buffer as a stock 9'( solution, to be diluted to 9(, 1( or '.1( prior to use. $o prepare a 9'( stock solution, dissolve 1>* grams of +a7l and II grams of sodium citrate in O'' ml of water. Ad"ust the p: to >.' with 1 + :7l and bring to a final volume of 1 liter. For use, as a 1( SS7, dilute 1 part 9'( stock with 1O parts distilled water. For a 9( Cissolve '.5 grams of pyronin J in 1'' ml of acetone. P" an& 2.>2 grams of <2 P" in 32 ml of &istille& 0ater.

A&& 2.? grams of crystalline serum albumin an& a&Fust the *olume to a final 122 ml 0ith

SS7, dilute 1 part 9'( stock with O parts water. +chiffIs 6eagent -issol*e 2.3 grams of basic fuchsin in 3? ml of &istille& 0ater. A&& 1.> grams of so&ium metabisulfite an& 1?.2 ml of 1 4 Cl. Place the solution in separatory funnel an& sha:e at 2 hour inter*als for a perio& of appro(imately 12 hours. A&& 222 grams of a cti*ate& charcoal$ sha:e for 1 minute an& filter the clear solution. 7f the solution is still pin:$ a&& another 122 grams of charcoal an& sha:e for an a&&itional minute. /ilter an& store in a &ar: bottle. +olution shoul& be clear Dno pin: colorationE for use. +cott solution -issol*e 2.2 grams of so&ium bicarbonate an& 22.2 grams of magnesium sulfate in 0ater to a final *olume of 1 liter. A&& a pinch of thymol to retar& the gro0th of mol&s. +-+ 6efer to +o&ium lauryl sulfate. 1J +-+;Electrophoresis 6unning %uffer -ilute ?J Tris;)lycine buffer to 1J an& a&& 1.2 gram of +-+ per liter of 1J Tris;)lycine. The p shoul& be 3.3 after &ilution. 2J +-+ +ample %uffer -issol*e 1.?2 grams of Tris base$ 2.2 grams of +-+$ 22 ml of glycerin$ 2.2 ml of ;mercaptoethanol an& 1 mg of bromophenol blue to a final *olume of 122 ml 0ith 0ater. +iliconi!e& pipettes Pasteur pipettes can be siliconi!e& by soa:ing them in a bea:er containing ?G D*'*E &ichloro&imethylsilane in chloroform for about 1 minute. 6emo*e$ &rain an& rinse se*eral times 0ith &istille& 0ater. %a:e the pipettes at 132 K C for 2 hours an& cool before use. Dichlorodimethylsilane and chloroform are both toxic and olatile. !se only in "ro"er fume hood and #ee" all flames a$ay from $or# area. Insure that all silicone and chloroform are remo ed from glass$are before "lacing in an o en. +il*er nitrate solution Dfor electrophoresis stainingE -issol*e 2.1? grams of 4a" in 1?2 ml of 0ater. A&& 3.? ml of concentrate& 4 "

an& bring to a *olume of 222 ml. +eparately$ &issol*e 2.2 grams of sil*er nitrate in a final

*olume of 12 ml. 8ith constant stirring$ a&& 3.2 ml of the sil*er nitrate to the 222 ml of 4a" '4 " .

This solution shoul& be prepare& imme&iately prior to use$ an& use& 0ithin 32 minutes. Dis"ose of this solution $ith co"ious flushing. It becomes ex"losi e u"on drying. +# agar me&ium D+lime #ol& me&ium of +ussmanE -issol*e 12.2 grams of glucose$ 12.2 grams of peptone$ 1.2 gram of yeast e(tract$ 1.2 gram of #g+" $ 1.? grams of < +o&ium acetate D#8 32.24E 1M Cissolve I9.') grams of sodium acetate to a final volume of 1 liter with water or buffer. '.'9 M Cissolve 1.5) grams of sodium acetate to a final volume of 1 liter with water or buffer. +o&ium acetate buffer 1 M p: *.> $o O9* ml of 1 M sodium acetate, add >* ml of 1 M acetic acid. P" $ 1.2 gram of <2 P" an& 22.2 grams of agar to a

final *olume of 1 liter. eat to &issol*e the agar$ autocla*e an& &ispense to petri plates.

+o&ium a!i&e D#8 @?.22E '.'1 M Cissolve '.'5* grams of sodium a&ide to a final volume of 1'' ml with water. '.?OS ,w8v- Cissolve '.?O grams of sodium a&ide to a final volume of 1'' ml with water or buffer. +o&ium barbitol '.9S ,w8v- Cissolve '.9 grams of sodium barbitol to a final volume of 1'' ml with water. +o&ium bicarbonate D4a C" #8 34.2E '.1 M Cissolve '.I) grams of +a:7; to a final volume of 1'' ml with water or buffer. +o&ium caco&ylate buffer '.9 <repare a '.9 M solution of cacodylic acid, sodium salt ,M. 1*O.O1-. Cissolve ?.9'

M p: >.)

grams of cacodylic acid, sodium salt to a final volume of 1'' ml with water. Ad"ust the p: to >.) with :7!.

Cacodylic acid contains arsenic. %andle "ro"erly. +o&ium carbonate D#8 12@.2E 9S ,w8vCissolve 9.' grams of sodium carbonate to a final volume of 1'' ml with '.1 + +a;:. '.1 + +a;: 1sed for !owry <rotein assay. +o&ium chlori&e D#8 ?3.44E M For a molar solution of sodium chloride, dissolve *I.)) grams of +a7l to a final volume of 1 liter with water or buffer. For corresponding dilutions, multiply the weight by the molarity re3uired. For e/ample, for '.'* M, multiply *I.)) by '.'* or 9.O9 grams8liter. S For S solutions, they are invariably w8v. For a 1 S ,w8v- solution, dissolve 1.' gram of +a7l to a final volume of 1'' ml with water or buffer. Multiply the weight by a corresponding change in S for other concentrations. 9'',?'',)'' m;sM ;smoles for +a7l are calculated as twice the molar concentration. $hus, a 9'' m;sM solution would be .1'' M +a7l. !ikewise, ?'' m;sM would be .1*' M and )'' m;sM would be .9'' M +a7l. +o&ium citrate D#8 2>4.12E '.'O M Cissolve 9.5* grams of sodium citrate to a final concentration of 1'' ml with water. +o&ium citrate'formal&ehy&e Dfor sil*er staine& proteinsE -issol*e ?.2 grams of so&ium citrate in 322 ml of 0ater$ a&& ?.2 ml of concentrate& formalin D3AG formal&ehy&e solutionE an& &ilute to 1 liter 0ith 0ater. +o&ium &eo(ycholate D-eo(ycholic aci&$ so&ium salt #8 3>2.?3E '.1*S Cissolve 1*' mg of deo/ycholic acid, sodium salt to a final volume of 1''

,w8v-

ml with water. " #8 242.1@E

+o&ium &ithionite D4a + " H 2 .1 mg8ml

Cissolve 1' mg of sodium dithionite in 1'' ml of water "ust prior to use. Alternatively, to reduce a solution, the dry powder can be added as needed. Sodium dithionite should be stored at 49' G 7 .

+o&ium fluori&e D4a/ #8 42.2E '.1 M Cissolve ).9 grams of +aF to a final volume of 1 liter with water. +o&ium lauryl sulfate D+-+ or +5+ #8 233.33E '.1S ,w8v1'S ,w8vCissolve '.1 grams of SCS to a final volume of 1'' ml with water or buffer. Mi/ by gentle stirring, do not shake. Cissolve 1' grams of SCS to a final volume of 1'' ml with water.

SDS should not be inhaled in its "o$der form. &hen $eighing' use a mas#' or better' a hood. +o&ium malonate D#8 124.2E '.5 M Cissolve 9.)O grams of malonic acid, sodium salt, to a final volume of 9* ml with water or buffer. +o&ium perchlorate D4aCl" H " #8 142.4AE

1 M Cissolve 1).'1 grams of sodium perchlorate to a final volume of 1'' ml with water or buffer. +o&ium phosphate$ monobasic D4a 1M P" " #8 13A.>>E

Cissolve 1).'1 grams of sodium perchlorate to a final volume of 1'' ml with water or buffer.

'.'1 M Cissolve 1.?I grams of monobasic sodium phosphate to a final volume of 1 liter. +o&ium phosphate$ &ibasic D4a P" H A " #8 2@3.2AE

1M '.9 M

Cissolve 95I.'> grams of dibasic sodium phosphate to a final volume of 1 liter. Cissolve *?.51 grams of dibasic sodium phosphate to a final volume of 1 liter.

'.'1 M Cissolve 9.5I grams of dibasic sodium phosphate to a final volume of 1 liter. +o&ium phosphate buffer These are the most common buffers use& in biology. They are pro&uce& by a&&ing equimolar solutions of < P" an& 4a P" . Equal *olumes of the t0o 0ill yiel& a p

of A.2$ 0hile so&ium phosphate 0ill increase the p . 7ncrease& *olumes of potassium phosphate 0ill &ecrease th e p . The p can be a&Fuste& from ?.4 to 3.2. 7f a p of A.2;3.2 is &esire&$ start 0ith ?22 ml of so&ium phosphate an& a&& potassium phosphate 0hile stirring an& monitoring the p 0ith a p meter until the &esire& p is reache&. 7f a p of ?.4;A.2 is &esire&$ start 0ith ?22 ml of potassium phosphate an& a&& so&ium phosphate until the &esire& p is reache&. Typically$ the molarity of the buffer 0ill range from 2.21 to 2.1 #. Use the appropriate molarity of < an& 2.? # 4a P" an& 4a P" . That is$ if 2.2? # buffer is &esire&$ use 2.? # < P"

P" as &irecte& abo*e.

+o&ium potassium phosphate buffer 6efer to +o&ium phosphate buffer. +o&ium pyrophosphate D4a P " H 12 " #8 44@.2@E

1' mM Cissolve '.))5 grams of +a < ; c 1': ; to a final volume of 1'' ml with water. +o&ium succinate D#8 2A2.1@E '.5 M Cissolve 15.9 grams of succinic acid, sodium salt to a final volume of 1'' ml with water or buffer. +orbitol D#8 132.1AE '.?? M Cissolve 5'.19 grams of sorbitol to a final volume of 1 liter with water or buffer. +orenson phosphate buffer

Refer to sodium phosphate buffer. '.9 M p: >.* Cissolve 9).1) grams of +a :<; and ).'I grams of B: <; in I'' ml of water. Cilute to a final volume of 1 liter.

+ubbing solution Dsli&esE 6efer to chrom alum gelatin. +ucrose D#8 342.3E 1.' M Cissolve ?).9 grams of sucrose to a final volume of 1'' ml with water or buffer. For other molarities, multiply the weight by the re3uired molar concentration. For e/ample, for '.9* M sucrose, weight ?).9 / '.9* or I.** grams to a final volume of 1'' ml. )'S ,w8vCissolve )' grams of sucrose to a final volume of 1'' ml with water or buffer. Cilute this solution for lower percent re3uirements. #f using for sucrose density gradients, the sucrose should have '.1 ml of diethylpyrocarbonate added, the solution brou ght to a boil for ?4* minutes and cooled before use. $his will eliminate R+Aase, which would otherwise be a contaminant of the solution. Store all sucrose solutions in a refrigerator +ulfuric Aci& D 2+"4 #8 >3.23E Caution: Sulfuric acid is extremely caustic and $ill cause se ere burns. It must al$ays be added to the $ater' $hen ma#ing dilutions. !"on addition to $ater or alcohol' heat $ill be generated $hile the solution $ill contract in olume. !se extreme care in handling this acid. Concentrate& 2+"4 is 1A.3 # or 3?.@ 4. 1.2> 4 A&& 32.@ ml of concentrate& sulfuric aci& slo0ly$ 0ith constant stirring$ an& 0ith a&equate protection from splashes$ to appro(imately 322 ml of 0ater. Cool an& ma:e up *olume to 1 liter 0ith 0ater. +ulfurous aci& Dfor /eulgen 6eactionE A&& 1.2 ml of concentrate& Cl an& 2.4 grams of so&ium bisulfite to 122 ml of &istille& 0ater. This solution shoul& be ma&e fresh prior to use. 7t &oes not store 0ell. +0abbing &etergent /or tissue culture purposes$ use a non;to(ic &etergent &esigne& for surgical scrubbing. e.g. Phisohe($ %eta&ine or equi*alent. /or most routine s0abbing$ A2G D*'*E ethanol is sufficient an& has the a&*antage that it 0ill lea*e no resi&ue.

TE#E- D4$4$4I$4I;tetramethylethylene&iamineE Catalyst for PA)E. Use &irectly an& a&& 12 l TE#E- per 1? ml of gel solution. Tolui&ine blue '.1S ,w8vCissolve '.1 grams of toluidine blue in 1' ml of ethanol and add water or citrate buffer ,p: 5.I4>.9- to a final volume of 1'' ml.

Trichloroacetic aci& DTCA CCl3C"" #8 1@3.4E (xtremely caustic acid. %andle $ith care. >9S ,w8vCissolve >9 grams of $7A to a final volume of 1'' ml. $7A is hydroscopic and will readily absorb water. $he solid crystals will become li3uid if the stock bottle is placed in warm water, with a loose cap ,melting point *>4*I G 7. #t is easier to handle as a li3uid. Storage of solutions greater than ?'S ,w8v- are not recommended as decomposition is rapid. $herefore these solutions should be made as needed. Tris buffer $here are many variations on the basic $ris4:7l buffer combination, most of which are commercially available. Solutions with DC$A are known as $D buffers, while solutions with DC$A and acetic acid are known as $AD buffers. $he terminology varies with the author, with $ris buffer being used to mean $ris4:7l solutions. Sigma 7hemical 7o., St. !ouis, carries a full line of the buffers marketed under the tradename of $ri&ma ,base and :7l-. $he basic buffer is a combination of $ris ,tris,hydro/ymethyl-aminomethane- and :7l acid. $hese are sometimes referred to as $ris4base and $ris4:7l solutions. $ris buffers should not be used below a p: of >.9 or above a p: of O.'. $ris buffers are also e/ tremely temperature senstive. Cirections are given for room temperature ,9* G 7-. $he p: will decrease appro/imately '.'9I units for each degree decrease in temperature. 1 Cissolve 191 grams of $ris in I'' ml of distilled water. Ad"ust the p: with diluted from this stock or mi/ed as combinations of lower molarities of $ris and :7l. M concentrated :7l. Cilute to a final volume of 1 liter. !ower re3uired molarities can be

#t is important to measure the p: at the temperature an d molarity that will be used in the final analysis. Tri;)lycine buffer *( Cissolve 1*.1 grams of tris base and >9.' grams of glycine to a final volume of 1 liter. For use, dilute 1 part *( buffer with ) parts water. Trypan blue '.9 S ,w8vTrypsin '.9*S '.9*S Cissolve '.9* grams of crude trypsin in <%SA to a final volume of 1'' ml. 7old sterili&e by filtration. Alternatively, purchase pre4diluted crude trypsin, sold as 129*' which is pre4 sterili&ed as well. +ote2 .hen using trypsin for tissue disaggregation, it must be subse3uently inhibited by the use of serum in the culture media, or by the addition of soya bean trypsin inhibitor. Trypticase soy broth A&& 1A.2 grams of trypticase peptone$ 3.2 grams of phytone peptone$ ?.2 grams of so&ium chlori&e$ 2.? grams of &ipotassium phosphate an& 2.? grams of glucose to 1 liter of 0ater. A&Fust the p to A.3$ an& autocla*e. T0een 22 or 32 DPolyo(yethylene sorbitan mono;oleateE 1S ,v8vAdd 1.' ml of $ween to O' ml of water. Mi/ and dilute to a final volume of 1'' ml with water. +ote that $ween is e/tremely viscous and care must be taken to accurately pipette 1.' ml. .ipe the outside of the pipette before dispensing. Uranyl acetate D#8 424.1>E *S ,w8vCissolve *.' grams of uranyl acetate to a final volume of 1'' ml in *'S ,v8vethanol. Store in the dark at room temperature. Allow at least 9) hours for the uranyl acetate to completely dissolve. $his solution will keep for about ? months. Cissolve '.9 grams of trypan blue to a final volume of 1'' ml with water.

Urea D#8 @2.2@E 9.* M Cissolve 1*.'9 grams of urea to a final volume of 1'' ml with water or buffer. 1' M 1) M ,iability stain 6efer to Trypan blue Cissolve 5'.'5 grams of urea to a final volume of 1'' ml with water or buffer. Cissolve I).'I grams of urea to a final volume of 1'' ml with water or buffer.