Techniques in Cell Biology Manual New1

Techniques in Cell Biology
TECHNIQUES IN
CELL BIOLOGY
ANIL KUMAR
DINESH PANDEY
SONU AMBWANI
G.K. GARG
DEPT. OF MOLECULAR BIOLOGY & GENETIC

ENGINEERING
COLLEGE OF BASIC SCIENCES & HUMANITIES
G.B. P.U.A. & T., PANTNAGAR-263 145
U.S. NAGAR, (INDIA)
CONTENTS
I. MICROBIAL TECHNOLOGY
a. Maintenance of aseptic conditions and handling during microbial
culture practices.
b. Techniques to obtain pure culture of microorganism from mixed
culture
c. Inoculation of pure culture by streak plate and pour plate
methods
d. To perform simple/ direct staining and indirect/ negative staining
and Grams staining of pure culture isolates
e. Identification of unknown bacterial cultures by biochemical tests
II. CELL/TISSUE CULTURE TECHNOLOGY
a. Plant cell culture :
i. Seed culture of Brassica/ Tobacco seeds
ii. Callus culture or shoot regeneration from hypocotyls of
Brassica or Tobacco seedlings
b. Animal Cell culture :
i. To culture human fibroblast cells
III. BIOPROCESS ENGINEERING & ENVIRONMENTAL
BIOTECHNOLOGY
a. Immobilization of yeast cell by formation of agar beads
b. Immobilization of yeast cell by formation of sodium alginate
beads.
c.
Demonstration of fermenter and glucose fermentation process
d. Bioremediation of heavy metals from industrial effluents

IV. IMMUNOTECHNOLOGY
a. To perform Haemagglutination test to determine blood groups.
b. To detect pregnancy by the presence of hCG in urine by latex
particles agglutination inhibition test
c. Demonstration of gel diffusion/ouchterlony
d. Widal slide test for the detection of Salmonella antibodies in
human serum
e. Lateral flow Immunochomatography Test Strip for the Qualitative
Detection of Treponemal Antibodies in Syphilis patients
f. Extraction of total proteins from wheat seeds
g. To perform SDS polyacrylamide gel electrophoresis of the
protein samples
h. Western blotting
V. DNA TECHNOLOGY
a. Isolation of genomic DNA from plant , animal and plant cells
b. Isolation of plasmid DNA
c. Quantitation of genomic and plasmid DNA
d. Restriction digestion
TECHNIQUES IN CELL BIOLOGY

Cells are small and complex. It is hard to see their structure, hard to
discover their molecular composition, and harder still to find out how their
various components function. What we can learn about cells depends on the
tools at our disposal, and major advances have frequently occurred from the
introduction of new techniques. To understand contemporary cell biology,
therefore, it is necessary to know something of its methods/techniques. Thus,
the course Techniques in cell biology has been introduced for the students of
Biotechnology and related discipline.
Cell biology is a specialized branch or biological sciences dealing with
the structure, function and uses of microscopic organisms/cells, such as
prokaryotic cells, Viruses and bacteria, Eukaryotic cells; actinomycetes, fungi,
algae, protozoa, plant and animal cells.
This course has been designed to acquaint students with cell culture
techniques of microbes, such as microorganisms, plant and animal cells and
to apprise them with the importance of microbes in our daily life and the vital
roles they play in the ecology of life on earth. This course covers a wide
spectrum of exercise such as microbiological techniques, Bacterial culture,
plant tissue culture, Animal cell culture and immunological techniques.
There is, as in all sciences, a need for basic equipment (instruments,
tools, glassware and miscellaneous items) much of which can be found in any
cell biology laboratory. Following is a list of basic requirements which a cell
biologist/microbiologist require in the laboratory of microscopic examination,
isolation or culturing and identification of a microbe as well as to study its
structure, function and application.
A.
Instruments and Applications

i.
Microscopes and immersion oil
ii.
Photomicrographic camera
iii.
Laminar flow safety hood
iv.
Microbiological inoculation chamber
v.
Autoclave
vi.
Pressure Cooker
vii.
Incubators
viii.
Refrigerator
ix.
Oven
B.
C.
x.
Water bath
xi.
Bunsen burner or spirit lamp
xii.
Hot plate/heater
xiii.
Centrifuge
xiv.
pH meter
xv.
Spectrophotometer
xvi.
Camera Lucida
xvii.
Quebec colony counter
xviii.
Balance
xix.
Homogenizers for grinding of specimens
xx.
constant temperature water bath
xxi.
Tripod with asbestos mat
xxii.
Haemocytometer
xxiii.
Counter
xxiv.
Stop watch
Tools
i.
Transfer needle
ii.
Inoculating loop
iii.
Dissecting needles
iv.
Forceps
v.
Scissors
vi.
Ocular micrometer
vii.
Stage micrometer
viii.
Burette set up
ix.
Thermometers
x.
Scale
Glass wares
D.
i.
Petri dishes
ii.
Conical flasks
iii.
Culture tubes without screw caps
iv.
Screw capped tubes for media
v.
Beakers
vi.
Funnels
vii.
Graduate cylinders
viii.
Graduate pipettes
ix.
Capillary pipettes
x.
Dropper bottles for staining reagents
xi.
Screw-capped bottles for stock reagents
xii.
Glass microscopic slides
xiii.
Depression (concave) slides
xiv.
Glass cover slips
Miscellaneous
i.
Culture media, Agar, peptone, Beef extract, yeast

extract, plant and animal cell culture media
ii.
Test tube rack
iii.
Cotton plugs
iv.
Wooden sticks, with and without cotton scabs
v.
Rubber bulk for pipettes
vi.
Glass marking pen
vii.
Tube for labels for sealing plates
viii.
Stains and staining apparatuses
ix.
Parafilms
x.
Aluminum foil
xi.
Butter paper/bags
xii.
Nail polish/wax for sealing microscopic mounts
xiii.
Disinfectant (savlon, phenyl etc.)
xiv.
Discard containers
xv.
Facilities for disinfecting hands
xvi.
Distilled water
xvii.
Syringes and needless
xviii.
Bottling paper and lens tissue
xix.
Tissue paper
xx.
Rubber bands
xxi.
Muslin/cheese cloth
xxii.
Pipette can (tin or glass)
xxiii.
Petri dish can
xxiv.
Cotton or gauze masks
xxv.
Test tube cap
xxvi.
Drawing pencil
xxvii.
Match box
xxviii.
Surgical gloves or rubber gloves
xxix.
Mask
xxx.
Apron
USEFUL DATA OF CELL BIOLOGY

1.
2.
3.
4.
5.
6.
Number of cells present in Human

1 cm3 volume contains
Number of lymphocyte in Human
Number of chromosome
Size of DNA present in a chromosome
DNA content
pgm/Cell
7. Number of base pair in a DNA
8. 1 K. base pair
Kilodt.
9. 1 x 10-12 gm or/ pgm DNA
kbp
10. 1 kb pair can coded
Amino acid
11. 260 nm absorbance give rise
DNA
12. 1 g/ml DNA have
phosphate
13. RNA content
pgm/Cell
14. 1 kbp mRNA will coded
15. 1 AA
16. 10 kdt protein coded by
pair
1014 Cells
109 Cells
1012
46 (44+2)
5 mm
6.4
5.8 x 109
660
9.1 x 105
333
50
g/ml
3.0
10
3.4 kdt
111 dt
270 base
DEFINITIONS
1.
Sterilization
Means complete destruction of all forms of life various method of
sterilization are as follows:
Day heat:
One hr at 160C to 180C is hot air sterilizer

Use Glass ware, surgical instruments etc.
Intermittent: 30 minutes in flowing steam 100C for 3 consecutive days.

Use Media tissue, liquids, solids.
Moist heat: 15-30 minutes at 121C at 15 psi pressure
Use Glassware, media
Filtration:
Use media, liquid samples
Radiations: Use Surface sterilization
2.
Disinfection
Means the destruction of pathogenic microorganism.
3.
Disinfectant
Is the chemical substance which is used to destroy (kill) disease
producing
agent.
4.
Antiseptic
Is an agent that opposes sepsis i.e. stops growth of microorganism.
5.
Antibiotic
Are the substance produced by one microorganism to kill the other
microorganism.
6.
Sanitizer
Is an agent that reduces the microbial population to safe level.
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SUGGESTIONS & REGULATIONS

General Suggestions and Information
1.
Wear a coat, smock, or apron to protect your clothing.
2.
Before each laboratory period, read over the exercises to be done and
plan your work carefully, known how each exercise is to be done and
what basic principles it is intended to convey.
3.
Each laboratory meeting will begin with a short discussion and

instruction period. Dont begin work until you have received your
instructions. Ask questions when you do not understand the method
and purpose of any experiment. Good laboratory technique depends
primarily on knowing what you are to do.
4.
Properly record all observations at the time they are made laboratory
examinations will cover both the information given by your laboratory
instructor and that contained in the manual, as well as you own
observations and deductions. Answer the questions following each
exercise on the report sheet.
Laboratory Regulations
1.
Sponge off the top of your laboratory desk with germicide solution to
both the beginning and the close of each laboratory period.
2.
Keep your desk free of non-essential materials at the time and at the
end of the period leave it free of all materials and equipment.
3.
Place all solid waste material in the waste cans and all dirty glassware
in the trays. Your laboratory grade will depend to some extend on your
techniques, orderliness, and cleanliness.
4.
Because many of the microorganisms with which you will be working

are potentially pathogenic it is imperative to develop aseptic techniques
in handling and transferring them. Avoid any hand to mouth operation
such as smoking, eating, or moistening labels with the tongue.
5.
Report immediately all accidents such as cuts, burns, or spilled

cultures to your instructor, take all precautions to avoid such accidents.
Make yourself familiar with the equipments incubators, centrifuges and

microscopes etc. If you have problems, approach the course director or
course instructors.
Good laboratory work habits will help you and the course a ground
success. Follow the following guide lines very strictly. These will protect you
and you experiments.
11
1.
No eating, drinking or smoking in the lab.
2.
No storage of food in the lab.
3.
No mouth pipetting. Use appropriate pipetting aids that are available.
4.
Wear gloves for certain experiments.
5.
Work cleanly and in an organized manner. Wipe tissue culture hood

bench with 70% ethanol.
6.
Most important, label every thing you use with your initial, date and
name of the reagent, buffer or medium.
Procedures for Handling Cells and Medium

-
Use sterile glassware and pipettes.
When you open a glass bottle, before and after use flame the
mouth in a Bunsen flame.
Flame glass pipettes.
Use bulbs to control pipettes. DO NOT MOUTH PIPETTE. This is to

protect both you and the cells from contamination.
NEVER INSERT a pipette which may have contacted cells back

into your stock bottle of medium. Use of fresh pipette. Thus, to
change the medium on a dish, the procedure is as follows:
-
Pre-warm the medium and serum to 37C in water bath.
Wipe bottles with filter paper and transfer to the tissue culture
hood (which you should have been wiped with ethanol,
equipped with pipettes, beakers for waste etc.).
Open bottles, flame tops, replace caps loosely so that they wont
fall off.
Transfer desired quantity of serum to the medium or mix in

separate (sterile) container with the help of a sterile pipette. Use
rubber bulb for pipetting and flame the pipette before use.
Move dish(es) of cells to the hood.
Using sterile, flamed, but not plugged, Pasteur pipette, aspirate

the medium flame the Pasteur pipette between dishes.
Use a separate pipette for different cells, Dont do too many

dishes at a time.
12
Using a sterile flamed pipette, transfer desired amount of

medium and serum to the dish(es). Dont reuse the pipette.
Return dishes to incubator without shaking to avoid spill over of

medium.
Reflame tops of bottles and close tightly. Close pipette cans.
Work only in designated tissue culture area.
Wipe surfaces with ethanol before starting.
Wipe bottles dry (e.g. if they have been standing in a water bath
before moving them into tissue culture hood.
Discard used medium, time/expired and especially contaminated

when you open a new bottle of medium or serum, write the date
on it and indicate how much has been removed. If you add
anything to it, indicate this on the bottle. If there is only a small
amount left in a bottle, discard it.
After you have finished, remove your belongings; put them away
or discard properly. Wipe the working surfaces with ethanol.
Cleaning of Glassware
It is extremely important to use clean glassware for culture purpose.
The following procedure ensures clean surface for culture flasks, tubes, Petri
dishes and other glassware.
1.
Immerge all glassware in 2-5% detergent (eg. Extran/Merck)

immediately following use for 20-24 hr. Heating considerably
accelerate the cleaning process.
2.
To remove resistant dirt soak the glassware in a solution containing

100 ml of potassium dichromate in a gallon of concentrated sulfuric
acid for 4 h.
3.
Rinse thoroughly in tap water.
4.
Rinse in hot water.
5.
Soak in distilled water for at least 3 h.
6.
Rinse in distilled water and dry in a hot air oven.
13
Sterilization
1.
Sterilize surgical instruments, glassware, Petri dishes, pipettes,

beakers etc. and aluminum foil after wrapping in aluminum foil for 2 h
in a hot air oven at 200C.
2.
Sterilize culture media, distilled water and other stable mixture in glass
containers with cotton wool plug and caped with aluminum foil or caps
in an autoclave at a pressure of 15 psi for 15 min.
3.
Solutions of unstable substances (eg. IAA) can be filter sterilized using

appropriate Millipore filters and added to the culture medium after
autoclaving.
4.
All sterile operations should be carried out in a Laminar Flow cabined

to sterile room. The cabinets are available in different sizes. They are
movable and eliminate the necessity of a separate room. Fins on the
cabinets are often operated continuously and pre-filters replaced or
cleaned bi-monthly. Alternatively a less expensive inoculation hood
fitted with UV lamps can also be used.
Sterilization and lab ware/material preparation:

Now a days, a lot of tissue culture plastic ware is available as
disposable pre-sterilized single use materials. In spite of these laboratory
level, sterilization needs to be done for glassware, pipettes and for the
purpose of decontamination of infected tissue culture use is listed in
Table-I.
Table-I
Sterilization methods
Method
Time
and Use
Model of action
Disadvantage
temperature
Dry heat
One
hour
at Glassware,
160C to 180C in Surgical,
a hot air sterilizer Instrument
etc.
Thirty minutes in Media,
flowing
steam tissues,
(100C) for 3 Liquids,
consecutive days Solids
Oxidizes bacterial proteins Cannot be used

and nucleic acid
for
media
sterilization
Moist heat
Fifteen to thirty Glassware

minutes at 121C media
at 15 psi
Coagulates proteins
microorganisms
Filtration
Ambient
Intermittent
Media
liquids
samples
Heat shock the material for Tedious,

the first 30 minutes and if consuming
spores are present and
they will germinate and the
vegetative cell will be
destroyed at the next
cycle.
time
of Some
nutrients
are not stable to
these conditions,
eg. lactose.
Filters uniform pore from Pores permit virus
and mycoplasma
pore size of upto 0.22 .
of flow through
14
Toxic gases Ethylene

sterilization under
pressure
oxide Large
slight surface
area
pieces of
particularly.
a)
Ultraviole Media
t (UV)
liquids
b)
Ionizing Y solids
or X rays)
pharmaceu
ticals and
surface
sterilization
Lethal gas kills all life Flammable,

forms; equipment plastic toxic
ware.
Radiation
Causes
genetic
mutations interferes with
metabolism
and
respiration, and leads to
death.
Does
not
penetrate
glassware
easily; requires
access to a core
or cathode ray
tube, mutation
inducing.
15
MICROBIOLOGICAL TECHNIQUES
INTRODUCTION
Microorganisms are all round us and according to Louis Pasteur Life
would not along remain possible in the absence of microbes. However,
some micro-organisms cause disease in humans, other animals and plants.
They were friends as well as adversaries.
As co-habitants on the planet earth, micro-organisms plays significant
role in the lives of other organisms. They are present every where in air, in
water and soil. Close interaction between microbes and humans existed from
time immemorial. Man was quick to tame them and use them to his
advantage, for improving agriculture productivity, in controlling food shortage
and producing safe and nutritious foods, to check diseases, as supplements
to the depleting energy resources, for environmental explorations and of
course as powerful tools of research to unravel the mysteries of the living
organisms.
Microorganisms were first discovered by Antony Van Leeuwenhock
who called them animalcules. Louis Pasteur laid microbes in the spoilage in
wine. Robert Koch was the first one to show that diseases are produced by
micro-organisms. Soon a host of discoveries followed and several industries
came up based on them yeast is used in the leavening of bread and in the
manufacture of wine and beer. It is also used for fermenting sugar into
alcohol, as well in the production of organic acids, amino acids, enzymes and
vitamins. The discovery of penicillin by Alexander Flaming revolutionized
modern medicine. The discovery of a host of antibiotic like streptomycin,
tetracycline, erythromycin, chloramphenicol etc. soon followed which are now
widely used for the control of diseases. Microbial enzymes are used
worldwide for industrial purposes.
This, in the closing yeast of this century, when the sciences of
microbiology has seemingly reaches its pinnacle, it ourselves where has all
this led us to? Has it resulted in a better world? A less hungry world? Have
we controlled all diseases? Have we been able to improve the quality of life?
Has it resulted in a cleaner and healthier planet? Or to the end of the world
and to conquering new planets to inhab? Yes, a little of all this o doubt, and
just enough perhaps to open new areas of investigations. Techniques now
being available for manipulating living organisms in a genetic and
biochemical sense have immense potential. The possibilities now before us
for the construction of bacterial strains finely tuned to the tasks of human
welfare give us a clue to the further possibilities.
In this section we shall review briefly some of the principle
microbiological techniques used to study micro-organisms their culture,
aseptically handling, requirement for growth. Isolation, identification
characterization preservation, mutant study and basic bacterial and phage
genetics.
16
Introduction to Microscopy
Microscope:
Microscope enables us to see microorganisms and their structure
otherwise invisible to the naked eye. Microscopes are of two type:
(A)
Light Microscopy
In which magnification is obtained by a system of optical lenses using
light waves.
(B)
Electron Microscopy
It uses a beam of electron in place of light to produce the image.
Specimen examined by either transmission or scanning electron
microscopy.
Resolving power
The ability to distinguish two adjacent points as distinct and separate.
Numerical aperture
The angle subtended by the optical axis and the outermost rays still
covered by the objective is the measure of the aperture of the objective.
N.A. + n sine
Maximum N.A. for a dry objective is less than 1 oil immersion objective
lens have an NA value of slightly greater than 1.
Limit or Resolution
The limit of resolution is the smallest distance by which two objects can
be separated and still be distinguishable as two separate objects:
d
2 NA
TYPES OF MICROSCOPY
BRIGHT FIELD MICROSCOPY
In bright field microscopy the microscopic field is brightly lighted and
the microorganisms appear dark because they absorb some of the light.
17
DARK FIELD MICROSCOPY

The effect produced by the dark field technique is that of a dark back
ground against which object are brilliantly illuminated.
PHASE CONTRAST MICROSCOPY
Phase contrast microscopy is exactly valuable for studying living
unstained cells and is widely used in applied and theoretical biological
studies.
18
LIGHT MICROSCOPY PRINCIPLES AND APPLICATIONS

INTRODUCTION
Microscope has been directly involved in emergence of several fields in
science. The science of microbiology being with the latter of Antony von
Leeuwenhock in 1967 to the Royal Society in England describing the minute
organisms he observed with the help of magnifying lenses. Human eye has a
limit within which only it can see two adjacent points separately. This power is
known as the resolving power of the 0.2 mm. Most of the micro-organisms
and cell components are very small and of micron size and cant be seen by
naked eye. So science was developing very slowly before the invention of
microscopes. Several milestones were laid in the development of light
microscopy and several new doctrines were developed with the help of it, the
first major one being the cell theory put forward by Schleiden and Schwaan in
1938 which resulted in the formal birth of cell biology. Microscopes have been
greatly helpful in identifying the course of several diseases and Louis pasture
and Robert Koch were able to suggest the causes and remedies for different
animal and human diseases. But the development of microscopy from the
infancy stage of simple use of lenses to advanced equipments like video
enhancement microscopy, florescence microscopy, etc. take a long time due
to the lack of knowledge about the character and properties of light. The
discovery of electromagnetic wave nature of light has resulted in sudden jump
in the development of microscopy. This enabled scientists to develop new of
microscopy. This enabled scientists to develop new instruments like electron
microscope which is having very high magnification power to the tune of
2,00,000 times and very high resolving power of 1 nm where as for light
microscope these powers are 1500 times and 0.2 m respectively.
MICROSCOPY
These of microscope to study the microorganisms, which are
invisible to naked eye since their size is very small is known as microscopy.
We are able to see an object when light from a source falls in the object and it
reflects the light and when this light falls in the retina of our eye. But this is
possible with the help of the lens we are having in the eye which focuses the
rays in retina. The microscope is an optical system for magnification and
illumination and it contain a lens or a series of lenses with a source of
illumination.
PRINCIPLES OF LIGHT MICROSCOPY
LIGHT
To know how microscope is working we must first know about the
characteristics of light. Light is a form of energy and it has an electromagnetic
wave character. It follows a straight line path but undergo deviation when it
passes from a medium to another medium which is known as Refraction. It
can also be reflected with a mirror and when an object is put on the path of
19
light you can see dark and lighter bands is put the image of the object. This is
due to the curvature of light rays and the phenomenon is known as diffraction.
When 2 waves of light of different wavelength reach a point they interfere with
each other and result in a new wave which will be according to the phase of
the waves and this phenomenon is known as interference. Visible light when
passes through a prism it get divided into light of different wavelengths and is
known as dispersion. The above said qualities of light have a definite role to
do in microscopy. Light has a fixed speed in a medium, this also affect the
resoling power of a microscope.
HOW IMAGES ARE FORMED
Lenses are devices which cause a beam of light to coverage or diverge
as passing through it (or a transparent medium bounded by atleast one
spherical surface). According to nature of the surface of lenses there are
different types of lenses like:
(i)
Biconvex
(ii)
Planocovex
(iii)
Concavoconvex
(iv)
Biconcave
(v)
Planoconcave
(vi)
Convexo concave
The image formed by a lens or a group of lenses depend upon the

focal length of the lens, distance of object form the lens numerical aperture of
the lens and refractive index of the lens.
MAGNIFICATION
Magnification power of a lens is its ability to increase the size of the
image of the object. Generally it is measured as the ratio of size of image to
the size of the object. Before going into details of magnification we must know
two properties of the lens the focal length of power and refractive index.
Focal length of a lens is the distance between optic centre and
principal focus (the point of convergence of divergence of rays). The
reciprocal of focal length in meters is known as power of the lens is expressed
in diopters. As focal length increases the power decreases and vice-versa.
Refractive Index (RI) of a lens is the ratio of the speed of light in
vacuum to its speed in the lens medium. This ratio determines how much the
light rays are bent while it enter and exit the lens medium from or into air.
Magnification can be defined in another way also as the ratio of angle
subtended at the eye by the image formed at the least distance of distinct
vision to the angle subtended at the eye by the object kept at the same
distance. The total magnification of a microscope is found by multiplying the
magnifying power of the objective by that of the eyepiece. The lens system
near to the objective lens, called objective produces a magnified real image
which is further magnified by the eyepiece. The greatest useful magnification
of a microscope is that which makes the smallest visible objects clearly
visible. Theoretically it is possible to magnify the objects infinitely using more
number of lenses.
20
b)
In case of compound microscope
The total magnification

Magnifying
Magnifying power of eyepiece X

power of objective
NUMERICAL APERTURE AND RESOLVING POWER

Resolving power is the ability to distinguish two adjacent objects as
separate and distinct images rather than a single blurred image. The
resolution power of human eye is 0.2 mm which means eye can not
distinguish between 2 points which are distant by less than 0.2 mm. The
resolution power is influenced by several factors.
1.
Wavelength of the light used for illumination
Resolving power is inversely proportional to the wavelength of light

used. If you use a light of smaller wavelength or higher frequency higher
resolution can be obtained.
2.
Numerical Aperture
Resolution power is directly proportional to the numerical aperture.

Numerical aperture is defined as the function of the effective diameter of the
objective lens in relation to its focal length and the light bending power or
refractive index of the medium between the specimen and the objective. This
property of an objective helps in resolving fine details. The higher the entire
value of NA of optical system including condenser and objective lens, the
greater is the resolving power.
Numerical aperture also decides certain other things like light
transmission i.e. the amount of light passed through an objective lens. It also
decides working distance. As the N.A. increases working distance decreases.
The flatness of the field of view i.e. uniform brightness and depth of field is
also inversely related to N.A.
You will face certain problems when high power is used since the
refractive index of air is different to that of lens medium a lot of light rays are
bent and many rays reflected from the specimen are refracted at a higher
angle and they completely miss the objective. By interposing immersion oil
which is having refractive index nearly same as glass we can decrease
refraction and can give more resolution power and a clear image. Since for a
dry lens n = 1 and since the absolute maximum value that can have is 90
the maximum value of NA must be:
1 x Sine 90 = NA
Hence all objectives with an aperture greater than 1 must be of oil
immersion type and greater the refractive index of the medium used for
immersion the large the aperture can be.
21
Resolving power is given by the formula

Re sovling Power
Diameter of smallest structure visible

Numerical aperture
So the maximum resolution power possible by light microscopy

is 0.22 m with oil immersion objective.
WORKING DISTANCE
It is the distance between objective lens and the object. Focal length
and working distance are directly related i.e. as focal length decreases or
power increase the working distance also decreases and vice-versa. It is
possible to use the oil immersion since the working distance is small due to
very small focal length. Generally in compound microscopes 3 types of
objectives are provided low power (10 x) high power (40 x) and 100 x oil
immersion type.
HOW SPECIMENS DETAILS ARE VISIBLE

In case of plant tissues just by taking a thin section of tissue and
observing under microscope it is possible to see the cells. A thin section is
essential to allow light to pass through the specimen. For getting more
contrast to the specimen stains are used. According to the nature of the
specimen different types of stains are used either they will enter the specimen
and stain the cells completely or differentially or stain the surrounding and the
cells remain colourless known as indirect staining. Now a days fluorescent
chemicals are added as stain so that they will bind to cell and emit
fluorescence which can be microscope effective staining and preparation of
specimens are very important. Complex staining methods are there where
tissues are treated with different stains for fixed time interval. This methods
give best contrast e.g. Gram staining.
TERMS IN MICROSCOPY
ACHROMATIC
A lens which brings light from 2 parts of the spectrum (red and blue
wavelengths) to the same focus thus reducing chromatic aberration. It is the
most common type used in microscope. If you use an achromatic with white
light, colour fringes may appear on the margins (outer) of the image because
this objective cant bring all the wavelengths to an acceptable focus range. If
monochromatic light is used as in phase contrast microscopy the image will
be much better and sharper.
22
BARREL FOCUS
A microscope in which the body tube (barrel) moves and the stage is
stationary during focusing.
BRIGHT FIELD ILLUMINATION
White light illuminates a transparent or translucent specimen that is
either naturally colored or stained and which appears dark against a bright or
white background.
BODY TUBE LENGTH
The distance between the inserted position of the objective and the top
of the body tube-usually 160 mm. If objective and body length are
mismatched it will lead to spherical aberration.
COAXIAL
Focusing arrangement where the find and coarse focus knobs and
mounted together on a common axis.
CONDENSER (SUBSTAGE) / ABBE CONDENSER
It provides an even cone of light that illuminates the specimen. Light
from the condenser coverages on the specimen, passes through it and
diverges from an inverted illumination cone that is captured by the objective
lens. Abbe condenser is most commonly used. Numerical aperture of
condenser should be equal or more than the NA of the highest objective i.e.
1.25 to 1.32 for a 100 x objective. The resolving power of the optical systemcondenser, objective, ocular lens-is limited by the lowest NA value of its
individual components.
CHROMATIC ABERRATION
Failure of a lens to bring light of different wavelengths to a common
focus.
DARK FIELD ILLUMINATION
An optical technique in which the specimen is seen as a bright
objective against a dark background.
DIOPTER ADJUSTMENT
Adjustment ring on the eyepiece that find positions eye lens element to
accommodate visual acuity, bringing an object into sharp focus, will not
accommodate astigmatism.
DIN
23
Abbreviation for deutsche Industries Norman, an industry standard for

optics, assuring you a high quality lens system.
EYEPIECE IDAPHRAGM
The round field of view that is seen when looking in a microscope
whatever is placed on the diaphragm of eyepiece will be superimposed on
and in focus with the specimen image.
FIELD OF VIEW
The area visible through the eye piece when the microscope is in
focus.
FLAT FILE OPTICS
Optically corrected to eliminate field curvature. Flat field optics are
particularly suitable for examination of photomicrography of large viewing
areas. The prefix plain or plano is used before the name of the objective lens
with its characteristic.
IMMERSION OBJECTIVE
It is used to achieve high magnification and high resolving power. The
most commonly used is oil when NA of objective exceeds 1.0. It is placed on
the cover slip. Only if the condenser and objective are oiled to slide you will
achieve full N.A.
IRIS DIAPHRAGM
Leaf diaphragm mounted beneath or between within the condenser
controls the size of illumination cone.
KOEHILER ILLUMINATION
The technique provides for a uniformly illuminated field from nonuniform light sources such as coiled filament lamps.
MICROMETER DISC
Glass disc with scale or grid mounted to the eye piece diaphragm and
used for quantitative measurement of object.
PLANOCONCAVE MIRROR
Usually two sided (50 mm diameter) with one side flat and the other
curved. Always use the plane surface with substage condenser. Use concave
surface for very low NA objective when no condenser is used.
PARCENTERED
24
All the elements of the optical system are aligned on a single axis
minimizing aberration.
PERFOCALITY
The property of microscope system when the subject stays in focus
when the objective lenses are changed.
PLANACHROMAT
An achromat lens corrected for flatness of field.
Optical techniques of revealing the structural features of microscopic
transparent objects whose varying but invisible differences in thickness result
in varying difference in the phase of transmitted light. This phase differences
are converted to visible intensity differences when part of the transmitted light
has its optical path changed by about 1/3rd wavelength.
POLARIZER
A transparent material which absorbs all vibrations of light passing
through it except those in a single plane.
RACK AND PINION
The system gears used for raising or lowering the stage or barrel when
focusing.
RETRACTILE OBJECTIVE (XR)
Spring loaded so that minimal damage is done to microscopic slides
when objective is ranked down too far. Designated XR for higher
magnification, low working distance objectives.
SLIP (SAFETY) CLUTCH
Mechanism built into the focus system that prevents gear stripping if
the coarse focus is forcibly ranked down beyond its normal stop.
SPHERICAL ABERRATION
Failure of a lens system to image the central and peripheral rays at the
same focal point.
STAGE FOCUS
A microscope style where the stage moves and the body tube is
stationary during focusing.
ZOOM LENS
25
It is a lens system that provided for variable magnification capacity. The

object stays in focus through the full magnification range.
SIMPLE AND COMPOUND MICROSCOPES AND THEIR PARTS
Simple microscope
The hand lens or magnifying lens falls in this category and operates
upon this principle. The image of an object is projected and enlarged but not
inverted. The object should be placed at any point within the focus. We can
observe only the gross from of the cells with this lens.
Compound microscope
The magnification of compound microscope is achieved in 2 stages. A
real image of the object is first formed by the objective and an image of this
image is formed by the eyepiece which is virtual and inverted and so cannot
be used to be real under conditions of projection.
COMPONENTS OF MICROSCOPE
Stand
It is mechanical frame to carry to optical system of instrument and is
divided into 2 parts foot and arm. Foot is horseshoe shaped and has 3 contact
pads on the underside to give a stable support as surfaces which might not be
truly flat and sufficient to give balance to microscope in inclined portion. Arms
carry three optical units and the focusing system.
BODY TUBE
On this the eye piece and the objectives are mounted and the distance
between them can be adjusted.
Coarse adjustment
This is used to focus the image by rack and pinion.
Fine adjustment
After focusing with a screw and nut mechanism fine adjustment with
about 70 threads to inch. The result is small movement of objective by the
turn of the knob.
Objective changer
This is a rotating nosepiece carrying upto 4 objectives with an indexing
spring mechanism to ensure its position in line with that of microscope.
Substage
26
To hold the condenser.

Mirror
Double sided mirrors with one side plain and other concave is used to
provide illumination. If concave is used due to desirable convergence
accurate focusing of light on the specimen will not occur.
Stage
It is square or rectangular plate of great rigidity. It is usually treated with
reagent resisting finish. Spring clips are provided to keep the specimen in firm
contact to the stage surface. By rack and pinior it can be moved slowly go get
every part in the field of view. It can also be removed to view Petri plates.
Sales and verniers
Two vernies and scale are used on running sough north and other east,
west and north and other east-west and can be used as finding device.
LENSES
It is of two types objectives and eyepiece. Lenses has optical centre
which is on the principal axis on the lens and all the rays passing through this
point emerge without deviation. In a simple microscope with a single biconvex
lens, 200-300 times magnification is possible.
Objectives
They are lenses near to object and produce inverted real image. They
are of different types such as follows.
a)
Achromatic lenses
This is used to nullify spherical and chromatic aberrations. This is

achieved by cementing together a convex and concave parts and one made
of crown glass and other flint glass so that both errors are cancelled each
other. Such lenses are also called doubled. They are of moderate NA, hence
moderate resolving power and long working distance and flat field of view.
b)
Apochromatic
It is a combination of triplet of lenses made of suitable material so that

the aberrations can be decreased by the maximum. They are perfect in colour
so used in colour photomicrography.
c)
Fluorite
27
Intermediate between achromatic and apochromatic objectives but

nearer to latter. It is made of fluorite ground and polished. They cant correct
chromatic aberration perfectly. They have high NA.
d)
Flat field objectives
High NA is used in photomicrography and flat field is obtained with less

NA so better resolution.
e)
Spring loaded objectives and parfocalled objectives which are

explained earlier
Another classification is dry, water immersion and oil-immersion

according to their use in air, water and oil respectively. Both high power and
low power objectives can be used with uncovered objects but 4 mm objective
is very much sensitive.
EYEPIECE
It is the lens near to eye and give second magnification and image is
virtual. It is of different types such as:
a)
Huyghenian eyepiece
It consists of 2 plano convex lenses with a metal diaphragm in between

them located at the focal plane of the top or eye lens. The bottom lens or field
lens collect the rays of light coming up from the objective and brings them to
focus in the plane as diaphragm. Pointer eyepiece is some variety of this
provided with an adjustable pointer and compensation eyepiece is one with a
correction for achromatism.
b)
Holoscopic eyepiece
i)
Telaugic eyepiece: as one to correct the curvature of the field.
ii)
Complex type: This is of compensation type but of special design with

a large eyelens, a raised eye point and a rubber guard over the run of
the eyepiece so that a spectacle wearer can easily rest his lens against
the rubber guard and then see the whole of the field of view without
moving his head.
iii)
Rainsden and Kellner: Plane of the focus of eye lens lies just below
the file lens external to the eyepiece. The allows easy positioning of
reticules and hairlines and so used in micrometry.
iv)
Projection eyepieces: They are hughenian type but with an

achromatic lens mounded in an adjustable sleeve. This image can be
projected and used in photomicrography.
28
v)
Parfocal eyepieces: Eyepieces of tubular collars of different lengths

so arranged to view the object in focus after focusing even if the
eyepiece is changed.
As the eye piece power increases the size of field decreases. So for
better resolving power and depth of field the highest power of objective is
used with the lowest of eyepiece.
CONDENSER
Condenser is used to illuminate the object at the point on which the
object is focused and to fill the field of view with uniform illumination. There
are different types of condensers as:
a)
Sample abbe
It consists of 2 large diameter planocovex lenses of considerable

thickness, air separated with an adjustable iris diaphragm mounded at the
back of focal plane.
b)
Three lens abbe

They consist of 2 plano-convex lenses.
c)
Achromatic and Aplanatic

They do correction of chromatic and spherical aberration respectively.
d)
Oil immersion type
To get most number of rays concentrated on object distance between

condenser and underside of slide is replaced by oil which prevent total
internal reflection.
e)
Special condenser like macro for photomicrography
Dark ground for dark ground illumination, Trilux for phase contrast,
Auxillary for Koehler illumination, etc. are also used.
The effective aperture of any objective-condenser combination is the
mean of N.A. of the objective and the aplanatic aperture of the condenser i.e.
the six of cone, which is free from spherical aberration. The light source can
be built in and it is of limited adjustability or preferably external and movable.
HOW TO USE THE MICROSCOPE
1.
Identify the objective lens to be used and put it in proper position and
fully open the condenser and adjust the mirror to get the maximum
light.
2.
Place a specimen slide on the stage and looking through the eyepiece
turn the coarse focusing knob when a blurred image is obtained and
29
bring the specimen into fine focus with fine adjusting knob. Never focus
downward while looking through the eyepiece. Adjust the iris
diaphragm bright light to cover the whole field.
3.
After adjusting with low power and then high power dry adjective or 100
x using the oil. Place a drop of oil at the centre of the specimen or
cover glass, locate the specimen with low power and then use 100 x
and focusing is done by the same way do not allow objective to touch
the slide and just the diaphragm to get clean image. Each time after
use, the oil is wiped off with tissue paper and moistened with xylol.
A finely focused image should be clearly visible without superimposition

and amount of light will be optimum slides. After viewing the object keep the
best possible from the dirt free and slides are washed and dry and keep the
microscope covered with polythene cover. Keep the lens clean by wiping it
with dirt free linen paper moistened with xylol.
SPECIAL TYPES OF LIGHT MICROCOPY
1.
DARKFIELD MICROSCOPY
It provides a useful means of looking at wet mounts of unstained

specimens and detecting very small structures by reflected and detracted
light. It is performed by illuminating the specimen with the hollow cone of light
such that only the light detracted by object in the field of view is transmitted up
the microscope tube to the eye or camera, the beam forming the cone of light
focused on the specimen one at too low an angle to be captured by the
objective. The result is a field of bright objects against dark background low
power and high power adjectives can be used in a dark field mode by using
an ordering condenser enquired with a filter carrier below the condenser
mount and an angular cone of light is produced which can be focused on the
object without any direct rays entering the objective. There must be good
intensity of light and focal length should be shorter for high power this slides
are needed so that condenser can them are high light intensity, oil immersion
contact throughout, funnel stop in the objective, central mount and a special
dark ground condenser.
2.
It is system of joining contrast in a translucent specimen without the

help of stains and has the advantage of using high resolution optical
components. It also needs high intensity of light. A green filter is used to
reduce the unavoidable achromatic aberration. Phase contrast works because
a phase annuls is inserted at the lower focal phase of the condenser and
because of phase plate is incorporated in the back of focal plane of the
objective. An image of the annulus will be thus formed on the phase plate.
Since light beams are detracted by the specimen to go through all the
objective field and some go directly through the specimen and the phase plate
ring, both take part in forming the image. It is essential to centre the annulus
plate in back focal plane of the objective. The specimen for phase microscopy
30
must be as thin as possible and must be mounted in fluid or gel and under a
cover slip to reduce heterogeneity of the background of the image. Light is
passed through the cell depending on the refractive index of the cell. So
different parts of various intercity pass light at different rate so this
difference gives the phase difference. This is used to visualize living cells.
3.
INTERFERENCE MICROSCOPY
It is used in finding out the structure of the cells. Interference

microscope produces separate object and reference beams, rather than
forming an image from the direct and diffracted elements of a single beam
from a condenser annulus as in phase contrast microscopy. It gives internal
morphological details of the cells. Using this we can see the movements
involved in mitosis, cell migration etc.
4.
FLUROESCENCE MICROSCOPY
In this the phenomenon of fluorescence is used which is the property

displayed in certain substances whereby, if they are illuminated with short
wavelength (UV) they are able to convert the short wavelength which is
invisible to long wave, visible light. So when such substances are added to
cells with the help of other protein like antibodies they specifically bind certain
sites and when illuminated with UV they produce fluorescence which can be
varied by the microscope as colored spots while other area remaining usual. It
is widely used to detect molecules and give structure of the cell. The
illumination can be different types like (i) transmitted light through a substage
condenser (ii) dark field illumination and incident illumination. Flurescent
microscope uses 2 set of filters to filter light falling on another filter is used
which will permit only the longer wavelength to pass to the objective. Common
dyes used for this are fluorescent and rhodamine which gives green and red
colors respectively.
5.
POLARIZED LIGHT
Light is a form of energy and contain a series of waves at right angles

to the direction of travel. When this is passed through certain prisms the light
become plane polarized all others being eliminated and they are called
polarizer. This is used to find whether an object is optically active or not, by
passing the plane polarized light through the solution of object and measuring
the direction of turning of the light. This is with the help of analyzer.
6.
PHOTOMICROGRAPHY
This is a method taking the picture of the image of the object with the
help of camera attached to the eyepiece. Also video cameras can be attached
to get live films of cell migration. There are 3 types of photomicrography.
a)
A roll film camera back and shelter together with an integral

device that includes the ocular and beam splitting prism with a
31
side viewing arm to allow accusing forms the working unit. The
whole device rests on the microscope tube.
b)
An old fashioned light tight bellows is sometimes used. It is

located on a stand on which microscope can also be placed.
Also there is a plate carrier to place the file.
Success of photomicrography depends on specimen quality and

focusing, good optics properly aligned and illuminated and choice of color
filters and camera free of vibrations and good quality film and processing and
proper panchromatic ASA 60-100 and for phase contrast microscopy ASA
300-400 is needed. Photomicrography is helpful in proper recording of the
details of cells and microorganisms and their activities.
7.
STEREOMICROSCOPY
In effect there are 2 microscopes of its own objective and eye piece
and 2 microscopes have in inclination separation of 15, the images
presented to the eyes differ as they do in normal unaided vision and a true
stereoscopic picture is seen i.e. 3 dimensional. These are also called
binocular microscopes. For this flat field of view as large as possible is
needed and a long working distance and distortion for clear images, maximum
depth of field are needed so special optics are used and have images are
erect giving natural sense. In zoom stereo microscopes there is an extra set
of lenses zoom lenses by which you can vary magnification and field of
coverage and focus of the lens system remains the same. This is also used to
observe surfaces of materials and sections are not essential. As the value of
this microscopy is in depth of focus and field of coverage which is best at low
power objectives we use lowest possible objective and highest possible
eyepiece to produce desired magnification.
8.
CONFOCAL SCANNING MICROSCOPY
This used to view unsliced live specimens 2 dimensionally and with

high power laser object is illumined at a particular point at a particular depth
and is viewed. All other light sources are prevented, fluorescence emitted
from illuminated material is collected and brought into an image at the entry
point of a suitable light detector. Through a pinhole this light is focused and
other portion become out of focus. But using electronic techniques like image
processing we can get cleat pictures.
Light sensitive video camera are used to detect dim light details which
cant be seen so it is possible to view camera produce digitalized image which
can be recorded and interference contrast microscopy helps to view a single
microtubule clearly which have a diameter of 0.025 m now-a-microscopy,
high contrast and VIM-video enhancement microscopy, high contrast and
VIM-Video intensification microscopy which amplifies low light images are
available.
LIMITATIONS OF LIGHT MICROSCOPY
32
1.
There is maximum limit of resolving power with light. Light cant resolve more
than its wavelength 0.4-0.7 m in one of visible light. Because of wave nature
light doesnt follow exactly the idealized path (straight line) but travel through
an optical system in slightly different routes so that they interfere with each
other and cause optical diffraction effects. If 2 rays reading a point are not
exactly in same phase they produce complex interference effects by
mismatching of crests and troughs of the wave. At high illumination and
magnification the evenly illuminated light of uniform wavelength appears as a
set of parallel lines where as set of concentric rings. So the image formed will
be blurred and theoretically with a NA of 1.4 and violet lights of (0.4 m) limit
of resolution in 0.2 m. Although it is possible to enlarge the image as much
as one wants it is never possible to resolve the two objects in the light
microscope that are separated by less than about 0.2 m such objects will
appear as one.
2.
Chromatic aberration: Since different wavelengths are focused at different

points dispersion occurs and color fringes occurs and it hinder to view the
object in color this corrected by using achromatic lenses.
3.
Spherical aberration also result in blurred image and this can also be
corrected to an extent by using combination of lenses of adjusted refractive
index this is possible.
4.
Also the field view is sometimes curved and this hinder the power of
resolution.
5.
There is limitation for magnification also (upto 1500 times).
CONCLUSION
Light microscopy has developed a lot from the times of Anton von
Leuwenhock to the end of 20th century where by the help of electronic image
processing fine quality pictures can be taken. But light microscopy has been loosing
its glamour with the evolution of electron microscopy and its improved applications
which give high magnification and resolution power. Even then light microscopy is
widely used in the labs and in microbiology and cell biology studies and to view live
specimen. Anyway the science of microscopy flourished through the advances in light
microscopy.
REFERENCES
1.
Alberts et al. Molecular Biology of the cell (1994) Garland Publishing

Company, Newyork. p. 139-149.
2.
Casartelli, J.D.; Microscopy for students (1965) Mc-Graw Hill Publishing

Company Ltd. London P 10131.
3.
Frielder David Physical Biochemistry Applications to Biochemistry and

Molecular Biology (1976). W.H. Freeman and Company Sanfransisco P
21-50.
33

INTRODUCTION
When a small biological object, such as a microbial cell is observed in
the living state, it is normally suspended in an aqueous medium compressed
into a thin layer between a slide and cover slop. The perception of such an
object depends on the fact that it displays some object degree of contrast with
the surrounding medium as a result of the fact that less light is transmitted
through it than through the medium. This decreased light transmission is
caused by two factors: light absorbed by the cell and light refracted out of the
optical path of the microscope by a difference in refractive index between the
cell and the surrounding medium. The degree of contrast can be greatly
increased by staining procedures. Before staining, the ells are sometimes
fixed, by treatments designed to minimize postmortem changes of structure.
For light microscopy, heat is the most commonly used fixative.
So to overcome all these difficulties and to observe the cells in living
state, phase contrast microscope was developed by a German optical worker
Prof. F. Zernike.
Phase contrast microscopy
Phase contrast microscopy is technique which enables us to see very
transparent objects, which are almost invisible by ordinary transmitted light in
clear detail and in good contrast to their surrounding and to detect very small
differences in thickness or density within the object. This is achieved without
altering the object by staining or other processes so that we can see living
cells and tissues in their natural state.
Principle
Phase contrast microscopy is based on the fact that the rate at which
light travels through the objects is inversely related to their refractive indices.
Since the frequency of light waves is independent of the medium through
which they travel, the phase of a light ray passing through an object of higher
refractive index than the surrounding medium will be relatively retarded.
These retarded waves are out of phase with the reminder of the light waves
but the difference is not large enough to be detectable with ordinary light
microscope. The phase microscope amplifies these phase differences and
converts them into difference in light intensity thereby increasing the contrast
between the cells and its environment.
Phase Contrast
If we have a very transparent object whose density or thickness is
insufficient to create a phase difference of about one half of a wavelength
between the direct and diffracted rays, the object can barely be seen and it is
in such cases the phase contrast comes to the resume.
34
The phase microscope differs from the conventional light microscope in

having annular diaphragm in the substage condenser and a diffraction plate at
the rear focal plane of the objective lens system. Annular diaphragm permits
only a ring of light to pass upward through the condenser and the object. At
this point is mounted the phase plate. This consists of a disc of glass in one
face of which is an annular groove or depression. Any light passing through
this groove as at AA, has less glass to penetrate than light passing through
the remainder of the plate as at BBB. We see that rays passing through B,
having more glass to penetrate will be retarded in relation to other rays
passing through AA.
As light rays pass through the area-surrounding object as well as
through the object, some rays are retarded and thrown out of phase. Both the
deviated and undeviated light waves pass as a cone of light into the objective
lens system, where they pass through the diffraction plate.
In phase contrast we form an image of the light source annulus at AA,
the annular groove of the phase plate, and the rays producing the image are
all the direct rays. They rays which are scattered by the object the diffracted
rays, will pass through the remainder of the phase plate BBB. The retardation
imposed by the phase plate, which is one quarter of a wavelength, added to
the slight initial retardation impose by the object itself due to its optical density
and thickness, usually about one quarter of a wavelength, amount to the
desired one half of a wavelength retardation necessary to produce an image
of good contrast when these two sets of rays meet and interfere with each
other. This has been done by retarding the diffracted rays and so altering their
phase relationship to the direct rays. In other words, we have obtained good
contrast by a phase alterations. Hence the name of this technique: Phase
Contrast.
DARK FILED ILLUMINATION
They are many objects which because of their transparency, cannot be
seen easily by the method using transmitted light. In such cases, an increases
in contrast between the object and its surrounding is of more help and dark
ground illumination is used to provide this contrast.
Principle
When light impinges on a small object, some light is scattered, making
the object appear luminous and thus visible against a dark background. It
permits the detection of objects so small as to otherwise provide insufficient
contrast.
Working
Dark field illumination employs a special type of condenser, which
forms a Hollow cone of light. This cone of that no light ray directly enter the
objective lens. As the apex of the cone strikes the object light rays are
35
scattered or reflected from it into the objective lens of the microscope. This
one sees a brightly illumines particle or cell against a dark background.
Advantages
By providing contrast, the dark field microscope, is useful in the study
of living cells. With dark field illumination the presence of bodies, whose size
is below the resolving power of the light microscope can be detected. This is
because light emitted from an object can be seen even though the object itself
is not discernible. This technique while it demonstrates the shape, size and
presence or absence of motility of the object, shows only the outer envelop;
no internal structure is visible by dark ground. Phase, contrast on the other
hand, allows us to see the interior of these very transparent object.
UV MICROSCOPY
INTRODUCTION
Scientists have come a long way since Anton Von Leeuwenhoek Ist
sighted his animalcules. The formally unknown world of microbes is now
accessible to the common man, through that indispensable aid-the
microscope.
Light Microscopy-both simple (as that used by von Leeuwenhock) and
compound (used by latter scientists), reached its height of development in the
latter part of the 19th century. The light microscope has two essential parts
objective and the eyepiece. No major changes in the eyepiece resulted in a
satisfactory improvement of the compound microscope but improvement on
the objective side by oil immersion system and use of apochromatic lenses,
helped reach the resolving limit of the microscope (0.2 ) by 1890.2 scientists
responsible for this were- Carl zeiss and Ernest Abbe.
Thus scientists had reached a major stands till at this point of time. It
was the subsequent efforts made from them on that led to one of the most
advanced forms of microcopy-ultraviolet and Fluorescent Microscopy.
PRINCIPLE OF UV MICROSCOPY
The major drawback of the light compound microscope is its resolving
limit/limit of resolution. The only way in the 1900s that scientists could think of
to increase the resolving power and thus reduce the limit of resolution was
to reduce the wavelength of light. This was because the value of N.A. i.e.
numerical aperture was relatively stable at around 1.2 to 1.4 and could be
changed to every little extent.
Visible light lies in the range of 4000-7000 A 248 wavelength. Light of
lower wavelength thus comes in the ultraviolet spectrum i.e. 150-3900 A.
36
Characteristics of UV Microscopy
Ordinary glass starts to absorb heavily from the 340 nm range
downwards. Virtually all transparent plastics have a similar transmission limit.
Therefore, it becomes impossible to form an image with glass lenses for UV
light.
Materials like quartz (transparent for UV down to 200 nm), fluorite
(transparent down to 185 nm) or lithium fluoride are in sue for construction. It
has been shown possible to construct ultraviolet microscopes with objectives
of short focal length and high N.A. In 1904, A Kohler was the I st to construct
such a quartz objective.
Light Source: Use has to be made to a light source emitting sufficient
amount of radiant energy in the ultraviolet region.
The specimens have to be mounted between quartz object slide
(usually made smaller and less thick than ordinary glass slides, 25 x 37.5 x
0.5 nm) and a quartz cover glass which has to be thicker than glass ones
(0.025 mm). The mounting medium and contingent immersion fluid should be
transparent for UV light use is often made of anhydrous glycerin.
Limitations of UV microscopy
1.
UV light as an imaging agent entails a series of complications-the need

for quartz system makes it very expensive.
2.
Though there was a measurable gain in resolving power, the

theoretically expected gain e.g. : of a factor of 2 for 275 nm instead of
550 nm) was seldom obtained due to factors like stray light,
deficiencies in corrections in objectives, problems with contrast and
illumination.
3.
Ultraviolet radiation can cause damage to the cornea of the eye.
4.
Suitable sources of radiation are difficult to make for light of wavelength

below 250-350 nm ranges.
5.
The specimen itself shows progressive absorption and even quartz

object slides can no longer be used.
6.
Air gradually absorbs much of the UV such that the microscope has to
be placed in the vacuum system or in an atmosphere of nitrogen.
7.
Cells are highly sensitive for ultraviolet light of certain wavelengths.
Advantages
1.
Ultraviolet light in the region of 260-320 nm is subjected to strong

selective absorption by certain biological substances.
37
Caspersson showed that biologically important proteins (280-320

nm) and nucleic acids (265 nm) show natural absorption for UV light
of the indicated wavelength. This is used as a major asset of UV
microscope i.e. the revealing of distribution and quantitative
analysis of materials which show selective absorption in 250-380
nm region i.e. UV micro-spectrophotometry and micro-photometry.
2.
As contrasts in cells are related entirely to natural absorption of certain

substances without any pretreatment of cells, it is possible to perform
UV microscopy on unfixed cells.
Applications
1.
Microspectrometry and microfluorometry
2.
Microspectrophotometry and microphotometry for analysis of

nucleic acids, proteins. This also has been applied for study of the
metabolism of uric acid in certain yeasts.
Photomicrography can also be used to register images as these cannot

be perceived by human eye. Images are now being perceived by image
converters.
FLUORESCENCE MICROSCOPY
Phenomenon of Florescence
Florescence can be defined as the phenomenon displayed when
certain substances are struck by electromagnetic radiation absorb a part of it
and re-emit it at a greater wavelength.
The process of emission is a short one and takes only a few
milliseconds during which atoms struck return to their ground state. In contrast
to fluorescence phosphorescence, depending on other types of transition has
a much longer emission period. This phenomenon is of no importance for
microscopy.
Molecular aspect of Fluorescence
When a molecule is exposed to suitable radiation, it gets excited to a
higher electronic level. If the ground state of the molecule is a singlet state
then the excited is also a singlet state because singlet-singlet transitions are
allowed transitions, whereas singlet triplet transitions are forbidden transitions.
If his excited singlet state is a stable state, then there are much less chances
for a secondary reaction. When conditions are such that the probability if
intermolecular collisions is less and the excited molecule is unable to transfer
its energy either to other molecules/convert it into kinetic energy, then the
excited molecule relaxes through a radioactive process within 10 -6 seconds
and the absorbed energy is re-emitted in same/different wavelength. This
phenomenon is termed fluorescence. This is instantaneous.
38
Electronic level possesses several vibration levels, the excited

molecule may or may not return to its original vibration level. If there is a
change in the vibration level, the emitted light has a different wavelength.
Phosphorescence is due to intersystem crossing when the excited
molecule during vibration relaxation transits to the triplet state. Since this is a
forbidden transition the molecule takes time to return: reason for its being a
delayed fluorescence.
Types of fluorescence
a.
Primary fluorescence/ auto-florescence- it occurs by natural properties

of certain substances.
b.
Secondary, fluorescence, induced by substances with fluorescent

properties which have been attached to certain components of other
specimens egs. : acridine orange staining for DNA and RNA.
c.
Immuno florescence: A phenomenon of supreme importance in

diagnostic microbiology today. This is a phenomenon in which an
immunological agent i.e. the antibody previously conjugated to a
florescent marker like fluorescing is used to detect specific antigens or
sites, even microtubule structures.
Fluorochromes
There are certain chemical substances having fluorescent property
used for the purpose of staining and as dyes. The advantage in using these
chemicals is that they are needed in very, very low concentrations such as 1:
500-1; 100,000 with distilled water. Depending on their nature in water, they
are divided into:
1.
Alkaline fluorochromes: Acridine orange, auramine, acridine yellow,

corphosphine, berberine sulphate, netural red, acriflavine hydrochloride
etc. Acridine group is that containing most used fluorochromes.
2.
Neutral fluorochromes: Rhodamine B.
3.
Acidic fluorochromes: Primuline fluoroscein, sodium fluorescein,

pepsin, erythrosine.
MAXIMA FOR ABSORPTION

FLUORESENT AGENTS
Acridine orange
Auramine
Fluroescein isothiocyanate
Lissamine rhodamine B.
AND
EMISSION
SPECTRA
Absorption
Maxima (nm)
Emission
Maxima (nm)
267; 492
440
280, 325, 495
350, 575
540
550
520
595, 710
OF
39
Fluorescence Microscope System

Components:
1.
Light source: Generally mercury discharge lamps which give out high
intensity of UV fluorescent light. Incandescent filaments for blue light
fluorescence. Light yield should be high. Ultimate yield in flux, of
fluorescent light is only of the order of 0.1% of light emitted by light
source.
High pressure mercury lamps have powerful spectral line sin proximal
ultraviolet especially at 366 nm and in visible blue violet near 405 and
435 nm. Recently, period xenon burners are also being used.
2.
Excitation filters/Ist Barrier filters: These are place at the entry of light
and lets through only blue light of 450 and 490 nm absorbs the rest.
3.
Mirror: This can not be of the conventional design as light from the UV
region is only partly reflected by silver. Therefore, specially designed
mirror is used having aluminum covered with thin layer of silver.
Now a days bean spitting mirrors, i.e. dichroic mirrors as developed by
Ploem (1967) are used. This has an interference coating which has a
high reflectance for the light passed by an excitation filter but is
transparent for the fluorescence light of longer wavelength. Thus even
this acts as a filter.
4.
Barrier filter: Placed near eyepiece to remove stray fluorescent

illumination. It only transmits light of longer wavelength emitted by the
object/specimen. This can be of low absorption since the mirror also
functions effectively as a barrier.
Confocal scanning microscope

This is a recently developed form of microscopy which enables a direct
way of visualizing a specimen in 3D. Electronic imaging methods make it
possible to focus on a chosen plane in a thick specimen while rejecting light
that comes from out of focus regions. From a series of such images-3D
images are obtained.
The microscope is used with fluorescence optics. But instead of
illuminating the entire object, the optical system at any instant focuses a spotlight into a single point at specific depth in the specimen. A very bright pin
point source of illumination is required-laser is used.
Fluorescence emitted from the point is collected; brought to an image
at the entry port of a suitable light detector. Pin hole aperture is placed at the
detector, at the site that is confocal with illumination pin Hoe-precisely where
rays from illuminated point come to focus. Thus this light converges on
aperture-enters the detector.
40
Out of focus light can not enter. To build a 2-D image, data from each
point on the plane of focus is collected sequentially by scanning and are
displayed.
This is used to resolved structure of numerous complex 3-D objectsnetwork of cytoskelecal fibres in cytoplasm and arrangements of
chromosomes and genes.
Applications
1.
Immuno fluorescence is flurescein antibody staining in diagnostic

microbiology: In this technique fluorescent dyes are combined with
antibodies i.e. substances that combine with specific microorganisms
(antigens). These are labeled antibodies. These labeled antibodies are
mixed with a suspension of eg: bacteria. The preparation is then
examined by fluorescent microscopy. Those cells which have been
linked to the antibody are now visible. This is the fluorescent antibody
technique. Theoretically it is possible to identify single bacterial cells by
this procedure eg: For poliomyelitis-Mahoney stain-fluorescein.
This techniques permits detection of various aggregates smaller than
Negri bodies. Even species of Salmonella typhosa can be identified
from S. Virginia.
2.
Fluorescent analogue cytochemistry

In this techniques to introduce membrane impairment molecules
into & living cell, whether antibodies or normal cell proteins-these are
tagged with fluorescent label and microinjected into the cell.
Purified protein coupled to fluorescent dye microinjected and
fate of injected protein followed by fluorescence microscope as cell
grows and divides eg. if tubulin is labeled with a dye that fluoresces red
microtubule dynamics can be followed second by second.
3.
Chromosome analysis in cytogenetics:

When it is possible to localize certain substances in a cell, it is
possible to detect minute quantities of the substance using fluorescent
dyes. Reigler was able to demonstrate 10-5 gm DNA in single cell.
4.
Microflurometry
To measure fluorescence/total amount of fluorescent material in the cell
eg: acridine orange to DNA
5.
Flow system analysis:

Large number of cells measured in few minutes/flow microfluorometry,
flow in capillary across a beam of excitation light. Emitted pulses
sensed by photomultiplier used in leukemia blood analysis.
41
Reference
1. Alberts. B. Bray D., Lewis, J.; Raff. M.; Roberts K.; Watson J.D. Molecular
Biology the cell : Garland Publishing Inc. 1994 Third Edition.
2. James J. Light Microscopic Techniques. M.N. Medical Division, 1993.
3. Pelczer M.; Kreig; Chan. Microbiology. Tata Mc. Graw Hill, 1993
4. Rodina, R. Aquatic Microbiology, 1972.
5. Stanier R.; Ingrahm, Wheelis General Microbiology, 1987.
ELECTRON MICROSCOPY
INTRODUCTION
No one person can be singled out as the inventor of electron
microscope. The invention is a product of large no. of inventions and
discoveries in Physics. A culmination of many scientific advancements finally
lead to the advent of Electron Microscope. Before its development several
microscopes like light Microscopy, Fluorescent Microscopy, Phase contrast,
Microscopy, Interference microscopes were invented.
By these microscopes the maximum resolution that can be obtained is
0.2 m. Before this dimension however Words inside the cell seems to be
particularly interesting. It was the dream of every biologist to get hold of a
device with better power of resolution. U.V. radiation has slight advantage.
Although the radiation is visible, the picture produced by it can be visualized
by a radiation transformer (fluorescent layer, photo plate). However the
increase in resolution produced by this is relatively small. The resolution can
be increase by a factor of 1000 by using electromagnetic waves X-rays,
rather than light. But the problem for this radiation to be used in microscopy is
that there are no sufficiently strong deviating mirrors and lances elements one
could not build a useful X-ray microscope for atomic resolution. This is
because of lack of interaction to the rays with the specimen. In case of X-rays
interaction is extremely weak. The same physical law that permits to
transilluminate a human body would let a bacterium appear completely
without contrast.
Then came the concept of e-rays to be used in microbiology. As for as
interactions are concerned e-rays are almost ideal. Since with medium
voltage. Each is only a few thousand of nm. One need not worry
about the restruction of resolution by diffractions. Furthermore e-ray can be
influenced in the direction of their propagation by electrical and magnetic field.
A source of e-rays provided to means. By this, our vision can be extended
thousand fold. By this thus the simplest forms of life-viruses can be observed
and even the building blocks the macromolecules. Thus electron microscopy
has significance as it provides us with the opportunity of studying matter
molecular level.
HISTORICAL BACKGROUND
The major events which contributed to the development of electron
microscope are:
42
(1)
Discovery of electron by J.J. Thomson as negatively charged particles

who termed them as thermions
Charge of eMass of e-
=
=
-1.6 x 1019C
-9.1 x 10-31 kg
(2) De Broglies hypothesis that moving charge particles like e- have wave
nature similar to electromagnetic waves.
(3) Bush gave a theoretical hypothesis that e-beams can be focused by
employing electrostatic field and thus laid the foundation of Electron
optics.
(4) Knoll and Ruska Published the first result obtained with electron
microscope by using magnetic radiation. Although the resolution
obtained was inferior to that of light microscope, the real beginning in
electron microscopy was accomplished, and the I st electron micrograph
of non-self illuminous specimen had been obtained.
This Ruska built the Ist TEM:
(5) Knoll demonstrated the feasibility of the SEM and three years later a
prototype was built by Von Ardenne.
(6) Siemens produced the Ist commercial TEM.
(7) Rebertson described the trilaminar structure of cell membrane, seen for
the Ist time in the electron microscope.
BASIC NTHEORY OF ELECTRON MICROSCOPY
(1)
Wave nature of e-beam

De Broglic in 1924 proposed that e-beam have wave nature:
h
1
X
m
v
h
m
v
KE
v
=
=
=
=
=
=
...........1
Wavelength of matter wave

Planks constant = 6.63 x 1034 J/S
Mass of particle
Particle velocity
mv2 = Ve ..2
Pot. difference through which e-bean has been
accelerated.
charge on e.
Combining eq (1) and (2) we get
12.3
V
43
This shows that the bean is dependent upon the voltage through which
has been accelerated.
This high velocity e-owing to their small wavelength might provide
suitable form of radiation of fulfill the requirement for high resolution
microscopy.
(2) Resolution
Defined as ability to distinguish between two points
or lines separated by as short a distance possible.
Abbe gave an eq.
d
NA
n
d
0.612
0.612
n sin
NA
wave wavelength of image forming radiation
=
=
=
=
Numerical aperture = n sin

Refractive index of medium
Diameter of the image
1.2 aperture angle
This example expressed the magnitude of diffraction effect and

provides numerical value of limit of resolution any aberration free optical
system. Significance of d is that two points will just be resolvable if they are
separated by a distance greater than d.
Thus to obtain maximum value for resolution the limit or resolution or d
should be as minimum as possible and to obtain minimal value of d should
be as small as possible and n and sin should be large. But since, the values
of sin and n cant be increased significantly beyond a certain value, the only
factor that can be altered is
R.P. of eye = .25 mm, R.P. of light mic = .25 m, R.P. of e- = 2.5 A.
Thus R.P. of e.m. = 100 times of light mic microscope.
Development of Magnetic lens
Comparison between optical lens and magnetic lens
Lenses are used to bend rays of light o a bean of e- so that they are
deflected in predictable manner from their original path. Lenses do so
because the transparent material of which they are made of cause slowing
down of the velocity of light rays during its passage through the lens.
The lens with shorter focal length is a strong lens Convex lenses are
Converging or Magnifying or Positive. Concave lenses are diverging or
negative lens.
44
Unlike light lays in light microscope the electron does not change its
velocity as it passes through the magnetic field and only gets deviated by a
force acting on it owing to the surrounding magnetic field.
In case of optical lenses refraction takes place at the interface between
the lenses and the immersion medium which is air in case of light microscope.
In case of magnetic lenses the e= get defected whole time while their
passage through the magnetic field refraction is continuous and there is no
sharp interface between the magnetic field (refracting medium) and the
immersion medium (the vacuum within the electron microscopic column).
Principle
When a current is passed through an axillary would coil it behaves like
a hollow bar magnet. Part of the magnetic field run inside the coil parallel o
the axis, part is radiated out in specie outside the coil. The only useful part of
B is that inside the coil which is parallel to axis and the external field of the
solenoid is simply wasted.
The efficiency of a simple solenoid lens was improved by enclosing the
energizing coil in a sheath of soft iron. Soft iron has the property of
concentrating the lines of force in magnetic field thus becoming itself
magnetized by induction. As the current in the solenoid is increased, strength
of B is increased and so the induced magnetism in soft iron sheath and hence
strength of the lens increase or its focal length decreases.
Focusing action of uniform magnetic field
An e- enters the uniform portion of the magnetic field of a long solenoid
at an oblique angle. When its reaches the point O which lies on the axis of
coil its trajectory will be determined by its vector components (horizontal and
vertical). This simultaneous actie forces produce curricular forward motion i.e.
a helical path which is tangent to the axis. If there had been no B than the ewould have moved optical counter part functions by forcing an e-which is
diverging from the axis back into the axis.
It the number of e-having same axial velocity, the pitch of their helices
will be the same and all electrons passing through a given point will after one
revolution come together at the same time.
Hence in this way magnetic field can produce an erect image of an
object placed in its path which rendered visible by fluorescent screen.
Magnification
M of light Mic =
1000 times
M of Electron Mic = 1,00,000
= 1000 x Magnification by light
microscope
45
Different parts of Electron Microscope

Electron Microscope consists of 3 systems
(1)
(2)
(3)
Illuminating system
Imaging system
Image Translating system
I.
Illumination system has following parts
(i)
Electron Gun
The gun acts as an electrostatic lens and as has following parts.
(a)
Filament
It is a thermionic cathode, usually V shape tungsten wire and can be

electrically heated to incandescence to act as electron source.
(b)
Shield
It is equivalent to a grid of triode and is an aperture cylinder. A small

potential difference is maintained across filament and shield. It acts as a
focusing electrode.
(c)
Anode
An aperture disc and attached to high positive terminal voltage. The

emitted e-are accelerated in the space between to be filament and the anode
by the potential difference across the gap.
There are basically two methods of connecting the accelerating
potential to the electron gun.
In Non biased system a shield and filament are connected through a
pair of Balancing resistors directly to V terminal of high voltage supply.
In Biased system the filament is connected to the high voltage supply
through both a Bias and a pair of balancing resistors while the shield is
attached directly to the high voltage supply.
(e)
Self Biased gun
In practice almost all microscopes currently manufactured have

incorporated the biased system in their electron source and the type usually
used in called the self biased gun. In such a system the shield aperture acts
as a strongly converging lens.
(2)
Condenser lens
The basic function of the condenser lens is to focus the electron beam
emerging from gun into the specimen to permit optimal illuminating conditions
46
for visualizing and recording the image. This depends upon the condenser
lens, focal length.
The condenser lens of Electron Microscope is a relatively weak lens
having a focal length of the order of few cms which can be adjusted over a
considerable range of values.
c
fc
fc
S
=
=
=
=
S
s fc
Condenser aperture angle

radius of condenser aperture
focal length of the condenser
distance from the specimen to the lens
Intensity of illumination: As in case of light microscope, the intensity of

illumination is directly proportional to the square of aperture angle.
I
I
K
c
II.
=
=
=
=
k c 2
Intensity of illumination
an Instrument constant
Condenser aperture angle
Imaging system has follows parts

It consists of objectives lens and one or more projectors lens.
(i)
Objective lens
It forms the initial enlarged image of the illuminated portion of the

specimen in a plane that is suitable for further enlargement of the projectors
lens. Their f length is very short = 1.5-5 mm i.e. it is a stronger lens. The
magnification of the image produced is 100.
(2)
Projector lens
The projector lens as the name implies serves to project the final
magnified image on the screen or photographic emulsion. This lens has a
construction similar to objective lens except that it has no provision to
accommodate the specimen holder. The pre image obtained from the
condenser lens acts as a object for projector magnification is 100.
Projector lens has follows criteria:
(1)
Board range of focal length.
(2)
The great depth of focus of the resulting high magnification system.
47
(3)
(3)
A difference in manifestation and a less serious effect of its

aberrations on resolution.
Intermediate lens
May be employed in between objective and projector lens for further

magnification. It has variable focal length.
III.
Image Translating System
The information the e microscopy provides in the form of variations of

e-intensity. The e-image is converted into a visible light image by the
following.
(1)
Fluorescent Observation Screen
Instantaneous and continuous transformation of the electron image into

visible image is done by fluorescent materials which have the property of
emitting light of greater wavelength when bombarded by e-beam. A mixture of
Zn and Cd is generally used.
(2)
Photographic Recording
A photographic negative of the final electron image is produced. A print

of image on the film is known as electron micrograph and serves as a
permanent record.
Operation of Electron Microscope
Operating Adjustment
The operating adjustment consists of following steps:
Lens
Usual E.M. adjustment
Condenser
Aperture diameter of condenser and condenser

current controlled.
Objective
Objective current control.
Projector
Projector and/or Intermediate current control

and/or pole piece interchange.
Photography
Routine procedure followed is:
(1)
The field of view selected at low magnification and at low beam

intensity and preliminary centering and focusing is carried out.
(2)
The magnification is raised so that described level of resolution of

detail in the e-image is obtained.
48
(3)
The intensity of illumination raised but only up to adequate level for

vertical focusing and reasonably short exposure.
(4)
The photograph is than taken without changing the condenser setting.
Methodology of Electron Microscopy

Tissue to be studied is removed from the site and cut into small pieces
within one or two min. cutting should be done rapidly.
The size after slicing should be less than 1 mm. Immediately after
slicing transferred to fixative.
(2)
In site fixation
Tissues can be fixed in situ in the living an aesthetic animal to minimize

the effects of both cutting on unfixed tissue and of anoxia resulting from
cutting off circulation.
(3)
Perfusion
Tissues that the too delicate to with stand anoxia like that on CNS are
subjected to in-situ vascular perfusion of the tissue.
a)
Osmium tetraoxide fixation
OSO4 is widely used in majority of studies as fixative. It reacts with

unsaturated lipids which is evident by the blackening of the tissues. OSO 4
acts to stabilize proteins of the cell by reacting at the polypeptide side groups
of tryptophan and histidine thereby linking protein chains together.
No. of factors are critical for good fixation.
i)
pH of fixative
7.2 7.6 is recommended
ii)
Buffering medium
Acetate veronal buffer of Michaelis was used before. Now S-collidine

and phosphate are used.
iii)
Tonicity of fixative
Optimum is 0.4C.
iv)
Duration of fixation
Optimum is from 30-90 min. but it dependents upon the nature of

specimen.
49
(B)
Formalin
Useful for plant tissue for 10-15 min.
(C)
Glycerol dehyde
Very useful in studying cellular ultra-structure esp. organelles like

microtubule.
(D)
Permanganate
For study of membrane system in plants
Dehydration
Various dehydrating media employed
Ethyl alcohol
Acetone
Propylene
Embedding
The various embedding medium used are :
i)
Methacrylate
Easy to handle with good rate of penetration through the tissue

capacity to be cut to thickness in the range of 500-1000 A248 with relative
case.
ii)
Water soluble medium
Three water soluble agents has been used Glycol methacrylate,

Aquon (eposey resins component) and Durcupan. Aq. Sol of these media are
used immediately after fixation both as dehydrating agent and than in pure
form as infiltration and imbedding agents.
iii)
Polyester resin are also used
Microtomy
Improvements in the method of thin sectioning were needed to take full
advantage of the increased resolving power of electron microscope.
Principle of Microtomy
In order to efficiently cut successive thin sections an ultramicrome must
structurally meet following requirements.
(1)
All of its movements must be free of vibrations of magnitude of the

order of .01 rev.
50
(2)
Should have very little static friction
(3)
The specimen should pass the knife edge once only
Ultramicrotome consists of a horizontal bar to the front end of which is

attached the specimen bolder. This bar is moved forward b advanced
mechanism, knife is mounted in front of instrument and cuts sections by
repeatedly moving the specimen past the knife edge.
Knives can also be used for microtomy but must be able to fulfill
following criteria:
i)
The edge should have redices less than section thickness required.
ii)
Resistant to chemical decomposition
iii)
Hard and tough
iv)
Made by homogenous material
v)
Should be physically stable.

Glass knives and diamond knives are usually employed.
Section thickness
Contrast of e-microscope and section thickness are related as follows:
C
C
d
P
V
ed
V ( o ) 1 / 2
=
=
=
=
=
Contrast
object thickness in A
Effective density
Voltage
objective aperture angle
This dic. aperture angle and increase in object thickness increases

contrast. However, as object thickness increases and voltage decrease
chromatic aberration rather than contrast sets the limit of resolution. Therefore
depending upon particular properties of both specimen and microscope
compromise is made for maximum resolution.
Kinds of Electron Microscopes
(1) TEM = Transmission Electron Microscope
TEM can only image a specimen thin enough to transmit a substantial
proportion (50-90%) of the electrons indicent on it. The electrons transmitted
by the specimen are focused to form a magnified image on a fluorescent
screen.
51
(2)
SEM = Scanning Electron Microscope
High velocity electrons strike the surface of a solid object in the

vacuum. Electrons are either reflected of emitted from the surface. Reflected
e-are called as back-scattered primaries are deflect through large angle by
interadtion with atomic nuclei. Secondary electron of very low velocity are
produced due to ionization of the specimen atoms by the incident primary
atoms. A picture of the surface may be built upon a synchronous display tube
in the light either of back scattered primaries or of emitted secondaries.
Advantages of E.M.
(1)
In the determination of particle size and shape.
(2)
Some of the industrial problems have been studied like structure of

textile fibres, purification of lubricating oils, composition of paints and
paper, surface of metals, plastic etc.
(3)
The concentration of colloidal particles in suspension can also be

quantitatively measured.
(4)
Used indirectly in the quantitative analysis of nucleation and crystal

growth.
(5)
In the field of medicine and pathology used in the study of virus and
bacteria.
(6)
In the determination of no. of protein molecules in virus.
(7)
Due to shadow casting method one can get good outlines and profiles
of viruses, bacteirophages and macromolecules.
Disadvantages of Electron Microscope

(1)
The intense electron beam acting for a considerable length of time

destroys the specimen in many cases and beam can serve as
reducing agent.
(2)
The electron beam is unable to penetrate through metallic surface;

hence study of metals is carried out by an indirect method called
replica method.
52
APPLICATION OF ELECTRON MICROSCOPE

The electron microscope is an instrument which utilized electron as a
source of illumination for observing objects at a great magnification. Because
of its high resolving power, it is widely used for visualization of biological
specimen. Various types of electron microscope had been developed, they
are1231.
SCANNING ELECTRON MICROSCOPE (SEM)

TRANSMISSION ELECTRON MICRSOSCOPE (TEM)
FREEZE-FRACTURE AND FREEZE ETCH. E. MICROSCOPY
SCANNING ELECTRON MICROSCOPE (SEM)
Scanning electron microscopy combine the mechanism of electron

microscopy and television, scanning electron microscope has been used to
examine the surface of microorganisms in great detail. SEM became
commercially available only in the early 1960s after over the 3 decades of
intensive researches by knoll, Von Ardene, Zworykin, Hayes and many others.
SAMPLE PREPARATION FOR SEM
(leave, pods, insects, fungi, grains, and other tissue)
1.
Select appropriate location and size of the leaf, fix it immediately in 5%

Glutaraldehyde in phosphate buffer (0.1 M, 7.3 pH). Apply, mild
vacuum so as to remove air for effective, infiltration for fixative.
Glutaraladehyde used as a strong protein cross linking fixative. To
retain biological activity (e.g. Antigenicity) a weak cross linker
paraformaldehyde can be used. Glutaraldehyde is a dialdehyde that
react primarily with free amino group of proteins, resulting in cross
linking of adjacent protein molecules. It provides excellent structural
preservation, due to formation of methylene bridges, as follows-
Cross linking by chemical fixation

2.
Sample are fixed for 7 hrs to over night and then washed thrice in the
same buffer at 15 minute interval.
3.
Post fix the samples with aquous OsO 4 for 4 hrs of more depending
upon the sample.
OsO4 cross links mainly with unsaturated fatty acid side chains
4.
Wash OsO4 with water thrice or more with 15 minute interval
5.
Dehydrate the tissue with 30-100% graded series of Acetone/ethanol or

Amyl acetate.
53
The purpose of dehydration is to replace all free water in the specimen

with a fluid which is miscible with water. Ethyl alcohol is widely used
because it does not harden the tissue and make it brittle for
subsequent ultra thin sectioning and a certain amount of OSO 4 remain
with tissue. This is reduced by the alcohol to black, insoluble OSO 2
which from a fine granular and colloidal suspension
6.
Dry the sample in a critical point drier suing liquid CO 2

Liquid CO2 used to avoid distortions due to surface tension affects
which cause the collapse of surface structure as in microvilli and
Pseudopodia
7.
The dried sample is mounted on a clean SEM stub having gun, Use
silver or graphite dag to aid conduction.
8.
Coat the sample with graphite to a thickness of 40 nm followed by

gold/gold palladium for a final thickness of upto zoom.
9.
Observe the sample at 5-15 KV and W.D. 15 mm.
WORKING OF SEM
The electrons originate at high energy (20,000V) from a hot tungsten or
lanthanum hex a boride cathode gun. These electrons are sharply focused,
adjusted and demagnetized by an arrangement of magnetic fields and lenses
which is analogous to the condenser of an optical microscope. Instead of
forming a side file as in optical microscopes or transmission electron
microscope, the electrons are made to form a needle sharp probe about 5.0
nm to 10 nm in diameter. The primary beam or probe acts only as exciter of
image, forming secondary electron that emerges from the surface of the
specimen. When the probe strikes secondary electron entering the detector,
strikes a scintillator causing it to emit light flashes that a photomultiplier
converts to an electrical current and amplifiers. The single is sent to a cathode
ray tube and produces an image like a television picture which can be viewed
or photographed.
The number of secondary electron reaching the detector depends upon
the nature of the specimens surface when the electron beam strikes a raised
area, the number of secondary electrons to reach the detector is large. The
contrast beam strikes a depressed area, numbers of secondaries appear
lighter on the screen and depressions are darker. A realistic 3D image of
microorganisms surface, with great depth of focus result.
ADVANTAGE
1.
2.
3.
4.
In SEM very thick specimen can be viaualize

In SEM specimen preparation is easily
In SEM picture can be seen in television
In SEM a 3D image formed
54
TRANSMISSION ELECTRON MICROSCOPY

Transmission electron microscope is an microscope in which the image
is formed form electrons that are transmitted through a thin specimen. The
TEM has a practical resolution roughly 1,000 times better than the light
microscope. TEM is used for the visualization of interval structure of the
smallest cell. The distinctive feature of the TEM is the specimen preparation
and nature of specimen.
SAMPLE PREPARATION FOR TEM
Fixation and Embedding
1.
The sample is cut when immersed in 3% Gluteraldehyde prepared in

0.1 M phosphate buffer pH 7.3 into pieces of 2 to 3 mm cube size and
the fixative is gently vacuum in filtrated into the tissue for 2-6 hrs or
over night.
2.
Samples are then washed throughout in the buffer several times over a
period of 2 to 3 hrs.
3.
Post fixation is carried out in 2% aqueous osmium tetra-oxide for 4 to 6

hrs.
4.
The post fixed samples turn black and brittle after sufficient time. The
tissue is washed carefully several times with distilled water till all
excess fixative is removed.
5.
The samples are dehydrated using a graded series of Acetone of

ethanol over a period of 3 to 5 hrs (30-45 minute each is 30, 50, 70, 90
and 100% ethanol/Acetone) side chains.
6.
The samples are then infiltrated with a 1:1 mixture of dehydration

agent: Exposyresin for about an hour. Various Epoxy resins are used to
embed the samples. Commonly Araldite, Epson 812 or spur resin are
used.
The purpose of embedding is to form a solid matrix for support during
subsequent procedures, such as sectioning.
7.
The samples are then infiltrated over night to 48 hrs. with pure resin
mixed finally placed in resin poured into flat silicon rubber moulds to
from block
8.
Blocks are formed by placing the mounds is 70C in cm oven for 24 to

48 hrs.
Ultra thin Sectioning / Microtomy

For the study of animal and plant tissue with optical microscope the
tissue are first soaked is a fixing solution then water, afterwards dehydrating
55
fluid and paraffin solvents. They are then heated and cooled and forms small
blocks. The blocks with the embedded tissue are cut into thin slices called a
microtomy. Each thin section is mounted on a glass slide, paraffin is removed
by some solvents and the tissue are then stained and examined with a
microscope.
A analogous procedure is used for making thin sections of bacteria and
other micro-organisms for TEM. Sine electron have very low penetrating
power, the section must be exceedingly thin of the order of 0.02 m for this an
ultramicrotome is used. The embedding material instead of paraffin is epoxy
resin, and after solidifying and warming, blocks are formed. These blocks are
cut by the knife with the edge of diamond.
SHADOWING
This technique is particularly useful in studying virus morphology,
bacterial flagella and plasmids. In this technique, it is coated with a thin film of
platinum or other heavy metal by evaporation at an angle of about 45% from
horizontal, so that the metal strikes the microorganisms on only one side. The
area coated with metal scatters electrons and appears light in photographs
while the uncoated side and the shadow region is dark.
STAINING
Since most of the constituent element in biological material are of low
mass the contrast of these material is weak is can be greatly enhanced by
staining with the salt of heavy metals (lead/tungsten/Uranium). These stains
are non specific i.e., it stain all the components. The two most widely used
section stain are uranyl acetate and lead citrate.
NEGATIVE STAINING
Negative staining is an excellent way to study the structure of viruses,
bacterial gas vacuoles and other similar material. The specimen is spread out
is a film with either phosphotungestic acid or uranyl acetate, heavy metal do
not penetrate the specimen but renders the back ground dark while the
specimen appears bright in photograph.
FREEZE PARACTURE AND FREEZE ETCH ELECTRON MICROSCOPY
Microorganisms prepared for ultramicrotome sectioning by chemical
treatment and embedding in plastic may show distortions and unnatural
changes due to the preparatory process. These can be eliminated to a great
extent by freezing the specimen instantaneously in ice at tem of -198 or low
there are two different methods.
1.
2.
Freeze fracture
Freeze etch.
56
1.
Freeze Fracture
Freeze fracture electron microscopy provides a way of visualizing the

interior or cell membranes. In this method fells are frozen at the temp of liquid
N2 (-198 C) in the presence of Cryoprotectant (Ant freeze) to prevent
distortion from ice crystal formation and then the frozen block is cracked with
a knife blade. The fracture plane often passes through the hydrophobic middle
of cell membrane. The resulting fracture forces are shadowed with platinum,
the organic material is dissolved away and the replicates are floated off and
viewed is the electron microscopes. (Shadowing is capable of providing high
contrast surface views). Such replicas are decorated with small bumps called
intra membrane practices, which represents large trans membrane proteins.
The technique provides a convenient and dramatic way to visualize the
distribution of such protein in the plane of a membrane.
FREEZE ETCH E.M.
Freeze etch electron microscopy can be used to examine either the
extenior or interior of cells. In this special device to cover the sample against a
copper block cooled with liquid helium and the frozen block is crack with a
knife blade. But now the ice level is lowered around the cells by the sub
limitation of ice in a vacuum as the tem. is revised (process called freeze
drying) the parts of the cells exposed by this etching process are then
shadowed as before to make a platinum replica. This technique exposes
structures in the interior of the cell and can reveal their 3D str.
Because of a metal shadowed replica rather than the sample
itself is viewed under vacuum in the microscope both freeze fracture and
freeze each microscopy can be used to study frozen unfixed cells, thereby
avoiding the risk of artifacts caused by fixation.
Reference
1.
Introduction to biophysical methods of protein and Nucleic acid Research

J.A. GLASSEL & DEUTSCHER.
2.
Practical electron microscopy for Biologist GEOFFREY A. MEEK
3.
Practical Biochemistry K. WILSON AND JOHN WALKER
4.
Fundamentals OF Microbiology FOR WISHER, HINSDILL, CRABTREE,

GOOD HEART.
5.
Molecular Biology of the Cell _ BRUCE ALBERTS, BRAY, LEWIS MARTIN

RAFF, ROBERTS, WATSON.
6.
Microbiology PRESCOTT. HARLEY, KLEIN
7.
An introduction to Microscopy The odorve GEORGE ROCHOW AND

EUGENE GEORGE ROCHOW.
57
Experiment No. 1
Objective:
To study the compound microscope and observe the

microbial slide.
Requirements:
Slides, Microscope, Oil etc.
Observations:
Different types of microbial slides were observed under

the 100 X power in microscope.
Sample Slide No. 1
BACCILLUS MEGATERIUM
1.
Rod like cells are visible.
2.
Chains of rod cells are present and such type of arrangement of cells is
called as bacillus from structure.
3.
In some cells oval shaped slightly lightish colour zone is present which
is called as endospore.
4.
Cells are violet is colour.
Sample Slide No. 2
E. COLI
1.
Cells are small rod shape
2.
No chain of cell is present
3.
Cells are pink is colour
58
Sample Slide No.3
SARICINA LUTEA
1.
Cells are oval shaped in group of 8 cells
2.
Only 4 cells are visible at a line.
3.
Cells are present in a 2 tear of 4 cells
4.
A outer sheath like, irregular covering is present where 4 cells are

enclosed.
Sample Slide No. 4
STAPHYLOCOCCUS
1.
Cells are spherical some what rounded in shape called cocci.
2.
Bunches of cells are present and such a arrangement of cells are

called staphylococcus arrangement.
Sample Slide No. 5
SACCHAROMYCES CEREVISEAE
1.
Yeast cells are oval in shape.
2.
In some cells are bud like appearance present which is a mode of a

sexual reproduction is a yeast.
3.
Smooth sheath like membrane is visible to surrounding the cells.
Sample Slide No.6 NOSTOC

1.
Oval shape cells arranged in a chain.
2.
In each filament a single transparent cells is present which is layer than

the remaining cells is heterocyst.
3.
Heterocyst is site of N2 fixation is these blue green algae.
59
EXPERIMENT NO. 2
Objective:
To determine the dimension of microorganisms by using

micrometer
Requirements:
Bacterial suspension, Microscope stage micrometer, stains

etc.
Background:
Bacteria are microscopic organism having size ranging from

0.2 m to 1 m. And they are so small and only visible by
the use of microscope, so measuring the bacterial size,
various methods are available and one of them is
micrometer count which givens very accurate size of
bacteria.
Observations:
Example
Least count = 10/6 = 1.67
1. Long rod (bacillus)
One bacillus cell covers 4 ocular divisions
So its size
4 x 1.67
6.68 m
60
EXPERIMENT NO. 3
Objective:
To observe motility of bacteria by hanging drop method.
Requirements:
Bacterial suspension, slide, inoculating loop.
Background:
E. Coli consists a peritrichoris flagella by which it moved

in a zigzag pathway. In small organisms such as bacteria
they have flagella which help it into movement in various
direction is response of different chemical/physical or
environmental stimulates. But in case of sarcinae, the
movement is also observed but this is due to H 2O molecule
and cell Brownian movement.
Observations:
Example: Prepare a bacterial hanging drop slide and motion of bacteria can
be observed
Sample I (Bacillus sp.)
Cilia are present on surface it exhibits a long to & fro
motion, this is due to cilia.
Sample II (E. Coli)
It also exhibit a ciliary movement because of cilia, one
present on all over the surface Brownian movement is
absent.
Sample III (Sarcinae sp.)
Random zigzag movement is present.
61
Microbial Technology
a. Maintenance of aseptic conditions and handling during microbial culture
practices
b. Techniques to obtain pure culture of microorganism from mixed culture
c. Inoculation of pure culture by streak plate and pour plate methods
d. To perform simple/ direct staining and indirect/ negative staining and
Grams staining of pure culture isolates
e. Identification of unknown bacterial cultures by biochemical tests
62
Contents
1. Bacterial Staining
2. Nutritional requirements for growth of bacteria
3. Study of Bacterial Growth
3.1
3.2
3.3
3.4
3.5
Doubling time
Bacterial Growth curve
Synchronous culture
Continuous growth culture
Growth Measurements
3.5.1
3.5.2
3.5.3
3.5.4
3.5.5
3.5.6
Direct counts
Colony Counts
Most Probable number
Biomass Measurement
Light Scattering (Spectroscopy)
Chemical Estimation
3.6 Total Viable count

3.7 Heat stable count
3.8 Octyl viable count
4. Growth of bacteria under Environment stress
4.1 Effect of Temperature
4.2 Effect of pH
4.3 Effect of osmotic pressure
4.4 Preservation of Laboratory Cultures
5. Koch,s postulates of bacterial and fungal pathogens
6. Culture sensitivity using different disinfectants and antibiotics
7. Biochemical Characterization of isolated microorganisms
8. Mutations and Mutants
9. Quantitation of viruses
63
1. STAINING TECHNIQUE
INTRODUCTION
Fixed, stained preparations are most frequently used for the
observation of the morphological characteristics of bacteria. Stains are used
to make characteristics of bacteria contrast with back ground and to make
differentiation of cells and localized of cellular materials.
The advantages of this procedure are that:
1. The cells are made more clearly visible when they are coloured.
2. Differences between cells of different species and within the same
species can be demonstrated by use of appropriate staining solutions
(differential of selective staining).
The essential steps in the preparation of a fixed, stained smear are:
(i)
Preparation of the film or smear.
(ii)
Fixation and
(iii)
Application of one or more staining solutions.
Microbiological stains
A large number of coloured organic compounds (dyes) are available for
staining microorganisms. These compounds are generally rather complex in
terms of molecular structure. On this basis they may be classified into groups
such as Triphenylmethane dyes, Oxazine dyes and thiazine dyes.
Type of Stains
Stains are of two types:
(a) Natural stains: Stains which are used in histological and general studies
of cells.
(b) Synthetic stains: Stains which are used in Molecular biological studies.
Synthetic stains are dyes having a benzene ring and
auxaphore or chromophoric groups.
A more practical classification for the cytological point of view is based
on the chemical behaviour of the dye, namely.
Acid, Basic or Neutral
An acid (or anionic) dye is one in which the charge on the dye ion is
negative. E.g. Nigrosine, Eosin, India ink.
A basic (or cataiornic) dye is one in which the charge carried by the dye
ion is positive. E.g. Methylene blue, crystal violet
A neutral dye is a complex salt of a dye acid with a dye base e.g.
Eosinate of methylene blue.
Acid dyes generally stain basic cells components and basic dyes
generally stain acidic cell components.
Basic Principle behind staining of microorganisms
The process of staining may involve ion-exchange reactions between
the stain and active sites at the surface of or within the cell.
e.g. the coloured ions of the dye may replace other ions on cellular
components. Contain chemical grouping of cell proteins or nucleic acids may
be involved in salt formation with positively charged ions such as Na + or K+.
64
Thus these peripheral areas of the cell carry a negative charge in combination
with positively charged ions. E.g. (Bacterial cell) (Na +)
Basic Terminology
Mordant
It is a substance which increases the affinity or attraction between the
cell and the dye.
Decolourizing agent
It is the substance which removes the dye from a stained cell
Counter stain
It is a basic dye of a different colour from the initial one. The purpose of
the counter stain is to give the decolourized cells a colour contrast different
from that of the initial stain.
Staining of bacteria
Following methods are commonly used.
1.
Simple stain technique
The coloration of bacteria by applying a single solution of stain to a fixed
smear is termed simple staining the fixed smear is flooded with a dye solution
for a specified period of time after which this solution is worked off with water
and the slide bottle dye. The cells are usually staining uniformly. However,
with same organisms, particularly when methylene blue is used, some
granules in the interior of the cell may appear, more deeply stained than the
rest of the cell, indicating a different type of chemical substance.
Procedure for simple staining
A small amount of bacteria is placed in a droplet of water on a glass
slide, then it is air dried. The slide is passed through a flame in a process
called heat fixing which fixes the cells to the slide, kills most organisms and
prepares them for staining.
Now the slide is flooded with a basic dye such as crystal violet or
methylene for a minute or so. The positively charged dye is attached to the
bacterial cytoplasm, which has a negative charge and staining taken place.
This is effective for negative cells; the stains do not easily penetrate spores.
Flow chart
Simple Staining
Bacterial Suspension
Smear
Air
Fixation
Heat
Crystal Violet
65
Wash with water
Dry
Observe under 100
2.
Negative Stain Technique
This technique works in a manner opposite to simple stain technique.
Bacteria are mixed on a slide with an acidic dye such as cargored or black
stain, nigrosine. The mixture is smeared on the face of the slide and allowed
to air dry. Because the stain carries a negative charge it is repelled by the
bacteria, which also has a negative charge the stain gathers around the cell.
Since a chemical reaction has not taken place, and because heat fixing has
been avoided, the cells appear less shriveled or distorted. They often appear
larger than stained cells and more natural.
Flow chart
Negative staining
Nigrosine
Smear
Fixation by air only
Observe under 100 X
Gram stain technique
A Danish scholar, Christain Gram in 1884 designed a differential
staining procedure, which differentiates between two kinds of bacteria. Gram
positive and gram negative bacteria. This procedure is called gram stain
technique. Different steps of Gram stain technique are as follows.
A thin smear of the bacteria is prepared on the slide. To the smear
crystal violet solution is applied for 30 seconds. The slide is then gently rinsed
in clear water and iodine solution is applied for 30 seconds. This is them
rinsed off. Then 95% ethyl alcohol is applied and this is renewed until all but
the thickest parts of the smear have ceased to give off the dye. This usually
takes from 20 seconds to one minute.
Microscopic examination of the slide will reveal that Gram positive
bacteria retain the violet iodine combination (Retaining of blue purple colour
even after alcohol wash). Where as Gram negative ones look the blue purple
colour after alcohol wash and will be of original colour. The species which
retain the stain are called gram positive where as that which yield the stain to
alcohol are called gram negative.
A counter stain a dye of same contrasting colour may be used to
clearly visualize the gram negative cells. The generally used counter stain are
3.
66
Eosin (red.), Safranin (red) Brilliant green or Bismarck brown. Each of these
colours the Gram negative species.
Flow chart
Gram Staining
Smear
Air
Fixation
Heat
Crystal Violet (30 Sec.)
Washing with water
(K/K1+I2) Lugals iodine (30 sec) (mordant)
Washing with water
Alcohol (25-40 sec.) most important step.
Washing
(Basic dye) Safranine (40 sec.)
Washing
Dry
Observe under 100
Gram negative (purple)
Gram negative (Pink)
67
Experiment No.4
Objective: To perform simple/ direct staining and indirect/ negative staining
Requirements:
Slides, Microscope, spirit lamp, inoculating loop, bacterial
culture, stains etc.
Background: In staining we used dyes to study the cell morphology.
Bacterial cell wall is slightly negatively charged structure. So
we used a basic dye for staining. Crystal violet is a basic dye
which contains a positive charge. It gives a colour basic group.
Bacteria contain a slightly negative charge on surface. Thus on
treatment with basic dye it takes a colour. While negative stain
also contain a negative charge so on treatment of bacteria with
acidic negative stain it will not gives a colour to bacteria. But
the back group of bacteria will observed as black colour and
bacteria are seen due to white transparent structure.
Type of staining
(i)
Simple staining
(ii)
Differential staining
(iii)
Structural staining
(iv)
Biochemical staining
Procedure:
I.
Simple staining (Direct)
Bacterial suspension
Dry Smear
Air
Fixation
Heat
Crystal violet (30 sec.)
Wash
Observe under 100 X
Observations:
Conclusion:
68
Procedure:
II. Negative/ Indirect staining
Nigrosine
Smear
Air dry only
Observe under 100 X
Observations:
Conclusion:
69
EXPERIMENT NO. 5
Objective: To perform gram staining
Requirements: Bacterial culture, inoculating loop, sprit lamp, crystal violet,
alcohol, water, lugals iodine, safranine, slide, etc.
Background: Gram staining is differentail staining. The stains are those
which enable one to differentiate two different groups of bacteria is a mixture
for instance Gram positive and Gram negative. This method was devised by a
Danish Scholar CHRISTIAN GRAM in 1884. Gram stain is differential stain
and it clearly differentiates a gram +ve bacteria from a gram ve bacteria.
Gram positive bacteria contains a very low amount of lipid (10-15%) and
Gram ve contain a enter lipo protein membrane so lipid content are very high
thats why on treatment of bacterial cell from alcohol which is a organic
solvent, dissolve all lipid from the membrane and make a holes in the wall that
all stain will comes out and bacteria and bacteria will not take a stain.
Procedure:
Smear
Air
Fixation
Heat
Crystal violet (30 sec.)
(Basic dye)
Washing with H2O
(Mordants)
Lugals iodine (30 sec)
Washing with H2O
(Removed excess stain)
Alcohol (25 40 sec)
Washing with H2O
(Basic dye)
Saffranine
Wash with H2O
Dry
Observed under 100x

Gram +ve (purple)
Gram ve (Pink)
70
Observations:
Conclusion:
Some examples:
Gram staining for E. coli, S. lutea and Bacillus megaterium were performed.
E. coli Pink
S. lutea purple
B. megatherium
Gram ve
Gram +ve
Gram +ve
71
EXPERIMENT NO. 6
Objective: To perform bacterial spore staining.
Requirements: Slides, Microscope, spirit lamp, inoculating loop, bacterial
culture, stains etc.
Background:
1. It is highly resistant due to thick spore wall the high resistance of spore
is due to dipcolinic acid (DPA).
2. The vegetative cell is killed at 600C + spore not even at 1000C
3. Spore requires special stain malachite green
Procedure:
Smear
Fixation by heat
Put a piece of filter paper on smear, keep the slide over a steam bath and add
malachite green at regular intervals so that filter paper is not dried
Wash
Safranin
Dry
Observe under 100 X
Observations:
72
2. NUTRITIONAL REQUIREMENTS FOR GROWTH OF BACTERIA

(Principles of Microbial Nutrition)
Every organism requires certain substances, drawn from their
environment, for the synthesis of their cell materials and for the generation of
energy. These substances are termed as nutrients. The nutrients preparation
on or in which a culture (i.e. a population of microorganisms) is grown in the
laboratory is called a culture medium.
A culture medium must therefore contain specific requirements of the
microorganisms for which it is designed i.e. all necessary nutrients required
for the sustenance of life. Although all the micro-organisms have the same
basis requirements but there is a diversity as to the use of organic and
inorganic and composition, depending on the species to be cultivated.
Thousands of different media have been proposed for their cultivation.
Some media contain solutions of inorganic salts and may be supplemented
with one or more organic compounds while other media contain complex
ingredients such as extracts or digests of plant and animal tissues.
DESIGN OF CULTURE MEDIUM SHOULD BE BASED ON SCIENTIFIC
PRINCIPLES I.E. THE PRINCIPLES OF NUTRITION
All the required metabolic elements can be supplied as nutrients in the
form of the cations of inorganic salts.
Specific mineral requirements, for example diatoms and certain algae
have a specific silicon requirement, supplied as silicates.
Specific chemical form of carbon, nitrogen sulphur and oxygen has
been provided depending on the need.
Now variety of nutritional patterns known to exist in the living world.
Nutritional classification is based on two parameters the nature of the
energy source and the principle carbon source.
Nutritional Classification
With respect to energy source, there are two classes.
Phototrophs
Use light as a energy source i.e. photosynthetic organisms.
Chemotrophs:
Use chemical energy source with respect to carbon source.
Autotrophs
CO2 as a principle carbon source.
Heterotrophs
Organic carbon source
There are four major nutritional categories by means of these criteria.
(i)
Photoautotrophs
Light as energy source CO2 as principle C-source.
These include photosynthetic organisms: Higher plant, algae, many
photosynthetic bacteria.
(ii)
Photoheterotrophs
Light as energy source, organic compounds as principal C-source e.g.
Purple and blue-green bacteria.
(iii)
Chemoautotrophs
Chemical energy source CO2 as principal C-source.
73
Energy is obtained by the oxidation of reduced inorganic compounds

such as NH3, NO2-H2, reduced form of sulfur CH 2S of light, grown in mineral
media strictly.
(iv)
Chemoheterotrophs
Chemical energy source, organic substances as principal C-source e.g.
all metazoan animals, protozoa, fungi and bacteria.
The role of oxygen in nutrition
Oxygen is universal components of cells and is always provided in large
amounts in the major nutrient (e.g. water and organic compounds). Many
organisms also require molecular oxygen (O2).
Obligately aerobic
Organisms that are dependent on aerobic respiration for the fulfillment
of their energetic needs and for which molecular O 2 functions as a terminal
oxidizing agents.
Obligately anaerobic
Do not require molecular oxygen to obtain energy. Molecular O 2 is a
toxin substance which either kills them or inhibits their growth.
Facultative anaerobes
Some micro-organisms able to grow either in the presence or absence
of O2 e.g. lactic acid bacteria (Anaerobic to aerobic in presence of O 2,
fermentative to respiratory), yeast (Aerobic to anaerobic, respiratory to
fermentative, energy yielding metabolism).
Microaerophilic
Some micro-organisms grow best at partial pressure of O 2 below 0.2
atomosphere.
Nutritional categories among micro-organisms
Two principal nutritional classes among organisms.
Autotrophs:
Which can completely use in-organic nutrients e.g. plants.
Heterotrophs:
Which require organic nutrients e.g. animals.
Osmotrophs:
Take up all nutrients in dissolves form
Phagotrophs
Take up solid food particles by the mechanisms termed phagocytosis.
The terms obligate and facultative are often used to indicate the
absence (or presence of nutritional versatility).
Additional pair of terms
Prototrophs
If a bacterium can grow in a minimal medium that is, it an synthesize all
necessary organic substances such as amino acids, vitamins and lipids from
simple precursors the bacterium is called a prototroph i.e. it can derive all
carbon requirements from the principal (source)
Auxotroph
If any organic substance other than a carbon source must be added for
growth to occur, the bacterium is called an auxotroph i.e. it requires one or
more organic nutrients in addition to principal carbon source e.g. growth
factors.
74
For example, if the amino acid praline is required in the growth

medium. The bacterium is praline auxotroph; the genetic symbol for a
bacterium with this phenotype is pro- A bacterium that doest not require
praline would be designated pro+.
The Methods of Microbiology
Cultures
Growing micro-organisms under more or less well defined conditions.
Pure or Axenic culture
A culture that contains only one kind of microorganisms.
Mixed culture
A culture that contains more than one kinds of micro-organisms
Two membered culture
If it contains only two kinds of micro-organisms and maintained
deliberately in association with one another
Preparation of pure culture involves not only the isolation of a given
micro-organisms from a mixed natural microbial population but also the
maintenance of the isolated individual and its progeny in a artificial
environment. In microbiology, we follow two kinds of operations.
i)
Isolation
Separation of a particular micoroganisms from the mixed population that
exist in nature.
ii)
Cultivation
The growth of microbial population in artificial environments (culture
media under laboratory conditions).
The terms obligate and facultative are often used to indicate the
absence (or presence of nutritional versatility).
Additional pair of terms
Prototroph
If a bacterium can grow in a minimal medium that is, it an synthesize all
necessary organic substances such as amino acids, vitamins and lipids from
simple precursors the bacterium is called a prototroph i.e. it can derive all
carbon requirements from the principal (source)
Auxotroph
If anyorganic substance other than a carbon source must be added for
growth to occur, the bacterium is called an auxotroph i.e. it requires one or
more organic nutrients in addition to principal carbon source e.g. growth
factors.
For example, if the amino acid praline is required in the growth
medium. The bacterium is praline auxotroph; the genetic symbol for a
bacterium with this phenotype is pro-A bacterium that doest not require praline
would be designated pro+.
The methods of Microbiology
Cultures
Growing micro-organisms under more or less well defined conditions.
Pure or axenic culture
A culture that contains only one kind of microorganisms
Mixed culture
A culture that contains more than one kinds of microorganisms
Two membered culture
75
If it contains only two kinds of micro-organisms and maintained

deliberately in association with one another
Preparation of pure culture involves not only the isolation of a given
microorganisms from a mixed natural microbial population but also the
maintenance of the isolated individual and its progeny in a artificial
environment. In microbiology, we follow two kinds of operations.
i)
Isolation
The growth of microbial population in artificial environments (culture
media under laboratory conditions).
These two operations are generally used for the study of virus,
bacteria, fungi, algae, protozoa and even small invertebrate animals as well
as isolation of cell or tissue lines derived from higher plants and animals
(tissue culture).
Three kinds of containers namely a test tube, a flask or a petridish are
most commonly used to cultivate microorganisms.
Inoculum
The microbial material used to seed or inoculate a culture vessel with
special precaution to maintain the purity of the culture.
Inoculum is introduced on a metal wire or loop which is rapidly
sterilized just before its use by heating in a flame.
Plating:
In lab jargon, a petridish containing a solid medium is called a plate
and the act of depositing bacteria on the agar surface is called plating.
Aseptic technique
Inoculation of sterile medium with a pure culture of micro-organisms
without contamination is commonly referred to as aseptic technique.
Growth Media or Culture Media
Construction of culture media
The primary goal of construction of culture media is to provide a
balanced mixture of the required nutrients in optimum concentration as well as
favourable environment for attaining good growth. Two basic requirements for
construction of media are:
ii)
The culture medium must contain those nutrients essential for the
growth of a given microbial culture.
iii)
The medium must at the same time provide suitable surroundings
for growth that is, the proper pH, osmotic pressure, atmospheric
oxygen and so on.
Requirement for carbon
Photosynthetic organism of bacteria uses oxidized form of carbon
(CO2) as the sole principal source of cellular carbon.
Reductive
CO2 = --------------- Organic cell constituents
Process
(Net input of energy)
All other organisms obtain carbon principally from organic nutrients and
they must supply the energetic requirements of the cell. Organic substrates
thus have a dual nutritional role as they serve as a source of carbon and as
source and energy are carbohydrate and its derivatives, fatty acids,
dicarboxylic acids, other organic acids, primary alcohols (ethanol, propanol,
76
butanol), Amino acids, other nitrogenous compounds and nitrogen-free ring

compounds.
Microorganisms can use single organic compound and variable
number of additional organic compounds for their purely biosynthetic function.
These addition organic compounds termed as growth factors.
Requirement for growth factor (s)
These must be provided as nutrients. Any compounds that an organism
requires as a precursor or constituent of its organic cell material and can not
synthesize from simpler carbon sources are collectively known as growth
factors. They fall into three classes (in terms of chemical structure and
metabolic function).
1) Amino acids-required as constituents of proteins
2) Purines and pyrimidines-required as constituents of nucleic acids
3) Vitamins-a diverse collection of organic compounds of certain enzymes
that form parts of the prosthetic groups are active centres.
Requirement for Nitrogen and Sulphur
Nitrogen and sulphur occur in the organic compounds principally in
reduced form as amino acids and SH groups.
Photosynthetic organisms and non-photosynthetic organisms

(bacteria and fungi) assimilate nitrates and sulfates as a source of
nitrogen and sulfur.
Some microorganisms are unable to bring about reduction and

this must be supplied in the reduced form as ammonium salts. Sulfur is
provided as sulfide or organic compound containing a SH group e.g.
cysteine.
N and S requirements can be met by organic nutrients that

contain two elements in reduced organic combination (amino acids or
more complex protein degradation products such as peptones). Such
compounds also provide carbon and energy sources.
Some bacteria can also utilize the most abundant natural N 2

source and assimilated by N2 fixation. It involves preliminary reduction
of N2 NH3 e.g. Rhizobium and other N2-fixing bacteria.
Requirement for favorable conditions
The control of pH
A given medium may be suitable for the inhibition of growth. Microbial
decomposition and utilization of compounds cause alterations in pH and may
become inhibitory. Thus, to prevent excessive change in ion concentration,
buffers are used e.g. phosphate buffers, bicarbonate buffers etc.
Avoidance of mineral precipitate
In culture medium mineral precipitation can be avoided by the addition
of chelating agents e.g. EDTA.
Control of O2
For obligate aerobic bacteria, O 2 is an essential nutrient. It is therefore
necessary to aerate the medium of liquid cultures by continuous passage of a
stream of air through culture.
77
The anaerobes which are killed by molecular O 2 can be cultivated by using

following techniques:
- Use of pre-reduced media by inclusion of reducing agents as cysteine,
thioglycollates, Na2S or sodium ascorbate which can be protected from
exposure to air.
- By evacuation of O2 and refilling with an inert gas e.g. N2.
- By chemical destruction of O2
- By combination of both methods
Provision of CO2
For photo autotrophs and chaemoautotrophs CO 2 is required for their
growth which may be provided by diffusion of CO 2 from the atmosphere into
the culture medium and by incorporation of NaHCO3 in the medium.
Provision of light
For phototrophic organisms (e.g. algae, photosynthetic bacteria) light is
an essential requirement. This may be provided by:
- Adequate illumination combined by control of temperature.
- Direct exposure to sub light should be avoided.
- Discontinuous illumination
- Fluorescent light has advantages.
- 750-1000 nm wave length of light is suitable for purple and blue green
bacteria.
Essentially all culture media are one or the other of two forms - Liquid or broth
media and solid media. On the basis of their composition there are two main
types of culture media.
A)
Synthetic medium
A medium composed of entirely of chemically defined nutrients of special
substances of known composition. It is also known as chemically defined
culture medium.
Synthetic medium are of four types.
Enriched medium
Used for a general purpose medium for a wide variety of
microorganisms
Selective media
Used for a selected microbe the selective media are those which
permit the growth of some specific group or type of organisms while
preventing the growth of others, thus facilitating bacterial isolation. The
selective action is brought about by the addition of certain chemicals in the
medium e.g. the dye crystal violet is selectively bacteriostatic for grampositive bacteria and is added into a selective medium and is added into a
selective medium for examination of enteries pathogens which are
predominantly gram-negative e.g. Mannitol-salt agar.
Differential medium
Used for differential isolation of microbes. A differential medium is that
which will cause certain colonies to develop differentially (i.e. differentiated)
from other organisms present by producing a characteristic change in the
bacterial growth and/or the medium surrounding the colonies. These media
are used for distinguishing among morphologically. These media are used for
distinguishing among morphologically and biochemcially related groups of
organisms e.g. blood agar.
78
Assay medium
Used for the assay of vitamins, amino acids and antibiotics. Roulins
solution and Martins rose Bengal medium.
B)
Complex medium
(Natural or empirical culture media) that contains ingredients of unknown
chemical composition. It is used for the cultivation of wide range or microorganisms. The natural media include milk, urine, diluted blood, vegetable
juices, meat extracts and infusions, peptone (digested protein rich in amino
acids that provide nitrogen and carbon). Now-a-days, group of living cells as
tissues or callus or an organ are being used for viruses and rickettsias.
Those media whose chemical composition is partially known are called
semi-synthetic media. Any medium which contains agar becomes a semisynthetic medium potato dextrose agar. Czapek-Dox agar, oatned agar, beef
peptone and nutrient.
These agar media are some of the semi-synthetic media.
Several media are available for use of microbiology (Table summarizes
properties of some of the commonly used media for growing various microorganisms).
Minimal medium
If the growth medium contains no organic compounds other than a
carbon source (such as sugar), it is called a minimal medium. A typical
minimal medium contains the ions, Na+, K+, Mg++, Fe++, NH3++, Cl-, phosphate
buffered to neutral pH and SO 4-2, and a source of carbon such as glucose or
glycerol.
Other metal ions are also required but in such small quantities that they
are usually provided as contaminants in the other salts used to prepare the
medium. The best source of carbon for most bacteria is glucose than with any
other single carbon source.
Classification
1.
Media may be grouped principally as follows:
Those media that require living cells or tissues the latter get parasitized by
them organisms to be cultured.
Those media that do not require living cells or tissues.
The media attain divided to two:
a)
Non-synthetic Media
A medium in which the exact chemical composition of each of the constituent
is not certainly known is referred to as non synthetic medium.
Potato dextrose agar (GM-25)
Soil Extract Agar (SM-1)
Oat Meal Agar (GM-24)
Malt extract agar (GM-19)
Waksman medium (GM-40)
b)
Synthetic media
A medium is which pure only chemicals in definite concentrations are used
is called synthetic medium. These media are useful for nutritional and
metabolic studies.
Czakeks Dox medium (GM-9)
Richards medium (GM-27)
79
Media may also be divided into 4 categories on the basis of consistency

i.e. their physical state; they are:
a) Liquid media
b) Semisolid media
c) Liquefiable solid media
d) Solid media.
b) Liquid Media
These media are used in liquid form
e.g. Nutrient broth
Skimmed milk
Peptone solution etc.
Agar are some of the semi-synthetic media.
c. Semisolid media
These media contain a smaller amount (0.5% or less) or agar which
imparts a custard consistency
e.g. cystine Trypticase Agar medium
d Liquefiable solid media
This medium is also called solid reversible to liquid medium these
media are prepared by adding suitable amount of gelatin or agar to the liquid
medium and remain solid when cool but become liquid when warm.
e.g. Nutrient agar medium
Nutrient Gelatin medium
Potato dextrose agar medium
e. Solid media
These media always remain solid
e.g. Potato slices
Coagulated Blood serum
Coagulated Egg
Trypticase Soy-Agar Medium
3. Media classified on the basis of their application or function as follows:
a. Cultivation media
b. Storage media
c. Enrichment media
d. Differential media
e. Assay media
f. Maintenance media
a. Cultivation media
Medium which is used for the general cultivation of bacteria e.g.
e.g. Nutrient broth
Nutrient Agar
b. Storage media
Medium in which bacteria are stored in Stock culture condition for
longer periods to provide a source of viable culture are called storage
medium.
e.g. Yeast Extract
Mannitol Agar Medium
80
c. Enrichement media
Enrichment medium is that in which nutritional environment is adjusted
in such a manner as to enhance selectively the growth of a certain bacterial
type with in a given mixed inoculums.
e.g. Addition of extracts of plant-animal tissues to nutrient broth of
nutrient agar media provides add
d. Differential media
e. Assay media
Certain media of prescribed composition have profound influence on
the bacterial cell with respect to formation of enzyme, toxin antibiotics are
other products.
e.g. Pyridoxine deficient growth medium
f. Maintenance media
These media are different from growth media and are required to
maintain the viability and physiological characteristics of bacteria.
DEFINITION OF TERMS
1.
Inoculum
Small amount of microbial culture used to inoculate a culture vessel for
the growth of the microorganisms in the culture under define condition.
2.
Subculture/ Passaging
Transferring culture from one vessel to another under aseptic condition.
3.
Culture
Growing microorganisms under desired condition.
4.
Axenic culture = pure culture
Growth of single microorganisms in the desired culture medium
5.
Mixed culture
Many microorganisms in a culture medium
6.
Cultivation
Growth of the isolated single microorganisms under desired conditions.
81
Experiment No. 7
Objective: To prepare nutrient broth, nutrient agar, agar slant, agar stab
and potato dextrose agar (P.D.A.)
Requirements: Measuring flask, distilled water, balances, peptone, beef
extract, potato, agar agar, pH paper, autoclave etc.
Procedure:
1.
Nutrient broth
Peptone
0.5%
Beef extract
0.3%
Final pH
7.0
2.
Nutrient agar
Peptone
0.5%
Beef extract
0.3%
Agar-agar
2%
3.
Potato dextrose agar
200 gm of peeled potatoes cut into chips + 500 ml distilled water, boil for
30 min and make up the volume upto 1000 ml. Add 20 gm dextrose + 20
gm agar-agar powder and sterilize at 15 lb/sq inch for 20 minutes.
Page 138
Preparation of nutrient media
Observations:
82
Experiment No. 8
Objective: To perform streak plate in order to obtained pure colonies of
bacteria.
Requirements: Petridishes, nutrient agar, inoculating loop, bacterial culture,
incubator, laminar flow, spirit lamp etc.
Procedure:
- Melt sterilized agar medium in a boiling water bath and cool to 45 0C:
pour the medium on to Petri dishes at least 12-15 ml is each: distribute
the medium throughout the dishes uniformly and let the medium
solidify. Do all this in aseptic conditions.
- Take an inoculation needle, sterilize it in flame and then cool by jabbing
it on to the agar.
- Take a small amount of the mixed culture at the tip of the needle and
start streaking on the surface of the medium.
- Streak several petridish-media from the same sample in different
fashions.
- Incubate petridishes at 300C for two days in an inverted position. This
results in the growth and multiplication of the inoculated
microorganisms in the form of isolated colonies.
- After 2 days of incubation, you will observe that the growing colonies
are more and more sparsely distributed towards the streak and. Pick
up well separated colonies and transfer than to agar slants as pure
culture.
Observations:
83
Experiment No. 9
Objective: Inoculation of pure culture of E. coli and Bacillus species in
Nutrient broth.
Requirements: Nutrient broth, bacterial culture, conical flask, laminar flow,
spirit lamp, inoculating loop.
Procedure:
1. 10 ml of nutrient broth (sterilized) is taken is a sterilized conical flask.
2. 1 ml of culture of E. coli and Bacillus species are aseptically added to
the 10 ml nutrient broth is laminar flow in front of flame.
3. Conical flask is corked and properly labeled.
Page no 116
Inoculation procedure
1 and 2
Observations:
84
Experiment No. 10
Objective: To perform the pour plate and serial dilution and count TVC
Requirements: Nutrient agar, sterilized test tube and water, bacterial culture,
petridish, etc.
Procedure: Make dilution of isolated bacteria by serial dilution method upto
10-6 and then from last 3 dilutions 10-1, 10-5, 10-6. Make plates by pour plating.
Observations:
Examples:
Dilution 10-6
1st plate
2nd plate
Mean
No. of cells
2.
Dilution 10-5
st
1 plate
2nd plate
Mean
No. of cells
3.
dilution 10-4
st
1 plate
2nd plate
Mean
No. of cells
1.
18 colonies
20 colonies
19
19 106 cells/ml
61
56
57.5
57.5 105 cells/ml
256 colonies
288 colonies
277
277 104 cells/ml
85
Experiment No. 11
Objective: To perform selective and differential plating
Requirements: Beaker, distilled water, reagent autoclave, cotton, aluminum
foil etc.
Procedure:
1.
Differential Medium
(bromocresol purple carbohydrate medium)
Tryptone
10 gm
Lactose
5 gm
Yeast
5 gm
K2HPO4
2 gm
Indicator
2 ml
Agar-Agar
20 gm
distilled water
1000 ml
2.
Selective media
(a) E.M.B. Agar
Peptone
10 gm
Lactose
5 gm
Sucrose
2 gm
K2HPO4
2 gm
Eosin yellow
0.4 gm
Methylene blue
0.06 gm
Agar
20gm
Distilled water
1000 ml
(b) Endo Agar
Peptone
10 gm
Lactose
10 gm
K2HPO4
3 gm
Na2SO4
2.5 gm
Basic fuschin
0.5 gm
Agar
20 gm
Distilled water
1000 ml
Observations:
86
3. Study of Bacterial Growth

Introduction
Microoraganisms exerts a great impact on natural ecosystem & human
affairs. Their role in transformation of organic matter and effect of ecosystem
was recognized only after 19th century.
The current chemical state of the elements on the earths outer surface is,
to a considerable extent, a consequence of chemical activities of living
organisms. The quantity of various gases in atmosphere and many other
compounds on the earths surface represents the net balance between their
rates of formations and utilization in biological & Geological processes. Such
transformations occur in all regions of Biosphere.
The composition of Biosphere is in more or less in static state, maintained
by a cyclic turnover of chemicals necessary for life, powered by continuous
input of Energy from the sun. The various steps in the cyclic turnover of
elements are brought about by different types of organisms. Thus continued
existence of any particular group of organisms depends on the chemical
transformations carried out by others. A break in the cycle at any point would
cause devastating effects on all life forms. All organisms participate in various
steps of the cyclic conversions, but contribution of microorganisms is
particularly important, both quantitatively & qualitatively.
There is over whelming contribution of microorganisms to these processes
of nature, which reflects the unequity of Microorganisms, their significant
contribution to the bulk of living material i.e. their biomass, their high rates of
growth and metabolism. Their collective ability to degrade a vast variety of
naturally occurring organic matter into inorganic forms, which is again utilized
by photosynthetic organisms.
Only minority of microbial species are pathogenic, i.e. they can proliferate
in human tissues causing infection and several disease. The discovery that
bacteria can act as a specific agents of infectious disease was made through
the study of Anthrax, a serious infection of domestic animals that is
transmissible to humans, and these discoveries led to isolation, cultivation
and characterization of the causative agents for the major infectious disease
to man.
Microbial processes have been used by human since prehistoric times in
the preparation of food, drinks, and textile. The increase in knowledge about
microorganisms, led to the improvement in many of them, but also to the
development of entirely new industries based on the use of microorganisms
that had previously not been exploited by humans.
It will not be an exaggeration to state that there is no life possible without
microoganisms. They are closely associated in major field like Agriculture,
Industries (like food processing), pharmaceutical etc. , lot of research work is
87
continuing with the help of various species of microorganisms, one of the

emerging field is Biotechnology.
But because of the very small size of microorganisms it is difficult to
quantify and qualify them. Therefore special techniques are developed for this
purpose, the first the foremost thing to do in this respect is the isolation of
microorganisms in pure culture. Once it has been isolated in pure culture
every aspect about them can be estimated? The following discussion is an
attempt to explain the methods used for the measurement of microbial growth,
together with various phases and modes of growth.
3. Bacterial Growth
Each group of organisms has to adapt itself during its evolution not
only on the nonliving environment, but also to the environment sometimes
involves the acquisition of special metabolic capacities that endow their
possessor of progeny with the unique ability to occupy a particular
physicochemical niche, and existing into a unique physiochemical Niche is a
highly effective means of meeting the challenges of biological competition.
Thus, the microorganisms have vast diversity.
From different natural environment different types of organisms can be
isolated by using specialized techniques in pure culture and their growth in the
pure culture could be monitored by applying.
a) Optimal conditions
pH = 7.0 temp = 370C and constant supply of nutrients
b) By applying stress
3.1.1 Chemical stress
Growth in presence of antibiotics, and thereby isolating mutants
resistant to a particular antibiotic or drug.
3.1.2 Physical stress
Growth of bacterial culture after they are given exposure to ultra violet
radiation for various time intervals. Finally, to isolate U.V. radiation resistant
strain???????????????????.
3.1.3 Environmental Stress
Effect of temperature
Effect of pH
Effect of osmotic pressure (by varying salt concentration)
Change in concentration of Glucose
Growth in the absence of oxygen (isolation of anaerobic bacteria)
When we provide microorganisms optimal conditions of growth i.e.
nutrients, physiological pH, temperature etc. They are in a state of balanced
growth i.e. the rate of increase of all the components of the population is
same (e.g. proteins, DNA, RNA etc.), or we could say that bacteria follows a
definite growth kinetics once they are adapted to particular environmental
conditions.
The Bacterial culture undergoing a balanced growth, mimic a 1 st Order
chemical reaction. E.g. of other 1st order reactions are
a) Nuclear reaction
88
b) Human population growth

I the 1st order reaction, the rate of chemical reaction in directly proportional to
the concentration of the reactants or amount of substrate present at any given
time. For bacterial growth, the substrate will be total no of viable bacterial
cells.
The chemical equation will be of the form:
dN
---- No.
dt
where, No. = no. or concentration of bacterial cells (viable) present at
any given time.
dN
---- = Rate of increase of bacterial cells in the culture media
dt
dN
---- = K No. Equation (1)
dt
where K =
constant of proportionality upon intetration of equation

(1), we get
dN
--- = K dt
dt
or
log e N = Kt + C Equation (2)

(1/x dx = log x + C)
Initially at t = 0
No. of bacterial cells = No.
Putting value of t and no. in Equation 2 we get Log eN = 0+C
= C = log e N = 0+ C
= C = log e N
Substituting the value of (C) in equation (2), we get
log e N = Kt + log e No.
N
Or log e --- Kt ----- (3)
No
( log x log y log x/y)
or K = 2.303/t log 10 N / No. ----- (4)
(loge X = 2.303 log 10x)
where K = rate constant for bacterial growth
= from equation (3) we could write
N
--- = eKt
No.
Or N = No. ekt - ----- (5)
89
Here, the equation (5) shows that the no. of bacterial cells
increases exponentially with respect to time, therefore, if we plot no. of
bacterial cells vs. time, the curve to be obtained should be an exponential
curve for the bacterial growth.
3.2 Doubling time
Also known as mean doubling time or generation time. It is defined as
the time required by the bacterial cell to double its concentration or amount.
At time t1 = no. of cell present are no.
And at time t2 = number of bacterial cells doubles its original
concentration i.e. 2 No.
Then t2 t1 = tg
Generation time of doubling time.
Putting values of initial and final concentration of bacterial cells in equation
(4).
2.303 N
Or K = ------ log 10 ---t
No.
N = 2 No. t = tg
2.303 2 N
Or K = ------ log ------tg
No.
2.303
K = ------ log 102
tg
0.693
K = -------tg
0.693
or tg = -------
(5)
tg
tg = generation time or mean doubling time.
Proceed to calculate
(1) The growth rate constant (K) of the culture.
(2) The mean doubling time = tg
2.303
N
equation (4) K = ------- log ----tg
No
Where t =
Time taken by bacterial cells from increasing their
concentration from No. to n cells.
N = May be taken as the concentration of cells/ml
But in the actual performance of this experiment different types of curves will
be encountered with several phases. Two curves (A) and (B) depict the
different phases of microbial growth in Nutrient media which depends on the
nature of bacteria. Particularly whether bacteria are spore former or non spore
former.
Curve A
90
Curve B
The curve A & B are drawn by plotting the growth with respect to time
for two different types of organisms in which their initial concentration in the
culture media is represented by No. This behavior is explained as follows:
3.3 Bacterial growth curve
1) Region A B
:
log phase
2) Region B C
:
Exponential growth phase or logarithmic phase
(log phase)
3) Region C D
:
Stationary phase
4) Region D E
:
Decline or death phase (only in curve B)
Between each phase there is a transition phase in the curved region also
called as Transitional periods.
When a culture proceeds from one phase of growth to another it does
not implies that all the cells are in identical physiological conditions (i.e. some
cells are remaining in original state of growth). Some time is needed for the
one logging behind to catch up with the others which have entered a new
phase.
3.3.1 Log period (phase)
Bacterial cells that are transferred from a natural source or from a
culture which was previously under the stationary phase, undergo a change of
chemical composition, before they are capable of initiating growth. This period
of adjustment is called log phase.
The period of adjustment implies that the bacterial cells are undergoing
some metabolic changes. For e.g. bacterial cell in this new environment may
be deficient in enzymes or co-enzymes which are needed for optimal
operation of cells chemical machinery.
If cells are picked from natural environment where they were facing
biological competition with other microorganisms they will take some time to
be adjusted in new environment.
The organisms are metabolizing but there is log in cell division. This log
phases is extremely variable in duration and its length is directly related to the
duration of the proceeding stationary phase. Greater is the stationary phase of
the culture, greater will be the log phase when the organism is grown in fresh
medium.
3.3.2 Log phase
Once the bacterial cells get adapted to the growth conditions, it divides
steadily at a constant exponential rate. Different strains of bacteria or different
91
species have got different growth rates and generation time (or mean
doubling time).
As given by equation (5)]
N = No ekt
The cells growing according to given equation are said to be in state of
exponential growth or logarithmic growth. It is also known as log phase.
Log e N = kt + log e No.
Kt
Log10 N = ---------- + log10 No.
---(6)
2.303
This equation is similar to equation of straight line.
Y = mx + c
On plotting the graph between log N and time we obtain a straight line
whose slope is K/2.303.
During log phase, the cells are in actively dividing conditions, as such
their machinery is operating at a maximum potential, hence these
enormous structural and physiological changes in cells are harvested
at different times. This phase is however the most uniform phase and is
commonly used for studies of microbial metabolisms.
To obtain better results, the experiments should be performed only from a
culture which is in logarithmic phase of growth.
3.3.3 Stationary phase
Microbial population seldom maintains the exponential growth at high
rates for log. The reason for this behaviour is obvious when one consider, the
consequence of exponential growth e.g. after 48 hr of exponential growth, a
single bacterium weighting about 10 -12 g. Having doubling time of 20 min. the
progeny produced has the mass = 2.210 31 g. This is nearly 4000 times the
weight of earth.
The growth of bacterial population is normally limited by two factors (1)
either by exhaustion of available nutrients or (2) by accumulation of
toxic products of metabolism as a result the growth declines and stops
after a transitional phase, giving the characteristics horizontal line on
the graph. Bacterial population remains constant with time.
The transition between the exponential phase and stationary phase
involves a period of unbalanced growth during which various cellular
components are synthesized at unequal rates, consequently cells in
the stationary phase have a chemical composition that is different from
the cells is the exponential phase and it depends on growth limiting
factors.
The cells in stationary phase are small as compared to cells in
exponential phase.
(cell division continues after increase in mass has stopped) and they
are more resistant to adverse physical (heat, pH, radiation) and
chemical agents.
3.3.4 Death Phase
Bacterial cells which area held in a non growing state for a longer time
eventually die. Death results from a no. of factors. Important ones are:
a) depletion of essential nutrients or the cellular reserves of energy.
b) Accumulation of inhibitory products or toxins.
92
Like growth death is also an exponential function and hence

death is an exponential decrease in the number of viable cells with
time. The death of bacterial cells is highly variable depending on.
Environment and particular organisms, for Enteric bacteria die

very slowly, while vegetative cells of certain Bacillus spp. die rapidly.
In spore forming bacteria (curve A) the death of all the cells dont
take place because one the vegetative cells find some hindrance for
growth they form spores, which are metabolically inactive and can
survive or exist under adverse circumstances.
Vegetative cells
Endospore
Spore
(metabolically inactive)
3.4 Synchronous growth culture
So far, growth pattern of populations of bacteria have been described
but from such studies we are unable to conclude about the growth behaviour
of individual cells, because the distribution of the cell size (and hence the cell
age) in most bacterial cultures is completely random.
Information about growth behaviour of individual bacteria can be
obtained by the study of the synchronous cultures. These cultures are
composed of cells that are all at the same stage of cell cycle, and
measurements made on such cultures are equivalent to measurements made
on individual cells.
3.5 Continuous Growth culture
If nutrients are not renewed the growth remains exponential for only a
few generation and after that stationary phase arrives. Such types of cultures
are called batch cultures.
Microbial population can be maintained in a state of exponential growth
over a long period of time by using a system of continuous culture. Here, the
growth chamber is connected to a reservoir of sterile nutrient medium, once
the growth has been initiated; fresh medium is continuously supplied from the
reservoir. The volume of the liquid in the growth chamber is maintained
constant by allowing excess volume to be removed, continuously through a
siphon overflow.
The condition to be maintained is
Rate of production of cells = rate of loss of cells
through growth
through overflow
3.6 Chemostat and Turbidostats
Continuous culture system can be operated as chemostats or as
turbidostats. In a chemostat the flow rate of nutrient media from reservoirs is
set at a particular value and the rate of growth of the culture adjusts to this
flow rate.
3.7 Growth Measurement
Bacterial growth in a culture can be measured on the basis of different
properties, some of the basic principles that are most commonly employed for
bacterial cells enumerations are as follow:
3.7.1 Direct counts
3.7.2 Colony counts
3.7.3 Most probable numbers
93
3.7.4 Biomass measurement

3.7.5 Light scattering (i.e. by absorption spectroscopy)
3.7.6 Chemical estimation of cellular components
3.7.1 Direct counts
These culture methods measure viable cells that grow in the media
provided. There are a number of methods that measure directly all cells in a
sample, living or dead. These direct methods are not as precise as culture
methods, but they are less time consuming. The few common methods are
given below:
3.7.1.1 Microscopic Enumeration
In this methods are measured volume of a sample is spread over a
given area of a slide. By making a microbial count in a known area and
multiplying by the appropriate factor, we can calculate the number of
organisms in the original sample. There are special counting chambers e.g.
the Petroff-Hausere slide that has cavities of known depth and volume,
marked off into squared areas. The no. of organisms in an area is counted
and the total number of bacteria in the sample can be calculated from the
proportion of the total volume known to be contained in the area. The
thickness of the fluid filling the squares is measured by focusing on bacteria
attached on top and bottom interfaces with the help of micrometer scale in the
microscope.
In bred method a known volume of sample is spread over an area of
one sq. cm on a slide and the film is dried, fixed and stained. The number of
microorganisms in a microscopic field are then determined and multiplied with
the area, same method is applied in Hawksly chamber.
3.7.1.2 Electronic Enumeration
Bacterial cells or particles may also be counted electronically the help
of counter. A sample is passed through a small orifice and from the measured
change in electrical resistance, not only the no. of particles, but also their size
can be determined. However, since this method measures any particle in a
sample, it is applicable only to aqueous suspensions of microorganisms.
Besides this there are certain other problems as well like production of pulse
comparable to noise produced in the instrument causing conclusion, failure of
cells to separate promptly from each other division, clogging of orifice etc.
3.7.1.3 Flow cytometery
It is a common instrument in hospitals, laboratories etc. a small
diameter stream of cells is encased in the stream of the fluid that is narrower.
The flowing stream is examined with laser light of different frequencies and
angles and the output of measurement circuits is used to detect when a
particle passes through. The design permits analysis of biomass.
3.7.2 Colony counts
The most common method of measuring the cell numbers is a plate or
colony count. This method is based on the theoretical relationship of one
bacterial cell giving rise to single colony and on the assumption that the no. of
colonies that develop on the agar plate corresponds to the original bacterial
count. There are various methods for counting these viable cells, or colony
forming units (CFU).
3.7.2.1 Dilution count
94
It used a serial dilution of a sample from which the dilutions are

inoculated into a series of tubes of liquid medium instead of being plated out.
On incubation, all tubes containing cells that have grown will be turbid, and
some tubes in which no growth takes place will be clear. From the highest
dilution of a sample that shows growth, the population is measured. Distilled
or tap water alone, usually should not be used because it is osmotically
hypotonic to all bacterial cells. A common general purpose diluents is
phosphate buffered saline with a protein usually gelatin or peptone.
3.7.2.2 Spread plate method
In this method, sample is pipetted onto the surface of solidified agar
medium in a petridish and the cells are distributed with a spreader. Optimum
dilution (so that countable colonies appear on agar surface) and temperature
for isolated colonies and growth is needed. All bacterial colonies are on the
agar surface.
3.7.2.3 layered plate method
In this method an additional layer of agar medium is poured over a softagar layer, containing the cells so that all the colonies are subsurfaced.
3.7.2.4 Pour plate method
In this method optimally diluted sample of bacterial cell is pipetted into
an empty sterile petriplate. Then molten agar at temperature of about 45 0C is
poured onto the sample, the contents are mixed by swirling and then the agar
is cooled to harden. The colonies of bacteria are formed after incubation
which are not in the same plane of agar surface.
3.7.3 Most Probable number
The concentration of viable cells can be roughly estimated by the MPN
method, also called the fraction negative method. It involves the mathematical
influence of the viable count from the fraction of multiple cultures that fail to
show growth in a series of dilution tubes, containing a suitable inoculum. It
involves making a number of dilutions in growth medium, and taking into
considerations the ones not showing growth. The distribution of cells show
poission ration. The mean no. plated at this dilution can be calculated from the
formulae.
Po = e-m
where m = mean number
Po = ratio of number of tubes with no. growth to the total number
of tubes.
3.7.4 Biomass measurement
A measure of the mass of bacterial cell constituents is frequently used
as the unit, for measurement of metabolic activity of morphological or
chemical constituents, i.e. biomass and cell numbers promote two basic
parameters of bacterial growth.
3.7.4.1 Wet weight
A nominal weight of bacterial cells originally in liquid suspensions is
obtained by weighting a sample in a pan, after separating and washing the
cells by filtration or centrifugation. A method for obtaining the actual wet
weight of the cells themselves is to correct the nominal wet weight of a
packed cell pellet by subtracting the experimentally determined weight of
95
diluents in interspatial space. Use of nonionic polymeric solute e.g. dextrans is

needed.
3.7.4.2 Dry weight
The dry weight of cell from a given volume of growth medium may be
determined as a measure of cell mass for such measurements. The culture is
harvested either by centrifugation or filtration, the cells are washed (to make
them free from medium), and their weight are determined after drying.
3.7.4.3 Water content
The amount of cell water in fully hydrated cells is equal to the
difference between the wet weight and the dry weight of cells.
3.7.4.4 Cell volume
It is determined by placing a standard quantity of liquid culture in a
calibrated centrifuge tube and measuring the volume of wet pellet after
centrifugation.
3.7.4.5 Wet and dry densities
The densities of bacterial cells may be obtained as either the wet
density, based on the total amount of solid and water contents or the dry
density based on solid content. Both densities are expressed in weight/
volume.
The wet density may be obtained by dividing the cell wet weight by cell
dry volume.
3.7.5 Light Scattering
They are techniques most generally used to monitor the growth of pure
culture. They can be performed quickly are non-destructive.
3.7.5.1 Turbidimetric method
It requires used of spectrophotometer to measure turbidity. In this technique
the sample is taken in a cuvate and the light of particular wavelength is made
incident light absorbed by a solution at a given wavelength is related to the
thickness of the absorbing layer (i.e. path length) and the concentration of
absorbing species. These two relationships are integrated into Beer Lambart
Law.
I0
Absorbency = log ---- = ecl.
I
I0 =
I = are incident and transmitted light intensity respectively
e = Molar extinction coefficient
c= concentration of solute
e = path length
The assumption that is made is that the incident light is parallel and
monochromatic and the solvent molecules are randomly oriented. The
expression log I0/I is called absorbance and also designated as A.
For bacterial culture before finding the concentration of cells in nutrient
media.
1.
Preparation of a blank, which is used as a reference to calibrate the

spectrophotometer. Use distilled water as a blank.
2.
Then calibrate the instrument for 100% transmittance, after
adjusting the zero knob to zero when switch is not pressed.
96
3.
Take the % transmittance for the nutrient broth containing no

bacterial cells (i.e. an autoclaved one). Finally calculate the %
transmittency for the bacterial culture (after proper shaking so hat there
is a random distribution of bacterial cells).
4.
Then subtract two optical density readings, to find O.D. for bacterial
cells at a particular concentration.
O.D. = 2 log T
Where,
O.D. = optical density or absorbance
T=
Transmittency of light (which is measured by the photo cell
directly)
The graph of optical density of absorbance vs. time can be plotted to
obtain the curve for bacterial growth in the culture.
3.7.5.2 Nephelometry
Although most of the light scattered by bacteria is nearly in the forward
direction in the transmitted light is measured, instruments that measure the
light scattered at 900C from the primary beam have also bean used to
measure bacterial concentration of scattered light at right angles are called as
Nephelometers. Rest all the principles used in a Nephelometer is same as
those of a turbidimeter.
3.7.6 Chemical Estimates
Chemical estimates of cell masses are made by measuring the amount
of some components characteristics of cells, for example cellular nitrogen,
protein, DNA, RNA or phosphorus. Under standardized condition, the amount
of these components provides a reasonably accurate determination of total
protoplast in a culture.
3.8 TOTAL VIABLE COUNT
It gives the total no. of organisms that are living in a given culture. This
can be calculated by using the procedures already discussed. The most
common is plating methods, either pour plating or surface spreading of media
with optimal dilution.
Total viable count is the counts the total no. of vegetative cells,
endospores and spores of bacterial, because endospores and spores are
capable of germinating under favourable conditions.
Total viable count = Number of (vegetative cells + endospores + spores)
Present in the culture media
97
Experiment No.
Objective: Turbidometric estimation of Microorganisms
Requirements: culture plates, test tube, photo colorimeter, tissue paper etc.
Procedure:
1. Make a quantitative plate count of a culture of bacteria, plating 10 -6, 107
and 10-8 dilutions of the culture; incubate the plates at 37 0C for 48 hrs.
2. Take 5 tubes, each containing 5 ml of sterile nutrient broth, use four of
these tubes to make four serial 1:2 solution of the culture.
a. Transfer 5 ml of culture to one of the tubes of sterile broth and
mix cell.
b. Transfer 5 ml of this first dilution to a 2d tube of sterile broth and
mix well.
c. Transfer 5 ml of this 2 d dilution to a 3d tube of broth, and after
mixing transfer 5 ml of the culture dilution to a 4 th tube of nutrient
broth the obtain final solution.
3. Use of photo colorimeter, with the 5th tube of sterile broth inserted in the
instrument. Set the galvanometer to read 100% transmittancy. Then
determine the optical density of the undiluted 48 hrs culture of bacteria,
as well as the four serially diluted cultures. Record the optical density
of each tube.
4. After determining the plate counts of these cultures plot the optical
densities of the various dilutions against the corresponding actual
bacterial count.
Observations:
S.No.
Dilutions used
OD
SPC
CFU/ml
98
Experiment No.
Objective: To Determine the Bacterial Growth
Requirements: purified bacterial culture, spectrophotometer etc.
Procedure:
1. Select a particulars organism, Bacillus or E. coli or any bacterial strain,
and inoculate this into a sterile 100 ml nutrient media in a 250 ml flask
by powering 2 ml of the original culture.
2. Incubate at 370C in incubator.
3. Take the optical density reading at 0, 5, 1, 2, 4, 12, 24, 48, 72, 96 and
120 hours.
4. Take the control reading before inoculating the nutrient broth.
5. Plot these O.D. against time on a graph (O.D. & bacterial cell
concentration).
Observations:
S.No.
Incubation time
OD
99
3.9 HEAT STABLE COUNT

Yeast, moulds, as well as some bacteria form spores under adverse
conditions, which hampers their growth. The spores of yeasts and moulds are
primarily reproductive spores, and several to many spores are produced per
yeast cell or mould plant.
In contrast only one bacterial spore in produced per cell. The bacterial
spores are heat resistant (also endospores), whereas the spores of yeast and
moulds are not, also the vegetative bacterial cells are susceptible to heat and
are killed. When the inoculum from bacterial culture containing vegetative
cells, spores and endospores is heated in a water bath to about 80 0C, only
vegetative cells will be killed. And when the heated incoulum (after cooling) is
plated to obtained viable count, the colonies that appear in the plate are those
of spores and endospores.
2.10 OCTYL VIABLE COUNT
Octyl alcohol kills the vegetative cells and endospores but not bacterial
spores. So by treating the culture with (0.5%) octyl alcohol and then by
subsequent dilution and plating we can count the spores.
Octyl viable count = no. of (spores) + No. of Endospores.
Total no. of Endospores = Heat stable count + octyl viable count
& Total no. of vegetative cells = total viable count octyl alcohol count
100
Experiment No.
Objective: To Determine the HSC, TVC & OSC.
Requirements:
Procedure:
(1) TVC
1. Take 1 ml of culture from broth culture and add it to 9 ml of sterile water to
prepare 10-1 dilution, like wise prepare dilution up to 10 -6
2. Inoculate 0.1 ml culture by using pour plate or surface spreading method.
3. Take the petriplates having 30-300 colonies and determine the count.
(2) HSC
1. Heat the culture in a tube at 800C for 5 minutes.
2. Prepare serial dilution from the heat treated culture
3. Inoculate these dilutions to nutrient agar
4. Inoculate the plates at 370C
5. Count number of colonies present in a plate and multiply with the dilution
factor.
35 colonies in 10-4 dilution
in original culture
Total no. of cells = 35104
(3) OSC
1. Treat the culture with 1% octyl alcohol (1.1 ml octyl alcohol rest water in
100 ml). Add 1 ml of octyl alcohol to 9 ml culture.
2. Prepare serial dilutions and inoculate in the nutrient plates and incubate it
for 24 hours.
3. Count the number of colonies and nulgiply it with the dilution factor as
described in above HSC procedure.
Observations:
101
4. GROWTH OF BACTERIA UNDER ENVIRONMENTAL STRESS

Microbes like all form of life, are profoundly influenced by their
surroundings. Environmental effects fall into three general categories:
Physical
:
The effect of temperature of extreme pressure, U.V.
irradiation etc.
Chemical
:
The need for food and response to poisons
(Antibiotics etc.)
Biological
:
The influence of coexisting species
(essentially chemical)
There is a particular optimum set of environmental conditions for each
living species but there is a wider range of tolerance in the microbial kingdom
as a whole than in higher from of life. Although vast majority of microbial forms
require the same conditions that are beneficial to most plants or animal cells,
still some extremist microbial populations exist in the world which survives in
extreme types of climatic conditions.
The series of exercises in this section will illustrate the response of
different bacterial strains; to various environmental conditions in continual
fight to control. Destructive and useful microorganisms, the most powerful use
have been the control of microbes environment. In the preservation of various
commodities useful in life we have used temperature, osmotic pressure, pH,
oxygen deprivation to check microbial growth. But their great adaptability and
versatility makes the control of microorganisms difficult.
4.1 Effect of temperature
Different types of bacteria have different requirements at which they
can grow and the temperature at which maximum growth of bacterial cell is
observed is called as optimal temperature. The temperature which hinders or
stops the growth of bacteria are called extreme temperature.
The rate of chemical reaction depends on temperature and given by
Arrhenius equation (Fig. 1.).
-H
Log 10V = ------------ + C
2.303 RT
V = reaction velocity
H = activation energy
R = gas constant
T = temp. in 0K.
This equation is applicable to bacterial systems also. But it is observed
that bacteria obey this equation for some intermediate temperature
only, as the extreme temperature approaches, there is an abrupt fall in
growth rate (Fig. 2). This is caused by the thermal inactivation for
proteins (enzymes) and possibly other cell structures like membrane.
As the temperature is raised above the optimum temperature for growth,
metabolic activity markedly increases, but at the same time the rate of
enzyme and protein break down (due to protein denaturation) markedly
increases, resulting in the damage and finally death of cells.
As the temperature is lowered, the enzyme activity and thus the growth
of the cell is lowered as predicated by Arrhenius equation. Bacteria will
continue to grow until the system freezes, however most of bacteria stops
growing at a temperature well above the freezing point. Every microorganism
102
has a precise minimum temperature of growth, below which growth will not
occur however long the period of incubation.
A. generalized arrhenius plot of
Arrhenius plot of growth rate
the relationship between velocity
of E.coli, BC range of temp.
of a chemical reaction and temperature where the growth curve is
linear above B or blow C the
temperature is extreme.
The numerical value of the cardinal temperatures (minimum, optimal and
maximum), and the range of temperature over which growth is possible varies
widely among bacteria. On the basis of the temperature range of growth,
bacteria are divided into four broad groups.
a.
Extremophiles or hyperthermphiles (which grow above 90 0C)
b.
Thermophiles: which grow at temperature above 55 0C.
c.
Mesophiles : They grow at mid range of temp (20-45 0C).
103
Experiment No.
Objective: To observe a growth rate of a microorganism and prepare a
growth curve.
Requirements:
Bacterial culture, spectrophotometer pipettes, test tube, tissue paper
etc.
Observations:
Sl. No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Time
Hrs.
O.D.
104
Experiment No.
Objective: To study the effect of temperature on Bacterial Growth
Requirements: Bacterial culture, spectrophotometer pipettes, test tube,
tissue paper etc.
Procedure:
1. Incuabte one whole set of 5 tubes with the culture of the organisms in
the sterile nutrient broth medium.
2. Incubate 5-tubes of each culture at 0, 5, 28, 37 and 50 0C.
3. Observe the growth by measuring the OD. Take nutrient broth as the
blank and measure the growth in interval of two hour.
4. Plot the O.D. reading vs. time in a group for each organisms at different
temperature.
5. Care should be taken to use nutrient broth without any organisms (i.e.
sterile) as the blank and all readings of OD are to be taken with respect
to it.
6. Phychrophile : they grow at 00C.
Observations:
Hrs.
0
2
4
8
16
24
34
00C
50C
RT 280C
370C
500C
105
4.2 Effect of pH
Most organisms have an optimum H+ ion concentration for growth,
however some of them can grow over a fairly wide region of pH. This pH
conditions are also effected by the metabolic activities of the bacteria for ex
yeast decreases the pH of the medium by production of alcohol. While some
putrefacture bacteria decomposes protein and produces basic amino acid
thereby increasing the pH of surroundings.
Since pH limits the kinds and type of microorganisms capable of
growing under a given conditions. Its control has a considerable practical
importance.
We know that a optimum pH is required for maintenance and function
of enzyme any change in pH effects the enzyme activity and since enzyme
activity is responsible of metabolism carrying in the organisms change in pH
affects growth and in extreme concentration of pH the organisms is unable to
survive because of the deactivation of enzymes.
Thus, pH concentration like temperature could be defined by
maximum, minimum and optimal pH concentration.
106
Experiment No.
Objective: To study the effect of pH on Growth
tissue paper etc.
Procedure:
1. Prepare nutrient broth culture of the given strain.
2. Prepare nutrient broth, maintained at different pH, 5, 6, 7, 8 and 9 in
separate flasks.
3. Inoculate 2.5 ml of inoculum into 22.5 ml of nutrient medium.
4. Keep in incubator at 340C.
5. Observe OD at different time intervals i.e. 24 hours, 48, 72, 96 and 120
hr.
1. Plot the graph of O.D. vs time for bacterial culture maintained at
different pH.
Observations:
Sl.
Time
No. Hr
1.
pH 5
O.D.
pH 6
O.D.
pH 7
O.D.
pH 8
O.D.
pH 9
O.D.
2.
3.
4.
5.
6.
7.
Result:
107
4.3 Effect of Osmotic Pressure on bacterial growth

At level optimal for metabolic activity, even when the so molarity of
surrounding environment varies over a relatively wide range.
Most bacteria with a rigid cell wall do not have to regulate their internal
osmotic pressure with precision because cell wall has a capability of
withstanding high osmotic pressure. Bacterial cell usually maintain their
internal osmotic pressure well above the of the media. This causes flow of
water inside the cell, a condition essential for diffusion of nutrient
substances and maintenance of outward pressure called turgor pressure.
When the osmotic concentration of the medium is considerably lower
than that of the cell (a hypotonic medium), excessive diffusion of water into
cell causes lyses of cells due to increased turgor pressure. Gram positive
are resistant to lyses due to rigid cell wall, but gram negative bacteria cell
lysis occurs. This phenomenon is called plasmolysis and if medium is of
high concentration i.e. a hypertonic medium. Water will leave the cell and
the volume cytoplasm decreases. Causing damage to the membrane in
Gram positive bacteria, this cause the cell membrane to pull away from the
wall, the cell is said to be plasmolyzed. Whereas in Gram negative
bacteria which have got only outer membrane contracts along with the
plasma membrane, and this damages the membranes.
Bacteria vary widely in their osmotic requirements. Some are able to
grow in very dilute solutions and some in solution saturated with salt.
Microorganisms that can grow in high osmolarity are called osmophiles. In
terms of their salt tolerance bacteria can be divided into four broad
categories.
1. Non halophiles : 0.0-4 (% g/100 ml) salt concentration)
2. Marine organisms : 0-5 (% g/100 ml)
3. Moderate halophiles : upto 20.5 (% g/100 ml) (salt concentration)
4. Extreme halophiles : upto 36 (% g/100 ml) (saturated)
Organisms
Concentration (%, w/v) in Ratio of intracellular to
growth medium of
extra cellular conc. Of
NaCl
KCl
Na+
K+
Nanhalophiles
Staphylococcus aureus
0.9
0.19
0.7
27
Salomella oranienbung
0.9
0.19
0.9
10
Moderate halophiles
Mirococcus
5.9
0.02
0.3
120
Halodenitrification
5.9
0.02
0.7
55
Vibro costicolus
Extreme halophiles
Sarcina morrhuae
23.4
0.24
0.8
64
Halobacterium salinarium
23.4
0.24
0.3
140
Osmotic tolerance The ability of an organism to grow in a media with wide
variety of osmolarities is by the adjustment of internal osmolarity so that it
always exceed, that of the medium. This in accomplished by different
phenomenon, each of which has a specific biochemical basis.
A hypotonic solution
A hypertonic solution
The osmotic pressure can be varied by the change in salt concentration.
108
Experiment No.
Objective: To study the effect of salt concentration on bacterial growth
tissue paper etc.
Procedure:
1. Prepare three sets of five test tubes which are having luria broth having
different concentration of NaCl.
[NaCl] 1.0%, 0.5%, 5%, 10%, 25%
2. Inoculate one whole set of five tubes
3. Incubate the culture at 370C
4. Observe the growth by measuring the O.D. in spectrophotometer at 24,
48, 72, 96 and 120 hrs.
5. Plot the O.D. readings vs. time on a graph paper for various salt
concentrations.
Observations:
S.No.
Incubation
period
OD
109
4.4 PRESERVATION OF LABORATORY CULTURES

A microbial culture may be preserved in order to avoid the necessity of
subculturing or of transferring it to fresh media at frequent intervals. To be
suitable any means of preservation should permit maximum survival of the
population with little or no genetic and physiological change. In principle all
methods depends upon bacteriostasis - That is maintenance of culture without
growth or reproduction. Most methods employ refrigeration or desiccation to
bring about such dormancy.
Short term preservation can be accomplished in a no. of different ways
depending aerobes may be preserved for months by refrigeration on agar
slope. With some facultative anaerobic preservation as refrigerates agar-stab
culture is more satisfactory.
Agar-slope (slant) an agar slant is prepared and then the pure
culture of organism is inoculated by streaking process with the help of needle.
During incubation growth of bacteria takes place. The exclude oxygen further
and to prevent evaporation, a seal of sterile mineral oil may be applied over
the agar slant. After which the slant culture is refrigerated. Strict anaerobes
are cultured and preserved in a strongly reducing medium such as
thioglycollate broth or chopped meat medium. If acid is metabolizing product
of the organism being preserved the medium employed should be well
buffered.
Although these refrigerated cultures prepared on the slant remains
viable for several month refrigeration methods are not suitable for long-term
preservation because such stocks must periodically be recultured for three is
possibility that cultures refrigerated for long periods may be frozen & dead.
Unfortunately many microbial species do not survive ordinary freezing or
dehydration. The common method for long term preservation of microbes is
the freeze drying technique called as Lyophilization. In this process the
microbial culture is suspended in a sterile milk blood serum or some equally
protective substance in a glass vial or tube quick-frozen with a mixture of dry
ice and alcohol then thoroughly desiccate while in the frozen state. Finally the
vial containing the dried culture is sealed.
Some bacteria which form spores, their spores can be harvested by
centrifugation and then they can be preserved. When these spores are
provided with favourable environmental, condition for growth they again
germinate & we get colonies.
110
5. A DEMONSTRATION OF KOCHS POSTULATES OF BACTERIAL AND

FUNGAL PATHOGENS
Introduction
When a pathogen is found on a diseased plant, the pathogen is
identified by reference to special manuals, if the pathogen is known to cause
such a disease and the diagnostician is confident that no other causal agents
are involved. Amongst the syndrome of symptoms which is the ball mark of a
particular disease one often sees a micro-organism which is presumed to be
the pathogen. To determine with certainty that this micro-organism is the
cause of the disease rather than some incidental contaminant it is necessary
to examine critically its relationship with the host plant. This dilemma was first
recognized in studies on microbial pathogens of man. In 1876 Robert Koch
provided the first experimental proof of disease causation of applying a set of
rules which have since come to be known as Koch's postulates. Koch's
considered that these rules must be satisfied before any micro-organism can
be regarded as pathogen.
The rules may be summarized as involving five step- wise operations,
outlines below:
1. The suspected pathogen must be consistently associated with the
some symptoms.
2. The organism should be isolated into culture, away from the host. This
preludes the possibility that the disease may be due to malignant
tissues or other disorders of the host itself.
3. The organism should then be re-inoculated into a healthy host.
4. Symptoms should then develop which are identical to those observed
in the original out break of disease.
5. The causal agent should be re-isolated from the test host into pure
culture and should be shown to be identical with the micro organism
initially isolated.
Even today these rules are still relevant, although we now appreciate that
they can not be rigidly applied in their original form to all types of pathogens.
The most important exceptions in plant pathology are those pathogens which
can not be grown in artificial culture i.e. the viruses and some biotrophic fungi.
The problem of isolating viruses from their host plants is generally overcome
by using indicator plants. These are alternative hosts which develop
symptoms which are specific for a particular virus. Healthy specimens of the
original host may then be re-inoculated. In addition, electron microscopy of
plant sap or of purified crystalline samples of the viruses, coupled with
serological techniques may be employed to investigate the types of virus
present at each step of the infection procedure.
The procedure of application of Koch's rule to non culturable fungal
pathogens presents less serious problems because they produce spores.
These can be removed from the host and used in re-inoculation experiments.
In many instances spore morphology is also a valuable aid to the identification
of the inoculated and re-isolated pathogens.
Disadvantages
Difficulties in satisfying these postulates may be experienced in cases
were symptoms result from mixed infections or when dealing with previously,
undiscribed disease agent. Not long ago, few pathologists would have
111
predicted the existence of pathogens such as viroids, which scarcely conform

to our preconceptions of a successful parasite.
Koch's rules are possible to implement, although not always easy to
carry out, with such pathogens as fungi, bacteria, parasitic higher plants,
nematodes, some viruses, some viroids and the spiroplasmas. These
organisms can be isolated and cultured or can be purified, and can then be
introduced into the plant to see if them cause the disease. With the other
pathogens, however such as some viruses, mycoplasmas, fastidious vascular
bacteria and protozoa, culture or purification of the pathogen is not get
possible, and the pathogen often can not be reintroduced into the pathogen
into the plant to reproduce the disease. Thus, with these pathogens, koch's
rules cannot be carried out and their acceptance as the pathogens of the
disease with which they are associated 1S more or less tentative. In most
cases, however,
112
Experiment No.
Objective: To demonstrate Kochs postulates in bacterial pathogens.
Requirements:
Procedure:
1.
Isolate the micro-organism, i.e. the pathogen from the diseased
part of the plant. For bacterial disease cut the damaged part and then
wash with water.
2.
Then place it in distilled water in an Petri plate and then cut or
piece it with sterilized glass rod the micro organism will get collected in
sterilized distilled water.
3.
Observe the suspension for pathogen under electron microscope.
4.
Then grow the culture by streaking in the agar medium. And then
incubate it.
5.
After incubation observe for colonies and see whether the
pathogens are some as they were in suspension.
6.
Then pick a colony from the agar plate then make it suspension in
distilled water then take a host plant which is diseased free.
7.
Then make an injury in the host plant or by giving an injection of
that suspension make the inoculation into the plant.
8.
Make the plant susceptible to that disease. After few days see for
any symptoms of the disease.
9.
When symptoms occur visualize the symptoms same for as that of
the original from which it was isolated.
10.
Then again isolate the pathogens from the disease plant and
observe under microscope the pathogens must be identical to that of
the original one which are isolated earlier.
For visualizing in animals
1. Take a mouse that was killed by bacteria injected into its peritoneum.
Open the abdomen of the mouse aseptically by washing the exterior
surface with ethanol and by using scissors sterilized with ethanol.
2. Insert a sterile loop into the peritoneal cavity to obtain the micro
organism and streak one blood-agar plate to isolate to pure culture. At
the same time, make a smear and gram stain the material obtained by
activity. Incubate the blood agar plate at 37C for 24 hrs.
3. Inoculate a tube of strep-base broth with a colony of the predominant
microorganism growing on the blood agar plate. Also streak two entire
blood agar plates with this same micro organism to obtain confluent
growth and aseptically apply sensitivity discs to one plate. Incubate the
tube and plates at 37C.
4. Flood the blood agar plate which does not have any sensitivity discs
with 2 ml sterile saline. Using a sterile glass spreader scrape the cells
from the plate into a sterile test tube.
5. Then inject one labeled mouse with 0.2 ml of the isolated culture.
Inject a second mouse with 0.2 ml of the isolated culture plus 0.1 ml of
the antibiotic to which the culture demonstrated sensitivity on the disc
plate.
6. Reinject your second mouse with 0.1 ml of the antibiotic for two
successive days. Observe the mice over a 24-74 hr period and record
113
the time when a mouse dies. Stain the growth from the peritoneum of
any mouse that dies to determine if it is similar to isolated one.
Observations:
114
6. DETERMINATION OF CULTURE SENSITIVITY USING DIFFERENT

DISINFECTANTS AND ANTIBIOTICS
Introduction
Disinfection generally refers to the use of; germicidal chemical agents
to destroy the potential infectivity of a material. . Micro organisms which cause
disease and destroys food materials should be controlled; many chemicals
both inorganic and organic are toxic to micro organisms and to control the
microbes responsible for deterioration and disease, and thousand of these
chemicals have been discovered.
Disinfectants are lethal to all sorts of cells. Because of this non
specificity it is not surprising that bacteria develop little resistance to these
agents. Classification as a disinfectant however, is some what arbitrary for at
a high enough concentration a great variety of compounds will inhibit bacterial
growth. Indeed whether a given substance is a meat or a poison to bacteria is
often simply a matter of concentrations. Thus oxygen, various salts, fatty
acids, some amino acids and glycerol, in high enough concentrations may be
batceristatic or even actively bactericidal to bacteria. Disinfectants act directly
on cell structures and thus do not require specific metabolic activities on the
part of the microbe.
Disinfection also means the freeing of an article from some or all of its
burden of live pathogenic microorganisms which might cause infection during
its use.
Uses
Disinfection, rather than sterilization is attempted in circumstances in
which sterility is unnecessary or sterilizing procedures are impracticable, yet
there is still some value of obtaining a partial or complete removal of non
sparing pathogens. It is highly impracticable to apply sterilizing procedures to
bedpans, baths, wash-basins, furniture, eating utensils, bed-cloths and other
somites that might spread infection.
ANTIBIOTICS
An antibiotic is a substance which destroys or interferes with the
reproduction of micro organisms. It is now possible to produce some
antibiotics by a semi-synthetic process. It has revolutionized the treatment of
infection, they carry the risk of causing side-effects or of encouraging the
growth of resistant micro organism. They should not be used unnecessarily
when several infective agents are available.
Bacterial resistance develops because antibiotics kill the most
susceptible strains, leaving the most resistant strains to multiply. Gram
negative bacteria can also in some circumstances transmit resistance to other
types of bacteria. To diminish the development of resistant strains, antibiotics
should always be given in adequate doses for an adequate period and should
be given in combination, if the emergence of resistant bacterial strains is
likely. Prophylactic antibiotics should be given only when specifically indicated
for instance to prevent rheumatic fever or bacterial endocarditis.
115
Experiment No.
Objective: To determine the sensitivity of the culture using different
disinfectants and antibiotics.
Requirements:
Procedure: Many chemicals, both inorganic and organic are toxic to micro
organisms and to control the microbes responsible for deterioration and
disease thousand of these chemicals have been discovered. The) chemical
agents simply inhibit microbial activities and growth or lethal and kill the micro
organisms. Inhibitory chemicals are usually termed antiseptics, the lethal are
known as disinfectants. And by using antibiotics which are responsible for
killing the specific micro organism we can control the destruction of food and
control the diseases, with are deadly to human beings.
1. Prepare a nutrient agar and autoclave it so as to sterilize the nutrient
medium.
2. Melt the agar and pour in Petri plates and then cool it to 45C, and then
inoculate the plates with micro organism one with Escherichia coli and
other with Bacillus
3. Then cut the filter papers into small circles and put into number of
disinfectants and antibiotics that are available. Allow it to drain and
place into the inoculated plate at an distance of 2" with the help of
sterilized forceps.
4. Then incubated the plate. Then record the relative effectiveness of
each antibiotics and disinfectants by measuring the diameter of the
inhibition zone.
Observations:
116
Experiment No.
Objective: Effect of various antibiotics on bacterial growth.
Requirements:
Observations:
Plate NO. 1 (Bacillus

pseudomonas)
S Streptomycine
P Penicillin
Plate No. 2 (Bacillus)
O Oxoteracycline
Ch Chloramphicol
Plate No. 3
+ = 5 mm
K Kanamycin
Results:
117
Experiment No.
Objective: Impact of different disinfectants on killing bacteria
Requirements:
Introduction: Use of antibiotics, antiseptics, disinfectants or immunization is
required to control the growth or check the growth of many pathogenic
microorganisms. Antiseptics are the chemicals which avoid sepsis or microbial
contamination. Disinfectants are the chemicals which when used at low
concentrations avoid the growth or entry or pathogen in a place. Antibiotics
are the substances produced by microorganisms and which act against
another kind or microorganism. Disinfectants are usually used for treatment of
inanimate objects whereas antiseptics are used at low concentrations for
treatment of animals objects.
Examples of disinfections:
NaoH (2%), Na2Co3 (4%), Calcium oxide, CaCl2, KMno4, phenol, Dettol.
Formalene, H2O2, HgCl2 (1:10000) Copper sulphate.
Examples of antiseptics: Aniflamin (1:1000) Crystal violet (1:100), Alcohol
(95%).
Antibiotics: Streptomycin, Penicillin etc. all these compounds disrupt
and disturb internal mechanism of microorganism. So their metabolic activities
are affected drastically. When these chemicals are applied on a bacterial
lawn, inhibition zone are formed on it depending on ability of these
compounds to inhibit microbial growth. Stronger the chemical against a
particular organism, more will be the diameter of inhibition cone formed by it
on bacterial lawn.
Procedure: Nutrient agar was made and placed in 2 plates then a bacterial
lawn was made on agar plates when they were cooled and agar was
solidified. Bacterial lawn was made near the flame. Then we cut the discs of
filter paper with the help of cutter and there discs were dipped in following four
disinfectants
(1) Savlon
(2) Formalin
(3) Dettol
(4) KMnO4
These discs were placed on solid surface of one agar plate.
We 0.1 M solution of sodium azide was made and diluted to obtain 10 -1,
-2
10 , 10-3 and 10-4 concentrations. Filter paper discs dipped in these dilutions
were placed on second agar plate.
These plates were kept in incubator but we did not get results because.
1. We placed plates in incubator which has temperature of 25 C only.
2. Bacterial lawn was not adequately made on agar plates.
So this experiment was repeated and this time we took following four
disinfectants for sensitivity test.
1. Dettol
2. Savlon
3. Mercurochrome
4. KMnO4
use of different disinfectants for use of different concentration
of
sensitivity test
Mecurochrome for sensitivity test
We performed this experiment in 2 parts
(1) To observe the sensitivity of different disinfectants.
(2) To observe the sensitivity of one disinfectant
concentrations.
at
different
118
We first made circular discs of filter paper and autoclaved them in a blank
Petri plates. Next we prepare nutrient agar plate and place 4 discs dipped in 4
different disinfectants over the media after making a bacterial lawn over the
agar plate.
In order to test the sensitivity of one disinfectant at different
concentrations Mercurochrome was diluted to obtain 10 -1, 10-2, 10-3
concentration and them sterilized discs were dipped in different
concentrations and placed over a agar plate after making a bacterial lawn
from pure culture with the help of bent glass rot.
The plates were incubated at 37 C for 24 hrs.
119
7. CULTURING OF ANAEROBIC BACTERIA

Introduction
The present atmosphere of the earth contains about 20% (v/v) of the
highly reactive oxygen. Many of the microorganisms are dependent on the
oxygen as a source of nutrient. The aerobes are dependent on O 2, the
facultative anaerobes use O2 if it is available, but they also can grow in its
absence, the anaerobes can not utilize oxygen.
Anaerobes are of two types the obligae anaerobes for which O 2 is toxic
and the aerotolerant anaerobes which are not killed to exposure to O 2. many
obligate aerobes cannot grow in oxygen concentrations greater than
atmospheric i.e. >20%v/v). Indeed some obligate anaerobes require O 2 i.e.
about 2-10%/v/v). Such type of bacteria are known as microaerophiles.
The effect of atmospheric oxygen on microbial growth is closely related
to the oxidation-reduction potential of the micro organism and culture medium.
All bacteria contain certain enzymes capable of reacting with O 2. The
oxidations of flavoprotein by O 2 invariably results in the formation of a toxic
compound H2O2. These oxidations produce small quantities of an even more
toxic free radical superoxide or O 2. The enzymes which convert them to water
and oxygen are not present in the anaerobic organism.
The oxidation reduction potential is a means of expressing the degree
of oxidation or reduction of a compound or an environment. A compound
having a high ratio of Hydrogen to oxygen i.e. higher than water is highly
reduced and has a negative oxidation reduction potential, and a highly
oxidized material has a positive oxidation reduction potential.
Anaerobic bacteria can be grown by eliminating free oxygen from the
environment or by establishing a low oxidation-reduction potential by
providing sufficient reducing materials. A considerable range of oxygen
tolerance exists among the Strict anaerobes. Some grow in air to a barely
visible extent, others require the complete exclusion of free oxygen.
The protection anaerobic cultures from free oxygen can be
accomplished by the following methods.
1. Expel the gas from culture media by boiling and then preventing its reintroduction by adding relatively solid barrier such as a layer of vesper
or mineral oil.
2. We can use sodium thioglycolate (HScH 2 COOHa) compound which
reacts with oxygen and maintains enzymes in the cell in a reduced
condition. The Brewer plate is especially designed to exclude oxygen
and at the same time to permit the development of surface colonies of
anaerobes.
3. Replacement of air by an inert gas in a widely used method and offers
a number of advantages. Agar plated or tubes to be incubated are
placed in an anaerobic incubator. The incubator is evaluated and
carbon dioxide, hydrogen or nitrogen is ruin in.
4. The burning of phosphorous is another chemical method for oxygen
removal.
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8. BIOCHEMICAL CHARACTERIZATION OF ISOLATED

MICROORAGANISMS
INTRODUCTION
An organism that has been isolated and studied must be given a name;
otherwise reference to it could not be made. Also, it is highly desirable that the
name applied to an organism by one person be understood by others.
Systematics bring order from chaos and to organize the many kinds to
bacteria into a coherent, understandable and useful system with three inter
related topics.
1. Classification
The arranging of bacteria with similar phenotypic characteristics
(Numerical taxonomy) or genotypic characteristics (the base
composition of DNA; its determination and significance, nucleic and
hybridization, nucleic acid sequence) into groups.
2. Nomenclature
The naming of bacteria according to internationally agree upon
principles.
3. Identification
The comparing of unknown organisms, with bacteria that have already
been classified, for the purpose of determining the identities (or) names
of the unknown organisms.
During the early years of bacteriology organisms were classified
entirely on the basis of morphology. The morphological characters
employed are:
(i)
Size and shape of nn organism
(ii)
Arrangement of the cells
(iii)
Presence (or) absence of well defined capsules.
(iv)
Presence (or) absence of spores.
(v)
Size, shape and position of the spore in the cell.
(vi)
Presence, number and arrangement of flagella.
(vii) Presence 9or) absence of characteristics granules.
(viii) Acid fastness, Gram reaction and other differential staining
procedures.
Classification based on phenotypic characters (Numerical taxonomy)
Numerical taxonomy
It is based on quantification of similarities and differences among
organisms. This was first suggested by Michel Adansonand is known as
Adansonian taxonomy. The underlying assumption is that, provided each
phenotypic character is given explicit weighing, it should be possible to
express numerically the taxonomic distances between organisms, in terms of
number of characters examines. For any pair or organisms, the calculation of
similarity can be made in two different ways. The similarity coefficient (Sj)
does not take into account characters negative for both organisms, being
based only on positive matches; the matching coefficient (S s) includes both
positive and negative matches in the calculation.
The data can then be transposed into a dendrogram, as a basis for
determining taxonomic arrangement in terms of numerical relationship.
121
Numerical taxonomy does not have the evolutionary cornotations of

phylogenetive taxonomy, but it provides an objective and stable basis for the
construction of taxonomic groupings.
Classification based on genotypic characters
DNA Base composition
Overall nucleotide base composition (moles percent g+c) for various
stains resolve many taxonomic problems i.e. strains having significantly
different DNA base composition do not belong in the same species.
Nucleic acid hybridization
These techniques allowed the entire genome of one bacterial strain to
be compared with that of other strains in terms of nucleotide base sequence.
A transition from native to denatured state is called denaturation. The
temperature at which DNA is half denatured is known as melting temperature
(Tm). At temperature near Tm, only duplexes between strands with a high
degree of complimentarily persist. When DNA preparations from who related
strains are mixed and treated in this manner, hybrid DNA is formed. This
technique is useful at species level and less useful at Genus level.
Nucleic acid sequencing
It deduced broader phylogenetic relationship among bacteria. The rate
of change of sequence of genes encoding 1675 r RNA is much less than that
of the bulk of the genome.
The major requirement of systematic is to learn about the bacteria as
much it is possible. Classification based on the above characteristics is
therefore inadequate and that more characteristics are necessary.
Biochemical reactions were, therefore introduced into the newer
classifications (eg: Production of indole and H2S, reduction of Nitrate- Nitrite
Ammonia - Nitrogen; fermentation of carbohydrates). As the present time
biochemical reactions are important in the classification of bacteria.
122
IDENTIFICATION OF UNKNOWN BACTERIAL CULTURES BIOCHEMICAL

TESTS
Microorganism like all living things may modify their environment to
some extent and utilize chemicals in solution as sources of energy and as
building blocks for growth and reproduction. All activities of cells are mediated
by enzymes, and in the complex chemical reactions of life the microorganisms
employs a large number of individual enzymes whose activities interlock. The
chemical end products of some of these enzymatic actions may be measured,
or in other cases the disappearance of certain substances from the medium
can be detected. By making a series of different tests a pattern of activity can
be established (which in turn reflects the enzymatic make up of the micro
organism) and the pattern aids in the identification and differentiation of the
microorganism from closely related species.
BIOCHEMICAL TESTS FOR BACTERIA
The tests described in this section are a fairly limited selection of tests
which are of general usefulness in the characterization and identification of
bacteria. Some organisms require the use of a medium specially adapted to
particular nutritional (or) osmotic requirements. In other cases, a selective
medium may be employed which not only allows the isolation of the organism
under investigation but also incorporates one (or) more biochemical tests. It is
important to check that each batch of a medium is satisfactory by using a
strain of bacterium known to give positive reaction. In addition, where
appropriate, an uninoculate tube or plate is to be incubated with each test in
order to detect false positive reactions due to impurities in or a deterioration of
medium or reagents.
A. REACTIONS INVOLVING PROTEIN, ANIMOACIDS AND OTHER
NITROGEN COMPOUNDS, INCLUDING TESTS FOR PROTEOLYTIC
ACTIVITY
Many bacteria can degrade a variety of products like proteins and
utilize the resulting peptides and amino acids to synthesize cell protein and to
provide a potential energy source for the cell. Microorganisms vary from
species to species with regard to their proteolytic ability and this feature can
be used to characterize a given species.
Although amino acids serve primarily as the basis they are also utilized
by the cell for other purposes. Amino acids may be degraded to yield energy
to the cell and they give a variety of end products for example NH 3, indole and
H2S are formed. Amino acids may be altered chemically to yield essential cell
components, including other amino acids.
The following exercises illustrate types or protein degradations and
some of the diverse ways in which microorganisms can utilize amino acids.
Since the pattern of amino acid degradation is characterization of a given
genus (or) species, there biochemical patterns may be used in microbial
characterization.
1. GELATIN HYDROYSIS
The protein, gelatin is obtained by the hydrolysis of collagen- a component
of connective tissue and tendons of animals. Gelatin is convenient as a
substrate to test for proteolytic enzymes in microorganisms.
Water solution of gelatin of the concentrations used in media for this
exercise are liquid at room temperature and solidifying in an ice bath. If the
123
gelatin has been hydrolyzed by the action of the microorganism being

tested, the medium will remain liquid.
Medium:
Nutrient broth with the addition of 10-15% gelatin, final pH 7.2, sterilize
by autoclaving for 20 min at 115C.
Procedure
1. Inoculate four tubes of nutrient gelatin with E.coli, Bacillus subtiles,
Streptococcus faecalis, and Proteus vulgaris.
2. Incubate at 37C together with a sterile tube of nutrient gelatin, which
will serve as a control. Thest at 2 days and upto7 days (or) until a
positive reaction is obtained.
3. To examine for hydrolysis, chill the tubes in ice water. The control tube
and tubes in which no hydrolysis has taken place will solidity.
Hydrolyzed gelatin will remain fluid.
Precautions
1. Care should be taken not to agitate the tubes while their contents are
liquid.
2. If liquefaction of the gelatin is proceeding slowly from the surface down,
as it often does, agitation can minimize the hydrolyzed with the
unhydrolyzed gelatin to give a combination that solidifies giving
negative and erroreous results.
Recording result
The liquefaction of gelatin by bacteria is the result of the action of an
enzyme known as gelatinase. It is an extracellular enzyme concerned with the
hydrolysis of the in diffusible protein prior to intracellular utilization.
- If the gelatin remains liquid, it indicates that the organism under
examination secreted a gelatinase into the culture medium.
- The extracellular nature of the enzyme may be demonstrated by
filtering a bacterial culture and adding some of the filtrate to a tube of
gelatin medium. The presence of the enzyme results in liquefaction of
the gelatin.
- The presence of a fermentable carbohydrate may result in a protein
sparing action. Under these conditions a test for gelatin liquefaction will
most likely be negative even though the organism under examination is
capable of hydrolyzing the protein. Therefore, non-carbohydrate media
should be employed to demonstrate. The test is of value in identifying
and classifying bacteria.
2. CASEIN HYDROLYSIS
Casein is the principal protein of milk. It exists as colloidal suspension.
Which gives milk its opaque whiteness. Many bacteria are equipped with
enzyme that hydrolyze thin protein into more soluble and transparent
derivatives. Protein breackdown, sometime called peptonization, is a useful
reaction in the identification of microbial species.
Casein is a protein capable of reacting with both acids and basis.
Some bacteria serete a rennin like enzyme capable of hydrolyzing casein to
soluble paracasein and a peptone like compound. The soluble paracasein
then reacts with the calcium salts in solution to give a precipitate of paacasein
(or) calcium paracaseinate. The clear liquid surrounding the curd of
paracasein is known as whey. This may be diagrammed as follows:
124
Casein + rennin
-----------------------------------------Peptone-like body
soluble paracasein
+
Calcium salts
Para casein
(Calcium paracascinate)
bacteria which ferment lactose very rapidly produce sufficient acid to

precipitate or curdle the casein. The clear supernatant liquid that separates
from the clot is known as when the acid produced is sufficient to stop further
growth of the bacteria.
Medium:
Milk agar, which consists of agar with the addition of skim milk to
10 percent, sterilized by autoclaving for 20 min at 115 o C. A mole opaque milkagar may be made by mixing 10 ml hot sterile skim milk, immediately before
pouring lthe plates.
Test reagent
Mercuric chloride solution, 1 percent hydrochloric acid or 1
percent tannic acid solution.
Procedure
1.
Pour two plates to skim-milk agar.
2.
Inoculate one plate with Escherichia Coli and the other with Bacillus
subtites, using short shokes of the loop over a limited area.
3.
Incubate at 37oC until the next laboratory period.
Recording result
Colonies of organisms that digest casein (proteolytic microbes)
will be surrounded by clear zones. Areas in which the casein has not
attacked will remain slightly opaque.
125
3. UREASE TEST
The genus proteins may be distinguished from some other gram
negative rods by its ability to produce large amounts to the enzyme
urease. The hydrolysis of urea by urease releases NH 3 as follows:
NH2
O=C
------ 2 NH3 + CO2

NH2
Since the production of ammonia raises the pH of the medium urease

activity may be detected by the change of the phenol redindicator to
purple.
1.
Inoculate a tube of urea broth with unknown culture.
2.
Incubate along with a control tube at 37oC for 24 hours.
Medium
User broth
Peptone 1gm; NaCl -5 Glucose 1 gm; KH 2PO4 -2gm; phenol red
0.012 gm; Urea 20gm; pH 6.8 6.9; Filter, sterilize the aseptically
transfer to tubes.
Recording result
The presence of a purple color is a positive test.
4. HYDROGEN SULPHIDE PRODUCTION TEST
The activity of some bacteria on sulfur containing amino acids
frequently results in the liberation of H 2S. common examples of this
reaction are found in the aroma of rotten eggs and in the blackening of
certain spoiled canned foods. In the latter instance, the blackening results
from a reaction between the H2S formed by bacteria and the metal.
Production of H2S by bacterial cultures can be demonstrated in
the laboratory if sulfide producing cultures are grown on media containing
salts of metals such as bismuth and iron. The darkening along the line of
the stab is caused by the formation of the metal sulfide.
Cystine and methionine are two sulfur containing amino acids
present in proteins. Practically all the information available on the effect of
bacteria on these acids has been obtained from cystine or its reduction
product cysteins.
Some organisms are capable of attacking cystine with the
liberation of hydrogen sulfide, others are unable to do so. The test is of
value in the identification and classification of bacteria.
Cystein does not occur in all proteins. Under anaerobic
conditions the cysteine is first reduced to two molecules of cysteine. Then
the cysteine is decomposed to hydrogen sulfide, ammonia, acetic acid and
formic acid.
126
COOH
COOH
CHNH2
CHNH2
CH2 S-S-
CH2
COOH
+ H2 CHNH2
CH2SH
Cystine
COOH. CH NH2. CH2SH +
HCOOH
Cysteine
Acetic Formic
Cysteine
2H2O
H2S + NH3 + CH3COOH +
acid
acid
Under aerobic conditions cysteine is dissimilated as follows
COOH. CH NH2. CH2SH + O HOOC . CO . CH2SH + NH3
CYSTEINE
HOOC. CO .CH2SH
H2S + Other products
H2S reacts with heavy metals to produce coloured compounds. The metal
salts are incorporated in solid media. The presence of H 2S is detected by a
darkening of the media.
Medium
Thiosulfate iron medium: Beef extract -3.0 gm; Peptone -30.0
gm; Peptonised iron 0.2gm; Sodium thiosulfate 0.025gm; Agar -30 gm.
Procedure
1.
Prepare step inoculation as Escherichea coli and proteus

vulgaris in two tube of thiosulfate from medium.
2.
Inoculate the stab cultures at 37oC for 48 hours.
3.
Observe for H2S production
Recording result
Production and liberation of hydrogen sulphide causes the
blackening of the lead acetate paper.
5. INDOLE PRODUCTION TEST
Indole is a nitrogen containing compound formed from the
degradation of the amino acid tryptophan by various bacteria. The
importance of the indole test has in the fact that only certain bacteria form
indole and that it can be readily detected chemically. Thus the degradation
of tryptophan is suitable differentiating reaction.
127
Pure tryptophan is not ordinarily used in the list medium. Instead

tryptone a digestion product of certain products is used as the substrate,
since it contains considerable amounts of tryptophan.
Medium
Peptone water (tryptone-1-2%; sodium chloride-0.5%;
final pH 7.2) dispensed in 5 ml amounts in test tubes and sterilized b
autoclaving for 15 minutes at 121 oC. Tryptone is the peptone to be
recommended for this test, since it is rich in tryptophan, one of the amino
acids destroyed by the usually methods of preparing peptones.
Test Reagent
Kovacs reagent is to be prepared, being rather more
sensitive due to the higher solubility of day complex in the amyl alcohol
layer.
Procedure
1.
Inoculate two tubes of 1% typtone broth one with Escherichia

coli and other with streptococcus.
2.
Inoculate a tube of 1% typtone btoth + 1% glucose with

Escherichia coli.
3.
Incubate at 37oC until the next laboratory period.
4.
Test each tube for the presence of indole, using the method of
Kovac,s.
Recording result
To the culture fluid (about 6ml) add .05ml kovacs reagent. Mix
well by rotating thee tube between the hands.
The alcohol layer will separate from the aqueous layer upon standing and
reddening of the alcohol layer with in few minutes indicates that indole is
present.
6. MICROBIAL REACTIONS IN LITHUS MILK
The fermentation of the lactose produces acid, which lower the
pH. As the acidity increases, the casein is curdled (acid curd) and is
accompanied by the sequeezing out of a liquid (whey) from the curd. If the
organism forms gas during fermentation of milk sugars, the gas may form
bubbles in the curd, sometimes to the extent that the curd becomes
furrowed or even term to shreds. This is knows as the stromy
fermentation of milk.
A differed type of curd is formed by some organisms which
produce a rennin like enzyme, whose action is to coagulate the casein. As
this reaction occurs at neutral pH, the curd is often called a sweet curd
Generally such bacteria are also proteolytic and through the proteolytic
128
digestion of the casein results in the formation of soluble products and

thus in the increasing transparency of the milk.
Procedure
1.
Filtered B.C.P. milk and transfer it into three test tubes 10ml
each.
2.
Inoculate the three test tubes with Baccilus, E. coli and

Pseudomonas.
3.
Incubate at 37oC.
Recording result
B.C.P. milk turn from purple to yellow in all the three test tubes.
B. REACTIONS INVOLVING CARBOHYDRATE AND OTHER CARBON
COMPOUNDS
1. AMYLASE PRODUCTION TEST [STARCH HYDROLYSIS]
Starch is a complex carbohydrate of the polysaccharide type. A
qualitative test for starch is the appearance of a blue color upon the
addition of a solution of iodine. When starch is hydrolysed, (decomposed
with the addition of water) the cleavage products dextrine, maltose and
glucose to list them in descending order of complexity do not give cold
reaction, this principle is used in testing for starch hydrolysis in this
exercise.
An organisms ability to hydrolyze starch is dependent upon the
presence of the enzyme amylase. Since not all microorganisms produce
amylase, the ability to hydrolyze starch can be used aid in the identification
of a given species.
a.
-amylase + starch
dextrin + - maltose
Medium
Starch hydrolysis may be tested with solid or liquid media,
although starch agfaris perhaps more convenient. This medium consists of
nutrient agar with addition of 0.2-1% soluble starch. The best results are
obtained by preparing layer plates which are prepared by pouring 10ml of
nutrient agar into each plate, allowing it to set, and then verifying this with
5ml of starch agar.
Test Reagent
Grams iodine solution as used for Grams stain.
Procedure
1.
Prepare two starch-agar plates and inoculate them by streaking

with short strokes over a limited area with cultures of
Escherichia coli and Bacillus sublites. Since the attempt here is
129
not to isolate colonies, the streaks may be confined to the large

center area of the plate. Avoid streaking all the way to edge so
that you will leave an uninoculated control cone to provide an
area of contrast after the plate has been developed with iodine.
The very centre of a Petri plate has a hump. Do not restrict your
sticks to this area alone.
Incubate at 37oC until the following laboratory period.
2.
Reading result
Flood plates with 5-10 ml of iodine solution. Unhydrolysred
starch forms a blue color with the iodine. Areas of hydrolysis therefore
appear as clear cones and are the result of pro amylase activity. Record
the width of any clear cone in mm from the edge of the colony to the limit
of clearing. Reddish brown cones around the colony indicate partial
hydrolysis of starch (to dextrins) which is the result of - amylase activity.
2.
FERMENTATION OF CARBOHYDRATES
Among the common products of carbohydrate breakdown by

microorganism are gases (for example CO 2, and H2). The types of
products formed, and the proportion of each, depends upon the species of
micro-organism as well as the particular carbohydrate being dissimilated.
Medium
The formation of acids can be readily detected by the pH
indicator in microbial growth media. In this experiment, media containing
the chemical bromocresol purple (BCP) which is purple at pH 6.8 and
yellow at pH 5.2 are used. In broth, gas formation may be detected
through the use of a vaspor seal (or) an inverted vial. (a Dusham tubs).
Gas production in agar medium is accompanied by the formation of gas
pockets, which appear as cracks in the agar.
This exercise illustrates a simple method, used to detect acid
and gas formation.
Procedure
1. 4 tubes each of the following media are taken.
a. Glucose broth with
fermentation tubes.
B.C.P. indicator
containing
Durham
b. Sucrose broth with

fermentation tubes.
B.C.P. indicator
containing
Durham
c. Lactose broth with

fermentation tubes.
B.C.P. indicator
containing
Durham
d. Inoculate a different tube of each medium with E. coli,

Streptococcus facales and Bacillus subites. The 4th tube in each set
is a control tube and is not inoculated.
Note:
Be sure to label each tube and be sure to place the cap

(or) plug
back on the same tube it came from.
130
2.
Using the culture of E. coli provided, inoculate:
(a)
A tube of glucose BC. P. broth covered with a vaspor (or)

agar seal.
(b)
A glucose BC. P. agar-shake culture
(c)
Incubate all tubes at 37oC
Recording result
At the next laboratory period observe the culture for acid and gas
production, and record the results in the table on the report sheet;
some bacteria for example, leuconostoc-produce only small
amounts of gas that cannot be detected by the Durham tube
technique the gas being lost from the surface of the medium. By the
use of vaspar seal technique, this problem is avoided, and small
amounts of gas can be detected.
3.
CITRATE UTILIZATION TEST

The ability to utilize citrate as a sole source of carbon and
energy can be used to distinguish certain gram negative rods.
Medium
Simmons citrate agar contain citrate as its only carbon and
energy source. Growth on this medium is a positive test for citrate
utilization. Certain organisms that give a positive test increase the
pH, changing the bromothymol blue indicator in the medium from
the green to blue.
Requirements: Bacterial culture of Bacillus, E. coli, pseudomonas;
Simmons Citrate agar.
Composition of Simmons citrate agar - per liter MgSo 4 0.2mg;
NaH2PO4 1.0gm; k2HPO4 1.0gm. sod. Citrate 2.0gm; NaCl 5.0gm;
Agar 15.0gm; Bromothymol blue 0.08gm.
Procedure:
1.
Inoculated a slant of simmons citrate agar with bacterial

cultures.
2.
Inoculated at 37oC for 48 hours.
3.
Observed the growth.
Recording result
Utilization of citrate and growth on citrate agar results in
an alkaline reaction, so that the bromothymol blue indicator in the
medium changes from the green to bright blue. When no growth
occurs and citrate is not utilized, the color of the medium remains
unchanged.
Bacillus, E. coli, Pseudominas are not citrate utilizing bacteria.
131
C.
MISCELLANEOUS TESTS
1. CATALASE TEST
Catalase is an enzyme found in most bacteria. It
catalyzes the breakdown of Hydrgen peroxide which release tree
oxygen gas.
Catalane + hydrogen peroxide
water + molecular oxygen.
In many cases, gases can be readily seen as a white

growth if a few drops of 3% H 2O2 are added to a microbial colony (or)
to a broth culture.
Catalase negative organisms tend to be anaerobic since cation
terminal respiratory enzyme reset with atmospheric oxygen to from
H2O2 which is tonic to the living cell. Perhaps the enzyme catalase is
essential for the aerobic growth of most micro organisms.
The enzyme catalase contains the hemeporphyrin
structure. This porphyrin ring structure is characterized not only of
catalase but also of the cytochrome respiratory electron carrier found
in aerobic forms of life and in the chlorophyll of all photosynthetic
cells.
Medium Nutrient broth nutrient agar.
Test Reagent
Hydrogen peroxide (10 volume concentration)
Procedure
1.
Inoculated the three bacterial cultures (Bacillus, E. coli, and

Pseudomonas) nutrient broth and incubate at 37oC.
2.
Prepared three nutrient agar slants in test tubes and streaked

the 3 bacterial cultures in 3 different slants and incubated at
37oC.
3.
Transfer a bit of growth from each slant to slide, added few

drops of Dissolve the colony. Then add 3% H2O2 and observe.
4.
Take three samples in test tube from nutrient broth add 3% H 2O2
and observe.
Recording result
Effervescence, caused by the liberation of free oxygen as gas
bubble, indicates the presence of catalase in the culture under test.
References
1.
Microbes in action
2.
Fundamental principles in Bacteriology A. J. Salle
3.
Bacterial metabolism- H.W. Dowelle
4.
Methods is microbiology
5.
General microbiology Stanier.
132
BIOCHEMICAL CHARACTERIZATION OF BACTERIA

A.
GELATIN HYDROLYSIS TEST

Peptone
0.5%
Beef extract
0.3%
Gelatin
4%
Tryptone
10 gm
Yeast extract
10 gm
K2HPO4
5 gm
Soluble starch
3gm
Microorganism inoculated in the medium after sufficient

time it is seen that medium is solidified or net. If medium solidified
then there is on organism synthesized proteolytic enzyme gelatinase
which hydrolyses the gelatin.
B.
CASEIN HYDROLYSIS TEST

Skimmed milk
2%
Agar
2%
HO4
Casein --
paracasein + Ca+2 --
Calcium paracaseinate
M. O
Calcium paracaseinate is formed by which around
microorganism white appearance will be lost. (Kanin also hydrolysed
the casein but in this case curd like appearance is seen).
C.
D.
H2S PRODUCTION TEST

Beef extract
3gm
Peptone
3gm
Peptonized iron
0.2gm
Sodium thiosulfate
0.25gm
Agar
3.0GM
Distilled water
1000ml
INDOLE PRODUCTION TEST

Tryptone
1-2%
NaCl
0.5%
pH
7.2
133
E.
UREASE TEST
Peptone
1gm
NaCl
1gm
KH2PO4
4gm
Phenol red
0.01gm
Urea
20gm
pH
6.8 6.9
Distilled water
1000ml
Na2
Urease
C=0
------
NH2
H2O
2 NH3 + CO2
Microorganism in which urease enzyme synthetized gives

the positive test urease hydrolyses the urea by which ammonia is
formed. Due to increase of ammonia pH is also changed (incl) and
thus to this high pH the red colour of phenol red into purple colour.
F.
CITRATE UTILIZATION TEST

Simonds Agar
MgSO4
0.2 gm
NaH2PO4
1 gm
K2H2PO4
1 gm
Sodium citrate
2 gm
NaCl
5 gm
Agar
15 gm
Bromomethyl
0.08 gm
Citrate utilization elevates the pH, they produced alkaline

substances. Green colour changes to blue.
134
Experiment No.
Objective: To identified the organism producing H2S gas.
Requirements: Beef extract, peptone, peptonized iron sodium thio
sulfate, Agar, Distilled water.
Principle: The activities of some bacteria frequently result in liberation
of H2S. common example for this reaction are found in the Aroma of
rotting eggs and is blackening of certain spoiled common food.
Blackening occurs due to the reaction between H 2O and compared
CH2SH
2H
H2N CH - COOH
-----
H2N CH + H2 2 ST
Cystine
Observations:
Result:
135
Experiment No.
Objective: To find out the presence of catalase in bacteria.
Requirements:
Principle: Catalase is an enzyme that is present in an aerobic
bacteria. It catalyses a break down of H2O2 in to H2O
H2O2 ----------
H2O
O2
H2O2 is a toxic for living organism and generally all aerobic bacteria
continues this enzyme. Presence of this enzyme in bacteria is easily
measurable. Take a bacteria culture into a test tube (1 ml) and a 2-3
drop of 3% H2O2 and if bacteria contains this enzyme then inginous
broth is obtained in the medium due to liberation of O 2.
Observations:
Fresh formation
Sample I
Yes
Sample II
Yes
Sample III
Yes
Sample IV
Yes
136
8. MUTATIONS AND MUTANTS

Introduction
Any permanent sudden and heritable change in the
sequence of bases in the bacterial genome is called mutation.
Mutations occur very rarely, one cell in 10 8 cells carries a detectable
mutation in any given, but exposure to certain powerful mutagens eg
nitro so guanidine increase the frequency of cells in a population that
assay a mutation in any given gene about 105 fold.
Classification of Mutations based on genotype
I.
Macrolesions
Mutations may involve a change that extends over a

number of base pairs or even a of genes. A macrolesion include variety
of changes in DNA.
A.
Deletions:
The complete elimination of a segment of DNA.
B.
Duplications: The tandem repetition of a segment of DNA.
C.
Inversions:
The inverting of a segment of DNA.
D.
Insertion:
Introduction of a new segment of DNA Within an

existing Sequence.
E.
Translocation:
another
Movement of a segment of a segment of DNA to

site in the genome.
II.
Micro lesions (Point Mutation)

Mutations may involve a change in only a single base pair of the
cells DNA
A)
Base pair substitutions

If a micro lesion involves a change in only a single base pair to
another.
1.
Transition:
Purine base of a pair is changed to another purine base.
2.
Translocation:
base.
B)
Purine base of a pair is changed to a pyrimidine
Frameshift
If the microlesion involves loss or gain of a base pair it is called

a frame shift mutation because it changes the reading frame of all codons of
the gene or operon distal to the point of mutation.
MUTAGENS
137
Mutagens are chemical or physical agents that increase the frequency

at which mutations occur in growing bacterial culture.
Physical Agents
These include various types of radiation. X=- rays cause breaks in
chromosomes that reform in a variety of ways, causing macrolesions.
Ultraviolet is absorbed by DNA and the energy so released causes
dimerization. Between adjacent pyrimidine residues on the same DNA strand.
The occurrence of these pyrimidine dimmers exposing a short region of single
stranded DNA on the opposite strand. The presence of these single stranded
regions induces the formation of a general DNA repair mechanism termed the
SOS system. This is a rapidly acting but inaccurate DNA mutations that follow
U. V. irradiation.
Chemical Agents
I.
Mutagens that associate with or become incorporated into DNA.
A.
Base analogue- Bg 5. Brornowail

Mutagens that become incorporated into DNA. Eg 2- aminopurine
causes transition mutations.
B. Intercalating agents Bg.
Mutagens that become associated with DNA eg. ICR 191
causes frame shift mutations.
Intercalating agents are plain molecules and can insert between the
stacked pairs of bases in the core of the DNA molecule. Such
incorporation distorts the backbone of the double helix in such a way
that frame shift mutations can occur when the distorted helix is
replicated.
II.
A.
Mutagens that react with DNA

Alkylating agents
These are most powerful known chemical mutagens which add

methyl or ethyl groups to the heterocyclic nitrogen atoms of the bases. By
these a variety of different types of mutations result. e.g. They cause
transition, transfusion and frame shift mutations.
NH
eg. Nitrosoguanidine
= N-N-C-NH-NO2
CH3
B.
Other DNA modifiers
eg.
1)
hydroxylamine (HONH2) reacts specifically with cytosine

converting it to 6- hydroxylaminouracil which pairs with
adenine, causing G/C to A/T transitions.
2) Nitrous acid is less specific in its action and reacts with all bases
(A,G and C) that contain amino groups, thereby converting the
138
amino groups to hydroxyl groups. This causes both A/T to G/C

and G/C to A/T transition mutations.
Phenotypic Consequences of Mutations
Many mutations inactivate indispensable gene product and
therefore kill the cell. But many others inactivate gene products that are
not essential under all conditions of growth and these are not essential
under all conditions of growth and these are not lethal to the cell. Cells
carrying later type of mutations can be maintained in culture and they
differ phenotypically from their unmutant parents (wild type strains).
Other mutations that alter the target protein of an antibiotic can
render the cell resistant to the antimicrobial agent. Still other mutation
strains might have lost some nonessential capacity, such as motility.
Mutant Methodology
A mutation alter or eliminates the functioning of a particular gene
particular gene product; by observing the effect of genotypic change on
the cells phenotype, one can deduce the cellular functions of the gene
product. This process is called mutant methodology.
Mutation Rate
The mutation rate can be defined as the probability that any one
cell will Mutate during a defined interval of time. Mutation rate is thus the
number of mutations per cell per generation.
139
Experiment No.
Objective: To study the effect of U.V. light on % survival of bacteria.
Introduction: Mutagens are chemical or physical agents that increase
the frequency at which mutation occur during growth of a culture. Among
the physical agents various types of radiations are powerful mutagens.
-rays cause breaks in chromosomes that reform in variety of ways
causing most types of macro lesions. Radiation damage is conveniently
generated by a U.V. light source such as 15 or 25 W germicidal lamps. irradiations from a 60CO source is also convenient method for DNA
damage.
Sunlight is normally injurious to most bacteria. This effect is
primarily due to the U.V. portion of the spectra which increase the rate of
mutation. U.V. light is observed by the DNA and the energy so released
causes dimerisation between adjacent pyrimidine residues on the same
DNA strand. The occurrence of these pyrimidine dimmers triggers a
repair mechanism that excises the pyrimidine dimmers excising a short
region of single stranded DNA on the opposite strand. The presence of
these regions induces the formation of a general DNA repair mechanism,
termed SOS system. It is a relatively inaccurate DNA repair system that
fills in the gaps opposite to the single stranded regions. This error prone
repair, introduces the mutations that follow U.V. irradiation. The irradiated
bacterial cell cannot reproduce and hence dies.
The maximum absorption of U.V. by DNA is at the wavelength of
254nm. Maximum mutagenicity also occurs at 254 nm. Suggesting that
U.V. induced mutations process is mediated directly by absorption of
U.V. by purines and pyrimidines. The two major products of U.V.
absorption by pyrimidine are pyrimidine hydrates and pyridine dimmers.
Many evidence suggest that thymine dimerization is probably the major
mutagenic effect of U.V.
Products of U.V. irradiation
Hydrotes cause mispairing of bases during replication.Cross linking of
adjacent thymine molecules to form thymine dimmers, which block
replication.
Requirements: Apparatus and materials U.V. light apparatus, patri

plates, pure culture of bacteria (E. Coli)
Chemicals Nutrient agar of composition Peptone = 0.5 gm
Yeast extract = 0.3 gm
Agar = 1.5 gm
Distilled water =100 ml
140
Procedure:
Make 10-6 dilution of the culture
Pour 1 ml of this dilution into 5 small sterile Petri plate
Label these plates 0 sec, 5 sec, 10 sec, 15 sec, 20 sec
Expose these plates with their cover removed under UV light
for the interval of time marked on them.
Add proper amount of melted and tempered agar and mix it
Incubate all plates at 37oC for 48 hr
Count and record the colonies and calculate the percentage
of survivors in the irradiated sample.
Observations:
General Comments: The virtues of radiation treatment are several fold:

1.
The dosage is easily controlled by varying the exposure time

and intensity of the source.
2.
Radiation generates a wide variety of lesions across a genetic

target and consequently a broad spectrum of mutational events
including base substitutions, frameshifts and deletions.
3.
There is no hazardous chemical to handle or dispose off.

A drawback of radiation mutagenesis is that the lesions
generated by U.V. are not efficiently fixed as mutations unless
the cells are capable to carrying out SOS repair.
Precautions: U.V. radiation is a potent mutagen and human

carcinogen. To avoid the exposure to U.V. radiations, wear U.V. opaque
glasses and cover hands and fore arms.
141
Experiment No.
Objective: To study the effect of Photoreactivation.
Introduction:
Although certain wavelengths of visible light are beneficial still
sunlight is normally injurious to most bacteria. This effect is primarily due to
rays of the ultraviolet spectrum, particularly wavelength between 2400 and
3000 .
2650 is also the peak of absorption of UV light by DNA and
radiation of this wavelength affects mutation rates, bacterial transformations
and U.V. radiation appears to act on genetic material to produce pyrimidine
dimmers on the same DNA strand or between different DNA strands. The
pyrimidine bases absorb the energy of the U.V. light in the energized state
link with adjacent pyrimidine bases interfering with the proper replication and
function of the DNA molecule. Therefore irradiated bacterial cell cannot
reproduce and die.
The bactericidal effect of exposure of ultraviolet light can be
educed by immediate subsequent exposure to certain wavelength of visible
light
(3650-4500 ). This reversal of killing action of ultraviolet
light is called photo reactivation. In this visible light activates an enzyme that
cleaves the pyrimidine dimmers, which result from U.V. irradiation, allowing
DNA to resume its normal functioning. Exposed to visible light immediately
after being exposed to visible light will be many times of a population
exposed solely to U.V. light. However there will always be some cells whose
damaged cannot be repaired by this process of photo reactivation.
Procedure:
1)
Plate 10-6, 10-7 and 10-8 dilutions of a 24 hour culture on agar from the
colony counts these plates you will determine the average number of
cells per milliliter in the original culture.
2)
Place 2 ml from the 10-6 dilution flank into each of four small, sterile
Petri plates. Label these plates 5 sec, 10 sec., 15 sec and 20 sec.
3)
Place each plate with the cover removed, under the U.V. light for the
length of time marked on it.
4)
Immediately following its exposure to the U.V. light, prepare a 10 -7

dilution of each sample by transferring a 0.1 ml aliquot to a sterile Petri
plate adding the proper amount of method and tempered agar and
mixing by suruling. Label each new plate with word irradiated, the time
of exposure, and the dilution.
5)
Transfer a 1.0 ml sample from each plate irradiated in step 3 to a

different sterile test tube; label each with the time of irradiation.
142
6)
Place the four tubes 6 inches away 500 watt bulb for 30 minutes. If the
light source you are given does not have a fan attached for cooling,
place the tubes in a beaker of ice.
7)
Plate 10-7 dilutions of each sample exposed to light, label each plate
with a word photo reactivated, the time of U.V. irradiation and 10 -7.
8)
Incubate all plates at 30oC for 48 hours.
9)
Following incubation count and record he number of colonies on each

plate. Calculate the percentage of survivors in the irradiated and photo
reactivated samples.
10) Plot the number of survivors versus time for the irradiated and
photoreactivated samples.
143
Selection of spontaneous mutants

Mutant is an organism whose genotype differs from the wild type.
Mutations are the raw material of evolution, reducing the changes on which
selection can act.
The process of generating mutations is called mutagenesis.
Mutations sometimes arise spontaneously. This is called spontaneous
mutagenesis. Mutations can also be induced by mutagens which may be
chemicals or physical factors.
Because of the low rate of appearance of mutation bacterial
cells, special methods must be used to select them out & screen them.
(A)
Selection: is a condition that allows specific mutants to grow but not

the parent cells. Genetic selections are from a population of cells
eg. antibiotic resistant mutants can be selected by simply planting
large no of bacteria on solid medium containing the antibiotic. only
the resistant mutants can form colonies.
(B)
Screen: In screening for mutants on media on which both the

mutant and the parental cells grow, the phenotype of the mutation
can distinguished by the phenotype of parental cells.
Some bacteria are naturally resistant to certain antibiotics; others
can acquire resistance by mutations. There are two main ways in
ways which bacteria develop antibiotic resistance.
1)
Acquiring an enzymatic activity that directly inactivates the

antibiotic.
2)
Acquiring a mutation that modifies the target site of the

antibiotic, which prevents the antibiotic from interfering with
the normal function of the target site.
144
Experiment No.
Objective: To develop antibiotic resistant mutant
Requirements: Bacterial suspension, Tetracycline, Agar yeast extract,
peptone, streaking needle.
Principle: Antibiotic susceptible strains can be made mutant by various
means. The mutant can be developing by growing the culture at different
concentration of antibiotic. The concentration that can grow at higher conc. Of
antibiotic can be selected as the mutant.
Procedure:
1.
Prepare the agar gradient plate and solidify.
2.
Pour the antibiotic mixed agar (50 g/ml) on the already prepared
agar plate.
3.
Grow the bacterial culture by streaking on the agar plate.
4.
Incubate it 37oC for 48-72 hrs.
5.
Select the bacterial colonies that grow at higher.
6.
Concentration of the antibiotic culture the selected bacterial colony.
Observations:
Result:
145
Experiment No.
Objective: Selection of antibiotic resistant spontaneous mutants using
gradient plate method.
Requirements:
Principle
If a mutant cell, by virtue of the change that it has undergone, is
somehow especially suited to the environment in which it is formed its growth
may outstrip that of its parent culture and it may thus become dominant. The
spontaneous mutants that are resistant to antibiotics are readily detected
since they grow in concentrations of the antibiotics that would inhibit growth of
normal organisms.
Requirements: Pure culture of E. Coli, oxytetracy cline, nutrient agar, Petri
plate bent glass rod.
Procedure: We employ gradient plate method to isolate oxytetracycline
resistant mutants.
1)
Prepare a gradient agar plate

a) Till the sterile Petri dish by placing a glass rod or wood stock,
approximately 1/16 inch in diameter, under one side. Pour a
tube of melted trypticase soy agar into the Petri and allow the
agar to harden, slanting the plate just enough to cover the entire
bottom.
b) After the agar has solidified, place the dish in the normal
horizontal position. To a scanned tube melted agar (taken 100
ml) add 0.1 ml of oxytetracyline. Mix the tube well and pour the
melted agar on the surface of the gradient agar plate.
2)
Pipette approximately 0.5 ml E. coli culture on the agar surface.

Using a sterile glass spreader, streak the cultures over the agar
surface. Glass spreader is sterilized by dipping 95% alcohol,
flaming and cooling on the sterile surface of the gradient plate.
3)
Incubate at 37oC for 24 hours.
Observations:
146
Experiment No.
Objective: Transformation of DNA into competent E. coli cells.
Introduction: Transformation is the form of recombination in which free
DNA from a donar cell is taken up by a recipient cell and integrated into
the latters genome without the two cells ever coming into contact. Those
recipient strains that are capable of incorporating free DNA are called
competent. A natural competent is one which can act as recipient and such
bacteria are capable of undergoing natural transformations. Some bacteria
can be as exposure of cells to hi concentrations of divalent cations. Such
transformation systems are called artificial transformation.
Transformation is the only mechanism of genetic exchange
among prokaryotes that is chromosomally encoded. Inspite of this many
baceria lack natural transformation eg E. coli. The genetic exchange in
transformation is permanent and this method is employed in lab to bring
recombination; but it may be accruing innature as well.
Competence has been demonstrated only in some strains of a
few bacterial genera, and then only in cells at a specific stage of growth in
nutritionally minimal media. In this exercise we will extract DNA from a
tryptophan requiring, histicline synthesizing strain of Baccilus subtilus.
This free DNA will then be used to transform an auxotrophic strain of
Baccilus substlus, which is unable to synthesize the amoni acid histidine, a
histidine synthesizing strain.
Procedure:
A. DNA extraction
1)
Centrifuge the tryptophan- requiring culture of Bacillus

subtilus strain 168 provided.
2)
Aseptically d:, scard the supernatant and resuspend the cell

pellet in 5 ml of sterile saline- citrate solution. Pour the
solution into a 25 x 150 mm test tube.
3)
Add 0.5 ml of lysozyme solution to test tube. Min well.
4)
Shake gently at room temperature for 15 min. and observe

clearing or lysis. If lysis is not complete, add another 0.25 ml
of lysozyme.
5)
When lysis appears to be complete, add 5 ml of 4 M NaCl to

the test tube, mix gently and filter the solution through a
membrane filter (0.45 m pore size), collecting the filtrate in
asterile test tube. Test 0.1 ml of filtrate (containing DNA) for
sterility, by streaking on a tryptose blood agar plate. Table
the tube and refrigerate it. Incubate the plate at 37 oC for 24
hours.
147
B. Transformation
1)
To prepare competent cells, inoculate 1 ml of washed

suspension of a 5-hour culture of Bacillus subtilus (his, trp)
into a sterile 1.25 ml flask containing 4 ml of transformation
media (a minimal medium containing trp and his)
2)
Aerate this flask for by shaking for 1 hour.
3)
Pipette 0.9 ml of these competent cells into 3 sterile tubes.
4)
Label the three tubes A, B and C. To tubes A and B add 0.1

ml of the sterile DNA sample from a previous period. In
addition add 0.1 ml of DNA are (2 mg/ml) to tube B to tube C
add 0.1 ml of sterile saline citrate as a DNA less control.
5)
Aerate all tubes by shaking for 1 hour.
6)
Label three minimal-plus tryptophan agar plates A, B and

C. transfer 0.1 ml aliquots from tubes A, B and C to plates A,
B and C respectively. With a sterile bent glass rod spread
each aliquot over the agar surface of a plate. In addition
make 1/10 and 1/100 dilutions of the contents of tube A and
transfer 0.1 ml aliquots of these dilutions of minimal plus
tryptohan agar plates labeled A -100 and A-1000. again using
a sterile bent glass rod, spread these samples over the
surface of the agar plates.
7)
Incubate the plates at 37oC for 24-48 hour. Count the number
of transform ants at each dilution.
Observations:
148
Experiment No.
Objective: Genetic recombination of two strains of E. coli by conjugation.
Introduction: Genetic recombination is the exchange of genetic material
either by exotic means as well as by a straight forward conjugation process.
Conjugation is a process by which DNA can be transferred form a donar cell
to a recipient cell by cell to cell contact.
Conjugation has been demonstrated primarily in enteric gram
negative bacteria- including Escherichia, salmonella etc. and has been most
studied in E. coli.
There are two mating types in E. coli genetically determined by
the presence or absence in the cell of a small DNA element called the F
(fertility) factor.
a)
Cells tacking the F factor can only act as recipient: called F (or
female)
b)
Cells carrying F factors act as donars and are male cells. These
are of two types
[ F+ cell F particle is detached from the
cells chromosome Hf cells F particle is
part of the chromosome.]
In F+ cell only V- factor is transferred in conjugation, with the F - cell being

converted to a V+ cell in the process.
In conjugation if Hfr is one of the participants, chromosome 1 DNA is
transferred to the recipient cell. Complete chromosome transfer is rare V factor is not transferred: indicating that the F- particle is attached at the distal
end of the chromosome being transferred.
In this exercise we will observe genetic recombination resulting from
the conjugation of two strains of E. coli mutants1)
Strain C- 600 is an F- strain that requires threonine (T-) and Leucine

(L-) for growth; this is unable to ferment lactose (Lac -) and is
resistant to streptomycine (Ss).
2)
Strain Hfr 235

DONAR: does not require threonine (T +) or
leucine (L+) for growth, is capable of fermenting lactose (Lac +) and
is sensitive to streptomycine (Ss).
The genetic crossing of these mutants should result in
recombinant like the wild type E. coli from which the mutant
strains arose with respect to growth requirements.
Procedure:
You are provided with culture of E.coli, strains C- 600 and Hfr235, that have been grown in a complete nutritional medium, aseptically,
washed several times by centrifugation to remove nutrients, and concentrated
by resuspending in sterile saline to 1/20 of the original volume.
149
1)
With a sterile 1.1 ml pipette, transfer 1.0 ml of C- 600 suspension to

a sterile wasermann tube and 0.1 ml to the surface of a minimal
plus- streptomycin agar plate. Spread the 0.1 culture on the agar
plate with a bent glass rod as folowa:
a) Dip rod in alcohol, pass quickly through a flame, and allow
alcohol to burn off.
b) After cooling the spreader, using a circular motion, spread the
inoculum evenly over the entire surface of agar. Label this plate
NAC-600.
2)
Remove 0.2 ml of Hfr- 23suspensinon, trasnsferring 0.1 ml of Co600 suspension and pipette remaining 0.1 ml onto the surface of a
second minimal plus streptomycin agar plate. Using the glass
the inoculum over the entire plate. Label this second plate NA Hfr235.
3)
Mix the tube containing the two strains by rotating gently. Violent
mixing will interrupt genetic transfer. With the sterile 1 ml pipette
further mix by gently drawing the suspension into the pipette one
allowing it to flow out. Allow this mixture to rest on the bench for 3060 minutes. Remove some of the mixture with the same pipette and
add 0.1 ml of it to a third minimal plus streptomycin agar plate
spread in the same manner as above. Label this plate MAC- 600
plus Hfr-235.
4)
Invert and incubate the plates at 37oC.
Observations:
150
9. QUANTITATION OF VIRUSES BY PLAQUE ASSAY

Background: There are many methods for the enumeration of viruses.
Plant viruses are typically enumerated by the plant leaf local lesion assay:
a suspension of viruses is applied to the surface of a leaf along with an
ambrosia material that tears small holes in the walls of plant cells. Each
vision that enters a host cell initiates a local infection that spreads to
surrounding cells, creating a region of infection that becomes discolored
and thus easily recognized.
Before the development of techniques for culturing animal cells
invitro, animal viruses were usually enumerated by the pock assay
performed in developing chicken embryos: a sample containing viruses is
injected into a fluid compartment of the egg. These viruses absorb to cell
in one of the interval membranes of the developing embryo, producing
regions of infection termed pocks that can be recognized as being
thickened and discolored.
Another important method of enumeration of viral particles is by
Plaque Assay. The viruses are ultra microscopic too small to the viewed
with light microscope. They are particulate, not cellular; being more or less
macromolecules composed primarily of a nucleic acid genome either DNA
of RNA and protein. They are obligate parasites and use hosts machinery
for replication.
Basically the viral particle. Of version, is a nucleic acid core
surrounded by a protein coat or capsid composed of protein subunits or
cashmeres?
The viruses would have gone unnoticed during the early
development of microbiology except for the fact that they are infections
agents, evident from the symptoms of the disease they caused. Even
today, when are see a viruses with electron microscope, we still depend
for study primarily on the symptoms of experimental virus infections.
Bacterial viruses are called bacteria phages. As the bacterial
cells increase the viral particles are formed; the cells eventually disrupt
and release thee viruses into the medium. The plaques represent areas of
phage reproduction and lyses of the infected bacterial cells in the area.
151
Experiment No.
Objective: To quantitative viruses by plaque assay.
Introduction: Most commonly, animals and bacterial viruses are
enumerated by infecting host cells that growing in their layer on a medium
partially solidified by agar. An infected cell in such a culture establishes a
lock, spreading infection where, depending on the infecting virus, cells
either die or grow abnormally slowly. These infected zones termed plaques
differ in appearance from the surrounding cell layer. Plaques formed by
phages are usually clear, circular regions in a turbid layer of cells termed a
lawn; plaques formed by animal viruses are sometimes visualized
following application of a dye that stains line cells, but not those killed by
the viral infection.
Requirements:
Apparatus and material
Test tubes, pipette bacterial culture (E. coli), virus lysate,
Petri plates.
Chemical: TYG broth, TYG agar
Constituent:
Tryptone
5 gm
Yeast extract
3 gm
Glucose
1 gm
Agar
15 gm
Distilled water
1 liter, pH = 7
Procedure:
Took virus Lysated
Prepared its dilution ranging from 10-1 to 10-7
Prepared its dilution ranging from 10-5, 10-6 and 10-7 and pipette into molten
tubes of top layer agar (3.5 ml each)
By pipette we added .2 ml of overnight E. coli culture to each of the 6
tubes.
Poured the content of all six tubes into freshly made TYG agar plates and
spread it.
152
Incubated at 37oC overnight

Prepared control plate by inoculating only E. coli on TYG agar
Incubated at 37oC overnight
Observations:
General Comments
It is wise to plate each dilution in duplicate in case one of them
smears because of excess moisture. The most reliable liter is determined
from assay plates they contain between 50-400 plaques. At the time of
determining the phage lysate liter, it is wise to take some of the lysate and
sheak on a non selective L agar plate for enquiring that the lysate is free
from all viable donor bacteria.1
153
Experiment No.
Objective: Quantitation of viruses by Plaque assay (Bacterial lysis by
bacteriophage)
Principle: Bacterial viruses are enumerated by infecting host cells that are
growing in a thin layer on a medium partially solidified by agar. In infected
bacterial cell in such a culture establishes a local spreading infection
where cells either die or grow very slowly. These infected zones are called
plaques, formed by phages are usually clear, circular regions in a turbid
layer of cells termed a lawn. This technique of counting phage is called
plaque assay.
Theory: If 108 bacteria are spread on a rich plate, the colonies formed are
so close to each other that appear as a confluent, turbid layer of bacteria
called a lawn. Or the bacteria can be mixed into a small volume of warm,
slightly dilute, liquid agar, which is then poured into the surface of the solid
medium. The liquid known as top agar or soft agar, hardens, providing a
uniform lown of bacteria.
If a phage is present in the top agar, it can adsorb to one of the
bacteria in the agar; the infected bacterium lysis and releases about 100
phage, each of which adsorb to nearby bacteria. These bacteria in turn
release a burst of phage which can infect other bacteria in the cinity.
These multiple cycles of infection continue and after several house
phage will have destroyed all of the bacteria at a single localized area in
the agea, giving rise to a clear, transparent circular region in the turbid
confluent layer. This region is called plaque. As one phage forms one
plaque: number of bacterial virus added can calculated from the number of
plaques.
The efficiency of plating (EOP) is the fraction of phage particles
that can form a plaque. It is one for many phages but can less that 1 for
phages that make very small plaques. If phage lysates are stored, EOP
may decrease, as a result of accumulated chemical damage of penetration
of phage proteins. Storage of phage at 4 oC often reduces the loss of
phage viability. Addition of cations, glycerol and proteins also protects
phage from damage.
154
CONTENTS
ANIMAL (Mammalian) CELL CULTURE
I.
Introduction
II.
Cell culture techniques.
III.
IV.
A.
Cell biology
B.
Development of cell culture technique
C.
Handling of mammalian cell culture lies.
D.
Small scale culture of animal cells.
E.
Suspension culture.
F.
Anchorage dependent cells.
Cell culture: Organism and products.

A.
Primary cell culture.
B.
Genetic engineering
C.
Monoclonal antibodies
Cell culture media

A.
Introduction
B.
Components of cell culture media

Water
2.
Basal media
C.
Animal sera as medium supplements
D.
Defined media
E.
V.
1.
1.
Carrier proteins
2.
Insulin
3.
Growth and attachment factor
4.
Miscellaneous
Preparing and handling of media

1.
Quality control
2.
Sterility
Process design and operculum

A.
Introduction
B.
Suspension culture
C.
High density suspension culture
D.
Entrapped cell suspension reactors.
E.
Micro carrier culture
155
F.
VI.
Down stream processing
VII.
A.
Introduction
B.
Cell removal
C.
Concentration
D.
Purification
Regulatory aspects of products from Animal cell culture
VIII.
I.
High density perfusion reactor
A.
Biohazards associated with the cell culture.
B.
Regulatory environment.
References
INRODUCTION
The culture of animal cells has been a rather difficult field of

biology. Developments in several areas of biotechnology have brought
remarkable changes in this in the last few years. The discovery of bimolecular
with great potential for therapeutic or diagnostic benefit has provided
commercial motivation for rapid development of cell culture technology. At the
same time, advanced analytical techniques and methods for manipulation of
cell at the genetic level, along with an improved understanding of cell biology
at all levels, have given researchers new tools to help bring the benefits of
these discoveries to the public. This chapter will provide an over view of
current animal culture technology with an emphasis on animal biotechnology.
Developments in plant cell culture and organ culture have also generated
considerable interest.
II
CELL CULTURE TECHNIQUES
A.
CELL BIOLOGY
A discussion of cell culture technology should begin with the

animal cell in form and function, have been designed by millions of years of
evolution.
Malian cells have a fragile membrane, a high cell density and an
intricate circulatory system for supplying essential nutrients and removing
waste products, light control of the physical environment (temp, pH etc) and
control system for regulation by externally provided hormones and growth
factors individual cells are typically differentiated to serve specific functions
within a tissue or organ. This differentiation will suppress most of the available
genetic information and commit the cell to its particular course.
Aside from the use of animal cells in viral vaccine manufacture,
the main benefits of cell culture as a manufacturing system are derived from
the ability to assemble and than excrete large and complex proteins. These
complex proteins are often modified after translation by glycosylation or
carboxylation at specific locations. Bacteria do not possess the machinery for
such modification; yeast and other eukaryotic organisms do have some
156
capabilities in this regard but produce molecules different from the native
proteins. The animal cell culture now becomes the only practical method for
producing significant quantities of fully functional proteins. Even among
mammalian cells, the structure or post-translational modifications of a given
protein may vary when produced in different cell lines, the correlation of
functional activity with changes must be examined on a case-by-case basis.
B.
DEVELOPMENT OF CELL CULTERE TECNIQUE
Early work with cultured cells focused on normal or Primary

tissues and cell lines. Cell lines are generally isolated by grinding or chopping
on animal tissue, washing carefully and soaking a trypsin or collagens (if
necessary) to dissociate the tissue matrix (BASHOR, 1979). Ideally the cells
can be cloned or diluted such that a collection of cells can be cloned or
diluted such that a collection of cells can be recovered that has grown from an
individual cell. Many normal cells do not grow well under limiting dilution
conditions; as a result this procedure can select a cell line with characteristics
that differ markedly from the average properties of cells in the starting tissue.
Most normal animal cells can only reproduce for a limited
number of generations and do not survive in suspension culture; these
features limit their usefulness as producing cell lines. Those cells that do
become established in long-term culture (immortal cell lines) typically exhibit
chromosomal abnormalities and sometimes fail to respond to hormonal
signals and growth factors.
C.
HANDLING OF MAMMALIAN CELLS LINES
A well managed and thoroughly documented system for

cataloguing and handling cell lines is critical for meeting regulatory
requirements and establishing a reliable process. A sterile sample of the
producing cell lines should be preserved in vials or ampoules in liquid nitrogen
to form a master seed lot. One of the containers from the master seed lot
should then be expended and used of form a working seed lab. The frozen
seed lots and copies of the documentation should be maintained in at least
two separate locations. Both the master and working seeds lots should be
tested as necessary to ensure the integrity of the manufacturing process. Cell
cultures can contain aerobic or anaerobic microbial contaminants,
mycoplasma, fungal or viral contaminants. Cross contamination of cell lines is
also a major problem. The table below list procedures available for testing and
characterization of mammalian cells.
METHODS FOR ANALYSIS OF CELL LINES
Contamination Test Method
Result
Positive
Bacterial, fungal
Microscopy,
culture
Mycoplasma
Nutrient
immunofluorescence
Nutrient
broth Presence
of
cells
colonies turbidity in
broth
broth,
Mycoplasma
157
morphology,
immune
reaction hybridization to
mycoplasma DNA
Antibody
production
test
Detection of antibodies
coculture
with
sensitive
Cell
Viral: MAP testing
pathologic
effect
enzyme assay
Cytopathicity
Detecon o9f reverse
Retrovirus
transcriptase.
CELL LINE IDENTIFICATION TESTS

Karyatyping
Isozyme analysis
Characterization of products (esp. antibodies)
Nucleic acid hybrization
Immunofluorescent detection of cell surface antigens.
D.
SMALL SCALE CULTURE OF ANIMAL CELLS
Once a cell line of interest has been isolated and a seed lot
established, various techniques are available for culturing the cells at
laboratory scale. Cell cultures researchers recognized at an early stage that
tumor cell lines could be cultured simply by injecting the cultured cells into a
susceptible host animal. This technique is still important for the production of
antibodies form murine hybridomas.
In vivo antibody production begins with the injection of about 10 6
hybridoma cells into the peritoneal cavity of mouse. This procedure works
best if the mouse has been primed a week of so previously by the injection of
a mineral oil such as pristanc (HOOGANRAAD and WRAIGHT, 1986). After a
few weeks 10 ml aliquots of ascites fluid can be with drawn at regular
intervals, containing rather high concentrations of immunoglobulin. This in
vivo method suffers form poor reproducibility and contamination by murine
proteins and there is still proential for economy of scale when larger amount
of products are needed.
SUSPENSION CULTURE
In vitro cell culture techniques depend on the type of the cell to
be grown. Anchorage dependent cells require different techniques than cells
that have been adapted to suspension culture. Suspension culture is
conducted in Semi- continuous mode (repeated batch culture with exchange
of the bulk of the medium on the regular schedule) under conditions that allow
continual cell growth. This repetitive sub culturing procedure is common at all
scales i. e. wheat her in flasks or to full size production reactors.
Cell cultures should be maintained when possible under
conditions that do not stress that cells. This can cause selection of a
subpopulation of cells that may have low productivity or other undesirable
158
features. Stress covers a widevariety of potential problems. The most typical

being oxygen or nutrient limitation, accumulation of toxic metabolites in the
culture medium, changes in pH or over dilution. Non agitated cultures in
closed flasks usually show a reduction in net growth rate and then a cessation
of growth (called stationary phase at populations approaching 1.5 x 10 6
cells/Ml. semi-continuous culture will proceed most smoothly, however if the
medium is replaced well before 80% of the maximum concentration is
reached.
At small scales (10 to 50 ml of culture fluid), adequate nutrient
and oxygen supplies are easily maintained. The method of choice at this scale
is generally the tissue culture flask or T- flask: this is a rectangular prism with
a neck raised or inclined at an angle to keep liquid out of the cap when
typical sodium
Bicarbonate buffer (system) is used; one can control the culture pH with in a
tolerable range using an atmosphere enriched to about 5% in carbon dioxide.
The valium of the vessel occupied by the liquid is generally kept below 20% in
order to allow sufficient gas exchange through the gas liquid interface.
At volume large than about 30 ml, cell concentrations and productivity
can be improved through agitation of the culture medium. Spinner flasks are
routinely used; these are cylindrical glass vessels featuring a suspended
magnetic sire bar on a survival and side arms for addition or removal of
inoculums or medium components. Up to 50% of the flask volume can be
filled with the culture medium. Spinner flasks can be used for routine work in
volumes up to about 10 liters, about aeration the liquid surface become
limiting.
F.
ANCHORANGE- DEPENDENT CELLS
Anchorage dependent cell lines are also maintained in T-flasks

at small scale; a wider size range of T-flasks is used routinely because
spinner flasks do not provide the necessary surface area for cell attachment.
The maintenance of anchorage dependent cells requires new techniques and
more careful attention to the condition of the cells.
Cells provided with a compatible surface will attach, physically
spread into a flattened conformation and then begin to reproduce and spread
across the available surface. The attachment process depends on numerous
biological and physical factors such as the cell line (some cells produces their
own attachment factors), cell density and culture history, presence of
attachment factors in the medium, mixing conditions and especially the
characteristics of the surface (BARNGROVER 1986).Glass, polycarbonate,
and treated polystyrene are the materials in widest use as surfaces for the
culture of anchorage dependent cells. As the cells approach conflnce
(complete physical occupation of the provide surface) growth rates slow and
sometimes stop entirely. Many cell types, however continue to reproduce after
confluence is reached, forming multiple lays or shedding cells (which in some
cases remain viable) in to the medium.
Sub culturing of anchorage dependent cells requires removal
from the surface and reattachment after dilution into fresh medium. A solution
159
containing trypsin and EDTA is generally used to detach cells from surfaces.
Trypsin can have damaging effects on the cells. So this exposure should be
kept to the minimum, necessary for the cell detachment.
Anchorage dependent cell can be expanded to larger culture
volumes through the use of roller bottles. Roller bottles are typically plastic,
filled to about 20% of the total volume with culture fluid. The bottle is
inoculated and placed on a apparatus that slowly rolls the bottle, alternately
submerging the cells and exposing them to CO 2 up to 500 ml with other small
scale technologies the manufacture of biological products with roller bottles
becomes limited as material requirements increase.
The culture of anchorage-dependent cells can be expanded to
still larger volume through the use of micro carriers.
PRODUCTS OF MAMMALIAN CELL TECHNOLOGY
PRODUCT TYPE
USE OF PRODUCT
VACCINES:
Rabies
Rubella
Mumps
infection
Pregention
of
viral
Polio
MONOCLONAL ANTIODIES
Diagnostic kits
Detection of disease
In vitro therapy
body
targeting
of
certain
Tissues
In vitro diagnosis
Diagnosis of disease,
cancer
Immunity purification
biomolecules.
Purification
of
INTERFERONS
treatment
Prevention
and
of infection.
INTERLEUKINS
IL-1, IL-2
Anticancer, immune
potentiators.
GROWTH FACTORS
G-M-colony stimulating factor
Immune potentiator
160
Earythroprotein
Promotes blood cell

Formation
Fibroblast growth factor
Prometes tissue growth
Epidermal growth factor

tissue
Promotes
skin
and
Growth
BLOOD REGULATION PROTEINS
Tissue plasminogen activator
Dissolves blood clots
Prourokinase
Dissolves blood clots.
Auriculin
Blood pressure control.
Microcarriers can be added to spinner flasks, roller bottles, or other small

scale vessels to increase the available surface area.
CELL CULTURE
ORGANISMS AND PRODUCTS
This section will describe some of the products of commercial
importance and the cell lines normally used for production. The production
technology of choice can be influenced considerably by the special
requirements of the desired products.
A.
PRIMARY CELL CULTURE
The manufacture of cell culture products from the primary cells

is still of considerable commercial importance. Much of the cell culture
technology in current use was developed for the large scale manufacture of
final vaccines from animal cells; the use of primary cells is still being extended
to the manufacture of various biological products excreted from normal cr
transformed primary cells (HOPPS 1985).
Vaccines for prevention of human and animal viral infection are
generally produced by cultivating a large volume of susceptible cells, infecting
the cells with the pathogenic virus of interest, and collecting the newly
produced virus. If the virus is pathogenic, it is then inactivated in some
manner that retaina the immunogenic character of the viral proteins. Human
vaccines include poliomyelitis, influenza, measles. And mumps: veterinary
vaccines such as foot and mouth disease (FMD). Vaccines are also produced
in cell culture.
Interferon, actually a class of infection fighting molecules, is one
of the first cell culture products manufactured in commercial quantities. Alphainterferon is produced in namalwa cells (Human-B-lymphoblast) in suspension
culture at scales up to 8000 liters the largest mammalian cell culture vessels.
-
Plasminogen activators have thrombolytic capability and are

useful in allowing reperfusion of blood vessels after a heart attack.
161
Tissue plasminogen activator (t-PA) has been produced using

human normal and melanoma cells, and recombinant CHO cells.
Other molecules with thromblytic activity such as prourokinase can

also be produced by tumor cell lines.
Numerous other substances which are produced by

mammalian cell lines are being investigated as possible commercial
products. A partial list includes interleukins, stimulating factors and growth
factors, hormones such as erythropoietin and enzymes.
GENETIC ENGINEERING
Genetic engineering techniques can be used to produce molecules of
interest in mammalian cells, though genetic engineering of mammalian cells
presents several difficulties comparable to prokaryotic bacteria.
One can use genetic engineering techniques to over produce a
molecule already manufactured by the cell. A more common situation is the
use of viral vectors with a well characterized cell line for example. Chines
hamster- ovary cells (CHO) to produce the material of interest. The benefits of
working with a cell line with a well-known gnome and physiological properties
are substantial. There are no guarantees that CHO cells will perform the same
glycosylation and post-translational modifications as the original primary cells.
A recent development is the potential for the use of cell culture
to make novel molecules. This is designed through the use of protein
engineering techniques such as replacement, of a portion of the coding
DNA, site directed mutagenesis, or design of proteins that include active sites
combined from two or more other proteins. Such techniques have not yet
provided only visible examples of improved products; however the technology
is advancing rapidly and is a major focus of academic and commercial efforts.
C. MONOCLONAL ANTIBODIES
Antibodies produced by murine hybridoma have been reached considerable
Commercial importance despite the fact that the hybridoma technology was
developed as recently as 1975 (KOHLER and MILSTEIN, 1975,1976).
Hybridomas are the product of a fusion between a B-cell that provides the
ability to grow in suspension and reproduce indefinitely. Techniques for
generation and selection of hybidomas of various species have been
described by OSTERHAUS and UYTDEHAAG, 1985. typical antibody titers
range from 20 g/ml up to 200 g/ml.
At present, the most important application of monoclonal
antibodies is the manufacture of in vivo diagnostic kits : monoclonal
antibodies provide rapid response and specificity compared to most traditional
analytical techniques. Antibodies can also be used for in vivo diagnostic or
therapeutic use. These applications usually involve coupling of the antibody to
an imaging agent (e. g. indium-11-for radiological imaging) or a cytotoxic
agent.
162
It is possible that many of the problems with in vivo administration of

murine antibodies can be over come through the use of human human or
human- mouse hybridomas. at line present, human hybridoma technology is
poorly developed. Cell lines producing certain antibodies are difficult to be
produced (developed) and manipulate and the antibody yields have been low
relative to murine hybridomas.
The ability of monoclonal antibodies to find specific molecules
has also generated considerable interest in immunoaffinity purification
techniques. The widespread application of this technique will depend on
improvements in durability and the ability to revse the fixed antibody.
IV CELL CULTURE MEDIA
A.
INTRODUCTION
The application of cell culture to the large-scale production of biological

products has been limited, partly by slow development of suitable reactors but
also by problems encountered in developing low cost defined media able to
support cell growth. Bacterial media utilize fairly basic compounds to support
cell growth and product formation: nutritional needs are very flexible apart
from requiring balanced amounts of the required ions and elements. The
needs of animal cells are far more complex and variable.
The ideal cell culture medium would be a define formulation that
provides all the nutrients and factors in serum relevant to cell growth and
product formation. Unfortunately, some of the biochemical factors affecting
cell metabolism are unknown, unavailable or extremely expensive, even if the
ideal defined medium could be developed it would cost more than medium
containing
serum.
Depending on the product of interest and the cell line used for its
manufacture, it is smometimes possible to adapt the cells to simple serum-fee
or even protein free media without compromising product titres or quality.
Researchers involved in such media development efforts must consider the
fact that the cell line in use is probably undergoing both selection and
adaptive change to adjust to the altered media, for example cells with high
growth rates and low productivity can replace higher producing cells. The
product resulting from adaptation to a given medium should be carefully
compared to that produced by cells from the seeds bank using the original
medium, to ensure that glycosylation patterns and functional activity have
been altered during the adaptation process.
COMPONETS OF CELL CULTURE MEDIA
WATER
The first and important ingredient in a cell culture medium is
good quality water the importance of a highly purified and carefully monitored
water supply can hardly be over emphasized. Mammaliancell cells are
sensitive ti common imuritlies in water such as metal ions, bacterial
endotoxins, and organic molecules. Water used for cell culture should be
distilled at lleast once andpassed through successive ion exctiang and
163
activated cabon filtered to remove ions and activated cabon filtered to remove
ions and organic compounds. Once purified., the collected water should
ideally be maintained at high temperature and circulated to prevent growth of
microorganisms. Materials used for storage or circulation should not leach
metal ions or plasticizers into the water. The filters should have a schedule for
regular maintenance and replacement, and the water should be tested
regularly for conductivity, presence of organics and presence of pyrogens
from contaminating organisms.
(2)
BASAL MEDIA
Most cell culture media contain a mixture of salts, vitamins,

amino acids, coenzymes, and minerals generally celled basal media. The
most common formulas have an osmolality near 300 MOSM and are based
on animal cell nutritional studies. The common mediums are 199, DMEM,
F12, and RPMI 1640, used for large scale cell culture. Variations of these
formulas are numerous and can be expected to proliferate as media are
adapted to particular cell lines are requirements.
Most cell culture media use sodium bicarbonate as the pH
buffering system. Animal cells have a metabolic requirement for a small
amount of bicarbonate, however, the primary reason for using bicarbonate is
buffering of the medium pH. In closed systems or large flasks without
adequate potential for air exchange, organic buffers such as HEPES can be
used to improve pH control at the expense of possible harmful effects on the
cells.
The energy and material requirements for cell growth and
product formation and generally met by adding glucose and glutamine to the
culture medium. The patterns to biochemical utilization vary among cell lines.
Some cell lines have been observed to generate half of the ATP necessary for
cellular metabolism through the conversion of glutamine even when glucose is
available. Both glucose and glutamine can generate metabolic byproducts
that inhibit further activity. Glucose and glutamine both undergo glycol tic
processing with lactate as a product and the utilization of glutamine causes
the evolution of ammonium ion. Cell and product yields can be enhanced by
avoiding high concentrations of lactate and ammonia, through the use of
nutrient control strategies, substitution of alternative carbon sources, or
continuous perfusion of medium through the reactor vessel.
The addition of antibiotics to cell culture media to prevent
microbial contamination is a common practice, but one that should be
discouraged in a manufacturing setting. If used, antibiotics must be
completely removed by down stream purification before in vivo use of any cell
culture product as many people are strongly allergic to antibiotics., antibiotics
can also have the effect of suppressing but not eliminating microbial
contamination: for this reason all seed stock should be preserved from
cultures that have been maintained in Antibiotic free medium for several
weeks.
164
C.
Animal sera as Medium supplements
Animal serum is widely used in a animal cell culture at

concentration up to 15 vol % the optimal concentration and type of serum are
cell line dependent.
For much cell work, fetal ovine serum (FbS) appears to provide
the best overall combination of growth. Promoting activities and lack of
contaminating proteins. Serum serves several purposes in cell culture media:
aside from the obvious nutritional effect serum provides.
1)
Hormones and growth factors
2)
Carrier proteins for transport of ions
3)
Fatty acid, lipids and like substances
4)
Attachment factors to promote cell- surface interactions.
Serum also has detoxifying effect in that metal ions and even
nutrients that could inhibits cellular functions at full strength are instead bound
to albumin and other serum proteins.
FBS prices and supplies vary by season and in response to
economic pressures on the beef and dairy industries: one carit expect serum
supplies to keep pace with the current expansion in cell culture activity. Close
atension to serum quality is also necessary: concentrations of some
hormones and factors can vary as much as 10 fold in individuals lots of
serum. Some of this variability is unavoidable and influenced by factors such
as the diet of the source animals. Serum can contain trace amounts of
pesticides, antibiotics asd toxins ingested form plants and biological
contamination of the culture. Although filter sterilization of bacterial
contaminants is fairly reliable, but mycoplasma and viruses such as bovine
diarrhea virus are sometime present in filtered serum.
165
SELECTED COMPONENTS OF FETAL CALF SERUM

S. No.
Component
1.
PROTEINS
2.
3.
D.
Quality
Albumin
2300 mg/L
Transferrin
1790 mg/L
Hemoglobin
81 mg/L
LIPIDS AND FATTY ACIDS

Triglycerides
210 mg/L
Phospholipids
630 mg/L
Cholesterol
320 mg/L
HORMONES AND STEROIDS

Insulin
8 mg/L
Growth hormone
42 Pg/1
Progesterone
121 ng/L
Testosterone
261 ng/L
Corticosterone
67 ng/L
Deoxycorticosterone
13 ng/L
Dehydroepiondrosterone
270 ng/L
Aldosterone
10 ng/L
Cortisol
6000 ng/L
Estrone
23 ng/L
Estriol
17 ng/L
17B-Estradiol
11 ng/L
Thyroid stimulating Hormone
1790 ng/L
Parathyroid hormone
1929 ng/L
Follidestimulating
199 ng/L
Heutinizing hormone
900 ng/L
Platelet-derived growth factor
770 mg/L
Defined Media
Much of the early work with defined media was motivated by

efforts to study the behaviors of cells in response to individual component
such as hormones. HAM and Mckeehan (1979) Published a generalized
166
protocol for development of an optimal defined medium to support the growth

of any cell line. Following all the recommended steps would require years of
patient work with each cell line. One can achieve satisfactory result much
more quickly by recognizing that important effects of serum can be divided in
to a few major categories. Most serum-free media developed by individual
researchers or marketed commercially provide some means of addressing the
functions of each group.
1.
Carrier Proteins
Fetal bovine serum contains high levels of bovine serum

albumin (BSA) and transferring, BSA functions primarily as a carrier of fatty
acids, lipids and other small molecules and peptides in serum, some of which
are insoluble in native form. Albumin also may have the ability to detoxify
some inhibitors of cell function such as hydrogen peroxide. BSA from
commercial soureces is often bound to trace amounts of tatty acids and lipids.
These impurities may actually provide better result than use of purified BSA.
Transferring binds iron in solution and delivers it in to
transportable form to the cell membrane, serum- free medium typically contain
between 1 g/Ml and 50 g/Ml of added transferring. Transferring from
different source of serum can differ markedly in their ability to promote growth.
A recent study showed and ability of 9 given cells to bind the different form
transferring.
2.
Insulin
Most serum free media contain insulin at ferrins, insulin form

different source may differ in their ability to support growth. Insulin influences
many aspects of cell metabolism: some cell lines can be adapted to insulinfree media but lower yields and poor long term stability are usually observed.
3.
Trace elements
A wide variety of trace elements are known important to cell

metabolism, yet the provision of these components in basal media is
inconsistent. Water and basal media components often contribute sufficient
trace metals to support growth. One element that may be insufficient,
however, is selenium. Many investigators add amounts of the order of 10 -8 Mol
L-1 HAMILTON AND HAM F12 medium featuring the addition of a trace metal
mixture to avoid any potential for deficiencies.
Growth and attachment factors
Numerous hormones and growth factors have been used for
culturing cell lines in serum free media. A partial list includes epidermal
growth factor, growth factor, fibroblast growth factor, progesterone,
testosterone, and hydrocortisone the requirement for these factors in a serum
free medium is very much cell-line dependent. Highly differentiated cell lines
such as cell form endocrine organ typically require the provision of several
hormones or releasing factors to retain their natural functions, while highly
transformed line such as hybridomas require minimal supplementation. Most
of the published research in this field has described lab-scale investigations of
cell biology and media requirements: cell culture for commercial purposes
167
often occurs at sufficient scale that medium supplementation with purified

hormones is inconvenient or completely impractical.
5.
Miscellaneous
Numerous other medium ingredients have been tated and may

have value for a particular process. Some (such as lipids, fatty acids, amino
acids and Nucleic acids derivatives) are standard ingredients in available
basal media. Extra amount of these components may be added to meet
special nutritional needs or compensate for degradation. B-mercaptoethanol
is reported to enhance the stability of medium ingredients by preventing
oxidation and ethanolamine has been described as an essential ingredient in
serum free medium for hybridoma growth. Other ingredients such as methyl
cellulose and plutonic F68 have added to media to alleviate the effects of
physical shear, and phenol red is sometimes used to provide visual indication
of pH.
E.
Preparation and Handling of Media
The proper handling of cell culture media is largely a matter of

common sense and attention to details, with reorganization of the fact the
mammalian cells are relatively sensitive to impurities and deteriorated
components. Care should be taken to freeze or refrigerate heat labile
components, and to avoid unnecessary exposure to light to open air.
Sterilization of medium component by heat has long been
traditional in industrial fermentation processes, but the labile nature of many
ingredients in cell culture media has limited the use of this technique to salt
and stock solution used to make up complete media filtration has become the
method of choice for most large scale work. Filters with pore size sown
0.1m have been developed that are suitable for sterilization of any commonly
used media component filters use of sterilization of cell culture media should
be tested on a regular schedule, and should not leached any foreign materials
such as detergents and binders into the medium.
1.
Quality Control
The quality of any ingredients used in cell culture media should

be monitored closely, as trouble in this regard can cause costly delays and
wasted effort. Animal sera can introduce contaminants. Blood products such
as human transferring should be deactivated or tested for pathogenic human
viruses. In cases where cell line is particularly sensitive to the quality of serum
or other medium components, it may be necessary to pretest bulk lots with the
cells of interest before purchasing. Many medium components should be used
faitlly soon after thawing or formulation from powder: for example. Glutamine
in solution at room temperature will decay spontaneously to ammonia and
pyrodidoncarboxylic acid.
2.
Sterility
A low rate of contamination in an absolute requirement for a

successful cell culture operation. Both mechanical and operational problems
contributes to contamination. Mechanical problems can be reduced by the use
of double instead of single seals in reactor ressels, regular maintenance and
168
replacement schedules for critical parts such as sterilizing filter, and a solid
system design that minimizes dead legs and build up of steam condensate.
Operational problems can best development of clear and through
documentation of operating procedures. Automations of critical processes
such as sterilization can in some case help prevent operator errors and also
reduce variability in the process.
V.
PROCESS DESIGN AND OPERATION
A.
Introduction
Cell culture is unusual among biotechnology processes in that a

wide variety of reactor types have been used successfully for industrial
manufacture of cell culture product. The search for and optimum reactor
system must take into account the peculiarities of the cell line and product
being considered as well as factors not directly related to the cell culture
process it self, such a down stream purification requirements. The situation
can be stream purification requirements. The situation can be generalized by
stating tat the optimum reactor system is one that reliably produces
acceptable product in the amounts required. While minimizing cost from labor,
media, purification, and general operations. This is not necessarily the reactor
system that must closely imitate the cells natural environment. Traditional
fermentation processes have increased productivities enormously by creating
very unnatural environment for producing organisms.
In cell culture reaction future maximum cell densities varying
from spiner flask (2 x 10 6 per mL) to near densities observed in solid tissues
(greater then 1 x 108 per ml) achieved with several types of perfusion
reactors. Low density technology is simple and robust. High ensity reactor
offer much higher productivity for a given size of reactor but face a greater
nutrient and mainly taining adequate nutrient and oxygen supply throughout
the reactor volume. Process control requirements are also more stringent. A
high density reactor that loses its perfusion pump or pH control system will
generate 9 total environment very quickly compared to a suspension culture
reactor. Temperature control is rather easy to a achieve in either case due to
low metabolic rate of mammalian cells.
B.
Suspension Culture
Suspension culture of lymphocytes or hybridoma cells, for

example, is possible in small-scale fermeners of up to 10 mc 3 capacity, in
which cells are magnetically stirred gently. Alternatively, small scale air lift
fomenter may be employed. Very cell suspension culture has very limited
application, however, and has been surpassed in biotechnological application
by the development of micro carriers, which allows for large-scale suspension
culture of anchorage dependent cell lines is a common and practical
technology for manufacture of biological. Low density cell culture can be a
practical approach for large-scale manufacture of products from animals
cells.
Control system for suspension culture are rather simple.
Sterilizable pH and oxygen probes penetrating the reactor wall can provide
signals for automate feedback control of these variables. Maximum cell
169
densities achievable by un supplemented batch mode approach S x 10 6 cells

ML. Suspension culture can be conducted in either Semi = -continuous or
batch mode. Semi continuous mode has also been called semi batch, fedbatch and continuous culture.
Agitation can be provided either by traditional Rushton impellers,
marine impellers, or an air lifted configuration, stirred suspension culture of
mammalian cells has been described at 8000-liter scale and airlift suspension
culture at the 1000-liter scale.
Air lift cell culture used air bubbles both for agitation and
aeration for the culture. Stirred culture generally provide oxygen for the culture
through the use of sparged air: oxygen. Supplementation can be used if
necessary. Silicone tubing is also used inside the reactor for aeration.
One can improve call and product yields in suspension culture
through the use of nutrient feeds. A feed strategy can be developed by
analyzing medium samples from different stages of a production culture. The
continuous Cytostat operational mode may be helpful in maintaining the
cells in a particularly productive status.
C.
High density suspension culture
The productivity of suspension cultures in stirred reactors can be

improved by avoiding the wash out of cells two good methods are:
1)
Separation of the effluent, with recycle of a cell-enriched stream

back to the reactor
2)
The spin filters technique.
Cell recycle can be accomplished through the use of cross flow

membrane micro filtration. A membrane permits the flow of medium and
product but can retain cells without harming viability. Problems with
membrane recycle include clogging of pores with cell debris, and long term
sterility and performance. High speed centrifugation can also be used for
recovery of viable cells from spent medium.
Spin filter suspension reactor feature a rotation filter that will
passed medium but not cells, due tangential forces at solid liquid boundary
layer.
D.
Entrapped Cell Suspension Reactors
Researchers have attempted to improve the performance of

suspension culture through the use of immobilized or entrapped cells. The
most common technique involves production of small polymer spheres or
beads with mammalian cells either trapped inside during the polymerization
step, or seeded the beeds by mixing in the reactor.
Alginates, collagen and agarose have been used for entrapment
of mammalian cells. The polymer particles have an open, sponge-like
structure: ideally cells will populate the entire structure. Cell density up to 10 8
per mL beads have been reported. The beads can be suspended in reactors
at high densities, resulting in net cell concentration up to S- 107 per mL of
170
culture fluid. A fluidized bead reactor using weighted collagen beads has also
been usefull.
Tractors with entrapped cells have good potential for scal-up
because the beads can be placed in well mixed reactors. The technique is
also helpful in preventing shear damage when culturing sensitive cell types,
and in providing a micro environment with elevated concentrations of cellproduced growth factors.
The high density also introduces some problems. The cells are
no longer in a homogenous environment. As a result the nutrient and oxygen
conceration supplied to individual cells cannot be assessed by using probes in
the culture medium. Mass transfer limitations with in beads can creates a
central region without cell growth or necrosis of lightly packed cells once
beads is fully populated.
E.
Micro carrier culture
Micro carrier technology, in most common usage, involves

attachment of cell to non porous polymer beads with a diameter of 100 m.
Because cells reside only on the surface of micro carriers, the cellular
environment can be measured can controlled easily with pH and oxygen
probes in reactor vessel. New cell densities between 2 x 10 6 and 1 x107 can
be achieved, depending on the cell line used. The micro carrier should have a
density between 1.02 and 1.10 g/ml to allow easy suspension in stirred
reactor. Modified dextrins, polystyrene, s and other support materials have
given.
Large-scale micro carrier culture presents some unique
challenges. One complication is the large amount of inoculums required. Seed
culture must be prepared in roller bottles or removed from a smaller number
of confluent micro carriers. Bead-bead and bead-impeller collisions and
hydrodynamic shear forces, are reported to cause loss of viability, still, micro
carrier culture provide one of the few technique for anchorage- dependent cell
culture that dose not face obvious mass transfer limitations to scale up.
F.
High density perfusion
The cell culture reactors most similar to the native environment

are the high density perfusion ractors. These configurations have proven to be
very successful for small-scale research. Improvements is recent year have
brought some of these type of reactors up to productivities needed for largescale manufacture of cell culture products. Many types of reactors have been
used for maintaining a high cell density; most provide sufficient surface area
for culture of anchorage dependent cells. All these reactors culture cells at
densities high enough to inhibit or half growth. Ideally this will allow improved
product yields when medium is perfused past the cells.
Hollow fiber reactor has been used for high density cell culture. Most
reactors use medium flowing through fiber lumens to support cells in the
spaces between fibers. Typical designs utilize an altrafiltration membrane that
retains the product outside of the fibers. This product concentration effect can
171
be beneficial: hybridoma culture can typically produce several milligrams per

millimeter of the product. Higher viable cell densities and better over all
productivity can be achieved by using on open pore structure and collecting
toward density product from the lumen side of the fibers.
Hollow fiber reactor generates very high cell densities. This can crate
nutrient limitations and subsequent cell sheath. Some techniques have been
developed to correct this situation, e.g.; regularly reversing the flow direction,
imposing cyclic pressure variation to force medium flow across the fibers, or
providing for circulation of cells on the shell side of the fibers.
A simple and effective technique for culturing anchorage dependent
cells is the use of a ceramic support with interior channels. Cells attach to the
surfaces of each channel and are perfused with medium from an external
reservoir. This technique also faces engineering limitation to scale-up.
Columns packed with glass beads have also been used for culture of
anchorage dependent cells. This reactor configuration would appears to suffer
from scale up difficulties as well. However, the technology has reportedly
been used with success at scale up to 100 liters.
Other successful designs have incorporated large membrane filter for
perfusion of entrapped cells: A static maintenance reactor design used
hollow fibers to aerate the interior of a high density reactors and flat porous
membranes above and below the culture chamber that supply and removed
medium but retain cells with in the reactors.
VI.
Downstream processing
A.
Introduction
Most downstream operations for mammalian cell culture products are
familiar from manufacture of other biological products depends greatly upon
end use. For example, antibodies manufactured for in vitro diagnostic kits may
follow cell removal with one step precipitation process. Cell culture products
for human intravenous injection must undergo extensive purification and
testing.
B.
Cell Removal
Some type of cell culture reactors provide a cell free product perfusion
reactors entrap most cells but debris and whole cells are still present in the
effluents suspension culture broth contains between 10 6 and 107 cells per mL,
and some reactors required recovery of product from broth with even higher
cell densities.
Centrifugation is a common and reliable technique for removing cells
from the product containing medium. Two approaches are possible: low speed
centrifugation and high speed centrifugation that attempts to pellets these
suspended cell components. Cell removal can also be accomplished using
microporous membranes. Cross flow filtration and improved membrane
technology have established membrane filtration as a reliable large scale
operation.
C.
Concentration
Cell culture products can be concentrated by several techniques.
Ammonium sulfate precipitation is a popular small scale technique, however,
the process is difficult to control at large scale and the protein phase change
sometime causes loss of activity.
172
The most popular concentration technique is ultrafiltration. The cell free

product stream is passed across a membrane that passes water and salts but
retains large molecules. cross flow filtration units with molecular weight cut
offs between 10000 and 10000 daltons are suitable for concentration of most
product stream. One can prepared the product for uncoming chromatographic
purification at this point by diafiltration with the buffer to be used in the next
purification step.
Adsorption and affinity columns can also be used for concentrating cell
culture products.
D.
Purification
Well established protein purification techniques are suitable for
purification of cell culture products. Ion exchange chromatography is the
dominant technique for large scale protein purification. Affinity purification
HPLC and FPLC techniques are becoming increasingly important as new
methods are developed and support materials improved.
VII
A.
Regulatory aspects of products from animal cell culture

Biohazards associated with cell culture
The primary hazard to be considered in cell culture operations are the
potential for viral contamination of working cell line, and the possible presence
of harmful nucleic acids in the final product. One can reduced most of the
concern of microbial and viral contamination through testing of the master
seed stocks. The safety tissues and available procedures have been
described HOPP. Once a clean cell line has been established, viral
contamination is still there by several routes. Viruses can be introduced from
the physical environment from the cell culture media or through expression of
an endogenous viral sequence integrated in the host cell chromosome.
B.
Regulatory Environment
The regulation of biological product manufacture using animal cells has
for the most part been a function of regulation are based on earlier experiment
with viral vaccines. In recent years agencies have responded to developers in
biotechnology by issuing new regulation and guidelines.
VIII.
references
1.
Animal cell Biotechnology vol. 2
2.
Mommalian cell technology W.G. Thilly
3.
Bashor, Methods in Enzymology 58
4.
Large scale mammalia cell culture by J. Feder and W.R. Talbert.
173
COMPONENTS OF BASAL MEDIA

Medium
DMEM
199
RPMI`1940
AMINO ACIDS
Arginine
4.0 E-4
3.3 E-4
1.1 E-3
Cysteine
6.3 E-7
Halfcysteine
4.0 E-4
6.8 E-4
4.2 E-4
Glutamine
4.0 E-3
1.0 E-4
2.1 E-3
Histidine
2.0 E-4
1.0 E-4
9.7 E-5
Isoleucine
2.0 E-4
3.8 E-4
3.0 E-4 ()
Leucine
8.0 E-4
3.8 E-4
9.1 E-4 ()
Lysine
8.0 E-4
3.8 E-4
2.2 E-4
Methionine
2.0 E-4
1.0 E-4
2.0 E-4 ()
Phenylalanine
4.0 E-4
9.1 E-5
3.0 E-4 ()
Threonine
8.0 E-4
1.7 E-4
5.0 E-4 ()
Tryptophan
8.0 E-5
2.4 E-5
9.8 E-5 ()
Tyrosine
4.0 E-4
1.1 E-4
2.2 E-4 ()
Valine
8.0 E-4
1.7 E-4
4.3 E-4 ()
Alanine
5.6 E-4 ()
Asparagine
3.3 E-4
Aspartate
1.5 E-4
4.5 E-4 ()
Glutamate
1.4 E-4
9.1 E-4 ()
Glycinene
4.0 E-4
6.7 E-4
1.3 E-4
Proline
3.5 E-4
1.7 E-4
Serine
4.0 E-4
2.9 E-4
4.8 E-4 ()
SUGARS, NUCLEIC ACIDS AND INTERMEDIATES
Glucose
5.6 E-3
5.6 E-3
1.1 E-2
Pyruvate
1.0 E-3
Actate
6.1 E-4
Ribose
3.3 E-6
Deoxyribose
3.7 E-6
Adenine
4.9 E-5
AMP
5.8 E-7
ATP
1.8 E-6
Guanine
1.6 E-6
Hypoxanthine
2.2 E-6
Xanthine
2.0 E-6
Thymine
2.4 E-6
Thymidine
Uracil
2.7 E-6
LIPIDS AND DERIVATIVES
Cholesterol
5.2 E-7
Choline
2.9 E-5
3.6 E-6
2.1 E-5
Innosital
3.9 E-5
2.8 E-6
1.9 E-4
Linolic acid
3.0 E-7
VITAMINS AND CO-ENZYMES
Ascorbic acid
2.8 E-7
-
F12
1.0 E-3
2.0 E-4
1.0 E-3
1.0 E-4
3.0 E-5
1.0 E-4
2.0 E-4
3.0 E-5
3.0 E-5
1.0 E-4
1.0 E-5
3.0 E-5
1.0 E-4
1.0 E-4
1.0 E-4
1.0 E-4
1.0 E-4
1.0 E-4
3.0 E-4
1.0 E-4
1.0 E-2
1.0 E-3
3.0 E-5
3.0 E-6
1.0 E-4
1.0 E-4
-
174
Biotin
Folic acid
9.1 E-6
a
Amino Benzoic acid
a-lipoic acid
Nicotinic acid
Nicotinamide
3.3 E-5
Pentothenic
1.7 E-5
Acid
Pyridoxine
Pyridoxal
2.0 E-5
Riboflavin
1.1 E-6
Thymine
1.2 E-5
Vitamin B12
Vitamin D
Vitamin K
Vitamin E
Vitamin A
INORGANIC IONS (TOTAL)
Calcium
1.8 E-3
Magnessium
8.1 E-4
Potassium
5.4 E-3
Sodium
1.6 E-1
Chloride
1.2 E-1
Phosphate
9.1 E-4
Sulphate
8.1 E-4
Nitrate
7.5 E-7
TRACE ELEMENTS
Iron
2.5 E-7
Cobalt
Copper
Zinc
-
4.1 E-8
2.3 E-8
3.6 E-7
8.2 E-7
2.3 E-6
7.3 E-6
3.0 E-8
3.0 E-6
1.0 E-6
2.0 E-7
2.0 E-7
4.2 E-8
8.2 E-6
1.0 E-6
3.0 E-7
1.0 E-7
1.2 E-7
1.2 E-7
2.7 E-8
3.0 E-8
2.5 E-7
5.8 E-8
2.0 E-8
3.5 E-7
4.9 E-6
5.3 E-7
3.0 E-6
3.7 E-9
-
3.0 E-6
1.0 E-7
1.0 E-6
1.0 E-6
-
1.8 E-3
8.1 E-4
5.4 E-3
1.5 E-1
1.3 E-1
1.0 E-3
8.6 E-4
1.2 E-6
4.2 E-4
4.1 E-4
5.4 E-3
1.4 E-1
1.1 E-1
5.6 E-3
4.2 E-4
8.4 E-4
3.0 E-4
6.0 E-4
3.0 E-3
1.5 E-1
1.4 E-1
1.0 E-3
1.0 E-6
-
4.1 E-7
-
3.7 E-7
-
3.0 E-6
1.0 E-6
1.0 E-8
3.0 E-6
*e.g. 2.5 E-7 means (2.5 10-7 moles)
175
ANIMAL CELL AND TISSUE CULTURE

The foundation of animal cell and tissue culture was laid at the beginning of
the present century when cells could be shown to survive and divide in vitro.
Ross Harrison (1907) later Alexis Carrel (1912) used tissue and embryo
extract as culture media. They successfully demonstrated that animal cells
can be grown indefinitely in vitro. The following terms are used for animal
culture techniques.
*Cell culture
*Tissue culture
*Organ culture
ORGANOTYPIC CULTURE
If the explants maintain its structure and function in culture it is
described as organotypic culture.
HISTOTYPIC CULTURE
If cells is culture reassociate to create a 3-D structure, irrespective of
the tissue from which it was derived, it is described as a histotypic culture.
Different areas of research in biotechnology depend largely on tissue
culture. These areas are :
1. Production of antiviral vaccines
2. Cancer research
3. Cell fusion techniques
4. Genetic manipulation
5. Monoclonal antibodies
6. Production of pharmaceutical drugs
7. Chromosome analysis of cells derived from womb
8. Study of the effect of toxins and pollutants
9. Use of artificial skin
10. Study of the function of nerve cell.
ADVANTAGES AND DISADVANTAGES OF THE USE OF ANIMAL TISSUE
CULTURE
ADVANTAGES
1. Controlled physiochemical environment (pH, temp. osmotic pressure,
O2, CO2 etc.)
2. Controlled and defined physiological conditions (constitution of medium
etc.)]
3. Homogencity of cell types (achieved through serial passaging)
4. Economical, since smaller quantities of reagents are needed than in
vitro.
5. Legal, moral and ethical question of nimal expt. Ion are avoided.
DISADVANTAGES
1. Expertise is needed, so that behaviour of cell in culture can be
interepreted and regulated.
176
2. Ten times more expensive for same quantity of animal tissue therefore
reason for its use should be compelling.
3. Unstable aneuploid chromosome constitution.
TISSUE CULTURE FACILITIES
Minimum requirement (essential)
1. Sterile area
2. Preparation area
3. Storage area
a. Liquids ambient, 40-200C
b. Glass ware
c. Plastics
d. Small items
e. Specialized equipment
f. Chemcials
Desirable features (beneficial)
1. Filtered air (air conditioning)
2. Hot room with temperture recorder
3. Microscope room
4. Dark room
5. Serivice bench adjacent to culture area
6. Separate sterilizing room
7. Separate sterilizng room
8. Cylinder store
Useful additions
1. Piped CO2 and comapred air
2. Store room for bulk plastic
3. Containment room for biohazard work
4. Liquid N2 sotrage tank (-2001).
TISSUE CULTURE EQUIPMENTS
Minimum requirement (essential)
1. Incuabtor
2. Sterilizer (Autoclave pressure cooker)
3. Referigertor
4. Freezer (fr -200C)
5. Inverted microscope
6. Soaking bathc or sink
7. Pipette cylinder
8. Pipete washer
9. Water purifier
10. Bench centrifuge
11. Liquid N2 freezer (1,500-3000 ampules)
12. Liquid N2 sotrage flask
Desirable features (beneficial
1. Laminar flow hood
2. Cell counter
3. Vaccum pump
177
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
CO2 incubator
Coarse and fine balances
pH meter
Osmometer
Phase contrast and fluorescence microscope
Ortable temperature recorder
Roller racks for roller bottle culture
Magnetic stirrer
ipette dirve
Pipette plugger
Auto pipette.
Useful additions
1. -700C
2. Glasswater washing machine
3. Closed circuit TV for inverted microscope
4. Colony counter
5. High capacity centrifuge
6. Cell sizer
7. Time lapse cinemicrographic equipment
8. Interference contrast microscope
9. Polythene bag scaler (for packing of long term storage)
10. controllled rate cooler
11. Filling for freezer records and catalogues
12. Centrifugal elutriator centrifuge and rotor
13. Fluorescence activated cell sorter
14. Densitometer
15. Density meter
THE SUBCULTURE ON WHICH CELLS GROWN
Cells shown to require attachement for growth are said to be
anchorage dependent. However, cell which have under goen transformation
frequently become anchorage independent and can grow in suspension.
Substrate may be adhesive (glass, plastic palladium etc. or a metallic
surface) or nonadhesive (agar, agarose, methocel).
Class as a substrate
Glass is the most commonly used substrate in the form of slides,
converslips, flasks and test tubes.
Disposable plastic as asubstrate
Poly styvene is often used as the most common substrate. Others are
poly vinyl chloride (PVC), polycarbonate, polytetrafluorethylene or feflon
(PTFE), thermanox (TPX).
Palladium and metallic surface as substrate
For 90% of animal cell and tissue cultures, glass or plastic is employed
as a substrate. However, other substrate have been usedfor specialized
applications e.g. Palladium diposited on agarose (using electron microscopy
shadowing equipment) was used as a substrate for growth of fibroblast and
glia stainles steel discs and other metallic surfaces were also used.
178
CULTURE MEDIA FOR CELLS AND TISSUES

Culture medium is one single most important factor for culturing cells
and tissues. It provides.
(i)
The optimum conditions of factors like pH 0 p etc.
(ii)
The chemical constituents, which the cells or tissuesare
incapable of synthesizing.
Animal cell need either a completely natural medium or an artifical
medium supplemented with some natural products.
1.
NATURAL MEDIA
a) Plasma dots
b) Biological fluids e.g. scrun
c) Tissue extracts
2.
DEFINED MEDIA
Synthetic media have been designed for a variety of tissues and purposes
like
(i)
(ii)
(iii)
(iv)
Media for immediate survival

Media for prolonged survival
Media for definite growth
Media for specilized functions
1) RPMT-1640
(Moore et al., 1950)
2) DMEM
(Delbeccos)
3) MEM
4) F12
5) TCM
(Ham, 1965)
6) IMDM
7) CMRL 1066 (Parker et al., 1957)
These media are serum media
Serum free media
The use of serum in culture media has several disadvatnages, which
led to the developments of many serum free media. Some of the
disadvantage include the following:
(i)
Serum varies from batch to batch and determiorates within one year
(ii)
Changing serum batch requires fresh testing.
(iii)
If more then one cell types one used, each may require different
serum batch.
(iv)
When cell culture is used for down stream processing to recover
cell products, the presence of srum is an obstacle to purification.
(v)
Serum increases the cost of the medium, since its cost is 10 times
the cost of its constituents if used as a substitute.
(vi)
Serum may stimualte undesirable groth and may even inhibit
growth in some cases.
The major advantage of serum free media is the ability to make a medium
selective for a particular cell type.
179
Example of serum free media

1. MC DB 110
2. MC DB 202
3. MC DB 402
4. MC DB 153
5. Iscoves
6. LHC
Selection of a suitable medium
Cells or cell line
1.
Hemopoietic cells
2.
Human
diploid
fibroblast
3.
Human leukemia
4.
Human tumours
5.
Mouse leukemia
6.
Mouse
erythroleukemia
7.
Mouse myloma
8.
Hela cells
9.
Glial cells
10.
Continuous cell lines
Medium
RPMI, 1640
Eagles mem
RPMI 1640
RPMI 1640, DME
Fischers RPMI
SF12, RPMI 1640
RPMI, DME
Eagles meme
MEM
Eagles Meme
STERILIZATION OF EQUIPMENT AND APPARATUS

Item
Sterilization
Disposable tips for micropipettes
Autoclave
Filter Millipore
Autoclave
Glass ware
Dry heat
Magnetic sterrer bar
Autoclave
Pasteur pipette
Dry heat
Agar
Autoclave
Amino acid
Filter
Antibodies
Filter
DMSO
Self sterilizing
EDTA
Autoclave
Glucose-20%
Autoclave
HELPES
Autoclave
HCI in
Filter
Glucose 1 2%
Filter
NaHCO3
Filter
NaOH In
Filter
Phenol red
Autoclave
Serum
Filter
Trypsin
Filter
Vitamins
Filter
Water
Autoclave
180
Experiment No.
Objective
To isolate single cell suspension from spleen and liver rat to culture
than in DNEM
Requirement
Medium, NaHCO3, bacterial filter, automatic pipette, Laminar flow,
inverted microscope petridishes, Milk bottle, culture plate etc.
Principle
Single cell culture can be isolated by either mechanical dispersion or
by enzymatic method. In mechanical disruption recovery is low and possibility
to damage. Cells may also be damaged by enzymatic activity.
Procedure
(i) Media preparation
We prepare DMEM medium by bacterial filter. After filtration we add 1%
NaHCO3 and antibiotic. NAHCO3 is acts as a buffering agent.
ii) Kill the experimental animal (Mice) and dip it into 70% alcohol
iii) Dissect the animal from ventral side and remove the liver and
spleen
iv) Mechanical disaggregation: In this method tissue is carefully
sliced by operation blade and the cells which spell out are collected or
cells may be repeatedly pipetted.
v) Enzymatic disaggregation: Crude trypsin is the most common
enzyme used for disaggregation for the following reasons.
It is tolerated by a variety of cells
It is effective for various tissues
Expose the cells to the warm enzyme for a minimum period;
dissociated cells are collected every half an hour and the trypsin is
removed by centrifugation
vi) Incubate the culture in CO2 incubator
181
Experiment No.
Objective
Counting of total cell viability by Trypam Exclusion Test
Requirement
Floemoaytometer, cell culture, trypem blue Microscope (Polyvar),
Pasteur pipette.
Principle
Trypam blue is the stain which can stain only dead cells the viable cells
can not be stain by this due. So it is very useful stain to distinguish viable and
dead cells in a population. By using this stain we can determine the half life of
a cell line.
Procedure
i)
Take 2 ml of culture and add 1 ml of trypen blue in it
ii)
Keep the stain and culture mixture for 2 minute
iii)
Charge the sample to moemocytometer
iv)
Observe the cells under polyvar microscope
Observation
The viable cells looks colourless and dead cells appear in blue color
Discussion
Trypan blue a blue coloured stain capable of entering into dead cells
and dead cells appear blue in color. Not permit the entry of trypen blue. So
that they appear colourless under microscope.
Hepatocyte - Emzymatic means
Total no of cells
= 20 x 16 x 5 x 2 x 10
= 320 x 102 =32 x 104
No of dead cells
= 4 x 16 x 5 x 2 x 10
= 64 x 102 = 32 x 104
2.54 104
Viability
=
___________ x 100
3.2
104
Mechanical means = 80%
Total no of all = 30 x 16 x 5 x 2 x 10
= 4.8 x 104
0.8
4
No of dead cell
= 4 x 10 , Via =________ x 104 x 100
4.8
= 16.6%
182
Experiment No.
Objective
To culture MACROPHAGES in RPMT medium
Requirement
RPMI medium, NaHCO3 bacterial filter, automatic pipetter, laminar flow,
inveted microscope petridishes, Milk plate, culture plate etc.
Procedure
i)
Media preparation
We prepare RPMT medium and filter by bacterial filter. After filteration
we add 1% NaHCO3 and antibiotic. NaHCO3 is acts a buffering agent.
ii)
Macrophage separation
We inject 3 ml of sodium thioglycolate (1%) in the peritoneum cavity of
white rat. After the injection, macrophages of blood get excited and
accumulated in the peritoneal cavity. After 2 days we take out the
macrophages the cavity after disscting the mouse from ventral side wash the
cell with buffer.
iii)
Macrophage culturing
We transfer the macto phages in the RPMI medium and them into well
culture plate. Incubate it into CO2 incubator.
Observation
See under inverted microscope. macrophages are part of body defence
mechanims they act as phagocytes or antigen presenting cells. They usually
gets accumulated at the site of injection we can induce macrophages
production artificially by infecting any foreign particle. Intraperitonial injection
of sodium thioglycolate will induce the macrophages.
183
Experiment No.
Objective
To form animal cell lines of
a)
Cymphocyte cells from peripheral blood/ spleen
b)
Splenocyte from spleen
Experimental out line
Sacrifice mice
Remove spleen
Sieve spleen cells through wire mesh wash cells
Viable cell count with trypan blue
Cell count with hematocytometer
Materials
Balanced salt solution
Acetic acid counting solution
Trypan blue vital dye
70% ethanol
Squeeze bottle
Small/ Large surgical scissors
Curved forceps
Rubber policemen
Stainless steel wire mesh
Glass petridesh
Hoemocytometer
Pasteur pipettes
Pre lab preparation
1. Balance salt solution (BSS)
Stock # 1 10x
Dextore
- 10gm
K2HPO4
- 0.6 gm
0.5% phenol red
- 20ml
Dissolve in 100ml distilled water
Stock # 2
CaCl2 . H2O
1.86 gm
KCl
4.00gm
NaCl
80.00 gm
MgCl2
1.04 gm
MgSO4.7H2O
2.0gm
Dissolve in 1000 ml distilled water
Mix 10 ml of stock 1 and 10 ml of stock 2 bring upto 100ml with distilled
water. The pH would be 7.2 7.4
Method
1.
Sacrifice the mice
2.
Make a cut through the loose skin by pulling gently upwards and
suign the blunt scissors on the skin flap. This will expose the
peritoneal wall.
3.
Pull gently in opposite direction on the two sides of the skin incision
to expose a wider area of the peritoneal wall
4.
With the small surgical scissors make a cut over the spleen to
expose it through the puritoreen
184
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Group the spleen with the forcep and pull it gently

group a folded corner of the stainless steel wire mesh with the
hemostat. Insert the mesh into the petridish and add about 10 ml of
BSS
Put the removed spleen on the wire mesh with a rubber policemen.
Rub the spleen back and forth orier of mesh continue until all the
psleen dissociates
With a sterile pasture pipette bring the cell suspension up and down
several times to dissociate large clumps pipette the cell suspension
into the glass centrifuge tube.
Allow the cells to settle for 5 minutes
Centrifuge at 1000 rpm
resuspend the cell pellet
Add 0.1 ml of tryptan blue soln to 1.8 ml of acetic acid solution. Add
0.1 ml of the cell suspension and mix thoroughly
Fill hemo cytometer chamber with a drop of mixture
Dilution factor = 20
volume factor = 10,000
Record the grid count
185
Experiment No.
Objective
Stain the viable cells (lymphocyte) and observation requirement
Cell suspension stains, spirit lamp, micro scope etc.
Procedure
1. Wrights Stain
Smear
2 drop wrights stain (2.3 minute)
Add distilled water (8-10 minute)
Wash with running water
Air dry and observe
2. Giemsa stain
Smear
Fix in methyl alcohol (3-5 minute)
Dry in air
Dilute giemsa stain (9 dw:1 GS) 30 minutes
Wash with distilled water
Air dry and observe
Result Lymphocyte
Round/oval nuclei covering almost all
parts of the cell nucleus deep purple
blue with 1-2 nucleoli
Cytoplasm is very small and stain sky
blue color
Monocyte
Largest cell of blood Kidney/
shaped cytoplasm relatively
lympholyte
Plae cells with pale staining
nucleus
with
chromatin
reticulum
bean
than
round
being
186
Experiment No.
Objective
Formation of gradient by diffusion method
Requirement
Sucrose (12%, 25%, hypodurmic syringe etc.)
Procedure
This is the simplest method of forming gradients, layers of medium
solution at different densities are manually layered on top of each other and
either used in this form (discontinuous) or allowed to stand for a period of time
for diffusion to occur produce a continous gadient. Syringe fitted with a large
bere needle is usually used to introduce known volumes of solution of
medium. The least dense solution is added first followed by more dense
solution which when added, slowly displace the less dense solution. This is
repeated to produce a discontinuous gradient
187
Experiment No.
Objectives
Discontinuous isopycnic separation of blood cell.
Requirement
Blood, Isotonic pereal solution tube, centrifuge etc.
Principle and procedure
Granulocyte can be purified from human blood by a 2 step
discontinuous percoll gradient 4 ml of isotonic pereall with a density of 1.10
g.ml is overlayered with 4 ml of a 1-077 g/ml percoll solution creating a 2 step
gradient. 4 ml of calf blood anotr layeed on the gradient and centrifuged for 20
minutes at 2000-3000 rmp. Lymphocytes and monocytes bond at the interface
between the blood and the 1.077 g/ml percoll the granulocyte band between
the two percall solution and the red cells pellet to the bottom of the tube.
Experiment No. 18
Objectives
Immobilization of yeast cell by formation of poly acrylamide beads.
Requirement
Aerylamide, N.N.- medhylae bis acrylamide, M./M. tetra ethylene
methol diamine (TEMD) potassium persulfate, ammonium persulfate, amlyl
alcohol.
Procedure
10ml of washed yeast cell suspension added to 12.75 ml of eq solution
of acrylamide, N2 gas passed to derive out oxygen. Since O 2 is an inhibitor of
polymerization. Add a layer of amyl alcohol. Add catalyze system i.e. 2 ml of
2.5 potassium/ammonium per sulfate then add 2.25 ml of 5% TEMED with
constant stirring. Mixture is poured rapidly into a solid verticle glass chamber
0.2mm thickness keep the chamber for cooling at 10 oC water bath kep it for 1
hr. Remove the 2mm thick sheet and cut it into 4 x 5mm and finally washed
the blocks with phosphate buffer. Air dry it to room temperature.
188
Experiment No.
Objective
Immobilization of yeast cell by formation of agar beads.
Requirement
Agar (2.5%), growing yeast cell culture, phosphate buffer, toluene,
chloroform, hypodermic syringe.
Principle
Living cells use energy for its survival as well as multiplication. If cell
multiplication is stopped, all the available energy will be used for its survival
and the substrate will be converted to product. If the cell is incorporated on a
solid support it growth will stop and the substrate will be converted to product.
Procedure
Take 25ml of 2.5% agar solution, and autoclaved it. Take 10ml of the
yeast suspension and washout with phosphate buffer. After washing melt agar
at 45-50oC and the yeast cell to the agar under aseptical condition take
toluene and chloroform in a beaker in the ratio of 3:1 in cold, pour the agar
drop by drop with the help of syringe in the liquid. Small beads are formed.
Take out the beater and wash it with phosphate buffer and dry it keep at 37 oC
for 24 hrs.
Observation
Place the yeast is glucose solution. After some days, glucose is
converted into ethanol. Test the glucose by Benedicts reagent.
189
Experiment No.
Objective: Immobilization of yeast cell by formation of sodium alginate beeds.
Requirement
3% sodium alginate, phosphate buffer, 0.025M, anhyd CaCl 2.
hypodermic solution.
Procedure
The procedure of sodium alginate bead formation is exactly some as
agar bead. Sodium alginate is taken it 40 ml phosphate buffer and melted in
flask and then autoclaved. After autoclaving. Mix yeast cell suspension
(previous washed.) Mixed well. pour into 2.5% calcium chloride (saturated) by
using hypodermic sofringe drop by drop. Take out the beads from the solution
and washed with buffer solution air dry at 37 oC.
Observation
Place the beads in glucose solution after some days. Yeast converts
the glucose into alcohol.
190
INDEX
S.No.
Content
1.
Introduction
2.
Tissue culture: Application
3.
General methodology
Page
3.1.3 Preparation of stock solution

4.
Special techniques
4.1
Fusion of protoplasts
4.2
Products of plantlets from suspension culture
5.
Agrobacterium mediated transformation in plants
5.1
Agrobacterium
mediated
transformation
in
tobacco/pigenonpea
6.
Embryogenesis, organogenesis
6.4
Somatic embryogenesis
6.5
organogenesis
6.6
Plant regeneration directly from cultured explants
Reference
191
PLANT TISSUE CULTURE : AN OVERVIEW

I.
II.
Introduction
Agriculture, being the mainstay of our economy, received so much
impetus from the immemorial. By and large and domestication of wild
plants depends on the genuine effort of human being and we are now a
logway away from the traditional farming practices. Land being the limiting
factor in the cultivation, combined efforts of agromonists, plant breeders,
plant pathologists and entomologists etc. have helped a lot to increase the
grain yield. Of late, it was felt by the scientific community that, traditional
breeding practices can no larger increase the grain yield. So they began to
look upon the new found wisdom of biotechnology or for this stagnation in
grain yield. No plant biotechnology programme will be complete without
tissue culture as it is the only method for propagating these genetically
engineered propagules which might have failed otherwise. This aspect has
been reported by Levy (1981) who stated the micropropagation through in
vitro techniques the advantage of satisfactorily propagating genetically
superior phenotypes.
Tissuec culture: Applications
2.1
Production of true to type plantlets

Tissue culture may be best described as clonal propagation as the
plantlets produced are clones to each other. This aspect has been exploited in
seed anther or v\ovules and later subjecting them to chromosome doubling by
the application of colchicines for the production of either homozygous of
heterozygous diploid. This isolation and culturing of haploid lines is normally
possible only through tissue culture
2.4
Embryo rescue technique
The production of intergeneric or interspecific hydrids is practically
impossible due to the defective chromosome pairing which results in lose of
viability so in these cases, as soon as the fertilization is over, embryo is
separated and allowed to grow in artificial media which would have been
otherwise repelled by the motherplant.
2.5
Somaclonal variation
In single cell culture, when we are dealing with a very large population
sometimes it may happen that one plantlet out 10 4 or 105 may deviate from
parental lines and show novel phenotypes. If these novel phenotypes are
agronomically desirable traits then they may be propagated further or else
discarded. But sometimes, these variants may revert back to parental lines.
2.6
Mutation breeding
In single cell culture or tissues by the application of suitable mutagens,
evolved lines can be developed. This techniue, for plant breeders, is a very
handy tool as they can be used for the isolation of herbicide resistant and
other stress tolerant varieties.
2.7
Protoplast fusion
Instead of dealing genome, scientists now deal with protoplast which is
a cell without cell wall. By suitable means cell wall as dissolved and
protoplasts of two different in suitable media and allowed regenerate into
192
planlets. Even though the method sounds simple it is very tedious. As it was
often found, it final product resulted is of an undesirable variety (eg. pomato,
obtained by the fusion of potato and tomato).
2.8
Propagation of transgenics
Transgenic crops which are the fruits of biotechnology are propagated
only by tissue culture because most of them had lost their viability.
III General Methodology
3.1
Media
The success of any tissue culture practise depends on the careful
selection of media. By the scrupulous work of available. This include MS
(Muradhige and Skoog, 1962), Woody Plant Medicines, WPM (Loyed and
Mcoum, 1980) ambroges these media and manipulate them by varying the
salt concentration and growth regulation.
3.1.1 Chemical composition of media
Compound
Amount Mg e-1 MS
NH4NO3
1650.00
H2NO3
6.20
CaCl2.2H2O
440.00
CaCl2.6H2O
0.025
CaNO3
0.00
FeSO4.7H2O
27.80
MnSO4.7H2O
22.30
MgSO4.7H2O
370.00
Na2EDTA
37.30
KCl
1990.00
KNO3
0.00
KH2PO4
170.00
NaMoO4.7H2O
0.25
ZnSO4.7H2O
10.60
Vitamins
Glycine
2.00
Nicotinic acid
0.50
Pyridoxine HCl
0.50
Thiamine HCl
0.10
Other
Inositol
100.00
Sucrose (in % w/v)
3.00
Agar ()
0.80
WPM
400.00
6.20
96.00
0.00
0.00
27.80
22.30
370.00
37.30
0.00
990.00
170.00
0.25
8.60
2.00
0.50
0.50
0.10
100.00
2.00
0.80
3.1.2 Composition of stock solution for ms medium

Stock No. Chemical
I
NH4NO3
KNO3
KH2PO4
MgSO4.7H2O
II
CaCl2.2H2O
Stock Conc.
50x
50x
Quantity
82.5 (ge-1)
95.0
8.5
18.5
22.0
193
III
IV
V`
Na2EDTA
FeSO4.7H2O
MnSO4.7H2O
ZnSO4.7H2O
H3BO3
KI
Na2MoO=
COCl2.6H2O
CuSO4
Vitamins
Glycine
Nicotinic acid
Pycidoxine-HCl
Thiamine HCl
100x
100x
3.7
2.8
22.3
1.06
0.62
0.83
0.83
0.025
0.025
200mge-1
50.00
50.00
50.00
10.00
3.1.3 Preparation of stock solution

Each stock should be prepared separately. For this each of the
chemicals mentioned in the above description to be weighed accurately and
dissolve in 200ml of distilled water taken in a beaker. The contents were then
made upto one litre by adding distilled water. The stock solutions should be
properly labeled indicating the stock number found date of preparation and
stored in chamber coloured bottles under refrigerated conditions.
Stock solution of the salts have to be prepared once in three months
and that of vitamins once every month. The stocks should be abandomed
whenever contamination is noticed.
The iron stock (stock number III) precipitases readily. To avoid this Na2
EDTA and FeSO4. 7H2O were dissolved in separate beakers with
approximately 200ml distilled water. Both beakers were placed on hot plates
and bring them to the point of boiling. Then FeSO4.7H2O solution should be
added slowly to Na2EDTA (never vice versa) over a 15 minute period with
constant stirring. The mixture should be allowed to cool to room temperature
in dark and then to be refrigerated. If the stock is cloudy, it is to be discarded.
3.2
Preparation of media
Quantity of stock solution required for preparation of media 1 liter MS
Stock No
Quantity (in ml)
I
20
II
20
III
10
IV
10
V
10
The specified quantities of stock solution should be pipetted into a
1000ml beaker in which about 1/3 rd distilled water is taken. The required
quantity of sucrose and myoinositol is to be added and dissolved. Make up
the volume to 1 liter and adjust the pH to 5.7 5.8 weighed quantity of agar
should be dissolved by boiling. About 20ml of the culture media should be
taken in precleaned culture vials and plug tightly with non-absorbent cotton.
The media is to be sterilized by autoclaving at 15 psi pressure for 15-20
minutes
194
3.3
Collection and preparation of explants

Explants should be collected with care since they will affect the quality
of plantlets regenerated. The motherplants should be so taken that, middle
aged plants that too without defects, free of diseases and pests should be
selected for explants. Explants collected were surface sterilized using
mercuric chloride, chlorine water, calcium hypochlorite etc. at varying
durations depending on the tissue selected. They were later washed with
sterilized distilled water, dried and cultured under aseptic conditions in a
klenzoids laminar air flow cabinet and put for incubation at 252 oC and light
intensity varying with species and tissue used.
Special techniques
Isolation and culture of protoplasts brassica /nicotiana
Introduction
Plant protoplasts provide the starting point for many techniques aimed
at the genetic modification of plant cells and whole plants. The isolated
protoplasts, a wall less cell can be induced to fuse with protoplasts of different
species thus providing the opportunity for the creation of noval plant hybrids
(somatic hybridization)
1)
The protoplast is also an ideal recipient of foreign DNA, often in the
form of plasmids and providing incorporation and expression ensure, such
transformation can lead to the production of genitically modified plants.
2)
Protoplasts can also incorporate larger partials (organelles) thus
facilitating the exchange of extra techniques can not by definition, be
described in isolation and are deliberately linked therefore, to subsequently
recover the whole plants
Back ground
One of the most significant developments in the field of plant tissue
culture during recent years has been the isolation culture and fusion of
protoplasts the techniques are especially important because of their teaching
implications in studies of plant improvement by cell modification and somatic
hybridization. Isolated protoplasts also offer the means of tackling various
fund omental research problems in experimental plant biology. This is realized
because of the totipotent nature of plant cells. The literature accumulated so
far has revealed that protoplasts is culture can be induced to undergo intra
and inter specific fusion to form a somatic hybrid and also to take-up foreign
organelles and genetic materials. Protoplasts have also been used to
investigate various problems in plant physiology. This fascinating field of
research, though still informative stages as for as the technology is converted
has awakened the interests role in opening up new vistas and has awakened
the interests of plant physiologists, pathologists, molecular biologists and
cytogeneticists. It is envisaged that in future years protoplasts will be one of
the most frequently used research tools for tissue culture studies.
Plant and cell requirements for protoplast isolation
1.
Growth of plants
The genetic and physiological status of the green house grown plant
can influence the ultimate yield and viability of protoplasts. An example of
green house conditions suitable for petunia is given below:
195
a.
Germinate seeds of petunia in the light on soilless compost a t

20-30 C
b.
Grow the seedling in the green house at 20-25 oC wit an
illumination of 10,000 lux and 0-16 hours day length.
c.
Transfer the seedlings to seed trays and grow as futher for 5-6
weeks under the same conditions plants 8-10 cm high, with 7-10 fully
expanded leaves are used as a source of protoplasts. N.B. Plants in
bud are never used.
Pest control
Two of the main causal agent of bacterial contamination through leaf
damage once white fly and red spoiler.These can be controlled by a
sequential treatment withvarious fumigants including DdT/ lindane.
o
Growth of cell suspensions

Cellsuspensions to be used as a source to protoplasts should be
their exponential phase of growth.At thin and cellulosic in nature thus
requiring minmum incubation with enzyme mixtures.
Growth of Seeding
(1)
Surface sterilize seeding in 0.1% mercuric chloride for 10 minutes.
(2)
Wash thououghly in sterile tap water
(3)
Transfer the seeds aseptically to petri dishes cohtaining MS mediu
without phytohormones but including 0.6%agar and 0.2 % sucrose.
(4)
Germinate the seeds in the dark or light (2000 lux) at 25 0C.
Isolation and cultur3e of mesophyll protoplasts from tobacco shoot
culture
Plant species
Nicotiano glauca x N. congsdoffi
Origin of protoplasts Mesophyll cells
Enzyme mixture
Macerozyme (0.4% + cellulase onazyka (4%) in
0.6 M
sucrose.
Culture medium
NAGATA and TAKEBE + NAA (3 mg/l)+6-BAP (1
mg/l)
Growth response
Somatic hybrids
Steps
1. Take two to four fully expanded leaves of a 6 week old shoot culture
under sterile conditions and place them in a 9 cm plastic culture dish
wet the leaves thoroughly with enzyme solution and remove the mid
ribs.
2. Cut the leaves into pieces and transfer leaf pieces bottom side down in
to a 9 cm dish containing 12 ml enzyme solution. Seal the dish with
peafilm and incubate over night at 25oC in the dark wihtout shaking.
3. entlyagitate the digest release of protoplasts from leaf debris inaided by
smoothly pumping once the suspension up and down a 10 ml pipette
with a wide opening takeup through a 100 m stainless steel mess
seave on a 100ml glass beaker. Add 6-8 ml of Ku medium to thedish
and disrupt remaining tissue by carfully pumping it up and down the
196
pipette. Sieve this suspension to collect both fractions in the glas

beaker.
4. Mix the protoplast suspension gently an distribute in to two 14 ml sterile
plastic centrifulge tubes. Carefully overlay thesuspension with 1 ml w5
solution. Spin for 7-10 protoplasts at the interphase w3iht a 2 ml
pipette, taking as little as possible of the loer phase transfer the
protoplasts to one new 14 ml centrifuge tube combining theprotoplasts
from twointerphases.
5. Remove the protoplasts at the interphase with a 2 ml pipette,taking as
little as possible of the lower phase transfer th protoplasts to one new
14 ml centrifuge tube combining theprotoplastw from two interphases.
6. Gradually add 8-10 ml W5 solution, close the tube and resuspend th4
protoplats by gently titting thecapped tube. Pellet the protoplasts.
Remove the supernatant repeat this step.
7. Resuspend protoplast pellet in 5-10 ml. w 5 solution. To determine
protoplastyield with a 1 ml pipette take up 100l aliquot of the
protoplasts suspension, add 900 w5 solutionto it and count the
protoplasts in a hemocytometer.
8. Pellet the protoplats. Remove the supernatant and resuspend
protoplast pellet inmedium K3 to yield a suspension with a cell density
of 1x 106 protoplast/ml .
9. Place 0.5 ml of the protoplat suspension in a 6 cm falcon petridish. Add
4.5 ml prewarmed 1: 1 mixture of media K3 and H containing 0.6% sea
plaque agarose. Mix gently and allow to set.
10. when midium is solidifide seal thedishes with parafilm and culture the
protoplasts fro 24 hr in the dark at 24 oC, followed by 6 days
incontinuous dim light where first and mutliple cell divisionoccur.
11. cut theagarose containing the dividing Protoplasts quadrants and place
these in 50 l of medium A, ina 9 cm plastic culture vessel. Incubate at
24oC in continuous dim light on a rotary shaker at 80 rpm.
12. After 102 weeks a loan of protoplast- derived colonies is formed within
th agarose sectors floating in liquid medium A in bead type culture
under continuous shaking and the same culture conditions.
Solutions
1. Enqyme solutin: Dissolve 1.2% (w/v) cellulase onoquka R 10 and
0.6%(w/v) macerozyme R10 in K4 medium ( K3 with 0.4 instead of 0.3
m sucrose).
Spin down to pellet starchcontaminating the enzyme preparations.
Adjust to pH 5.6 withkOH and filters- sterilize store at 4 oC for no longer
than 3-4 weeks.
2. Washing Solution: W5 : Dissove 15.4M NaCL 125 mm CaCl2, 5 mM
KCL and 5 mM glucose in distilled water, adjust to pH 5.8 with KOH
and autoclave store at room temperature.
3. Media: The compotion of the media used at final concentrations of
their individual ingradients is medium A, Medium H, MediumK 3
Medium Ms morpho.
4. Stock Solutions for preparation of media: for all couture media wsed,
prepare macro elements as 10 fold concentrated stocks. Micro
elements, vitamins, and organic acid stock solutions are 100 fold
197
concentated in all cases.For preparation of 1000 ml microelement

stock, distilled water each. Mix dissolved solutions and heat at 60 o C.
After cooling down, add remaining stock ingradients dissoved in
distilled water and complete to stock solution of organic acids for
medium H only stock solutions of carbohydrates are 10 fold
concentrated and wihtout glucose for medium H. 1000 fold
concentrated but fold concentrated but without sucrose for medium A.
5. Medium Preparation: Prepare the couture media using stock solutions
and dilute them glass distilled autocluave. Keep sterile liquid media A,
N, and K3 in autoclaed glass bottles at 4oC . medium K4 is prepared as
medium K3
but contains 0.4 M sucrose final concentration.
Prepare 1:1 mixture of media K 3 and H solidifide with 0.6% (w/v) low
getting tempeerature sea plaque agarose. Autoclave dry agarose first, add
sterile K3 medium, melt in a microwave oven, and after cooling to 45 oC, add
filter steilized midium.
Adjust to pH 5.8 with KOH for medium MS add 0.8% (w/v) agar and
autoclave.Pour 100 ml fractions of melted meium in autoclaved couture glass
jars and store at room temperature.
To prepare medium MS morpho solidified with 0.6 (w/v) agarose, first
autoclave the agarose in three-fourths of required volume of distilled water
separately diluted corresponding stock solutions and other medium in
gradients to complete remaining fourth of final volume with distilled water and
after filter-sterilization, and it to the autoclaved, melted agarose to yield and
final medium pour 80 to 100 ml fractions of melted agarose to yield the final
medium in 9 cm sterile plastic culture vessels and is autoclaved culture glass
jars and store at room temperature.
Compositions of the Media used
Media A
Macro elements (mg/litre final concentration)
KNO3
1010
NH4NO3
800
CaCl2.2H2O
440
MgSO4.7H2O
740
KH2PO4
136
(NH4) succinate
50
Micro elements (mg/litre final concentration)
Na2EDTA
37.3
FeSO4.7H2O
27.8
H3BO3
3.0
KI
0.75
MnSO4.7H2O
10.0
ZnSO4.7H2O
2.0
CuSO4.5H2O
0.025
Na2NoO4
0.25
CoCl2.2H2O
0.025
Carbohydrates (g/litre final concentration)
198
Sucrose
30
Mannitol
50
Myo inositol
0.1
Hormones (mg/litre final concentration)
NAA
0.1
BAP
1.0
Vitamins (mg/litre final concentration)
Pyridoxin HCl
1.0
Thiamine HCl
10.0
Nicotinic acid
1.0
Media H
Macro elements (mg/litre final concentration)
KNO3
NH4NO3
CaCl2.2H2O
MgSO4.7H2O
KH2PO4
1910
600
300
300
170
Micro elements (mg/litre final concentration)

Na2EDTA
FeSO4.7H2O
H3BO3
KI
MnSO4.7H2O
ZnSO4.7H2O
CuSO4.5H2O
Na2NoO4.2H2O
CaCl2.2H2O
37.3
27.8
3.0
0.75
10.0
2.0
0.025
0.25
0.025
Hormones; mg/lt final concentration)

2, 4-D
NAA
BAP
0.1
1.0
0.2
Carbohydrates (g/litre final concentration)

Sucrose
Glucose
Mannitol
Sorbitol
Cellobiose
Fructose
Mannose
Rhammose
0.125
68.40
0.125
0.125
0.125
0.125
0.125
0.125
199
Ribose
Xylose
Myo inosital
0.125
0.125
0.1

Pyridoxin HCl
1.0
Thiamine HCl
10.0
Nicotinic acid
1.0
Folic acid
0.2
Ca Pautothenate
0.5
P-amino benzoic acid
0.1
Calcium chloride
0.5
Riboflavin
0.1
Ascorbic acid
1.0
Vitamin A
0.005
Vitamin D
0.005
Vitamin B12
0.01
B-Biotin
0.005
Organic acids (mg/litre final concentration)
Sodium pyruvate
5
Citric acid
10
Malic acid
10
Fumari acid
10
Other organics
Casein hydrolysate 250
Media K3
Macro elements (mg/litre)
KNO3
NH4NO3
CaCL2.2H2O
900
MgSO4.7H2O
250
(NH4)2SO4
NaH2PO4H2O
150
CaH PO4
250
250
250
50
Micro elements (mg/litre)

Na2EDTA
FeSO4.7H2O
H3BO3
KI
MnSO4.7H2O
ZnSO4.7H2O
CuSO4.2H2O
Na2NoO4.2H2o
CoCl2.2H2O
37.3
27.8
3.0
0.75
10.0
2.0
0.025
0.25
0.025
200
Carbohydrates (g/litre)
Sucrose
Xylose
Myo inositol
102.69
0.25
0.1
Hormones (mg/litre)
2,4-D
NAA
BAP
0.1
1.0
2.0
Vitamin (mg/litre)
Pyridoxin HCl
Thiamine HCl
Nicotinic acid
1.0
10.0
1.0
MS media
KNO3
NH4NO3
CaCl2.2H2O
MgSO4.7H2O
370
KH2PO4
1900
1650
440
170

Na2EDTA
FeSO4.7H2O
H3BO3
KI
MnSO4.7H2O
ZnSO4.7H2O
CuSO4.5H2O
Na2NoO4
CoCl2.2H2O
37.3
27.8
6.2
0.83
16.9
8.6
0.025
0.25
0.025
Carbohdyrates (g/litre)
Sucrose
10
Myo inositol
0.1
Vitamins (mg/litre final concentratino)
Pyridoxin HCl
0.5
Thiamine HCl
0.1
Nictonic acid
0.5
Other organic (mg/litre)
Glycine
2.0
MS Morpho media
201

KNO3
NH4NO3
CaCl2.7H2O
MgSO4.7H2O
370
KH2PO4
1900
1650
370
170

Na2EDTA
FeSO4.7H2O
H3BO3
KI
MnSO4.7H2O
ZnSO4.7H2O
CuSO4.5H2O
Na2NoO4.2H2O
CoCl3.2H2O
37.3
27.8
6.2
0.83
16.9
8.6
0.025
0.25
0.025
Carbohydrates (g/litre)
Sucrose
Myo inositol
30
0.1

Pyridoxin HCl
0.1
Thiamine HCl
0.1
Nicotinic acid
0.1
Ca-Pantothenate
1.0
Hypocotyle, stem and petiole protoplasts (e.g. Brassica napus)
(i)
For the isolation of hypocotyl protoplasts, germinate
sterilized seeds and cut the hypocotyl into 0.5-1.0 mm transverse
sections. Cut the stem of petiole tissue in a similar manner.
(ii)
Transfer to CPW 13 M for 1 hr and then to the enzyme
solution incuabte for 16-18 at 220C in the dark with slow shaking.
(iii)
Pass the incuabtion mixture through a 50 m nylon sieve,
washing the digested tissue pieces with small volumes of CPW 13M
medium. Spin at 100 g for 10 min in order to sediment the prtoplasts.
(iv)
Remove the supernatant resuspend the protoplasts in CPW
215 medium and spin. Protoplasts will collect at the surface.
(v)
Remove the protoplasts, transfer to the appropriate culture
medium and count.
4.1
Fusion of Proroplasts:
Protoplats fusionof plants involves four discrete atages.
(a) Isolation of protoplasts
(b) Fusion
202
(c) Selection and plant regeneration

(d) The analysis of regenerated plant material
Somatic hybridization provides a method wherby sexual incompatibility
can be bypassed.Since, therre are no barriers to protoplast fusion
incompatibility and therfore protoplast level. Followning fusion, heterokaryons
containing nuclei of two species in common cytoplasm regenerate a new cell,
enter division and nuclear fusion results in the formation of somatic cell hybrid
cell. Like the normal cell, the sometic hybrid cell is totipotent dand theerfore
capable of regeneration embryogenesis or organogenesis into whole hybrid
plants.It is terefore, an approach of immense value iin the general area of
plant breeding and plant improvement.
Many methods have been described for the induced fusion of
protoplassts, these differ mainly in relation to the necessary to visualize by
mrkers i.e. green mesophyll of other parental species. Heterokaryons are
identifided as those protoplat5s prossesssing chloroplasts ( from the
mesophy11 parent) in a richly cytoplasmic background ( from the
nonmesoplyll partner).
As an example, a protocol is described below for the fusion of leaf
protoplasts of Ptunia parodii with those of a cell suspension of albino Petunia
hybrida. Alternative fusion treatments are also described.
Stages invovled
- Isolation of protoplasts
- Fusion
- Selection and plant regeneration
- Analysis of regenerated plant material.
4.1.1 Poly Ethylene Glycol Method (PEG)
Adjust the denstities of protoplasts 2 105 ml-1 in MS medium
Mark each centrifuge tube at 4 and 8 ml level
Spin all tubes except viability cntrol and allow the protoplasts to
a.
b.
c.
settle.
d.
Remove the medium so as to leave the protoplats suspended in

a maximum of 0.5 ml of medium.
e.
Add 2 ml of PEG per tube and left for 10 min.
PEG- 30%(W/V)
Sucrose- 4%
0.01M CaCl2.2H2O
f. Dilution of PEG at 5 minute intervals by adding 0.5, 2.0,3.0 and 4ml
ofmS P1 DM medium per tubes. Resuspend the protoplasts after ech
dilution.
g. Spin ( 100 gm ,10 min) soas to sediment the protplasts and remive the
supernatant.
h. Wash theprotplats in M/S P1 2M medium.
i. Suspend the fusion treated protoplasts iin 1 ml of media.
j. Prepare twelve 2 cm petidishes containing 8 ml of M/S P 1 2 M agar.
k. Adjust thevolume of viability controls.
l. Dispension of protoplast.
203
m. Mix together the remaining 8 mleach of the two treatments and

dispense into two dishes.
n. Seal all dishes with film and iincubate.
Conditions
Vaies with species and variety
Temp
:25o -30o C
Light: - 1000 lux.
4.1.2. Plant regeneration
After 24-36 hours division starts beginning. A well established
colony will occur within 21 days. From 4 th week onwards colonies are
suficiently large for transfer, using the tip of a scalpel to an agar solidified
regenrationmediu,. In this stage colonies are maintained in petridishes and
may sometimes require subculturing roots are also induced.
PRECAUTIONS
1.
peerfoem ail manipulationsunder sterilize conditions.
2.
pipette very smoothly and avoid stromg sucking of the tube.
3.
in centrifugation steps, speed should be increased only
gradually.
4.
Density should be optimum.
4.2. Productio of Plantlets from Suspension Culture:
INTRODUCTION
Plant cell suspension celtures provide the biochemist with a
relativelhhomogenous populatio of cells. Readilyaccssible to exogenously
applied chemicals and growingunder defined aseptic conditions. Cell
suspension cultures are widely used as model systems for studying
pathways of secondarymetabolism, enzyme induction and gene material
for enzyme purificatio the lack of chlorophyll and carotenoid pigments in
most plant cell suspension cultures is of great benefit fro work involving
isolationof enzumes of secondary products. However, susensions cant be
maintaned for long periods of time,and are usually prepared fresh fo9r
each experiment.
Steps Involved
1. initiation of suspension cultures from callus.
2. Subculture and meaurement of growth.
3. Regeneration into free living plants.
1. Initiation of suspension cultures from Callus:
Most suspension cultures are obtained by tansfeer of friable callus
lumps of agitated medium of the same composition as that used for calus
growth. A relativelh large initial inoculum is advantageous as this will
ensure that sufficient single cells and / or small clumps are released into
themedium to provide a sutably high cell density for subsequent
204
growth.Agrtation rates on orbital shakers should b in the range of 30-150

rpm with an orbital motion stroke of 2-4 c,. At the first subculture into fresh
medium, remive large clumps of initial incoulum either by transferring the
material with a pipette or syringe of suitable orifice diameter to exclude
large cell aggregates.
1.
Aseptically transfer a volume of cell cultrue, which is
udeergoing rapid increase in cell density to a dialysis tube connected to
the end of a glass tube of equal diameter.
2.
Place the dialysis tube in a conical flask containing fresh
couture medium such that the bottom of the glass tube is level with the
surface submergd. The top of the glass tube may be held in the neck of
the conical flask with a cotton wool plug.
3.
Incubation in an orbital shaker, the extent of conditioning of
the fresh medium will depend upon
a)
The density of actively growing suspension
b)
The relative volumes of suspension and fresh medium.
c)
The time of incubation.
Subculture and Measurement of Growht
Cell suspension cell cultures usually require a regular subculture at
more frequent intervals than the callus cultures transfer of a suitable size
inoculum to fresh meidum using either a ) Pipettes b) autoclaves metal
syringes or c) transfer by simply tipping culture into the new vessel upto a
graduation mark to ensure approximately constant inoculum size.
The growht of cell suspensioncultures may be monitored by
measurement of one of more of the following parameters.
1.
packed cell volume
2.
Cell number
3.
Wet and dry weights
4.
Protein or DNA content
5.
medium conductivity
6.
Cell viability
Protocol
Production of free living Plants
1.
2.
3.
4.
5.
Subculture 2-4 g of embryonic callus into a 250 ml Erlenmeyer

flask containing 25 ml of mS Liquid medium supplemented with 0.45
M 2.4D place theflask on an orbital shaker and agitate at 50 rpm.
Inclubate under 1000 lux illumination using a 16h photoperiod at
o
25-29 C .
initially, subculture using a 1:1 dilution ratio by pipettin off onehalf of the medium and replaceing ith fresh medium after 2 weeks.
Subculture using a 1:4 dilution ratio using large month pipettes,
transfer a 5 ml of suspension to 20 ml of fresh medium every 10-18
days. Generally repidly proliferatin cultures are subcultured every 10
days. Cultures can be maintained in tis manner for several years.
Asepticallyaspirate off 2,4-D containing medium and transfer the
suspension callus to Ms liquid medium to uniform embryo pooulation
205
can be produced by sieving the callus suspension through 43-200 m

stainless steel mesh and culturing onMS liquid medium.
6.
Initiate the plantlet formation via embryogenesis by subculturing
0.5-1 cm2 pieces of callus to the surface of mS agar medium.
7.
Incubate thecultures under 16 h photoperiod conditions ( 1000
lux) at 25-29oC. Within 2 weeks culturesw will exhibit theformation of
visible embryos and green plantlets.
8.
Agter 4-8 weeks, individual plantlets can be treased a part from
the other callus- embryo-plantlet mass an recultured separately or halfstrength MS agar medium.
9.
Incubate the cultures under 5000 lux ilumination using 16h
photoperiod conditions at 25-29oC . Within 4-8weeks cultures will
enlarge considerably and resemble seedling carrots
10.
otinue reculturing every 4-8 weeks until plantlets reachsufficient
size to be trasplanted to soil (~ 10-15 cm inlength), The selectionof
large well developed plantlets with functional shoot and root systems is
criticla fro success in obtaining free living plants.
11.
Transfer platntlets intact to distilled water for 10-15 min to avoid
dehydration and remove excess adhering medium Rinse plantlets three
times with distilled water spray, with 0.5%(W/V) benolate fungicide and
transfer to 7.6 cm2 diameter platic pots or fifty pent pots containing
sterile peatmiss and vermiculets in 1:1 (V/V) ratio.Enclose plantlet and
container with two interlocking cloear plastic polystyrens tubmblers to
maintain high humidityconditions.
12.
Administer weeklyapplicantions of 0.5% benolate sprays on
foliage to minimise fungal growth water the pots every other day with
distiled water and once a week with one forth strenght Hoaglands
solution during the first 1 or with 8000 lux light intensity under a 16h
photoperiod at Gradually acclimatise the plantlets to green house
covering.After 2 months, covers maybe removed and the plants treated
as normal seedlings.
Pit Falls
Suspension culture regenerated plantlets are similar to callus
regeneerated plantlets byt, regeneration thio suspension culture is a tedious
process.
4.
AGROBACTERIUM
MEDIATED
TRANSFORMATIONIN
PLANTS
Agrobacterium tumefarients causes crowngalls in plants. Crown galls
are tumors of plants that arise at the site of infectin.The tumour inducing agent
in Agrobacterium chromosome.Ti plasmids are large double- stranded circular
NA molecules of about 200 kb and 1 key exist as independent replicating unit.
Ti plsmids are maintained in Agrobacterium, because a part of the
plasmid DNA called T-DNA , carries the genes coding for the synthesis of
unusual amino acids called opines. The infected plant cell is indued to
synthesixe these opines. Selective advantage for Agrobacterium,. A second
set of genes in 7-DNA causes the unregulated growth of the plant cells.
206
Production of auxins. The third gene codes for an enzyme that hormones
cause t5he infected plant cell to divide and thus induces gall formation.
T-DNA, the part of the Ti-plasmid, which get integrated into the plant
chromosome can be used ofr genetic manipulationt. T-DNA is first cloned into
the standard E. coli.vector, and thepalnt gene subsequentlycloned into a
second cloning site carried in the vector. This intermediate vector4 is then
introduced into Agrobacterium containing intact Ti Plasmids. Re-Combination
occurs between the homologous plasmid, and on infectin of a plant with
Agrobacerium, the recombinant plasmid is trasferred to the plant cells. The E.
Plasmid because it becomes part of the ti plasmid.
Recombinant DNA technology has modifided agriculutre sector
completely virus resistant plants are prodced by introducing real coat proteins
into plants. Similarly the transfer of toxic gene from Bacillus thuringiensis
produced insect tolerant plant.
Very recently scientists at IARI. New Delhi developed transgenic
bringal with BT gene.
5.1 Agrobacterium mediated Transformationof tobacco/pigeon pea:
protocol
1. In Nicotiania tonacum
a)
Prepare leaf protoplasts
b)
culture the protoplasts in3.0 ml of liquid M/S p, 9M
medium over 12 ml agar-solidified M/S P, 9 M medium in 9 cm
petridishes. Incubats them at 25oC with an illuminatin of 700 lux.
c)
Agter 7 days transger the regenerating cells to

M/S P , 7M medium and inoculate with 0.4 ml of a 24 h culture of a.
tumefaciencs. For effective transformations the resulting mixture
should contain 5x 107 bacteria/ml and 0.8 x 10 2 bacteria per
regeneratin tobacco cell. Incubate for 36 h at 25 oC in the dark.
Selection for Transformed colonies

1.
Transfer the cells to fresh M/S P, 7 M

medium to which has been added 1.0 mg ml -1 carbenicillin.
2.
By subsequent transfer rreduce the 0.P to

3.5% mannitol and finallyafter 4 weeks to 6% mannitol or in M/S P1,
OM medium but still in presence of carbencillin.
3.
Place the cells on solidified M/S P1, OM

medium with antibiotic.After 4-6weeks , using a scalpel, transfer
individual colonies to agar solidified.M/50 medium and subculture after
every 4 weeks. Surviving colonies are trasform events.
5.1 Confirmatin of Transformationand Tumour Geneticity
207
Transformation events arising from protoplasts and regenerated cells

require a detailed biochemical analysis of the opine aminoacides. The
presence of these opines coupled Agrobacteriun and /or theTi-plasmid.a
simple test confirming the tumourous state, from the habituatee state involves
grafting of selected cultures.
Confirmationby grafting
-
Young shoots of greenhouse grown plants and surface

sterilize.
Take 5 cm lengths of shoots and insert some of the test

tissue into a 0.5 cm slit on each stem explant.
Seal the graft with a 3.0 cm length of sterile micropore

tape.
EMBRYOGENESTS, ORGANOGENESIS AND PLANT REGENERATION

6.1 Mode of plant regeneration
The term tissue culture has generated into an all encompassing,
convenient term to describe all types of steril plant culture, cells, tissues,
organs, embryos and plantlets. Plant regeneration through tissue culure can
be accomplished using one of three methods: embryo culture, somatic culture
of a zygotic embryo. Somatic or asexual embryogenesis is the production of
embryo-like structures from somatic cells. Thesomatic embryo is an
independent bipolar structure and is not physically attached to the tissue or
origin.
Plant development through organigenesis is the formation and
outgrowth of shoots from callus or initiation and outgrowth of axillary trends
generated from culture tips and their subsequent adventious rooting.
6.2 Explant factors
The term explant is used to describe the initial piece of the plant
introduded in vitro. For optimum success, explant must be obtained from
healthy vigorous plants. Pre-culture plant conditions may greatly influence the
subsequent growth of explants in culture. Practically any part of the plant can
be successfully cultured in vitro and can regenerate plantlets provide the
explant is obtained at the proper physiological stage of develoment.
Meristematic tissue should be given preference to others.
Following explant selection, the distinfection procedure are adopted.
Fungal and bacterial contamination in cultures is usually detectable in 1-14
days. Surface sterilization is adopted to combat with this.
Sterilant
Plant Part
Duration
NaOCl (0.2%)
Flowers, stem
10-20 minutes
208
NaOCl (0.25%)
Leaves
20 minutes
NaOCl (2.6%)
Roots
30 minutes
NaOCl (0.25-2.6)
Tap root
30 minutes
Iodine (3%)
Seeds
10 minutes
Hg Br2 (5%)
Seeds
10 minutes
6.3 Nutrient medium factors

A multitude of plant tissue culture media are suitable for plant regeneration.
The formulation derived by Murashige and Skoog (Table ) is the most
common medium employed in the plant tisseu culture field. Adverse chemical
discharge into the media like polyphenols which causes the browning of
media can be thwarted through frequent subculturing.
6.3.1 Media requirement for selected plant species caable of whole
plant regeneration in vitro
Species
Anthurium
Phoenix
Daetylifera
Brassica
campestris
Chrysanthemu
m norifolium
Cucurbita pepo
Oryza sativa
Triticum
aestivum
Zea mays
Prium sativum
Explant
Type
of Growthing conc : UM)
regeneration Callus
Shooting Rooting
Leaf
0-C
0.42,43.2 PBA
0.5 NAA
D
3.2 PNA
Axial bud SE-C
135 2,4- 0.54
D
NAA
13.9 Zip
Node
AS
4.4 BA
4.4 BA
0.44 BA
5.4 NAA
Shoot tip
0-C
5.4 NAA 0.11 NAA 0.054
NAA
Cotyledon SE-C
4.5
0.5
2,4-D
2, 4-D
Root
0-C
102, A- 0.054
D
NAA
Seed
0-C
4.5-9
5.7 IAA
0.054
2,4-D
NAA
Mesocotyl 0-C
67.8
0.054
2,4-D
NAA
Epicotyl
0-C
10.7
22.2 BA
NAA
1.11 IAA
4.7 BA
6.4 Somatic embryogenesis

The direct mode of somatic embryogenesis invovles the formation of
an asexual embryo from a single cell or group of cells on a part of the explant
tissue without an intervening callus phase.
209
PROTOCOL
CARROT
FOR
a)
b)
PRODUCTION
OF
EMBRYOGENIC
CALLUS
IN
Procurement of carrot seeds

15 minutes
sodiumhypochlorite solution
surface
sterilization
using
c)
Drying the seeds
d)
Place 1-3 seeds on the surface of MS agar
2.6%
e)
Incuabte the seeds in a culture room at 25-29 0C under a

16 b photoperiod until seeds germinate.
f)
Transfer germinating plant to a sterile petridish and cut

off the petiles. Place 1.3 pieces of petiole on the surface of MS medium
containing 0.45 M 2,4-D.
g)
Incuabte the cultures in compete darkness at 25-29 0C

for 4-8 weeks.
h)
Maintain the callus on agar medium by sub-culturing

0.5-1 cm pieces of callus to fresh medium every 3 weeks.
2
6.5 Organogenesis
The production of adventitious shoot in vitro is more common and
easier to control than the development of somatic embryos from cultured
explants. Plant production through organogenesis can be achieved through
one of three modes.
Production of adventitious organs from a callus derived from the
explant.
Emergence of adventitious organs directly from the explant without an
intervening callus phase.
Production of plantlets from outgrowth of axillary buds.
Protocol for the production of shoot from callus
a) Plants are grown in the greenhouse and leaf expalnts are used prior to
flowering. Leaves are taken either 1-2 h after sunrise or from dark
pretreated plants.
b) Wash leaves with soap and water, rinse with 40% ethanol and then
rinse with distilled water.
c) Surface sterilize leaves in 1% NaOCl 10 minutes.
210
d) Cut leaves using scalpels and transfer into MS containing 4.5 M 2,4-D
and incuabte.
e) Subculture pieces of callus obtained to MS containing 1 M 6-BAP to
initiate shoots.
f) Incubate at 25-290C under 16 h photoperiod at 1000 lux. Shoots will be
produced is 305 weeks.
g) Incubate cultures (shoots) in rooting media.
h) Transfer rooted plantlets.
6.6 Plant Regeneration Directly from cultured explants
The method of plant regenration is suited to herbaceous species using
leaves, corns, bulbs, stems, Rhizomes and tubers. Here explant is
established on a nutrient medium containing moderate levels of auxins and
cytokinins (to avoid callus production) and subsequently initiates shoot
organs. Callus production) and subsequently initiates shoot organs.
Multiplication is achieved through subdividuon of shooting clump and planting
out in separate vessels. This method should not be used to clone variegated
plant since the resultant plants will either show modified pattern of lose the
variegation completely.
6.6.1
Protocol for Regeneratino of plants via Direct

Organogensis for Begonia
a) Cut newly formed fully expanded leaves and accompanying petioles

from Begonia plants grown in the green house.
b) Disinfect leaves by treating with 5.25% NaOCl containing 0.5% tween20 for 10 min. Rise thrice with sterilized distiled water.
c) Section leaves into 5 mm lengths and place, with the proximal side
down, on the surface of MS, agar containing Nitsh & Nitsch vitamins,
1.8 M BA and 0.54 M NAA.
d) Incuabte 1000 lux using a 16 h photoperiod at 25-29 0C.
e) Subdivide each culture and transfer to the same medium after 4 weeks.
f) Root shoots by transferring MS with 0.5 M NAA.
g) Establish in soil.
CONCLUSION
Plant tissue culture is an inevitable method for propagation of plants. It
forms a very conversant tool in the hands of plant breeders, plant
biotechnologists etc. A detailed account of the general aspects of tissue
211
culture with merits and demerits is described. Apart from that various types of
plant regeneration techniques like callus culture, somatic embryogenesis etc.
has been dealt in detail. Culture media compositions, preparation of media
etc. had also been mentioned. Experimental protocol for protoplast fusion,
Agrobacterium mediated transformation had also found a palce in the present
discussion.
REFERENCES
Levy, L.W. 1981. A large scale application of tissue culture the mass
propagation of pyrethrum clones in Ecuador. Enus. Exp.Bot.21L: 273-395.
Lloyd, G and McCown, B. 1980. Commercially feasible microproparation of
mountain laurel (Kalmia datifolia) by use of shoot-tip culture.Proc. Int. Plant.
Prop. Soc. 30: 421-427.
Murashige, T. and Skoog, F.1962. A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol. Pant 15 : 437-479.
Rao, M.V.S., Rao, Y.V., Rao,Y.S. and Manga, V. 1988. Induction and growth of
callus in Aza diraehta indica. Crop Improvement 15(2) : 203-205.
Roy, S. K., Rahman, S.L. and Majumdar, R. 1990. In vitro propagation of jack
fruit (Artocarpus heterophyllus). J. Hort. Sci. 3(65): 355-358.
Watson, D.J., Gilman, M., Witkowshi, J., Zoller, M. 1992. Recombinant DNA
Iied., W.H. Freeman and Co., Newyork : 273-290.
212
I.
Objective: The generation of plantlets from callus materials
II.
required : Callus, media, flasks etc.
Procedure
1. Subculture 2-4 g embryonic callus into a 250 ml. Ebermeyer flask
containing 25 ml of MS liquid medium supplemented with 0.45 M 2,4D.
2. Incubate under 1000 lux illumination for a 16 hour photoperiod at 25280C.
3. Subculture using a 1 : 1 dilution ratio.
4. Transfer ml of suspension to fresh media every 10-15 days. Cultures
can be maintained in this manner for several years.
5. Tranfer the callus into MS liquid media for inducing further stages of
asexual embryogenesis.
6. Initiate the plantlet formation via embryogenesis by subculturing small
callus pieces on MS agar medium.
7. When individual plantlets arise, separate them and culture on halfstrength MS medium.
8. Continue subculturing for a period of 4-8 weeks till the plantlets are
ready for planting out.
III.
Objective: To fuse protoplasts by PEG method
Procedure
1.
Adjust the densities of protoplasts in culture to 2 10 5 /ml in M/SP1 9

M.
2.
Mark each centrifuge tube at 4 ml and 8 ml level.
3.
Spin all tubes, except the vaibility control and pelletize the protoplasts.
4.
Remove the medium so as to leave the protoplasts suspended in a

maximum of 0.5 ml of medium.
5.
Add 2 ml of PEG per tube.
6.
Dilute the PEG at 5 minute intervals by adding M/S i 1 PM medium

standards.
7.
Spin (100 g, 10 min) so as to sediment the protoplasts and remove the

supernatant.
8.
Wash the protoplasts and resuspend it.
9.
Prepare petridishes with M/S P1 9 IX agar medium
10. Dispense the protoplasts into the petridishes.

11. Mix together the remaining 8 ml each of the two selfed treatments and
dispense into two dishes 8 ml/dishes.
213
12. Seal all the dishes with parafilm and maintain at 25 0C with continuous
ilumination of 700 lux.
IV.
Objective:
tobacco/pigeon pea
Agrobacterium
mediated
transformation
of
Procedure
1. Prepare leaf protoplasts.
2. Culture the protoplasts in 3.0 ml of liquid M/S P 1 9 M medium over agar
at 250C.
3. After 7 days, transfer regenerating cells to M/s P 1 7 M medium and
inoculate with 0.4 ml of a 24 h cultue of agrobacterium tumefaciens.
Selection of Transformed Colonies:
1. Transfer thecells to fresh M/s P1 7 M medium to which has been added
carbencilin.
2. By subsequent transfer reduce the osmotic pressure to 3.5% mannitol
and finally after 4 weeks to 0% mannitol as in M/s P 1 9M medium.
3. Plate the cellls on solidified M/s P 1 O M medium with antibiotiec. After
4-6 weeks transfer individual colonies to agar medium.
Confirmation of Transformation by Grafting
1. Take 2-3 week old seedlings grown in green house.
2. Take 0.5 cm of the test tissue and insect the scion.
3. Seal the graft with a sealed microtape.
4. Insect the stem base into agar-solidified MS medium with 0.1 mg 1 -1
Mas in 6 OZ power round jars.
Transformed tissues will proliferate while non transformed tissues will not.
214
V.
PLANT TISSUE CULTURE
Objective
To germinate the Brassica seeds in MS medium under aseptic
conditions.
Requirement
Reagents, Atuclave, milk bottle, beakers, flasks, measuring cylinder,
Culture bottle, etc.
Principle
In tissue culture maintanance of aspectic condition and aspetic
handling has prime importance. Any contamination during handling will
destory all material and our objective will be failed. The Brassica seeds can
be sterilized by using usrface sterilizing agents such as 0.1% HgCl 2, for 2
minutes 1% sodium hypochloride or 70% alcohol.
Procedure
(1)
Media preparation
MURASHIGE AND SKOOG MEDIUM

Constituents
Soluntion 1
100 X
1.
NH4NO3
1650 mg/lt
8.25 g/50 ml
2.
KNO3
1900 mg/lt
9.5/50 ml
Solution 2
100 X
CaCl2.2H2O
440 mg/lt
2.2/50 ml
Solution 3
100 X
1.
MgSO4.7H2O
370 mg/lt
1.85 g/50
2.
KH2PO4
170 mg/lt
0.55 g/50 ml
Solution 4
100 x
1.
Na2EDTA
370 mg/lt
0.1865 g/50 ml
2.
FeSO4.7H2O
27.8 mg/lt
0.139 g/50 ml
Solution 5
1000 x
1.
H3BO3
6.25 mg/lt
0.3125 g/50 ml
2.
MnSO4.H2O
22.5 ,g/lt
1.15 g/50 ml
1.
215
3.
ZnSO4.7H2O
8.6 ,g/lt
0.43 gm/50 ml
4.
KI
0.83 mg/lt
0.041 gm/50 ml
5.
Na2MoO4
0.025 mg/lt
0.00125 gm/50 ml
6.
CuSO4.5H2O
0.025 mg/lt
0.00125 gm/50 ml
7.
CoCl3.6H2O
0.025 mg/lt
0.00125 gm/50 ml
Solution 6
1000 X
1.
Nictonic acid
0.5 mg/lt
0.025 gm/50 ml
2.
Pyridoxine HCl
0.5 mg/lt
0.25 gm/50 ml
3.
Thiamine HCl
0.1 mg/lt
0.005 gm/50 ml
4.
Glycine
2.0 mg/lt
0.1 gm/50 ml
BAP = 2 ppm = 10 mg; Sucrose = 30 mg/lt

NAA = 1 ppm = 5 mg
Procedure
1. Prepare MS medium andautoclave it.
2. Soak the brassica seeds in 0.1% HgCl2 for 2 minutes,
3. Wash the seeds in sterile wate for 2 to 3 times.
4. Aseptically tranfer 3-5 seeds to culture bottle and 6-10
5. Aseptically transfer 3-5 seeds to culture bottle and 6-10 seeds using
sterilized forceps.
6. Keeping the bottle in dark till radical emerges out.
7. Transfer the bottle to light after radical emergence.
Observation
The seeds took 2-3 days for germiantion and full sized plant (with 2
leaves) appeared on the medium after 5 days.
Experiment No. 2
Objective
Callus induciton from hypocotyl of Brassica seedling on callus inducing
medium
Requriment
Forceps, scalpel, medium, bottle spirit lamp etc.
Principle
216
Callus is homogenous undifferentiated mass of cells. Any plant part or

cell or tissue when inocualte into callus inducing media start multiplying and
produce a gorup of cells which are homogenous. The callus inducing medium
hasauxins and cytokinins in such as proportion that it is capabe of inducing
callus.
Procedure
1. Prepare the callus inducing medium and pour it into bottle.
2. Remove the seeldings from boiling tube in aspetic conditon and
aseptically separate the hypocotyl region.
3. Cut the hypocotyl region into small pieces using sterilzied scalpel.
4. Aseptically transfer few pieces of hypocotyl into the bottle containing
callus inducing medium.
5. Keep the flasks in dark.
6. Observe the callus produced after 6-7 days.
217
IMMUNOLOGICAL TECHNIQUES
Immunology is a rapidly developing field of study concerned with the
discovery of the mechanisms of the immune system, the development of
vaccines and the regulatory procedures for manipulating the immune
response.
Immune response may be cell mediated response (mediated by T.
Lymphocyte) of Humoral response (mediated by B. lymphocyte).
Antigen
An antigen is any substance which when introduced into the vertebrate
host stimulates the production of antiboides and reacts with preformed
antibodies.
Immunogenicity
Capacity to stimulate an immune response.
Specificity
Their ability to react specifically with immune antibodies.
Antibody
Antibodies are also termed as immjnoglobulins immunoglobulins having 2
light and 2 heavy chains Ab are classified into 5 classes according to their size and
heavy chain.
Heavy chain
Light chain
Mol. Wt.
IgG
K/
15,000
IgM
K/
900,000
IGA
K/
160,000/370,000
IgD
K/
180,000
IgE
K/
185,000
Antigen Antibody interaction

Epitopes of antigens interact with paratopes of antibody and form an
immune complex.
Ag + Ab Ag. Ab
[Ag Ab]
K1 = -------------
[Ag] [Ab]
K2 = --------------
[Ag] [Ab]
[Ag Ab]
218
K = Affinity constant
If K1 is more = more affinity
K2 is more = les affinity
Antibody is the measure of strength of antibody and antigen interaction
Cross reactivity
Ab may react with many other molecules other, then the antigen
agasint which it is raised. It is called cross reactivity and it will be raised due to
presence of poly functional binding sites.
Adjuvants
Adjuvants which may or may not be antigenic, increae the level of
circulating antibodies and thus improve immune responses.
Antigenic adjuvants
Gram ve bacteria (Borderella-pertusis) endotoxin of ram ve bacteria.
Acid fast microorganisms, phytonemagglutinins and antibodies.
Non-antigenic adjuvants
It includes potassium aluminium tarterate, calcium phospahte, mineral
oil, lanolin, surface active agents, calcium alginate, collodian, or acrylic
particles.
Freunds complete adjuvants
It is such a mixture which contain killed mycobacteria, an oil and an
emulsifier.
Affinity
The intrinsic binding power of a single antibody combining site with a
single antigen bidning site, which may be expressed interms of a single
binding constant.
Avidity
The net combining power of an antibody molecule with its antigen.
Antigen combining site or paratope
The site on the antibody that combinesspecifically with its
corresponding antigenic determinant. It usually involves only a few amino acid
residues antibody molecule but not usually directly linked to each other.
Antigenic determinant or epitope
A small site on an antigen to which a complementary antibody
molecule may be specifically bound through its combining site.
Antiserum
A serum containing antibodies agasint a specific antigen.
Avidin
A protein that specifically binds biotin with very high affinity.
219
Complement
A group of serum proteins essential for antibody mediated
immunolysis. Immunization is clonal expansion of immune cells. There are 2
types of immunization.
Active immunization
Here we use antigen. It is used as preventive or prophylactice
measure. In active immunization vaccine may be comitve agent or toid or
alttenuated vaccine.
Factors afecting antibody production
a) Dose of antigen
b) Nature of antigen
c) Mode of immunization
d) Immunoization schedule
e) Presence of immuno protentior
Dose of antigen B.S.A. 500 g
After immunization some non specific reactinos like enzyme activity
may degrade the antigen. Molecules that can induce non-specific
mechanisms of immune response is called as xenobiotics.
So we have to protect the antigen from non-specific immune reactions.
The protective agents are called immunopotentior or adjuvants.
e.g. Fruends ion plete/ incomplete adjuvants, Alum, MgSO 4
Mode of immunization
1. Intra pertionial (I.P.)
2. Intra dermal (I.D.)
3. Subcutaneous (S.C.)
4. Intra veinal (I.V.)
5. Foot Pad (F.P.)
6. Intramuscular (I.M.)
Immunization schedule
Ist
injection
0 day
IInd
injection
7 daysSC./IP
IIIrd
injection
14 days
SC./IP
IV
injection
21 days
SC/IP
injection
28 days
SC/IP
VI
injection
35 days
I.V.
SC. at multiple sties
220
Mode of Bleeding
1. By ear (Marginal vein of pinnae. Dialate by xylene)
2. From eye (Retro orbital bleeding)
3. From heart (cardiac puncture method)
4. Juglar vein.
221
Experiment No.
Objective
Observe agglutination reactions by blood grouping
Requirement
Blood, antiserum, mixing rod, slide etc.
Principle
Agglutination form a clump instead of using bacterial cells to
demonstrate agglutination, whole human red blood cells can be employed.
There are 2 types of antigen present on blood corpuscles.
Blood Group
Antigen
Antibody
Genotype
1.
IaIa/Ia/Ia
2.
IbIb/IbIb
3.
AB
ab
4.
IaIb
IoIo
Ab
Whatever Ag, a person has in his cell the corresponding Ab is lacking

in the serum; this obviously must be so. Since if a person of groupo A had the
antibodies agaisnt A in this serum his cells would be agglutinated when an Ag
is not present, the corresponding Ab. B present. This a person of group A has
no Aba as he has no Ag B but does have Ab : b.
The blood groups are of great importance. Knowledge of their
existance has removed the hazards which were associated with blood
transfusion so that this valuable thereaputic procedure is commonly used.
The information received from blood grouping in some times used in
medico legal cases is case of disputed paternity or child mixing.
Possible effects of transfusion of blood
Donors
blood
group
O
A
B
AB
Receipients
Can
be
Agglutinates the blood given to
of
O
A
B
AB
O, A, B, AB
+
+
A & AB
+
+
B & AB
+
+
+
AB
Can
receive
blood
Remark
O
O, A
O, A
O, A, B, AB
Donor
Univ.
recipient
222
Observation Table
Name
Anti A
Anti B
Blood Group
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
223
Experiment No.
Objective
To detect pregnancy by the presence of B HCG in urine by latex
particles agglutinatino inhibition test.
Requirement
Sample, distt. H2O, black testing plate, Ab against GCG latex beads
coated with HCG, Mixing rods.
Principle
HCG is human chorionic gonadotropic hormone. It is released from the
placenta of a pregnant woman and can be collected from the urine of some.
HCG is a dipeptide glycoprotein. Containing 2 subunit and . It is soluble in
antibody. Hence the Ag Ab complex precipitation reaction is not so visible we
have to anpigy it. This is done by the use of HCG coated latex beads, in the
test reaction and positive test shows no agglutinatin, hence it is actually a
precipitation inhibition test reaction.
Procedure
1. Take immunological testing slide blakc in colour.
2. There were 2 circle outlines on the plate. On one add a drop of distilled
water to the other place a drop of urine sample.
3. Place a drop of antigen specific antibody near the drop of sample.
Mixed them with a mixing rod.
4. Now added a drop of the latex beeds coated with HCG to each circle.
Mixed them by moving the slide gently.
5. Observed the plate after 2 minutes.
Observation
In the circle with control; agglutination was visible beginning from
periphary of the urine sample there was no agglutination.
Result
The result implies the sample bears HCG and the donor of the sample
is pregnant.
Precuations
(i)
Do not use same end of the mixing rod for mixing control and
sample.
(ii)
Only a drop of the constituent shall sufficient for the test.
Discussion
The dark coloured plate is supplied in the kit enables use to visualize
the agglutination reaction which as such may not be visible on normal slide.
We can carry out the test even after 90 days of prenancy but is generally
carried out in the mid term of pregnancy to yield visible agglutination reaction.
224
HCG in sample acts as the antigen and react, pontanecously with

antibody provided with antibody provided (excess) and form Ag-Ab complex.
Now as we add HCG coated beads there out to be no agglutination as all the
AB have already formed lattice of complex with antigen and hence no
agglutination shows a the test for pregnancy.
GEL DIFFUSION /OUCHTERLONY
A major advantage of this type of gel diffusion assay is that more than
one antigen antibody systme in a mixture can be detected. It is important to
remember that whenantiboides one produced by injeecting an antigen into an
animal, many different antibody molecules reacting with different part of the
antigen will be found in the serum of the animal. In this serum referred to as
antiserum C is usually used as a source of antibodies in experiment. A single
antigen will combine with the antibody and it induces (its homologous
antibody) to form a single precipitin live in a gel diffusion assay. When two Ags
are present each behaves independently of the other. The position of the
precipitin lines wil depend primarily on the sizes of the antigens and their rates
of diffusion is the gel. Thus the number of precipitin lines indicates the number
of Ag-Ab system present.
Double diffusion, referring to the process described above in C both
Ag-Ab molecule diffuse from a well, is a simple but very useful procedure
invented by the Swedish scientist Auchterlony. It is used to compare antigens
for the number of indentical or cross reacting determinants. If a solution of Ag
in placed into 2 adjacent wells and the antibody is placed equidistant from the
2 antigen wells. The 2 precipitin lines that form will join at their closest ends
and fuse. This is called reaction of identity. When unrelated Ag and placed
inadjacent wells and the centre well in filled with Ab reacting with both Ag the
precipitin lives will form independently of each other and will cross. This is
called as a reaction of non-identity.
225
EXPERIMENT NO.
Objective
Demonstration of gel diffusion/ouchterlony
Requirement
1% purified Agar, phosphate buffer saline (pH 7.4), 4 microscopic
slides. Sodium azide, 95% ethanol will cutter, B.S.A. antibody.
Procedure
Preparation of 1% agar
1. For phospahte buffer saline, dissolve the following in 150 ml distilled
water
2.19 g NaCl
1.28 gm KH2PO4
2.06 gm Na2HPO4.7H2O
Adjust pH 7.2, add distilled water to make 250 ml
2. Add 2.5 gm purified agar in 250 ml water and 0.25 gm sodium azide
(bacteriostatic) heat to dissolve agar, and autoclaved
3. Cool the agar solution to 600C.
Preparation of slides with sample wells
Clean the slide by 95% alcohol and label it
Using a 5 ml pipet, pipet 4 ml agar carefully and place agar on slide.
Allow the agar to solidify. This will take approx. 10 minutes
Cut the 5 wells with well cutter.
Loading the sample
Fill the center will with 3 drop of antiserum using a transfer pipet.
Fill the outer wells with 3 drops of Ag.
226

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DEPT. OF MOLECULAR BIOLOGY & GENETIC