Practical Manual for the Cell Biology Experiments

EDITORS Jing Chen Jing Chen Xiujun Zhang Jie Zhao

Department of Biology North China Coal Medical University ,Tangshan, China 2013-07-22

CONTENTS Laboratory Apparatus.............................................3 Laboratory Safety.....................................................5 Guidelines for Laboratory Reports.........................8 Experiment 1. The Microscope..............................13 Experiment 2. Morphological Structure of Cells.22 Experiment 3. Osmosis and Diffusion...................27 Experiment 4. Identification of chemical constituents of the cell ...........................................31 Experiment 5 and 6. Cell culture...........................36

Laboratory Apparatus
Laboratory apparatuses include the following: 1. Glassware including Beakers, Flasks, Storage Bottles, Graduated Cylinders, Test Tubes, Pipettes, Burettes, Funnels, Petri Dishes, Staining jars, Slide Cabinets, Microscopic Slides & Cover Slips, Pestle & Mortar. 2. Lab tools: Dissecting Sets (Blades, Scalpel, Scissors, Needle, Forceps), Microbiological Loops, Syringes, Hand Lenses, Spatulas, Bunsen Burner. 3. Instruments: Microscope, Microtome, Balances, pH meter, Incubator, Oven, Autoclave, Spectrophotometer, Refrigerator, Centrifuge.

Basic Glass ware and Lab Tools

Laboratory Safety
Safety in the Laboratory should always is in your mind. Throughout this manual safety recommendations are given, below are some general consideration that anyone in a laboratory should know.

General laboratory safety precaution.

1. Follow all instructions carefully. Use special care when you see the word CAUTION in your laboratory instructions. Follow the safety instructions given by your teacher. 2. Determine the location of Fire Extinguishers, Chemical safety showers and Eye washers, Chemical Spill Kits, and alternative exit routes for lab evacuation. 3. Remember that smoking, eating, or drinking in the lab room is totally prohibited. 4. Wear lab aprons when working with chemicals, hot material, or preserved specimens. 5. Wear safety goggles when using dangerous chemicals, hot liquids, or burners. 6. Any chemicals spilled on the hands or other parts of the skin should be washed off immediately with a plenty of running water. 7. If you have an open skin wound, be sure that it is covered with a water proof bandage. 8. Never work alone in the laboratory. 9. Keep your work area clean & dry. 10. Turn of all electrical equipment, water, and gas when it is not in use, especially at the end of the laboratory period. 11. Tie back long hair. 12. Report all chemicals spills or fluids to your instructor immediately for proper clean up.

Special precautions for working with heat or fire:

1. Never leave a lighted Bunsen burner of hot object unattended. When an object is removed from the heat & left to cool, it should be placed where it is shielded from contact. 2. Inflammable liquid bottles should not be left open, not dispensed near a naked flame, hot electric element or electric motor. 3. Use test tube holders to handle hot laboratory equipments. 4. When you are heating something in a container such as a test tube, always point the open end of the container away from yourself & others. 5. Use only Pyrex glasswares for heating.
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6. Allow hot materials to cool before moving them from your lab station. 7. Make sure that Bunsen burner hoses fit tightly.

Special precautions for working with chemicals

1. 2. 3. 4. 5. 6. Never taste or touch substances in the laboratory without specific instructions. Never smell substances in the laboratory without specific instructions. Use materials only from containers that are properly labeled. Wash your hand after working with chemicals. Do not add water to acid. Instead, dilute the acid by adding it to water. Mix heat generating chemicals slowly.

Special precautions for working with electrical equipment.

1. Make sure the area under & around the electrical equipment is dry. 2. Never touch electrical equipment with wet hands. 3. Make sure the area surrounding the electrical equipment is free of flammable materials. 4. Turn off all power switches before plugging an appliance into an outlet.

Special Precaution for working with Glasswares and other laboratory equipments.
2. 3. 4. 5. 6. 7. 8. Become familiar with the names and appearance of all the laboratory equipments you will use. Never use broken or chipped glassware. Make sure that all glasswares are clean before you using it. Do not pick up broken glass with your bare hands. Use a pan and a brush. If a Mercury thermometer breaks, do not touch the mercury. Notify your teacher immediately. Do not aim the mirror of your microscope directly at the sun. Direct sun light can damage the eyes. Use care handling all sharp equipments, such as scalpels and dissecting needles.

Special precautions for working with live or preserved specimens.

1. If live animals are used treat them gently. Follow instructions for their proper care. 2. Always wash your hands after working with live or preserved organisms. 3. Specimens for dissection should be properly mounted and supported. Do not try to cut a specimen while holding it in the air. 4. Do not open Petri dishes containing live cultures unless you are directed to do so. 5. Detergents (detol 5 10%) should be used to sterilize and clean benches, glassware and equipment.
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6. Safety cabinet should be used while working with microbes. 7. Lab coats should be worn during the work in the lab. 8. Disposable items should be collected and autoclaved.

First Aid
1. Injuries: bleeding should be reduced using bandages; the wound should be cleaned with iodine alcohol mixture, and wrapped with sterile bandage. 2. Acid and fire burns: body burns must be washed immediately with tap water. Eye burns must be washed using eye washer, special cream for burns can be used. 3. Poisoning: if any toxic chemical is swallowed, the mouth must be sensed with water, in case of acid, milk is drunk, in case of alkaline, diluted acetic acid ( vinegar) can be used. 4. Skin contamination requires washing with water and removal of contaminated clothing, if the contaminant is insoluble in water remove with soap and water.

Guidelines for Laboratory Reports

General information
Each student is responsible for writing his or her own laboratory report, unless otherwise indicated by the laboratory instructor. The report is to be your own INDIVIDUAL responsibility, and you should share nothing other than your lab results with other students in the lab. Do not look at another students report as a model for your own. A laboratory report is a scientific discussion of the experiments or observations that you performed in your laboratory section. It should be written in a very specific and precise format as if it were to be submitted for publication in a scientific journal. Thus you should write it as if your audience were learning about what occurred in the laboratory for the first time. DO NOT assume that the reader knows what you mean, and do not use shortcuts in describing any part of the experiment. You must explain everything in detail. Some portions of a report may require that you report on observations, which may be best presented using colored pencils to indicate the results in a drawing.

Writing style
Your paper must be organized into complete sentences and paragraphs, with each paragraph beginning with a topic sentence that indicates the content for that paragraph. The paragraphs should be arranged in a logical sequential order that indicates what you did and how it relates to the topic as a whole. Your report will be read critically for scientific content as well as your ability to express yourself clearly and concisely. Remember that this is a scientific report, in which clarity and organization are important components. Be sure that you include and discuss only what actually happened, including your exact results. A scientific report MUST be truthful, even if the results were not those expected. You will have an opportunity to discuss what was expected and why your

results may have deviated from the expected results. You should keep track of what might have gone wrong so that your discussion will have value if your results are unexpected or different from those of other lab groups in your class. All discussion of what was done in the laboratory experiments must be presented in the past tense, since you are writing about what occurred at a previous time. The report should be written with as few words as possible to keep it clear and concise. Use active verbs to describe what happened, rather than passive. The use of we or I is acceptable to avoid the passive voice, but these terms should be kept to a minimum. If you must use scientific nomenclature to identify an organism used in the lab, genus and species names should be italicized (preferred) or underlined

Report format
The report will include the following components: (1) a cover page, (2) introduction, (3) materials and methods, (4) results, (5) discussion, and (6) references. Cover Page This should contain the title of the report; your name; the name of your lab partner(s); the due date of the reports submission; the laboratory section, time, and day; and the name of your instructor. Be sure the title of the report is very informative. It may even be a sentence that summarizes the results of the experiment. Introduction This gives an introduction to your experiment by providing a brief background about the topic and a context for your activities. This would include what information is known from previous studies and what further information you expect to obtain from your experimentation or observations. If you refer to your text or other references in citing these previous studies, be sure you give a proper citation in the text of your report and list the complete reference at the end of the report. Your introduction should begin with general statements about your topic, and then proceed to more specific statements, until you have given an overview of everything to be covered in your report. You should describe here what was done in a very general way, without the details that will be presented later in the Materials and Methods or Results sections. Another component of the introduction is a brief description of the specific questions that are being approached in your experiments. If you are testing a hypothesis in your experiment, you should state the hypothesis at the end of the Introduction section. Materials and Methods A description of the materials being used, possibly with diagrams or drawings if necessary, should begin this section. You should then provide a brief discussion of the methods used in the experiments, explaining how everything was done. If these methods are taken directly from your laboratory manual or another reference, you may refer the reader to them there rather than repeating them in your report. In this case, be sure to make the proper citation within the text, and also list the original in the References section of the report. If you refer to methods from another source, you must indicate any changes in those methods that were actually used in your experiments. If several experiments concerning the same topic are being discussed in your report, you may find that greater clarity is produced by discussing each experiment separately, perhaps with Roman numerals to identify each part, in Materials and Methods and again in Results.
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The purpose of this section is to explain what you did so that your reader can perform the same experiment using the materials and methods that you have explained. Thus, you must include any details that would affect the readers ability to repeat your experiment, but you should leave out irrelevant details that do not affect the repeatability of the activity. Be sure you include appropriate quantities, including size of containers, weight or volume of substances being used, temperatures, times of treatments, etc. Since this is a scientific report, quantities should be reported using the metric system, including temperatures in degrees Celsius. Results In this section you will begin with a text discussion of what results were obtained from each part of the experiment. You may make reference to data directly in the text of this section if there are not many numbers to report, or you may refer to any tables or figures in which the results are presented. A figure may be a graph, a diagram, or a picture, and each table or figure should have a detailed caption that explains its contents. Each table or figure should also have a number by which you can refer to it in the text of your results section. Be sure all required labels are included, such as labeling the X-axis and the Y-axis of a graph, or each column of a table. You may wish to include leader lines and identification labels on a diagram for greater clarity. Report ALL data you obtain as results in any experiments you perform. Do not omit data that do not support your hypothesis, or unexpected data that might result from following the methods incorrectly. DO NOT change any data to fit your expectations better. It is very important that scientific data be reported honestly, exactly as it is obtained, and unexpected results should be addressed in the Discussion section of your report. Types of data that you might include in this section are (1) any general observations you made during the experiment, (2) all quantitative results, usually shown in figures or tables, and (3) the results of statistical analysis of your results, if appropriate. Use the analytic methods indicated by your laboratory manual or your instructor, and present the data and tests in a figure and/or a table. Do not include raw data in this section, but summarize the data for greater clarity. If your instructor says you must include raw data and any calculations required to summarize the data, place the information in an Appendix after the Discussion section. The Results section should contain only the results, without discussion of why or how the results occurred. Since many of the details of the results are in tables or figures, you do not need to cover these points again in the text of this section. You should identify only the important points of each figure or table within the text. Discussion This is usually the most important portion of your laboratory report, since you are interpreting the results in light of the hypothesis that was being tested. Again, the information should be presented from the more general statements to the more specific details. Begin with your major conclusions that were obtained from your results, and tell whether the results support or do NOT support your hypothesis. Remember that you cannot prove a hypothesis true, so dont make such a statement. Your results will also probably not be sufficient to prove a hypothesis false, but you may have results that do not support the hypothesis. In this case, you need to explain
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how your results contradict the hypothesis, and perhaps compare your results with those of other students in your section (if their results are provided to you), or compare your results with the results of other studies to which you make reference. Remember to cite the studies in your discussion and give a complete reference to the book or article in your Reference section. After discussing your results and how they relate to the hypothesis and other work on the topic, you should try to offer an interpretation of why you obtained the results you did, especially if the results were unexpected. This is where you would discuss what you might have done incorrectly in the experiment, and how this would have produced the results you obtained. Remember also that you might get unexpected results even if you did everything exactly as specified in your experiment. Negative results are not necessarily WRONG. New experiments or improvements in the original design of the experiment may be suggested here as alternative ways of testing the same hypothesis. References If any literature has been cited in the text of your report, such as your laboratory manual, you must give a full reference to each book or article cited. Present these in alphabetical order, by the first letter of the first authors last name. Criteria for The Grading of Papers and Experimental Reports The maximum grade is a 100 and is a composite of three grades based on spelling grammar, and content. I. Spelling counts 10% of the total grade. Each different spelling or typographical error will usually result in a point deducted from the maximum. However, if one word is consistently misspelled, it will be deducted only once. Low grades in spelling can be avoided by keeping a dictionary on hand and proofreading your work before you submit it for review. II. Grammar counts 15% of the total grade. Each grammar error (wrong tense, poor sentence of paragraph structure) will usually result in a point deducted from the maximum. Low grades in grammar can be avoided by proofreading your work before you submit it and by writing practice essays. III. Content counts 75% of the total grade. The kinds of questions that are considered in evaluating content include the following: 1. Is your information accurate? 2. Is your discussion logical? 3. Did you transform the raw data into a more useful and appropriate format? 4. Do you adequately support your argument? 5. Do you adequately correlate and contrast your data to previous experience? 6. Do you support your conclusions with the appropriate statistical test(s)? Name______________________ Grades Spelling x 10% = . Grammar x 15% = . Content x 75% = . COMPOSITE GRADE .

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Experiment 1. The Microscope

Objectives::

This Exercise focuses on how to develop a working knowledge of the Microscope and its use. Students should identify the different parts of the Microscope. List and follow recommended procedures in using and caring for the Microscope.

Material

Compound Microscope Clean Microscope Slides Cover Slips Lens papers Sharp razor blades Newspaper letters Medicine droppers Scissors Distilled water Pond water Cork Xylene

Introduction:

Since an unaided eye cannot detect anything smaller than 0.1 mm in diameter, cells, tissues, and many small organisms are beyond our visual capability, so we need equipment to magnified objects which is too small to be seen with unaided eye. There are several types of microscopes but the only one used in this laboratory is the compound light microscope. The compound microscope (sometimes called the student microscope or light microscope); these microscopes are known as compound microscope because there are two magnifying lenses in the microscope. One magnifying lens is in the ocular or eyepiece, which further magnifies the image formed by the objective lens, and one, is in the objective. Each contributes to the magnification of the object on the stage. The total magnification of any set of lenses is determined by multiplying the magnification of the objective by the magnification of the ocular. The nose piece rotates the magnification of the microscope. Generally compound microscope magnifies from 40 x to 1000 x.

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Figure 1.1 A binocular compound microscope Parts of a microscope: The compound microscope is a delicate instrument composed of many parts that are accurately filled together (Figure 1.1). 1. Ocular of eyepiece lens. The ocular lens is the lens you look through, it is inserted at the top of the body tube. If your microscope has one ocular, it is a monocular microscope, if it has two, it is binocular. Its magnification is written on it. 2. Body tube. Body tube is the optical housing for the objective lenses. 3. Objective lenses. The objective lenses are a set of three to four lenses mounted on a rotating turret at the bottom of the body tube. The four objective lenses of your microscope and their magnifications are: Scanning lens 4X magnification Low power lens 10X magnification High power lens 40-45X magnification Oil immersion lens 100X magnification

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The magnification of the objective lens is written on the lens. Note: with the exception of the oil immersion lens all the objective lens is used dry. The magnification of oil immersion lens requires using the lens with special immersion oil for proper resolution. 4. Stage The horizontal surface on which the slide is placed is called the stage. It may be equipped with simple clips for holding the slide in place or with a mechanical stage, a geared device for precisely moving the slide. Two knobs, either on top of or under the stage, move the mechanical stage. 5. Condenser lens. Condenser lens system, located immediately under the stage, contains a system of lenses that focuses light on your specimen. The condenser may be raised or lowered using the condenser knob. An older microscope may have a concave mirror instead. 6. Iris diaphragm Iris diaphragm is located below the condenser or immediately below the stage in microscopes without a condenser. It functions in regulating the light intensity passing through to the stage. More light is required at higher magnification. 7. Light source The light source has an (ON/Off) switch & may have adjustable lamp intensities & color filters. 8. Base Base also called the supporting stand, rests on the bench. 9. Body Arm The body arm is used when carrying the instrument. 10. Nose piece Nosepiece is the mounting for the objective lenses which rotates to bring the desired objective into position. 11. Coarse adjustment Coarse adjustment knob is a large knob located at either side of the microscope which functions in controlling the distance between the objectives and the stage. Use the coarse adjustment only with the scanning (4X) & lowpower (10X) objectives. Why? So coarse adjustment is used for rapid focusing of the specimen until the specimen is roughly in focus & then left alone, in which the fine adjustment knob controls precise focusing of the object. 12. Fine adjustment Fine adjustment is a small knob located at either side of the microscope. This is used for the control of the object, precise focusing you should use just the fine adjustment knob with the higher magnification objective lenses; Because using the coarse adjustment knob with the higher objective lenses may damage the lens &/or the slide you are observing. Magnification: Compound microscopes consist of two lens system: the objective lens, which magnifies, & projects a virtual image into the body tube and the ocular lens, which magnifies the image further and projects the enlarged image into the eye. The total magnification of a microscope is the product of the magnification of the objective and the ocular. If the objective lens has a magnification of 5X and the ocular
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12X, then the image produced by these two lenses is 60 times larger than the specimen. Microscope safety cautions: 1. Always carry the microscope in an upright position using both hands. 2. Keep the microscope away from the edge of the table. 3. Always examine a slide first with the low-or medium power objective, never use the high power objective to view thick specimens. 4. Remove slide only after low-power objective has been rotated into viewing position, never when high power objective is in position. 5. Keep the stage dry at all times. A wet stage will prevent the slide from being accurately positioned. 6. When returning your microscope to its proper place in the cabinet always: Remove the slide from mechanical stage. Clean all lens surface and the stage. Rotate the nosepiece that the scanning lens is in place. Steps Used in viewing a slide: 1. Obtain a slide. 2. Check that the ocular and all objective lenses as well as the slide clean. 3. Use the coarse adjustment knob to obtain maximum working distance. 4. Place the slide on the stage, the slide should fit into the slide holder. Use the stage adjustment knob to move the slide over the hole in the stage. 5. Rotate the lower objective in place. 6. Use the coarse adjustment knob to obtain the minimum working distance. 7. Look through the ocular. Adjust the light with the iris diaphragm lever if necessary. Slowly turn the coarse adjustment knob until something comes into focus. Use the fine adjustment knob to sharpen the focus. 8. Using the stage adjustment knob move the slide around until you find an area you wish to examine more closely. Move the slide until the object you wish to examine is in the center of the field. 9. Rotate the high power objective in to place. Use the fine adjustment knob to sharpen the focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm lever if necessary. 10. Rotate the high power object halfway to the next position, place a drop of immersion oil on the slide, and then rotate the oil immersion objective into place. The objective should be immersed in the oil on the slide. Use the fine adjustment knob to sharpen the focus. Adjust the light using the iris diaphragm lever if necessary. 11. When finished viewing the slide use the coarse adjustment knob to maximize the working distance and remove the slide from the stage. If you are finished with the microscope clean the microscope and return it to storage. Procedure for cleaning a microscope: 1. Turn off the light. 2. Using the coarse adjustment knob to obtain maximum working distance and remove the slide from the stage. 3. Using lens paper cleans all the lenses starting with the cleanest first ocular, and objectives lens. 4. Clean any oil off of the stage using paper towels. 5. Rotate the scanning objective into place. Use the coarse adjustment knob to obtain minimum working distance. 6. Return the microscope to the appropriate storage area. Procedure for cleaning a microscope slide:
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Before placing a specimen on a slide, it must be clean, as any small foreign body might mislead the observation. If your slide is not clean, do the following: 1. Hold the slide from its ends by fingers of one hand. 2. Using a detergent liquid, rub the slide with one finger of the other hand. 3. Wash the slide under running tap water; rub again, until no trace of the detergent is left. 4. Rinse the slide with distilled water to remove the tap water. 5. Either blot dries the slide by placing it between two towel papers, or place in alcohol solution & keep until used. 6. Never touch the slide from the middle, clean slide always holds it from its ends. Preparation of microscope slide: Two types of slides are commonly used in biology classes: permanent slides and temporary wet mounts that you make. 1. Wet mounts Wet mounts are temporary slides that you prepare yourself. When doing a wet mount follow the procedure outlined below: Place the specimen (mixed culture, tissue, etc.) on the center of a clean slide. Add a drop of water or designated stain if required. (Note: liquid cultures do not require adding water) Place one edge of the cover slip on the slide near the specimen (This is done by holding the cover slide at a 45angle). Gently lower the cover slip on top of the specimen. Try to avoid trapping air bubbles. Blot an excess fluid with lens paper before you place the slide on the stage of the microscope. After you have made your observations the slide & cover slip should be washed, dried & replaced in their appropriate locations. 2. Permanent slides Permanent slides have been professionally prepared. They have often been stained to show better contrast of structure. A permanent slide may be a whole mount of the entire specimen or section of the material.

Procedures Exercise 1:

Make a wet mount using a hair from your skin as follows: 1. Pull out a hair from your skin, using a scissor cut the hair leaving about 2cm of the lower part containing the bulb. 2. Place this lower part of the hair in a drop of water in the center of a clean slide. 3. Place a glass cover slip on the specimen by holding the cover slip at a 45 angle to the drop of water and then slowly lowering the cover slip over the drop forcing trapped air out of the preparation (Figure 2). 4. Examine your preparation under the compound microscope use scanning and low poser of the microscope.

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Figure 2, Procedure of a wet mount

Exercise 2:

Make a wet mount of the letter e 1. Using microscope, slide, cover slip, water, scissors, and newspaper make a wetmount slide of a small-case letter "e". 2. Cut a small-case letter "e" from a newspaper. 3. Put the letter "e" on a microscope slide. 4. Using a dropper bottle, put a small drop of water on the letter "e". 5. Cover the letter "e" with a cover slip. 6. Look at the letter "e" using the low power objective lens. 7. What is the total magnification when using low power? 8. Draw what you see. 9. Rotate the nose piece to the medium power objective lens and observe the letter "e". 10. What is the total magnification when using medium power? 11. Draw what you see. 12. Rotate the nose piece to the high power objective lens and observe the letter "e". 13. What is the total magnification when using high power? 14. Draw what you see. In this lab you will set up your microscope and view what a simple letter e cut out from a piece of paper looks like. First let's examine the letter "e" with your naked eye and through a hand lens. Draw what you see in the circles below:

Exercise 3:
Use the microscope with a prepared slide: o Place your letter "e" slide, coverslip side up, on the stage. Use the low power
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o o o o

objective. Secure the slide with the stage clips. Turn on the light. Focus on the letter "e" using the coarse focusing knob. Draw what you see in the circle below. Try using the high power objective. After focusing (fine focus only), draw what you see.

Analysis Questions : What are some of the ways the e you see with the microscope is different from the e you see with the hand lens? If you are looking at the "e" through the microscope and you push your slide to the left, which way does the e in the microscope move? (Try this!) If you push the slide away from you, which way does the e in the microscope move? (Try this!) This phenomenon is known as inversion. Total Magnification of 40X Total Magnification of 100X

Observe the letter "e" with a higher magnification lens. The fine texture of the paper should be more visible. Additionally, some of the letter "e" is now missing from your field of view, since the specimen is too enlarged for you to see all of it at one time. This explains why you must carefully center small specimens within your field of view, before changing lenses. The higher the magnification of the objective lens, the smaller the field of view.

Discussion:
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1. Draw the structure of the hair bulb using the 4X and 10X magnification power. 2. Label the following drawing of a Microscope?

3. What is the function of each of the following: Coarse adjustment knob, Fine adjustment knob, Iris diaphragm. 4. How do you determine the total magnification of a set of lenses? 5. What objective should always be in place, both when beginning to focus and when replacing the microscope for storage? References: 1. Warren, D.D. (1999), Biological Investigations, 5th edition, State University, New York. 2. Tisdale et al. (1986), laboratory Studies in general Biology, Department of biological sciences, An-Najah National University, Nablus.

Total Magnification
Total magnification is calculated by multiplying the magnification of the ocular lens (eyepiece) by the magnification of the objective lens. Calculate the total magnification for each objective, and record your figures in a table. To calculate the total magnification, multiply the power of the ocular lens by the power of the objective lens. Your table should include the powers of both lenses and the total magnification.
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Microscope Value for each ocular unit at 4X (Scanning) Value for each ocular unit at 10X (Low Power) Value for each ocular unit at 40X (High Power) . . .

Total Magnification

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Experiment 2. Morphological Structure of Cells

The cell is the structural and functional unit of all known living organisms. It is contained within a limiting membrane and consists of various organelles suspended in cytoplasm. The morphological structure of cell is nearly correlated with its functions. In the lab, we will observe the morphological structures of several cells under microscope: Neuron Muscle cell Red blood cells and Lucocytes Sperm 1. Neuron Neurons (also known as neurones and nerve cells) are electrically excitable cells in the nervous system that process and transmit information. In vertebrate animals, neurons are the core components of the brain, spinal cord and peripheral nerves. Neurons are typically composed of a soma, or cell body, a dendritic tree and an axon. The majority of vertebrate neurons receive input on the cell body and dendritic tree, and transmit output via the axon. However, there is great heterogeneity throughout the nervous system and the animal kingdom, in the size, shape and function of neurons. Neurons communicate via chemical and electrical synapses, in a process known as synaptic transmission. The fundamental process that triggers synaptic transmission is the action potential, a propagating electrical signal that is generated by exploiting the electrically excitable membrane of the neuron. This is also known as a wave of depolarization. Anatomy and Histology

The slice of spinal cord

2. Muscle cell A muscle fiber, also spelled muscle fibre, also technically known as a myocyte, is a
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single cell of a muscle. Muscle fibers contain many myofibrils, the contractile unit of muscles. Muscle fibres are very long; a single fibre can reach a length of 30cm. Muscle fibres can be grouped according to what kind of tissue they are found in -skeletal muscle, smooth muscle, and cardiac muscle. The muscle cells of heart muscle tissue are called cardiomyocytes. Skeletal muscle is a type of striated muscle, usually attached to the skeleton. Skeletal muscles are used to create movement, by applying force to bones and joints; via contraction. They generally contract voluntarily (via somatic nerve stimulation), although they can contract involuntarily through reflexes. Smooth muscle is a type of non-striated muscle, found within the "walls" of hollow organs and elsewhere like the bladder and abdominal cavity, the uterus, male and female reproductive tracts, the gastrointestinal tract, the respiratory tract, the vasculature, the skin and the ciliary muscle and iris of the eye. The glomeruli of the kidneys contain a smooth muscle like cell called the mesangial cell. Smooth muscle is fundamentally different from skeletal muscle and cardiac muscle in terms of structure and function.

skeletal muscle smooth muscle Smooth muscle fibers are spindle shaped, and like all muscle, can contract and relax. In the relaxed state, each cell is spindle-shaped, 20-500 micrometers long and 5 micrometers wide. There are two types of smooth muscle arrangements in the body: multi-unit and single-unit. The single-unit type, also called unitary smooth muscle, is far more common. Whereas the former presents itself as distinct muscle fibers that are usually innervated by their own nerve fibers, the latter operate as a single unit and are arranged in sheets or bundles. Unitary smooth muscle is also commonly referred to as visceral smooth muscle because it is found in the walls of the viscera, or internal organs, of the body, including the intestines, ducts such as the bile ducts, ureters and oviducts, and most blood vessels. Unitary smooth muscle can be further divided into phasic and tonic. The cells that compose smooth muscle generally have single nuclei. The cells are generally arranged in sheets or bundles and connected by gap junctions. In order to contract the cells contain intracellular contractile filamentous proteins called actin and myosin. While the filaments are essentially the same in smooth muscle as they are in skeletal and cardiac muscle, the way they are arranged is different (and some regulatory proteins are different). The smooth muscle cell contains less protein than a typical striated muscle cell and much less myosin. The actin content is similar so the ratio of actin to myosin is ~6:1 in striated muscle and ~15:1 in smooth muscle. Smooth muscle does not contain the protein troponin, and caldesmon and calponin are significant proteins
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expressed within smooth muscle. As non-striated muscle, the actin and myosin is not arranged into distinct sarcomeres that form orderly bands throughout the muscle cell. However there is an organized cytoskeleton consisting of the intermediate filament proteins vimentin and desmin, myosin filaments and actin thin filaments. Actin filaments attach to the sarcolemma by focal adhesions or attachment plaques and attach to other actin filaments via dense bodies (acting much like Z-lines in striated muscle). Evidence indicates that smooth muscle myosin filaments are not bipolar with a central bare zone as in striated muscle, but is either side-polar or row-polar and have no bare zone. Some smooth muscle preparations can be visualized contracting in a spiral corkscrew fashion, and contractile proteins can organize into zones of actin and myosin along the axis of the cell. 3. Red blood cells and Lucocytes Red blood cells are the most common type of blood cell and the vertebrate body's principal means of delivering oxygen from the lungs or gills to body tissues via the blood. Human red blood cells are also known as RBCs, haematids, or erythrocytes (from Greek erythros for "red" and kytos for "hollow", with cyte nowadays translated as "cell"). A schistocyte is a red blood cell undergoing fragmentation, or a fragmented part of a red blood cell. The diameter of a typical human erythrocyte disk is 68 m, much smaller than most other human cells. A typical erythrocyte contains about 270 million hemoglobin molecules, with each carrying four heme groups. Adult humans have roughly 23 10 13 red blood cells at any given time (women have about 4 to 5 million erythrocytes per microliter (cubic millimeter) of blood and men about 5 to 6 million; people living at high altitudes with low oxygen tension will have more). Red blood cells are thus much more common than the other blood particles: There are about 4,00011,000 white blood cells and about 150,000400,000 platelets in each microliter of human blood. The red blood cells store collectively about 3.5 grams of iron, more than five times the iron stored by all the other tissues combined.

Erythrocytes: (a) seen from surface; (b) in profile, forming rouleaux; (c) rendered spherical by water; (d) rendered crenate by salt. (c) and (d) do not normally occur in the body.

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neutrophil

acidophil

basophil

lymphocyte monocyte blood platelet 4. Sperm The term sperm is derived from the word spermos (meaning "seed") and refers to the male reproductive cells. Sperm cells are the smaller gametes involved in fertilization. In these types of sexual reproduction, there is a marked difference in the size of the gametes with the smaller one being termed the "male" or sperm cell. A uniflagellar sperm cell that is motile is also referred to as spermatozoon, whereas a non-motile sperm cell is referred to as spermatium. Sperm cells cannot divide and have a limited life span, but they can fuse with egg cells during fertilization to form a totipotent zygote with the potential to develop into a new organism. The spermatozoa of animals are produced through spermatogenesis inside the male gonads (testicles) through meiosis. Sperm cells in algal and many plant gametophytes are produced in male gametangia (antheridia) through mitosis. In flowering plants, sperm nuclei are produced inside pollen.

Different types of sperm cells: human sperm A) spermatozoon (motile), B) spermatium (non-motile), C) fertilization tube with sperm nuclei.
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Exercise
In this investigation, you will compare the structures of a typical plant cell (onion) and a typical animal cell (your own!) Problem: How are plant and animal cells alike? How are they different? Materials: Forceps Medicine Dropper Elodea Leaf Water Microscope Glass Slide Coverslip Toothpicks Methylene Blue Stain Paper Towel Lens Paper Safety: Put on safety goggles. Always handle the microscope with extreme care. You are responsible for its proper care and use. Use caution when hadnling glass slides as they can break easily and cut you. Always use special caution when working with laboratory chemicals, as they may irritate the skin or cause staining of the skin or clothing. Never touch or taste any chemical unless instructed to do so.

Exercise 1: Human epithelial cells

Epithelial cells cover the body's surface and line its cavities. Obtain toothpick. Gently scrape the inside of yourcheek with the toothpick. Place the scrapings on a clean, dry slide. Add a drop of iodine stain and cover with a coverslip. Observe under the microscope, using the directions for focusing given above. Start with the scanning power objective to find some cells, then observe under both low and high power. 6. Locate the cell membrane, the cytoplasm, and the nucleus. 7. Make a drawing of what you see. Be sure to label the drawing with what it is, and what power you were using when you made your drawing. 1. 2. 3. 4. 5.

Exercise 2: Plant cells

1. 2. 3. 4. 5. 6. 7. 8. With your fingers, strip a thin, transparent layer of cells from a piece of onion. Place it gently on a clean, dry slide. Add a drop of iodine stain and cover with a coverslip. Observe under the microscope and draw what you see. Be sure to label your drawing. Locate the cell wall. Is a nucleus visible? Count and record the number of cells across the diameter of the high power field, both lengthwise and side to side. Using the number you calculated in exercise 3, calculate and record the length and width of an onion cell in micrometers. Draw what you see. Be sure to label your drawing.

Discussion:

In your lab report, please be sure to answer the following questions in your discussion section. Do not write them as question and answer, simply contain their answer in your report. 1. How are plant and animal cells different in structure? 2. How are plant and animal cells similar in structure? 3. Why are stains such as methylene blue used when observing cells under the microscope?

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Experiment 3. Osmosis and Diffusion

How substances get into (and out of) cells? Problem: What are some factors that influence the movement of materials through a semi-permeable membrane?
Osmosis: The membrane of the cell or of various subcellular organelles (chloroplasts, mitochondria) serves as a regulatory structure by controlling the entry or exit of substances into and out of the cell. The membrane may be permeable, impermeable or partially permeable to a given substance. In general, membranes are freely permeable to water molecules. In cells, water movement through the cell membrane is determined by the process of osmosis. Osmosis is the net movement of water across a partially permeable membrane from a region of high solvent potential to an area of low solvent potential, up a solute concentration gradient. It is a physical process in which a solvent moves, without input of energy, across a semi permeable membrane (permeable to the solvent, but not the solute) separating two solutions of different concentrations.

Fig. 1. Hypertonic, Isotonic and Hypotonic Diffusion: Diffusion is the net movement of molecules from a region of high concentration to a region of low concentration. This differs from osmosis which is the movement of water across a semi-permeable membrane from an area of higher concentration of H2O to an area of lower concentration of H2O.

Diffusion-Plasmolysis
The purpose of this activity is to investigate the effects of a hypertonic solution on the
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cells of the red onion. You will understand water that passes through the membrane of th plant cells. MATERIALS (per student): red onion epidermis, forceps, dropper, distilled water, 5% Sodium Chloride (table salt) solution, paper towels, microscopeor magnifying glass, slide or saucer, and cover slip. PROCEDURE: 1. Make a wet mount of the red onion epidermis. 2. Examine under 100X. When you have a clear view of several cells, switch to 430X. Make a colored drawing, properly labeled in the first circle on the data sheet (Fig. 2). of your cover slip while placing a small piece of paper towel along the opposite edge of the cover slip. The paper should draw out the water and draw in the salt solution. 3. Make sure that the salt solution has time to diffuse under the cover slip. 4. Observe the affects of the solution on the onion cells. Make a properly labeled, colored drawing of the cells' appearance in the second circle on the data sheet (fig. 3). 5. Replace the sodium chloride solution with distilled water and add it in the same way that the salt solution was added. Make a properly labeled, colored drawing of the cells' appearance in the third circle on the data sheet (Fig. 4).

Diffusion Through a Membrane

To study the cell membrane, you will employ a simple procedure. Experiments often utilize a simplified version of the situation that is being investigated, a model or a system that is acceptably similar to the actual case, but which is usually easier for the experimenter to control. Purpose: To investigate the diffusion of a substance across a semipermeable membrane. Materials:

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tincture of iodine cornstarch/water solution plastic sandwich bags (cheap is good) beakers or clear plastic cups

Materials: glucose test strip, cup or beaker. dialysis tubing, glucose solution, starch
solution, and water.

Procedure
1. Obtain a beaker (about 400 ml) or a cup as provided by your instructor and fill it approximately 2/3 full of water (preferably distilled water). 2. Use a glucose test strip to test the water for the presence of glucose. Record your results. 3. Take an approximately six inch length of membrane tubing and soak it in warm ater until it becomes pliable. 4. Tie one end and place about an inch of starch solution in the bag. 5. After putting the starch solution in the bag, add about an inch of glucose solution to the bag. 6. Use your fingers to squeeze the bag on the outside to mix the starch and glucose solution thoroughly in the bag. 7. Place 30-35 drops of Lugol's solution (iodine) in the water in the beaker or cup. (Enough iodine should be added until the water in the cup is light brown. 8. Test the bag to be certain that the starch solution does not leak from either end and that there are no holes in it. Wash the bag with water to remove any starch or glucose adhering to its surface; give special attention to the tied ends. 9. Place your bag which has now been tied on both ends and rinsed into the cup or beaker containing the iodine solution. 10. Wait for approximately 10 minutes and record your observations.

A "possible" arrangement to help you organize your data appears below.

Material Tested Result before running the experiment iodine (Lugol's) glucose solution starch solution Your procedure needs to include a drawing of the experimental setup for this activity with the components labeled. Result after running the experiment

Things to test or comment on in your data.

a.) Is there glucose in the water in the cup after 10 minutes? (Use the glucose test
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strip to test this.) b.) Was there a change in the color inside the bag? c.) Was there a change in the color of the water in the cup outside the bag? Hint: If there was a significant change, you did something wrong with this experiment.)

Questions which should be answered in your conclusion.

1. Did glucose leave the inside of the membrane and go into the beaker? How did you prove this? 2. Did starch leave the inside of the membrane and go into the beaker? How did you prove this? 3. Did iodine enter the membrane from the water in the beaker? How did you prove this? 4. Based on this investigation, form a tentative conclusion in reference to how the size of molecules influences their ability to diffuse through a semi-permeable membrane. Explain/justify your answer. 5. You didn't investigate the diffusion of water (osmosis) in this experiment, but if you did, do you think water would diffuse into the bag from the cup or out of the cup into the bag? How could you set up an experiment to test your hypothesis about the movement of the water

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Experiment 4. Identification of chemical constituents of the cell

Experiment 4.a: Identifying the presence of glucose in food items Experimental goal:
Identifying the presence of glucose in honey and onions.

Introduction
In this experiment you will identify the presence of glucose in food product using the blue colored, benedict solution. The copper ions undergo a reduction reaction in the presence of glucose and change color from blue to orange. Safety: Put on your safety goggles, lab apron and plastic gloves. Tie back long air.

Materials (per a team of 2)

1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. A spatula A plate with diced onions A jar of honey Powdered glucose Powdered sucrose 4 test tubes A bottle of water Benedict solution in a dropper bottle A waterproof marker

Procedure:
Mark the test tubes from 1 to 4. Using the spatula, put some powdered glucose in test tube #1. Add 1 ml water and shake test tube until glucose is dissolved. Repeat steps 2-3 with the, sucrose powder, honey and diced onion (use test tubes #2, #3 and #4). 5. Add 15 drops of the Benedict solution to the test tubes and swirl the test tubes gently. CAUTION: Benedict's solution is an irritant. Avoid skin /eye contact; do not ingest. Wash off spills and splashes with water for 15 min; rinse mouse with water. Call your teacher. 6. Put the tubes in a large beaker that contains boiling water (the beaker is standing on a heater). 7. Watch the changes in the Benedict solution color.

CAUTION: Boling water will scald, causing second-degree burns. Do not touch the beaker or allow boiling water to contact your skin. Avoid vigorous boiling. Handle test tubes with a test tube clamp. Immediately place the burned area under cold running water.

Results:
Write your results in the following table: Test Contents Water tube No. of test tube
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Benedict Color of solution solution

Benedict

1 2 3 4

glucose Sucrose honey Diced onion

+ + -

+ + + +

Analysis
1. 2. 3. In which test tubes does a change in color occur? What purpose does the powdered glucose serve? What purpose does the powdered sucrose serve?

Analysis answers: 1. Test tubes number 1,3, 4 . 2. The glucose serves as a positive control, showing that Benedict solution can be used as an indicator for glucose. 3. The sucrose serves as a negative control, showing that the Benedict solution does not change color in the presence of any sugar other than monosaccharides as glucose.

Experiment 4.b: Identifying the presence of starch in food items Experimental goal:
Identifying the presence of starch in various foods.

Introduction
To test for the presence of starch in various foods, we will use a Logol* solution whose color is brownish-yellow when no starch is present and a purple-blue color when starch is present. Focus on carbohydrates (sugars) Sugars are made up of carbon (C), hydrogen (H) and oxygen (O) atoms. The sugar molecules are differentiated by the number of carbon, hydrogen and oxygen atoms that make up a basic unit and in the number of basic units that each sugar molecule has (i.e. a simple sugar, a disaccharide and a complex sugar). The glucose molecule has a hexagonal ring shape with five carbon atoms (shown as numbers) and one oxygen atom. An OH group is bound to carbon atoms #1, #2, #3 and #4 and a CH2OH is bound to carbon atom #5.

Starch is made up of long chains of glucose molecules that are chemically bound to each other.

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*Logol Iodine dissolved in a potassium iodide solution: I2/KI.

Materials (per a team of 2)

1. 2. 3. 4. 5. 6. 7. A Petri dish with starch powder (dish #1) A Petri dish with diced potatoes (dish #2) A Petri dish with presoaked beans (dish #3) A Petri dish with diced bread slices (dish #4) A Petri dish with banana slices (dish #5) A Petri dish with cucumber slices (dish #6) A dropper bottle filled with a 0.1% Logol solution.

Procedure: Safety: Put on your safety goggles, lab apron and plastic gloves. Tie back long hair.

Add two drops of the Logol solution to each plate and shake gently. Watch the changes in the logol color.
CAUTION: Lugol's iodine solution is irritating to skin and eyes and can stain clothing. Wash off spills and splashes with water for 15 min; rinse mouse with water. Call your teacher.

Results:Write down your results in the following table

Dish number 1 2 3 4 5 6 Contents of dish Color of Logol solution

Analysis
1. In which foods was a color change observed? 2. What purpose does the cucumber slices serve? 3. What purpose does the starch powder serve?
Analysis - answers: 1. Color change can be observed in the potato, beans, bread and banana. 2. The cucumber slices serves as negative control, showing that the color of the Logol will not change in any food. 3. The starch powder serves as a positive control , showing that the Logol can be used as an indicator for starch.

Experiment 4.c: Identifying the presence of protein in food items Experimental goal:
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Identifying the presence of protein in milk powder and tuna.

Introduction
The Biuret reaction: In order to identify proteins in a solution, we will be using a basic solution of copper ions (Cu++), called Biuret solution, whose color is blue. The copper ions are reduced when they bind to carbon atoms on amides. Amides, or peptide bonds, are the bonds that bind amino acids in proteins. This reduction reaction changes the color of the solution from blue to purple.

Materials (per team of 2)

1. A spatula 2. Albumin (OVA/BSA). 3. Milk. 4. A can of tuna. 5. Egg yolk. 6. Sucrose powder. 7. 6 test tubes. 8. A bottle of water. 9. Biuret solution #1, 6M NaOH in a dropper bottle. 10. Biuret solution #2, a 1% CuSO4 solution in a dropper bottle.

Procedure:
Safety: Put on your safety goggles, lab apron and plastic gloves. Tie back long hair. 1. Number the test tubes sequentially from 1 to 6. 2. Put some albumin then add 10-20 drops of Biuret solution #1. Gently stir the test tube 3. Add 10-20 drops of Biuret solution #2. Gently stir the test tube 4. Incubate for 5 minutes at room temperature. 5. Repeat steps 2-4 with 1 ml milk, tuna chunks, egg yolk and sucrose powder. Dissolve the sucrose in 1 ml water. Use test tubes #2, #3 #4 and #5, 6. Repeat steps 2-4 with albumin, but instead of adding the Biuret solutions, add an equal volume of water (use test tube #6).

Results:
Write down your results in the following table: Test Contents of test Biuret Water tube No. tube solutions 1 2 3 4 5 6 Color of Biuret solution

Analysis
1. In which test tube did you see a change of color from blue to purple?

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2. Explain the importance in using sucrose powder and water instead of using the Biuret solution? 3. There is a linearic correlation between the intensity of the purple color and the amount of protein in solution. The more peptide bonds are in solution, the more intense the purple color. Can you propose an experiment that will quantify the amount of protein in solution?
Analysis-answers 1. In test tubes 1-4, a change in color from blue to purple can be seen. 2. The sucrose powder is used as a negative control, indicating that the Biuret reaction identify protein and does not change its color in the presence of every material. The use of water instead of Biuret solution is a control that indicates that the change in color is a result of the interaction between the protein and the Biuret reagent. 3. The amount of protein can be quantified by using a spectrophotometer. A spectrophotometer is an instrument that measures the light that is absorbed by the sample. As the intensity of the color increase, less light will go through the sample.

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Experiment 5 and 6. Cell culture

Introduction
One of the major breakthrough in studying mammalian cell biology was the ability of replicate cells outside the living organism. This allowed for one of the areas for modern biotechnology to take hold. Many important therapeutic proteins are produced by cell culture. The monoclonal antibody used in research and several medical tests requires that the antibody producing hybridoma to be grown in culture. Although, sometimes difficult, mammalian cell culture is a highly valued technique for research in the modern biology laboratory.

Growth in a mammalian cell culture

The growth of mammalian cells in artificial environments requires several different components and good aseptic techniques. Since the environment is artificial, the investigator can strictly control the environment. This environment usually requires specific temperature, gas environment, pH, specific nutrients, and a growth surface. We will be using a human carcinoma cell line. It has some specifics that allow us to carry out our experiments with greater ease but still requires many of the same features. Temperature-usually body temperature (37) and needs to be tightly controlled. Gas Environment-usually requires 10% CO2 and humidity. The CO2 is important in many cell cultures for maintaining proper pH. Our cell line does not require CO2. The humidity is important in the loss of fluid due to evaporation. pH- Very important in cell cultures and is followed by the color pH indicator Phenol Red (you used this in the microbiology labs). If the culture is too acidic, it will change to an orange and later to a yellow. This can indicate bacterial or fungal contamination. If the color of the media goes to a purple red color, the culture is becoming alkaline. This indicates the culture is not able to maintain proper pH usually due to lack of CO2. Nutrients- the normal amino acids, salts, sugars, vitamins, minerals, and very importantly, serum. Serum contains growth factors that are needed for inducing the cells to grow. Many cell lines are serum independent but we will use one that is dependent on serum. Growth surface-Many of the mammalian cell cultures require a surface to grow on. In our case, the plastic bottom of the plate will be the surface that the cells attach to. Some cell lines actually require specific proteins to be coated on the plate before they will grow.

Basic viewing of cell cultures

When you look at a cell culture for health (of major importance if you are interested in studying them), you can do a couple of quick checks to see if the cells are doing well or if you have problems.
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pH- as mentioned above, look at the color of the media. If it is orange or purple red, you may have some problems starting. Cell attachment- most cells should be spread out on the surface of the culture flask. You can check this with the inverted microscope. If you have round cells floating in the media, the culture could be having problems. Confluence-cells do not like growing over each other. You can get an idea if the cells are ready to pass to new culture flask or plate by looking at the confluence. Look in the inverted microscope and figure out how much of the plate or flask is covered by cells. Half of the plate would be considered 50% confluence. You should pass when you get to 80% or more.

Aseptic Technique

The most important aspect of mammalian cell culture is the use of aseptic technique. You can have the best preparation in the world but if you cell culture is contaminated with bacteria or fungi, the culture will be quickly overrun by the contamination. I will go into aseptic technique with you in great detail in the laboratory.

Procedure

The first day will be based on transferring you cell culture to new plates. To do this, you must break the adhesion between the cells and the culture flask and then transfer the cells into new culture flasks and allow them to attach. I will do one set of plates to show you the procedure. Passing Cells to New Culture Flask 1. After sterilizing the area with ethanol, pipet out the old media from the culture flask and pipet the waste media into the waste container. 2. Wash the monolayer of cells with 5 ml of calcium, magnesium free phosphate buffer (CMF-PBS). This is done by pipetting in the CMF-PBS and rocking back and forth. Pipet out the CMF-PBS into the waste container. This is done to wash away the calcium and magnesium. Remember how these divalent cations are needed for certain adhesion proteins. When you remove these cations, you loosen the cells. 3. Add 1 ml of 1 X Trypsin/EDTA solution to the flask and rock back and forth a couple of times. The trypsin is a protease that cleaves the adhesion proteins keeping the cells on the culture flask. Check the cells in the inverted microscope every couple of minutes for the presence of rounded up cells. 4. When sufficient number of cells are rounded up and loose from the culture flask, add 10 ml of L-15 ++ media (Leibovitz 15 with 10% fetal calf serum and antibiotics) to the flask. The media contains trypsin inhibitors to stop the trypsin reaction. 5. Pipet the cells up and down several times to resuspend the cells in the new media.

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6. Pass cells (pipet the cells) evenly to two new flasks. Label cultures with date of passage and place back in the incubator. The cells should attach to the surface to the culture flask in a couple of hours. If you are around, you can check the flask on the inverted microscope. 7. The cells will need to be fed every other day and passed to new flasks at least once before next week. I will have a sign-up sheet for student to come in and check you cultures. Resuscitation of Frozen Cell Lines 1. Read Technical data sheet to establish specific requirements for your cell line. 2. Prepare the flasks as appropriate. Label with cell line name, passage number and date. 3. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container. 4. Still wearing protective clothing, remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e.g. 37C for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety cabinet. 5. Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid. 6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO. 7. Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere. 8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary. Cryopreservation of Cell Lines 1. View cultures using an inverted microscope to assess the degree of cell density and confirm the absence of bacterial and fungal contaminants. 2. Bring adherent and semi adherent cells into suspension using trypsin/EDTA and re-suspend in a volume of fresh medium at least equivalent to the volume of trypsin. Suspension cell lines can be used directly. 3. Remove a small aliquot of cells (100-200uL) and perform a cell count. Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing. 4. Centrifuge the remaining culture at 150g for 5 minutes. 5. Re-suspend cells at a concentration of 2-4x106 cells per ml in freeze medium. 6. Pipette 1ml aliquots of cells into cyroprotective ampules that have been labeled with the cell line name, passage number, cell concentration and date. 7. Place ampules at 80C overnight. 8. Frozen ampules should be transferred to the vapor phase of a liquid nitrogen
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storage vessel and the locations recorded. References: http://www.sep.alquds.edu/biology/scripts/Biology_english/ http://slohs.slcusd.org/pages/teachers/bsmith/ http://snhs-plin.barry.edu/cell-biology-lab/cell_biology_lab.htm http://www.umanitoba.ca/Biology/lab3/

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