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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 4 .

Follow the safety instructions given by your teacher. 1. eating. 9. 8. especially at the end of the laboratory period. When an object is removed from the heat & left to cool. Wear safety goggles when using dangerous chemicals. Inflammable liquid bottles should not be left open. If you have an open skin wound. 4. 11. 5. or drinking in the lab room is totally prohibited. Chemical Spill Kits. 6. Follow all instructions carefully. Report all chemicals spills or fluids to your instructor immediately for proper clean up. not dispensed near a naked flame. When you are heating something in a container such as a test tube. 12. Keep your work area clean & dry. 3. 5. Use special care when you see the word CAUTION in your laboratory instructions. be sure that it is covered with a water proof bandage. 5 . 7. Use only Pyrex glasswares for heating. hot electric element or electric motor. Never work alone in the laboratory. 2. 10. or preserved specimens. always point the open end of the container away from yourself & others. water. 4. Turn of all electrical equipment. Chemical safety showers and Eye washers. Throughout this manual safety recommendations are given. Determine the location of Fire Extinguishers. • General laboratory safety precaution. Wear lab aprons when working with chemicals. Tie back long hair. hot liquids. Any chemicals spilled on the hands or other parts of the skin should be washed off immediately with a plenty of running water. it should be placed where it is shielded from contact. 2. Use test tube holders to handle hot laboratory equipments. Never leave a lighted Bunsen burner of hot object unattended. or burners. and alternative exit routes for lab evacuation. below are some general consideration that anyone in a laboratory should know. • Special precautions for working with heat or fire: 1. and gas when it is not in use.Laboratory Safety Safety in the Laboratory should always is in your mind. Remember that smoking. 3. hot material.

Direct sun light can damage the eyes. 2.6. 4. 2. • Special Precaution for working with Glasswares and other laboratory equipments. 4. Never smell substances in the laboratory without specific instructions. Use materials only from containers that are properly labeled. 1. Become familiar with the names and appearance of all the laboratory equipments you will use. 5. 3. Wash your hand after working with chemicals. • Special precautions for working with electrical equipment. Do not try to cut a specimen while holding it in the air. 4. • Special precautions for working with chemicals 1. If live animals are used treat them gently. 3. • Special precautions for working with live or preserved specimens. Never touch electrical equipment with wet hands. Never taste or touch substances in the laboratory without specific instructions. Always wash your hands after working with live or preserved organisms. Do not pick up broken glass with your bare hands. Specimens for dissection should be properly mounted and supported. Notify your teacher immediately. 7. Use a pan and a brush. Follow instructions for their proper care. 5. such as scalpels and dissecting needles. 3. Instead. 6. 4. dilute the acid by adding it to water. Make sure that all glasswares are clean before you using it. 2. 1. Do not add water to acid. 5. Make sure that Bunsen burner hoses fit tightly. 3. Mix heat generating chemicals slowly. Do not aim the mirror of your microscope directly at the sun. Do not open Petri dishes containing live cultures unless you are directed to do so. Allow hot materials to cool before moving them from your lab station. Make sure the area under & around the electrical equipment is dry. Detergents (detol 5 – 10%) should be used to sterilize and clean benches. glassware and equipment. 6. 7. Never use broken or chipped glassware. Make sure the area surrounding the electrical equipment is free of flammable materials. Use care handling all sharp equipments. 6 . Turn off all power switches before plugging an appliance into an outlet. do not touch the mercury. If a Mercury thermometer breaks. 2. 8.

8. 7 . Lab coats should be worn during the work in the lab. Safety cabinet should be used while working with microbes. 7.6. Disposable items should be collected and autoclaved.

special cream for burns can be used. It should be written in a very specific and precise format as if it were to be submitted for publication in a scientific journal. and do not use shortcuts in describing any part of the experiment. A laboratory report is a scientific discussion of the experiments or observations that you performed in your laboratory section. Writing style Your paper must be organized into complete sentences and paragraphs. Skin contamination requires washing with water and removal of contaminated clothing. 4. and you should share nothing other than your lab results with other students in the lab. Poisoning: if any toxic chemical is swallowed. 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. milk is drunk. The report is to be your own INDIVIDUAL responsibility. Eye burns must be washed using eye washer. even if the results were not those expected. Some portions of a report may require that you report on observations. in case of acid. Injuries: bleeding should be reduced using bandages.First Aid 1. with each paragraph beginning with a topic sentence that indicates the content for that paragraph. the mouth must be sensed with water. the wound should be cleaned with iodine alcohol mixture. DO NOT assume that the reader knows what you mean. Acid and fire burns: body burns must be washed immediately with tap water. unless otherwise indicated by the laboratory instructor. Your report will be read critically for scientific content as well as your ability to express yourself clearly and concisely. 2. and wrapped with sterile bandage. 3. Thus you should write it as if your audience were learning about what occurred in the laboratory for the first time. A scientific report MUST be truthful. You will have an opportunity to discuss what was expected and why your 8 . including your exact results. Do not look at another student’s report as a model for your own. You must explain everything in detail. which may be best presented using colored pencils to indicate the results in a drawing. in case of alkaline. diluted acetic acid ( vinegar) can be used. Guidelines for Laboratory Reports General information Each student is responsible for writing his or her own laboratory report. Remember that this is a scientific report. Be sure that you include and discuss only what actually happened. if the contaminant is insoluble in water remove with soap and water. in which clarity and organization are important components.

your name. perhaps with Roman numerals to identify each part. be sure to make the proper citation within the text. All discussion of what was done in the laboratory experiments must be presented in the past tense. and day. the due date of the report’s submission. Another component of the introduction is a brief description of the specific questions that are being approached in your experiments. Cover Page – This should contain the title of the report. You should then provide a brief discussion of the methods used in the experiments. until you have given an overview of everything to be covered in your report.results may have deviated from the expected results. (4) results. should begin this section. The use of “we” or “I” is acceptable to avoid the passive voice. and then proceed to more specific statements. the name of your lab partner(s). 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. 9 . rather than passive. (5) discussion. in Materials and Methods and again in Results. be sure you give a proper citation in the text of your report and list the complete reference at the end of the report. and also list the original in the References section of the report. and the name of your instructor. but these terms should be kept to a minimum. If these methods are taken directly from your laboratory manual or another reference. Introduction – This gives an introduction to your experiment by providing a brief background about the topic and a context for your activities. It may even be a sentence that summarizes the results of the experiment. you should state the hypothesis at the end of the Introduction section. (3) materials and methods. Your introduction should begin with general statements about your topic. 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. genus and species names should be italicized (preferred) or underlined Report format The report will include the following components: (1) a cover page. the laboratory section. The report should be written with as few words as possible to keep it clear and concise. Be sure the title of the report is very informative. Materials and Methods – A description of the materials being used. Use active verbs to describe what happened. In this case. you may find that greater clarity is produced by discussing each experiment separately. This would include what information is known from previous studies and what further information you expect to obtain from your experimentation or observations. time. explaining how everything was done. you must indicate any changes in those methods that were actually used in your experiments. you may refer the reader to them there rather than repeating them in your report. If you refer to methods from another source. If you must use scientific nomenclature to identify an organism used in the lab. (2) introduction. If you are testing a hypothesis in your experiment. If you refer to your text or other references in citing these previous studies. and (6) references. If several experiments concerning the same topic are being discussed in your report. possibly with diagrams or drawings if necessary. since you are writing about what occurred at a previous time.

Types of data that you might include in this section are (1) any general observations you made during the experiment. such as labeling the X-axis and the Y-axis of a graph. Begin with your major conclusions that were obtained from your results. place the information in an Appendix after the Discussion section. and unexpected results should be addressed in the Discussion section of your report. DO NOT change any data to fit your expectations better. usually shown in figures or tables. without discussion of why or how the results occurred. Each table or figure should also have a number by which you can refer to it in the text of your results section. including temperatures in degrees Celsius. and tell whether the results support or do NOT support your hypothesis. so don’t make such a statement. temperatures. Use the analytic methods indicated by your laboratory manual or your instructor.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. Be sure you include appropriate quantities. and present the data and tests in a figure and/or a table. If your instructor says you must include raw data and any calculations required to summarize the data. and each table or figure should have a detailed caption that explains its contents. It is very important that scientific data be reported honestly. a diagram. (2) all quantitative results. A figure may be a graph. since you are interpreting the results in light of the hypothesis that was being tested. but summarize the data for greater clarity. or unexpected data that might result from following the methods incorrectly. Discussion – This is usually the most important portion of your laboratory report. weight or volume of substances being used. Do not include raw data in this section. Results – In this section you will begin with a text discussion of what results were obtained from each part of the experiment. you need to explain 10 . The Results section should contain only the results. times of treatments. Report ALL data you obtain as results in any experiments you perform. Since many of the details of the results are in tables or figures. you must include any details that would affect the reader’s ability to repeat your experiment. if appropriate. but you should leave out irrelevant details that do not affect the repeatability of the activity. In this case. Be sure all required labels are included. quantities should be reported using the metric system. Thus. exactly as it is obtained. the information should be presented from the more general statements to the more specific details. Again. You should identify only the important points of each figure or table within the text. or each column of a table. or a picture. and (3) the results of statistical analysis of your results. Your results will also probably not be sufficient to prove a hypothesis false. etc. Remember that you cannot prove a hypothesis true. You may wish to include leader lines and identification labels on a diagram for greater clarity. but you may have results that do not support the hypothesis. or you may refer to any tables or figures in which the results are presented. you do not need to cover these points again in the text of this section. Since this is a scientific report. You may make reference to data directly in the text of this section if there are not many numbers to report. including size of containers. Do not omit data that do not support your hypothesis.

Each grammar error (wrong tense. or compare your results with the results of other studies to which you make reference. Do you adequately support your argument? 5. Content x 75% = . III. 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. Spelling counts 10% of the total grade. Is your information accurate? 2. Do you support your conclusions with the appropriate statistical test(s)? Name______________________ Grades Spelling x 10% = . 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. Low grades in grammar can be avoided by proofreading your work before you submit it and by writing practice essays. Each different spelling or typographical error will usually result in a point deducted from the maximum. References – If any literature has been cited in the text of your report. Remember also that you might get unexpected results even if you did everything exactly as specified in your experiment. The kinds of questions that are considered in evaluating content include the following: 1. II. your results contradict the hypothesis. especially if the results were unexpected. poor sentence of paragraph structure) will usually result in a point deducted from the maximum. you should try to offer an interpretation of why you obtained the results you did. However. such as your laboratory manual. Grammar counts 15% of the total grade. and how this would have produced the results you obtained. you must give a full reference to each book or article cited. 11 . Do you adequately correlate and contrast your data to previous experience? 6. COMPOSITE GRADE . Low grades in spelling can be avoided by keeping a dictionary on hand and proofreading your work before you submit it for review. This is where you would discuss what you might have done incorrectly in the experiment. Grammar x 15% = . and perhaps compare your results with those of other students in your section (if their results are provided to you). and content. After discussing your results and how they relate to the hypothesis and other work on the topic. Is your discussion logical? 3. by the first letter of the first author’s last name. Did you transform the raw data into a more useful and appropriate format? 4. it will be deducted only once. if one word is consistently misspelled. Present these in alphabetical order. Content counts 75% of the total grade. Remember to cite the studies in your discussion and give a complete reference to the book or article in your Reference section.

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is in the objective. There are several types of microscopes but the only one used in this laboratory is the compound light microscope. The Microscope Objectives:: This Exercise focuses on how to develop a working knowledge of the Microscope and its use. The compound microscope (sometimes called the student microscope or light microscope). which further magnifies the image formed by the objective lens. and many small organisms are beyond our visual capability. The total magnification of any set of lenses is determined by multiplying the magnification of the objective by the magnification of the ocular. tissues. 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. 13 . Each contributes to the magnification of the object on the stage. and one. these microscopes are known as compound microscope because there are two magnifying lenses in the microscope.1 mm in diameter. The nose piece rotates the magnification of the microscope.Experiment 1. Generally compound microscope magnifies from 40 x to 1000 x. Students should identify the different parts of the Microscope. so we need equipment to magnified objects which is too small to be seen with unaided eye. cells. List and follow recommended procedures in using and caring for the Microscope. One magnifying lens is in the ocular or eyepiece.

Figure 1. 1. The ocular lens is the lens you look through. If your microscope has one ocular. Body tube is the optical housing for the objective lenses. 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 14 . 3. if it has two. Objective lenses. it is a monocular microscope. Its magnification is written on it.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). Body tube. Ocular of eyepiece lens. The objective lenses are a set of three to four lenses mounted on a rotating turret at the bottom of the body tube. 2. it is inserted at the top of the body tube. it is binocular.

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. Nose piece Nosepiece is the mounting for the objective lenses which rotates to bring the desired objective into position. move the mechanical stage. contains a system of lenses that focuses light on your specimen. An older microscope may have a concave mirror instead. Fine adjustment Fine adjustment is a small knob located at either side of the microscope. Body Arm The body arm is used when carrying the instrument. It functions in regulating the light intensity passing through to the stage. If the objective lens has a magnification of 5X and the ocular 15 . Condenser lens. Light source The light source has an (ON/Off) switch & may have adjustable lamp intensities & color filters. Two knobs. It may be equipped with simple clips for holding the slide in place or with a mechanical stage. precise focusing you should use just the fine adjustment knob with the higher magnification objective lenses. 11. in which the fine adjustment knob controls precise focusing of the object. 6. Magnification: Compound microscopes consist of two lens system: the objective lens. More light is required at higher magnification. a geared device for precisely moving the slide. The condenser may be raised or lowered using the condenser knob. 9. 12. Iris diaphragm Iris diaphragm is located below the condenser or immediately below the stage in microscopes without a condenser. Condenser lens system. 7. which magnifies. which magnifies the image further and projects the enlarged image into the eye. Because using the coarse adjustment knob with the higher objective lenses may damage the lens &/or the slide you are observing. Base Base – also called the supporting stand. & projects a “virtual image” into the body tube and the ocular lens. This is used for the control of the object. Use the coarse adjustment only with the scanning (4X) & lowpower (10X) objectives.The magnification of the objective lens is written on the lens. rests on the bench. Note: with the exception of the oil immersion lens all the objective lens is used dry. Stage The horizontal surface on which the slide is placed is called the stage. Why? So coarse adjustment is used for rapid focusing of the specimen until the specimen is roughly in focus & then left alone. The magnification of oil immersion lens requires using the lens with special immersion oil for proper resolution. located immediately under the stage. 5. 8. either on top of or under the stage. 4. 10. The total magnification of a microscope is the product of the magnification of the objective and the ocular.

The objective should be immersed in the oil on the slide. Procedure for cleaning a microscope slide: 16 . Always examine a slide first with the low-or medium power objective. Obtain a slide. 3. Use the stage adjustment knob to move the slide over the hole in the stage. Rotate the scanning objective into place. Use the fine adjustment knob to sharpen the focus. 2. If you are finished with the microscope clean the microscope and return it to storage. Procedure for cleaning a microscope: 1. and objectives lens. Rotate the high power object halfway to the next position. 6. Using the coarse adjustment knob to obtain maximum working distance and remove the slide from the stage. 2. Adjust the light using the iris diaphragm lever if necessary. 7. Adjust the light using the iris diaphragm lever if necessary. Clean any oil off of the stage using paper towels. Use the coarse adjustment knob to obtain the minimum working distance. 4. Remove slide only after low-power objective has been rotated into viewing position. Rotate the high power objective in to place. 4. then the image produced by these two lenses is 60 times larger than the specimen. Slowly turn the coarse adjustment knob until something comes into focus. Adjust the light with the iris diaphragm lever if necessary. 8. Use the fine adjustment knob to sharpen the focus. 5. 6. Look through the ocular. Use the coarse adjustment knob to obtain maximum working distance. 5. place a drop of immersion oil on the slide. Using lens paper cleans all the lenses starting with the cleanest first ocular. 5. never when high – power objective is in position. 9. Place the slide on the stage. 3. and then rotate the oil immersion objective into place. When returning your microscope to its proper place in the cabinet always: • Remove the slide from mechanical stage. Microscope safety cautions: 1. the slide should fit into the slide holder. 10. A wet stage will prevent the slide from being accurately positioned. Keep the stage dry at all times. Use the coarse adjustment knob to obtain minimum working distance. Using the stage adjustment knob move the slide around until you find an area you wish to examine more closely. • Rotate the nosepiece that the scanning lens is in place. 3. Return the microscope to the appropriate storage area. 4. • Clean all lens surface and the stage. When finished viewing the slide use the coarse adjustment knob to maximize the working distance and remove the slide from the stage. Do not use the coarse adjustment knob. Rotate the lower objective in place. never use the high – power objective to view thick specimens. 6. 2. Use the fine adjustment knob to sharpen the focus. Turn off the light. Always carry the microscope in an upright position using both hands.12X. Steps Used in viewing a slide: 1. Keep the microscope away from the edge of the table. Move the slide until the object you wish to examine is in the center of the field. Check that the ocular and all objective lenses as well as the slide clean. 11.

When doing a wet mount follow the procedure outlined below:  Place the specimen (mixed culture. Hold the slide from its ends by fingers of one hand. Either blot dries the slide by placing it between two towel papers. 5.  Blot an excess fluid with lens paper before you place the slide on the stage of the microscope. 3. (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 45ºangle).  After you have made your observations the slide & cover slip should be washed. until no trace of the detergent is left. 1. Examine your preparation under the compound microscope use scanning and low poser of the microscope. using a scissor cut the hair leaving about 2cm of the lower part containing the bulb. rub the slide with one finger of the other hand. tissue. Gently lower the cover slip on top of the specimen. 6. dried & replaced in their appropriate locations. 2.  Add a drop of water or designated stain if required. Place a glass cover slip on the specimen by holding the cover slip at a 45º 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). rub again. Permanent slides Permanent slides have been professionally prepared.) on the center of a clean slide. clean slide always holds it from its ends. or place in alcohol solution & keep until used. Rinse the slide with distilled water to remove the tap water. Preparation of microscope slide: Two types of slides are commonly used in biology classes: permanent slides and temporary wet mounts that you make. Wash the slide under running tap water. do the following: 1. 3. 4. 2. They have often been stained to show better contrast of structure. etc. A permanent slide may be a whole mount of the entire specimen or section of the material. Place this lower part of the hair in a drop of water in the center of a clean slide. 2. Try to avoid trapping air bubbles. Using a detergent liquid. 17 . 4. Procedures Exercise 1: Make a wet mount using a hair from your skin as follows: 1. as any small foreign body might mislead the observation. Wet mounts Wet mounts are temporary slides that you prepare yourself. Pull out a hair from your skin.Before placing a specimen on a slide. Never touch the slide from the middle. If your slide is not clean. it must be clean.

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. What is the total magnification when using medium power? 11. 10. 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. 2. What is the total magnification when using low power? 8. Cover the letter "e" with a cover slip. Rotate the nose piece to the medium power objective lens and observe the letter "e". Rotate the nose piece to the high power objective lens and observe the letter "e". 3. 4. Using a dropper bottle. water. cover slip. 6.Figure 2. Put the letter "e" on a microscope slide. scissors. 7. Draw what you see. Draw what you see. Use the low power 18 . put a small drop of water on the letter "e". 9. slide. Look at the letter "e" using the low power objective lens. coverslip side up. Cut a small-case letter "e" from a newspaper. 12. and newspaper make a wetmount slide of a small-case letter "e". Procedure of a wet mount Exercise 2: Make a wet mount of the letter “e” 1. What is the total magnification when using high power? 14. 13. on the stage. Using microscope. Draw what you see. 5.

since the specimen is too enlarged for you to see all of it at one time. Total Magnification of 40X Total Magnification of 100X Observe the letter "e" with a higher magnification lens. After focusing (fine focus only). The fine texture of the paper should be more visible. Turn on the light. 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. Secure the slide with the stage clips. some of the letter "e" is now missing from your field of view. Draw what you see in the circle below. Try using the high power objective. The higher the magnification of the objective lens. draw what you see. This explains why you must carefully center small specimens within your field of view. Focus on the letter "e" using the coarse focusing knob. which way does the e in the microscope move? (Try this!) If you push the slide away from you. Additionally. before changing lenses. which way does the e in the microscope move? (Try this!) This phenomenon is known as inversion. the smaller the field of view.o o o o objective. Discussion: 19 .

D. Your table should include the powers of both lenses and the total magnification. What objective should always be in place. 4. Calculate the total magnification for each objective. To calculate the total magnification. laboratory Studies in general Biology. Tisdale et al. Fine adjustment knob. (1986). 2. Department of biological sciences. Nablus. What is the function of each of the following: Coarse adjustment knob. and record your figures in a table. 2. Draw the structure of the hair bulb using the 4X and 10X magnification power. Label the following drawing of a Microscope? 3. How do you determine the total magnification of a set of lenses? 5. An-Najah National University. Iris diaphragm. D. (1999). New York. both when beginning to focus and when replacing the microscope for storage? References: 1. Warren. 5th edition. 20 . Biological Investigations. Total Magnification Total magnification is calculated by multiplying the magnification of the ocular lens (eyepiece) by the magnification of the objective lens. multiply the power of the ocular lens by the power of the objective lens. State University.1.

Total Magnification 21 . .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) . .

Morphological Structure of Cells The cell is the structural and functional unit of all known living organisms. However. The fundamental process that triggers synaptic transmission is the action potential. neurons are the core components of the brain. a propagating electrical signal that is generated by exploiting the electrically excitable membrane of the neuron. or cell body. Neurons communicate via chemical and electrical synapses. also technically known as a myocyte. In vertebrate animals. in the size. in a process known as synaptic transmission. It is contained within a limiting membrane and consists of various organelles suspended in cytoplasm. a dendritic tree and an axon. spinal cord and peripheral nerves. Neuron Neurons (also known as neurones and nerve cells) are electrically excitable cells in the nervous system that process and transmit information. is a 22 . This is also known as a wave of depolarization. we will observe the morphological structures of several cells under microscope:  Neuron  Muscle cell  Red blood cells and Lucocytes  Sperm 1. Anatomy and Histology The slice of spinal cord 2. there is great heterogeneity throughout the nervous system and the animal kingdom. The morphological structure of cell is nearly correlated with its functions. Muscle cell A muscle fiber. In the lab.Experiment 2. and transmit output via the axon. Neurons are typically composed of a soma. The majority of vertebrate neurons receive input on the cell body and dendritic tree. shape and function of neurons. also spelled muscle fibre.

Muscle fibres can be grouped according to what kind of tissue they are found in -skeletal muscle. They generally contract voluntarily (via somatic nerve stimulation). smooth muscle. usually attached to the skeleton. Unitary smooth muscle can be further divided into phasic and tonic. Muscle fibres are very long. the way they are arranged is different (and some regulatory proteins are different). Unitary smooth muscle is also commonly referred to as visceral smooth muscle because it is found in the walls of the viscera. the gastrointestinal tract. skeletal muscle smooth muscle Smooth muscle fibers are spindle shaped.single cell of a muscle. Skeletal muscle is a type of striated muscle. The smooth muscle cell contains less protein than a typical striated muscle cell and much less myosin. the respiratory tract. each cell is spindle-shaped. Skeletal muscles are used to create movement. and cardiac muscle. ureters and oviducts. the uterus. also called unitary smooth muscle. the skin and the ciliary muscle and iris of the eye. Smooth muscle is a type of non-striated muscle. The actin content is similar so the ratio of actin to myosin is ~6:1 in striated muscle and ~15:1 in smooth muscle. the latter operate as a single unit and are arranged in sheets or bundles. 20-500 micrometers long and 5 micrometers wide. although they can contract involuntarily through reflexes. can contract and relax. the contractile unit of muscles. Smooth muscle does not contain the protein troponin. The muscle cells of heart muscle tissue are called cardiomyocytes. The cells are generally arranged in sheets or bundles and connected by gap junctions. or internal organs. In the relaxed state. The single-unit type. a single fibre can reach a length of 30cm. including the intestines. male and female reproductive tracts. Whereas the former presents itself as distinct muscle fibers that are usually innervated by their own nerve fibers. of the body. by applying force to bones and joints. In order to contract the cells contain intracellular contractile filamentous proteins called actin and myosin. the vasculature. is far more common. The cells that compose smooth muscle generally have single nuclei. and like all muscle. While the filaments are essentially the same in smooth muscle as they are in skeletal and cardiac muscle. and most blood vessels. ducts such as the bile ducts. via contraction. and caldesmon and calponin are significant proteins 23 . found within the "walls" of hollow organs and elsewhere like the bladder and abdominal cavity. There are two types of smooth muscle arrangements in the body: multi-unit and single-unit. 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. Muscle fibers contain many myofibrils.

or erythrocytes (from Greek erythros for "red" and kytos for "hollow". 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. forming rouleaux.000 white blood cells and about 150. However there is an organized cytoskeleton consisting of the intermediate filament proteins vimentin and desmin. the actin and myosin is not arranged into distinct sarcomeres that form orderly bands throughout the muscle cell. Erythrocytes: (a) seen from surface. with each carrying four heme groups. A schistocyte is a red blood cell undergoing fragmentation. The red blood cells store collectively about 3. Red blood cells are thus much more common than the other blood particles: There are about 4. A typical erythrocyte contains about 270 million hemoglobin molecules. As non-striated muscle. myosin filaments and actin thin filaments. (b) in profile. Adult humans have roughly 2–3 × 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. 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). 3. and contractile proteins can organize into zones of actin and myosin along the axis of the cell.000–11. with cyte nowadays translated as "cell").5 grams of iron. Evidence indicates that smooth muscle myosin filaments are not bipolar with a central bare zone as in striated muscle. haematids. people living at high altitudes with low oxygen tension will have more). more than five times the iron stored by all the other tissues combined. (d) rendered crenate by salt. 24 . (c) rendered spherical by water. (c) and (d) do not normally occur in the body. or a fragmented part of a red blood cell. Human red blood cells are also known as RBCs. but is either side-polar or row-polar and have no bare zone.expressed within smooth muscle.000–400. The diameter of a typical human erythrocyte disk is 6–8 µm.000 platelets in each microliter of human blood. much smaller than most other human cells. Some smooth muscle preparations can be visualized contracting in a spiral corkscrew fashion.

there is a marked difference in the size of the gametes with the smaller one being termed the "male" or sperm cell. whereas a non-motile sperm cell is referred to as spermatium. Sperm cells in algal and many plant gametophytes are produced in male gametangia (antheridia) through mitosis. A uniflagellar sperm cell that is motile is also referred to as spermatozoon. In flowering plants. Different types of sperm cells: human sperm A) spermatozoon (motile). In these types of sexual reproduction. Sperm The term sperm is derived from the word spermos (meaning "seed") and refers to the male reproductive cells.neutrophil acidophil basophil lymphocyte monocyte blood platelet 4. The spermatozoa of animals are produced through spermatogenesis inside the male gonads (testicles) through meiosis. 25 . Sperm cells cannot divide and have a limited life span. B) spermatium (non-motile). C) fertilization tube with sperm nuclei. Sperm cells are the smaller gametes involved in fertilization. sperm nuclei are produced inside pollen. but they can fuse with egg cells during fertilization to form a totipotent zygote with the potential to develop into a new organism.

3. dry slide. and the nucleus. Place it gently on a clean. 7. Is a nucleus visible? Count and record the number of cells across the diameter of the high power field. Add a drop of iodine stain and cover with a coverslip. Exercise 2: Plant cells 1. 4. 5. as they may irritate the skin or cause staining of the skin or clothing. the cytoplasm. 2. 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. Never touch or taste any chemical unless instructed to do so. 3. Discussion: In your lab report. Why are stains such as methylene blue used when observing cells under the microscope? 26 . 6. You are responsible for its proper care and use. 1. simply contain their answer in your report. 8. both lengthwise and side to side. Be sure to label your drawing. Add a drop of iodine stain and cover with a coverslip. 5. using the directions for focusing given above. Observe under the microscope and draw what you see. 1. calculate and record the length and width of an onion cell in micrometers.Exercise In this investigation. 7. Exercise 1: Human epithelial cells Epithelial cells cover the body's surface and line its cavities. and what power you were using when you made your drawing. With your fingers. transparent layer of cells from a piece of onion. 6. strip a thin. Draw what you see. Locate the cell membrane. How are plant and animal cells different in structure? 2. Be sure to label the drawing with what it is. 4. Start with the scanning power objective to find some cells. please be sure to answer the following questions in your discussion section. Obtain toothpick. Observe under the microscope. Do not write them as question and answer. dry slide. Be sure to label your drawing. Always handle the microscope with extreme care. Gently scrape the inside of yourcheek with the toothpick. Use caution when hadnling glass slides as they can break easily and cut you. Using the number you calculated in exercise 3. Make a drawing of what you see. 2. Always use special caution when working with laboratory chemicals. Locate the cell wall. Place the scrapings on a clean. How are plant and animal cells similar in structure? 3. then observe under both low and high power.

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. In general. impermeable or partially permeable to a given substance.Experiment 3. Diffusion-Plasmolysis The purpose of this activity is to investigate the effects of a hypertonic solution on the 27 . Isotonic and Hypotonic Diffusion: Diffusion is the net movement of molecules from a region of high concentration to a region of low concentration. across a semi permeable membrane (permeable to the solvent. mitochondria) serves as a regulatory structure by controlling the entry or exit of substances into and out of the cell. up a solute concentration gradient. membranes are freely permeable to water molecules. 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. Fig. The membrane may be permeable. It is a physical process in which a solvent moves. water movement through the cell membrane is determined by the process of osmosis. In cells. 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. Hypertonic. 1. without input of energy. but not the solute) separating two solutions of different concentrations.

Examine under 100X.cells of the red onion. Replace the sodium chloride solution with distilled water and add it in the same way that the salt solution was added. 5. colored drawing of the cells' appearance in the third circle on the data sheet (Fig. Experiments often utilize a simplified version of the situation that is being investigated. of your cover slip while placing a small piece of paper towel along the opposite edge of the cover slip. microscopeor magnifying glass. Make sure that the salt solution has time to diffuse under the cover slip. colored drawing of the cells' appearance in the second circle on the data sheet (fig. but which is usually easier for the experimenter to control. Materials: 28 . Observe the affects of the solution on the onion cells. You will understand water that passes through the membrane of th plant cells. properly labeled in the first circle on the data sheet (Fig. Purpose: To investigate the diffusion of a substance across a semipermeable membrane. PROCEDURE: 1. paper towels. Make a properly labeled. 5% Sodium Chloride (table salt) solution. dropper. 3). 3. Make a colored drawing. a model or a system that is acceptably similar to the actual case. slide or saucer. Make a properly labeled. 4. When you have a clear view of several cells. and cover slip. The paper should draw out the water and draw in the salt solution. MATERIALS (per student): red onion epidermis. Make a wet mount of the red onion epidermis. distilled water. you will employ a simple procedure. Diffusion Through a Membrane To study the cell membrane. switch to 430X. 2). 2. forceps. 4).

Procedure 1. Use a glucose test strip to test the water for the presence of glucose. and water. 5. Use your fingers to squeeze the bag on the outside to mix the starch and glucose solution thoroughly in the bag. Result after running the experiment Things to test or comment on in your data. Take an approximately six inch length of membrane tubing and soak it in warm ater until it becomes pliable. give special attention to the tied ends. Wait for approximately 10 minutes and record your observations. Place your bag which has now been tied on both ends and rinsed into the cup or beaker containing the iodine solution. 9. 6. 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).) Is there glucose in the water in the cup after 10 minutes? (Use the glucose test 29 . 2. add about an inch of glucose solution to the bag. 4. Record your results. A "possible" arrangement to help you organize your data appears below. Place 30-35 drops of Lugol's solution (iodine) in the water in the beaker or cup. 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. (Enough iodine should be added until the water in the cup is light brown. glucose solution. 10.• • • • tincture of iodine cornstarch/water solution plastic sandwich bags (cheap is good) beakers or clear plastic cups Materials: glucose test strip. Test the bag to be certain that the starch solution does not leak from either end and that there are no holes in it. a. 3. Tie one end and place about an inch of starch solution in the bag. 8. cup or beaker. Wash the bag with water to remove any starch or glucose adhering to its surface. 7. dialysis tubing. After putting the starch solution in the bag. starch solution.

Did iodine enter the membrane from the water in the beaker? How did you prove this? 4.) Was there a change in the color inside the bag? c.strip to test this.) Questions which should be answered in your conclusion. but if you did. Based on this investigation. you did something wrong with this experiment. You didn't investigate the diffusion of water (osmosis) in this experiment.) b. Explain/justify your answer. 5. 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 30 . form a tentative conclusion in reference to how the size of molecules influences their ability to diffuse through a semi-permeable membrane. Did starch leave the inside of the membrane and go into the beaker? How did you prove this? 3. Did glucose leave the inside of the membrane and go into the beaker? How did you prove this? 2.) Was there a change in the color of the water in the cup outside the bag? Hint: If there was a significant change. 1.

6. #3 and #4). 2. 4. 5. 4. do not ingest. Wash off spills and splashes with water for 15 min. Avoid vigorous boiling. 3. 3. causing second-degree burns. 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. Identification of chemical constituents of the cell Experiment 4. 7. 8. rinse mouse with water. Introduction In this experiment you will identify the presence of glucose in food product using the blue colored.Experiment 4. CAUTION: Benedict's solution is an irritant. sucrose powder. Call your teacher. Repeat steps 2-3 with the. Add 1 ml water and shake test tube until glucose is dissolved. Immediately place the burned area under cold running water. Watch the changes in the Benedict solution color. 9. Materials (per a team of 2) 1.a: Identifying the presence of glucose in food items Experimental goal: Identifying the presence of glucose in honey and onions. Do not touch the beaker or allow boiling water to contact your skin. CAUTION: Boling water will scald. Avoid skin /eye contact. benedict solution. honey and diced onion (use test tubes #2. The copper ions undergo a reduction reaction in the presence of glucose and change color from blue to orange. Tie back long air. Safety: Put on your safety goggles. Add 15 drops of the Benedict solution to the test tubes and swirl the test tubes gently. Results: Write your results in the following table: Test Contents Water tube No. lab apron and plastic gloves. put some powdered glucose in test tube #1. Handle test tubes with a test – tube clamp. 7. 5. Using the spatula. Put the tubes in a large beaker that contains boiling water (the beaker is standing on a heater). 2. 6. 1. of test tube 31 Benedict Color of solution solution Benedict .

3. a simple sugar. Experiment 4. showing that Benedict solution can be used as an indicator for glucose. The sucrose serves as a negative control. #2.e. 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. hydrogen and oxygen atoms that make up a basic unit and in the number of basic units that each sugar molecule has (i.b: Identifying the presence of starch in food items Experimental goal: Identifying the presence of starch in various foods. Test tubes number 1. The sugar molecules are differentiated by the number of carbon. The glucose serves as a positive control. An OH group is bound to carbon atoms #1. 3. 32 . hydrogen (H) and oxygen (O) atoms. 2. Focus on carbohydrates (sugars) Sugars are made up of carbon (C). 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.1 2 3 4 glucose Sucrose honey Diced onion + + - + + + + Analysis 1. Introduction To test for the presence of starch in various foods. 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. 3. showing that the Benedict solution does not change color in the presence of any sugar other than monosaccharides as glucose. Starch is made up of long chains of glucose molecules that are chemically bound to each other. 4 . #3 and #4 and a CH2OH is bound to carbon atom #5. 2.

rinse mouse with water. lab apron and plastic gloves. 2. showing that the Logol can be used as an indicator for starch. Tie back long hair. 3. Wash off spills and splashes with water for 15 min. What purpose does the starch powder serve? Analysis . In which foods was a color change observed? 2.c: Identifying the presence of protein in food items Experimental goal: 33 . 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. Watch the changes in the logol color. 7. Add two drops of the Logol solution to each plate and shake gently. beans. 5. Color change can be observed in the potato. 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. The starch powder serves as a positive control . Call your teacher. 2. What purpose does the cucumber slices serve? 3. Procedure: Safety: Put on your safety goggles.answers: 1.*Logol – Iodine dissolved in a potassium iodide solution: I2/KI. CAUTION: Lugol's iodine solution is irritating to skin and eyes and can stain clothing. 3. Experiment 4. The cucumber slices serves as negative control. 4. bread and banana. Materials (per a team of 2) 1.1% Logol solution. 6. showing that the color of the Logol will not change in any food.

Introduction The Biuret reaction: In order to identify proteins in a solution. or peptide bonds. but instead of adding the Biuret solutions. Procedure: Safety: Put on your safety goggles. we will be using a basic solution of copper ions (Cu++). This reduction reaction changes the color of the solution from blue to purple. tube solutions 1 2 3 4 5 6 Color of Biuret solution Analysis 1. Tie back long hair. Dissolve the sucrose in 1 ml water. Materials (per team of 2) 1. 2. Biuret solution #2. Amides. Egg yolk. Repeat steps 2-4 with albumin. Sucrose powder. lab apron and plastic gloves. tuna chunks. whose color is blue. 3. are the bonds that bind amino acids in proteins. add an equal volume of water (use test tube #6). 1. Biuret solution #1. #3 #4 and #5. A spatula 2. Incubate for 5 minutes at room temperature. 7. A can of tuna. 5. Gently stir the test tube 3. The copper ions are reduced when they bind to carbon atoms on amides. A bottle of water. 6 test tubes. Results: Write down your results in the following table: Test Contents of test Biuret Water tube No. 9. In which test tube did you see a change of color from blue to purple? 34 . 4. Number the test tubes sequentially from 1 to 6. called Biuret solution. 6. egg yolk and sucrose powder. a 1% CuSO4 solution in a dropper bottle. Use test tubes #2. Milk. 5.Identifying the presence of protein in milk powder and tuna. Albumin (OVA/BSA). 6. 8. Put some albumin then add 10-20 drops of Biuret solution #1. Repeat steps 2-4 with 1 ml milk. Add 10-20 drops of Biuret solution #2. 6M NaOH in a dropper bottle. 10. Gently stir the test tube 4.

35 .2. 3. Can you propose an experiment that will quantify the amount of protein in solution? Analysis-answers 1. less light will go through the sample. 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. There is a linearic correlation between the intensity of the purple color and the amount of protein in solution. the more intense the purple color. A spectrophotometer is an instrument that measures the light that is absorbed by the sample. Explain the importance in using sucrose powder and water instead of using the Biuret solution? 3. The sucrose powder is used as a negative control. As the intensity of the color increase. In test tubes 1-4. a change in color from blue to purple can be seen. 2. indicating that the Biuret reaction identify protein and does not change it’s color in the presence of every material. The amount of protein can be quantified by using a spectrophotometer. The more peptide bonds are in solution.

minerals. sugars. and very importantly. This allowed for one of the areas for modern biotechnology to take hold. vitamins. Since the environment is artificial. the plastic bottom of the plate will be the surface that the cells attach to. • Gas Environment-usually requires 10% CO2 and humidity. the investigator can strictly control the environment. Cell culture Introduction One of the major breakthrough in studying mammalian cell biology was the ability of replicate cells outside the living organism. sometimes difficult. salts. specific nutrients. • Nutrients. pH. • Temperature-usually body temperature (37℃) and needs to be tightly controlled. If the color of the media goes to a purple red color. 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). In our case. Growth in a mammalian cell culture The growth of mammalian cells in artificial environments requires several different components and good aseptic techniques. This environment usually requires specific temperature. We will be using a human carcinoma cell line. This indicates the culture is not able to maintain proper pH usually due to lack of CO2. The humidity is important in the loss of fluid due to evaporation. 36 . Our cell line does not require CO2.Very important in cell cultures and is followed by the color pH indicator Phenol Red (you used this in the microbiology labs). and a growth surface. The monoclonal antibody used in research and several medical tests requires that the antibody producing hybridoma to be grown in culture.Experiment 5 and 6. • pH. Many important therapeutic proteins are produced by cell culture. serum. It has some specifics that allow us to carry out our experiments with greater ease but still requires many of the same features. Although. gas environment. mammalian cell culture is a highly valued technique for research in the modern biology laboratory. The CO2 is important in many cell cultures for maintaining proper pH. • Growth surface-Many of the mammalian cell cultures require a surface to grow on. If the culture is too acidic. you can do a couple of quick checks to see if the cells are doing well or if you have problems. it will change to an orange and later to a yellow.the normal amino acids. the culture is becoming alkaline. Serum contains growth factors that are needed for inducing the cells to grow. This can indicate bacterial or fungal contamination. Many cell lines are serum independent but we will use one that is dependent on serum.

add 10 ml of L-15 ++ media (Leibovitz 15 with 10% fetal calf serum and antibiotics) to the flask. you loosen the mentioned above. If it is orange or purple red. When sufficient number of cells are rounded up and loose from the culture flask. You can get an idea if the cells are ready to pass to new culture flask or plate by looking at the confluence. Wash the monolayer of cells with 5 ml of calcium. This is done to wash away the calcium and magnesium. You can check this with the inverted microscope. Confluence-cells do not like growing over each other. After sterilizing the area with ethanol. the culture could be having problems. look at the color of the media. You can have the best preparation in the world but if you cell culture is contaminated with bacteria or fungi. Pipet the cells up and down several times to resuspend the cells in the new media. You should pass when you get to 80% or more. If you have round cells floating in the media. Cell attachment. 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. pipet out the old media from the culture flask and pipet the waste media into the waste container. magnesium free phosphate buffer (CMF-PBS).most cells should be spread out on the surface of the culture flask. When you remove these cations.• • • pH. 5. Half of the plate would be considered 50% confluence. you may have some problems starting. Procedure The first day will be based on transferring you cell culture to new plates. 2. 37 . Look in the inverted microscope and figure out how much of the plate or flask is covered by cells. Check the cells in the inverted microscope every couple of minutes for the presence of rounded up cells. The media contains trypsin inhibitors to stop the trypsin reaction. 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. 4. 3. Remember how these divalent cations are needed for certain adhesion proteins. Passing Cells to New Culture Flask 1. I will go into aseptic technique with you in great detail in the laboratory. This is done by pipetting in the CMF-PBS and rocking back and forth. I will do one set of plates to show you the procedure. the culture will be quickly overrun by the contamination. To do this. Aseptic Technique The most important aspect of mammalian cell culture is the use of aseptic technique. Pipet out the CMF-PBS into the waste container.

Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.g. Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing. 5. Slowly. Suspension cell lines can be used directly. 5. dropwise. Pipette 1ml aliquots of cells into cyroprotective ampules that have been labeled with the cell line name. 2. Submerge only the lower half of the ampule. 3. 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. 3. pipette cells into pre-warmed growth medium to dilute out the DMSO. 7. Place ampules at –80ºC overnight. Remove a small aliquot of cells (100-200uL) and perform a cell count. passage number and date. 7. The cells will need to be fed every other day and passed to new flasks at least once before next week.usually 1-2 minutes. 8. 8. cell concentration and date. Allow to thaw until a small amount of ice remains in the vial . passage number. 37ºC for mammalian cells. View cultures using an inverted microscope to assess the degree of cell density and confirm the absence of bacterial and fungal contaminants. Transfer to class II safety cabinet. 4. Pass cells (pipet the cells) evenly to two new flasks. Label with cell line name.6. 6. 6. Prepare the flasks as appropriate. Cryopreservation of Cell Lines 1. Resuscitation of Frozen Cell Lines 1. 4. Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid. 7. Label cultures with date of passage and place back in the incubator. Frozen ampules should be transferred to the vapor phase of a liquid nitrogen 38 . The cells should attach to the surface to the culture flask in a couple of hours. Re-suspend cells at a concentration of 2-4x106 cells per ml in freeze medium. remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e. you can check the flask on the inverted microscope. 2. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary. I will have a sign-up sheet for student to come in and check you cultures. Centrifuge the remaining culture at 150g for 5 minutes. Still wearing protective clothing. Read Technical data sheet to establish specific requirements for your cell line. If you are around. 39 .sep. References: http://www.htm vessel and the locations http://slohs.umanitoba.