cell biology experiment

7/28/2019 Cell Biology Experiment

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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, China2013-07-22


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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

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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.

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Basic Glass ware and Lab Tools

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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 proofbandage.

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. Never taste or touch substances in the laboratory without specific instructions.

2. Never smell substances in the laboratory without specific instructions.

3. Use materials only from containers that are properly labeled.

4. Wash your hand after working with chemicals.

5. Do not add water to acid. Instead, dilute the acid by adding it to water.

6. 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. Become familiar with the names and appearance of all the laboratory

equipments you will use.3. Never use broken or chipped glassware.

4. Make sure that all glasswares are clean before you using it.

5. Do not pick up broken glass with your bare hands. Use a pan and a brush.

6. If a Mercury thermometer breaks, do not touch the mercury. Notify your

teacher immediately.

7. Do not aim the mirror of your microscope directly at the sun. Direct sun light

can damage the eyes.

8. 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.

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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 informationEach 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 thatyou 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 styleYour 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

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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. Thereport 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 formatThe 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, inMaterials 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 sizeof 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 youmay 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 ReportsThe 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 typographicalerror will usually result in a point deducted from the maximum. However, if one wordis 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, poorsentence of paragraph structure) will usually result in a point deducted from themaximum. Low grades in grammar can be avoided by proofreading your work beforeyou submit it and by writing practice essays.III. Content counts 75% of the total grade. The kinds of questions that are consideredin 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 previousexperience?

6. Do you support your conclusions with the appropriate statisticaltest(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 andits use. Students should identify the different parts of the Microscope. List and followrecommended procedures in using and caring for the Microscope.

MaterialCompound MicroscopeClean Microscope SlidesCover SlipsLens papersSharp razor blades

Newspaper lettersMedicine droppersScissorsDistilled waterPond waterCorkXylene

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 needequipment 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 thecompound light microscope. The compound microscope (sometimes called thestudent microscope or light microscope); these microscopes are known as compoundmicroscope because there are two magnifying lenses in the microscope. Onemagnifying lens is in the ocular or eyepiece, which further magnifies the imageformed by the objective lens, and one, is in the objective. Each contributes to themagnification of the object on the stage. The total magnification of any set of lenses isdetermined by multiplying the magnification of the objective by the magnification ofthe ocular. The nose piece rotates the magnification of the microscope. Generallycompound 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 areaccurately 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, ifit 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 rotatingturret at the bottom of the body tube. The four objective lenses of yourmicroscope 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 useddry.The magnification of oil immersion lens requires using the lens with specialimmersion oil for proper resolution.

4. StageThe 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 amechanical 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 asystem of lenses that focuses light on your specimen. The condenser may beraised or lowered using the condenser knob. An older microscope may have aconcave mirror instead.

6. Iris diaphragm

Iris diaphragm is located below the condenser or immediately below the stagein microscopes without a condenser. It functions in regulating the lightintensity passing through to the stage. More light is required at highermagnification.

7. Light sourceThe light source has an (ON/Off) switch & may have adjustable lampintensities & color filters.

8. BaseBase also called the supporting stand, rests on the bench.

9. Body ArmThe body arm is used when carrying the instrument.

10. Nose pieceNosepiece is the mounting for the objective lenses which rotates to bring thedesired objective into position.

11. Coarse adjustmentCoarse adjustment knob is a large knob located at either side of themicroscope which functions in controlling the distance between the objectivesand the stage. Use the coarse adjustment only with the scanning (4X) & low-

power (10X) objectives. Why?So coarse adjustment is used for rapid focusing of the specimen until thespecimen is roughly in focus & then left alone, in which the fine adjustmentknob controls precise focusing of the object.

12. Fine adjustmentFine adjustment is a small knob located at either side of the microscope. Thisis used for the control of the object, precise focusing you should use just thefine adjustment knob with the higher magnification objective lenses; Becauseusing the coarse adjustment knob with the higher objective lenses may damagethe lens &/or the slide you are observing.

Magnification:Compound microscopes consist of two lens system: the objective lens, whichmagnifies, & projects a virtual image into the body tube and the ocular lens, whichmagnifies the image further and projects the enlarged image into the eye.

The total magnification of a microscope is the product of the magnification of theobjective 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 thespecimen.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, neveruse the high power objective to view thick specimens.

4. Remove slide only after low-power objective has been rotated into viewingposition, never when high power objective is in position.

5. Keep the stage dry at all times. A wet stage will prevent the slide from beingaccurately 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 intofocus. Use the fine adjustment knob to sharpen the focus.

8. Using the stage adjustment knob move the slide around until you find an areayou wish to examine more closely. Move the slide until the object you wish toexamine is in the center of the field.

9. Rotate the high power objective in to place. Use the fine adjustment knob tosharpen the focus. Do not use the coarse adjustment knob. Adjust the lightusing the iris diaphragm lever if necessary.

10. Rotate the high power object halfway to the next position, place a drop ofimmersion 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 fineadjustment knob to sharpen the focus. Adjust the light using the iris diaphragmlever 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 finishedwith 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 bodymight 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 thedetergent 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 andtemporary wet mounts that you make.

1. Wet mounts

Wet mounts are temporary slides that you prepare yourself. When doing a wetmount follow the procedure outlined below:

Place the specimen (mixed culture, tissue, etc.) on the center of a cleanslide.

Add a drop of water or designated stain if required. (Note: liquidcultures do not require adding water)

Place one edge of the cover slip on the slide near the specimen (This isdone by holding the cover slide at a 45angle). Gently lower the coverslip on top of the specimen. Try to avoid trapping air bubbles.

Blot an excess fluid with lens paper before you place the slide on thestage of the microscope.

After you have made your observations the slide & cover slip shouldbe washed, dried & replaced in their appropriate locations.

2. Permanent slidesPermanent slides have been professionally prepared. They have often beenstained to show better contrast of structure. A permanent slide may be a wholemount of the entire specimen or section of the material.

ProceduresExercise 1:Make a wet mount using a hairfrom your skinas follows:

1. Pull out a hair from your skin, using a scissor cut the hair leaving about2cm of the lower part containing the bulb.

2. Place this lower part of the hair in a drop of water in the center of aclean slide.

3. Place a glass cover slip on the specimen by holding the cover slip at a45 angle to the drop of water and then slowly lowering the cover slipover the drop forcing trapped air out of the preparation (Figure 2).

4. Examine your preparation under the compound microscope usescanning 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 wet-

mount 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?


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?


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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objective.

o Secure the slide with the stage clips.

o Turn on the light.

o Focus on the letter "e" using the coarse focusing knob.

o 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 magnificationpower.

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, StateUniversity, New York.

2. Tisdale et al. (1986), laboratory Studies in general Biology, Departmentof biological sciences, An-Najah National University, Nablus.

Total MagnificationTotal 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 Total Magnification

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)

.

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Experiment 2. Morphological Structure of Cells

The cell is the structural and functional unit of all known livingorganisms. 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. NeuronNeurons (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

2. Muscle cell

A muscle fiber, also spelled muscle fibre, also technically known as a myocyte, is a

The slice of spinal cord

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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 notcontain 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 otheractin 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 1013 red blood cells at any given time (women have

about 4 to 5 million erythrocytes per microliter (cubic millimeter) of blood and men about5 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 todevelop 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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ExerciseIn 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 GlassSlide 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 cellsEpithelial cells cover the body's surface and line its cavities.

1. Obtain toothpick.2. Gently scrape the inside of yourcheek with the toothpick.

3. Place the scrapings on a clean, dry slide.

4. Add a drop of iodine stain and cover with a coverslip.

5. 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.

Exercise 2: Plant cells1. With your fingers, strip a thin, transparent layer of cells from a piece of onion.

2. Place it gently on a clean, dry slide.

3. Add a drop of iodine stain and cover with a coverslip.

4. Observe under the microscope and draw what you see. Be sure to label your

drawing.

5. Locate the cell wall. Is a nucleus visible?

6. Count and record the number of cells across the diameter of the high power

field, both lengthwise and side to side.7. Using the number you calculated in exercise 3, calculate and record the length

and width of an onion cell in micrometers.

8. 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 discussionsection. Do not write them as question and answer, simply contain their answer inyour 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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http://slohs.slcusd.org/pages/teachers/bsmith/ZZ%20%20Folder/Ron's%20Biology%20Web%20Page/Biology/Cells/cheek2.jpghttp://slohs.slcusd.org/pages/teachers/bsmith/ZZ%20%20Folder/Ron's%20Biology%20Web%20Page/Biology/Cells/onion3.jpghttp://slohs.slcusd.org/pages/teachers/bsmith/ZZ%20%20Folder/Ron's%20Biology%20Web%20Page/Biology/Cells/cheek2.jpghttp://slohs.slcusd.org/pages/teachers/bsmith/ZZ%20%20Folder/Ron's%20Biology%20Web%20Page/Biology/Cells/onion3.jpg


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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 apartially 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-PlasmolysisThe 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 magnifyingglass, 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 MembraneTo 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.

Procedure1. 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 glucoseadhering 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

Result after 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.

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 itemsExperimental goal:Identifying the presence of glucose in honey and onions.

IntroductionIn 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. A spatula2. A plate with diced onions

3. A jar of honey

4. Powdered glucose

5. Powdered sucrose

6. 4 test tubes

7. A bottle of water

8. Benedict solution in a dropper bottle

9. A waterproof marker

Procedure:1. Mark the test tubes from 1 to 4.

2. Using the spatula, put some powdered glucose in test tube #1.

3. Add 1 ml water and shake test tube until glucose is dissolved.

4. 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 withwater. 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 orallow 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

tube No.

Contents

of test tube

Water Benedict

solution

Color of Benedict

solution

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1 glucose + +

2 Sucrose + +

3 honey - +

4 Diced

onion

- +

Analysis1. In which test tubes does a change in color occur?

2. What purpose does the powdered glucose serve?

3. 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 itemsExperimental goal:Identifying the presence of starch in various foods.

IntroductionTo 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. A Petri dish with starch powder (dish #1)

2. A Petri dish with diced potatoes (dish #2)

3. A Petri dish with presoaked beans (dish #3)

4. A Petri dish with diced bread slices (dish #4)

5. A Petri dish with banana slices (dish #5)

6. A Petri dish with cucumber slices (dish #6)

7. 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 thechanges 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 tableDish number Contents of dish Color of Logol solution

1

2

3

4

5

6

Analysis1. 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 itemsExperimental goal:

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Identifying the presence of protein in milk powder and tuna.

IntroductionThe 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, orpeptide 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 tube3. 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

tube No.

Contents of test

tube

Biuret

solutions

Water Color of Biuret

solution

1

2

3

4

5

6

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 willquantify 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 incolor 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 theability of replicate cells outside the living organism. This allowed for one of the areasfor modern biotechnology to take hold. Many important therapeutic proteins are

produced by cell culture. The monoclonal antibody used in research and severalmedical tests requires that the antibody producing hybridoma to be grown in culture.Although, sometimes difficult, mammalian cell culture is a highly valued techniquefor research in the modern biology laboratory.

Growth in a mammalian cell culture

The growth of mammalian cells in artificial environments requires several differentcomponents and good aseptic techniques. Since the environment is artificial, theinvestigator can strictly control the environment. This environment usually requiresspecific 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 tocarry out our experiments with greater ease but still requires many of the samefeatures.

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 forinducing 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 culturesWhen 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 cellsare 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 TechniqueThe 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 thecontamination. I will go into aseptic technique with you in great detail in thelaboratory.

ProcedureThe 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 thentransfer 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 acouple 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 volumeof 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/
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.htmhttp://www.umanitoba.ca/Biology/lab3/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.htmhttp://www.umanitoba.ca/Biology/lab3/

cell biology experiment

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