uddd1104 cell biology lab manual 2013

Universiti Tunku Abdul Rahman (UTAR)

Faculty of Science - Department of Biomedical Science

UDDD1104 Cell Biology

Year 1 Trimester 1 & 2 & 3

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INTRODUCTION

1. The Scientific Method

Making observations

Generating hypotheses

Making predictions

Designing and carrying out experiments

Constructing scientific models

2. Practical Exercises

To get the most out of the practical exercises, you need to follow carefully the instructions

given. These instructions have been designed to provide you with experience in the following

skills:

Following instructions

Handling apparatus

Having due regard for safely

Making accurate observations

Recording results in an appropriate form

Presenting quantitative results

Drawing conclusions

3. Following Instructions

Instructions are provided in the order in which you need to carry them out. We would

advise that before carrying out the instructions, you read through the entire exercise. This will help

you to understand what you are doing and why you are doing it. In turn this will help you to

remember what you have learned.

Each practical exercise in the book begins with a few lines describing its purpose in most

cases the following headings are also used:

Procedure - numbered steps that need to be carried out.

For consideration - some questions to help you think carefully about the results you have

obtained.

Materials - a list of the apparatus, chemicals and biological materials you need.

4. Handling Apparatus

Biologists need to be able to use many different types of apparatus, for example,

potometers (to measure water uptake by plants), respirometers (to measure oxygen uptake or

carbon dioxide production), Petri dishes (for plating out bacteria and other micro-organisms) and





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the light microscope (to magnify specimens). Many of the practical exercises are designed to help

you derive the maximum benefit from a piece of apparatus.

5. Having Due Regard for Safety

Surveys have shown that science laboratories are among the safest places to be.

Nevertheless, this is no cause for complacency.

Always move slowly and carefully in a laboratory.

Never put your fingers in your mouth or eyes after using chemicals or touching biological

specimens until you have washed your hands thoroughly with soap and warm water, and dried

them.

Make sure glass objects (e.g. thermometers, beakers) cannot roll off tables or be knocked onto

the floor.

Wear safely goggles whenever there is a risk of damage to the eyes.

Situations of risk include:

Heating anything with a Bunsen burner (even heating water has its dangers).

Handling many liquids, particularly those identified as corrosive, irritant, toxic or harmful.

Handling corrosive or irritant solids.

Some dissection work.

Allow Bunsen burners, tripods, gauzes and beakers to cool down before handling them.

Never allow your own body fluids (especially blood and saliva) to come into contact with

someone else, or theirs into contact with you.

Keep long hair tied back and do not wear dangly earrings.

Do not allow electrical equipment to come into contact with water.

If you are unsure how to carry out a scientific procedure, ask.

Make sure you understand why you are going to do something before you do it.

Wear a lab coat when using chemicals or handling any biological specimens.

Follow exactly agreed procedures with regard to cuts, burns, electric shocks and other

accidents (e.g. with chemicals).

Follow exactly all specific safely instructions given in this book or provided by your teacher

for particular practical exercises (e.g. use of gloves, disinfectant)

With practice, these procedures should become second nature to you. They will enable you to

carry out practical work in safety.

6. Making Accurate Observations

In most cases the practical exercise will make it clear what you need to observe, e.g. the

time taken for a certain volume of gas to be evolved or the width of a sample of cells. Ensure that

you know how to use any necessary equipment before starting the practical. Think carefully about

the precision with which you will make your observations.





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7. Recording Results in an Appropriate Form

Results can be recorded in various ways. Often it is helpful to record raw data in a table.

Most data will be in the form of numbers, i.e. they will be quantitative data (also known as

numerical data). However, some data, e.g. flower colour, will be qualitative.

One form in which some biological findings can be recorded is a drawing. You don't need

to be a professional artist to make worthwhile biological drawings. If you follow the following

guidelines, a drawing can be of considerable biological value:

Ensure that your completed drawing will cover at least a third of an A4 page.

Plan your drawing so that the various parts are is proportion and will not be drawn too small.

Small marks to indicate the length and breadth of the drawing are a great help in planning and

a faint outline can be rapidly drawn to show the relative positions of the parts.

The final drawing should be made with clean, firm lines using a sharp HB pencil and, if

needed, a good quality eraser (not a white-out fluid). If important details are too small to be

shown in proportion, they can be put in an enlarged drawing at the side of the main drawing.

Avoid shading and the use of colour unless you are an excellent artist and they really help, for

example when drawing soil profiles.

When drawing structures seen with the naked eye or hand lens, use two lines to delineate such

things as blood vessels and petioles. This will help you to indicate the relative widths of such

structures.

When drawing low power plan drawings from the light microscope, do not attempt to draw

individual cells - just different tissues.

When drawing plant cells at high power under the light microscope, use two lines to indicate

the width of cell walls, but a single line to indicate a membrane.

Always put a scale on each drawing.

8. Presenting Quantitative Results

Presentation of data is all about using graphs or other visual means to make it easier to see

what your results tell you. The following four ways of presenting data are the most frequently

used in biology: line graphs, bar charts, histograms and scatter graphs (Figure 1).





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Figure 1 - Line graphs, bar charts, histograms and scatter graphs.





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9. Drawing Conclusions

Finally, you will need to draw conclusions. If your practical exercise has involved the

testing of a hypothesis, for example that the enzyme pepsin works better at low pHs than in neutral

or alkaline conditions, your conclusion should indicate whether the hypothesis has been refuted

(i.e. shown not to be the case) or supported. Of course, even if your hypothesis has been

supported, it doesn't mean that it has been confirmed with 100% certainty - in other words it isn't

proved. Science proceeds more by showing that certain ideas are wrong than by showing that

others are right (think about that!). Your conclusion might therefore include further ways of

testing the original hypothesis, or might raise new possibilities to be investigated.

Often you will only be able to arrive at your conclusions after statistically analyzing your

data.

10. Writing a Scientific Lab Report

Title

Communicate the subject investigated in the paper.

Introduction

State the hypothesis.

Give well-defined reasons for making the hypothesis.

Explain the biological basis of the experiment.

Cite sources to substantiate background information.

Explain how the method used will produce information relevant to your hypothesis.

State a prediction based on your hypothesis. (If the hypothesis is supported, then the results

will be.)

Materials and Methods

Use the appropriate style.

Give enough detail so the reader could duplicate your experiment.

State the control treatment, replication, and standardized variables that were used.

Results

Summarize the data (do not include raw data).

Present the data in an appropriate format (table or graph).

Present tables and figures neatly so they are easily read.

Label the axes of each graph completely.

Give units of measurement where appropriate.

Write a descriptive caption for each table and figure.

Include a short paragraph pointing out important results but do not interpret the data.





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Discussion

State whether the hypothesis was supported or proven false by the results, or else state that the

results were inconclusive.

Cite specific results that support your conclusions.

Give the reasoning for your conclusions.

Demonstrate that you understand the biological meaning of your results.

Compare the results, with your predictions and explain any unexpected results.

Compare the results to other research or information available to you.

Discuss any weaknesses in your experimental design or problems with the execution of the

experiment.

Discuss how you might extend or improve your experiment.

Conclusion

Restate your conclusion.

Restate important results.

Literature Cited

Use proper citation form in the text.

Use proper citation form in the Literature Cited section.

Refer in the text to any source listed in this section.

Acknowledgment

State any appropriate acknowledgement that you think is necessary.





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Experiment 1

Title: Measurement and The Compound Microscope

Part 1: The Measurement

Objectives:

After completing this exercise, you will be able to:

1. Define length, volume, meniscus, mass, density, temperature, thermometer;

2. Recognize graduated cylinders, beakers, Erlenmayer flasks, different types of pipettes, and a

triple beam balance;

3. Measure and estimate length, volume and mass in metric units;

4. Explain the concept of temperature;

5. Measure and estimate temperature in degrees Celsius;

6. Explain the advantages of the metric system of measurement.

Introduction:

One requirement of the scientific method is that results be repeatable. As numerical results

are more precise than purely written descriptions, scientific observations are usually made as

measurements. Of course, sometimes a written description without numbers is the most

appropriate way to describe a result.

Materials:

Rulers

Graduated cylinders

Beakers

Erlenmayer flasks

Pipettes

Triple beam balance

Thermometers

The Metric System:

Logically, units in the ideal system of measurement should be easy to convert from one to

another (for example, inches to feet or centimeters to meters) and from one related measurement

to another (length to area, and area to volume). The metric system meets these requirements and is

used by the majority of citizens and countries in the world. Universally, science educators and

researchers prefer it.

The metric reference units are the meter for length, the liter for volume, the gram for mass,

and the degree Celsius for temperature. Regardless of the type of measurement, the same prefixes

are used to designate the relationship of a unit to the reference unit. Table 1.1 lists the prefixes we

will use in this and subsequent exercises.

As you can see, the metric system is a decimal system of measurement. Metric units are

10, 100, 1000, and sometimes 1,000,000 or more times larger or smaller than the reference unit.





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Thus, it's relatively easy to convert from one measurement to another either by multiplying or

dividing by 10 or a multiple of 10:

x1000 x1000 x10 x100

meter ← meter ← meter ← meter ← meter

nano {liter micro {liter milli {liter centi {liter liter

gram → gram → gram → gram → gram

÷1000 ÷1000 ÷10 ÷100

Prefix of Unit (Symbol) Part of Reference Unit

nano (n) 1/1,000,000,000 = 0.000000001 = 10-9

micro (μ) 1/1,000,000 = 0.000001 = 10-6

milli (m) 1/1000 = 0.001 = 10-3

centi (c) 1/100 = 0.01 = 10-2

kilo (k) 1000=103

Table 1.1 - Prefixes for Metric System Unit

A. Length:

Length is the measurement of a real or imaginary line extending from one point to another.

The standard unit is the meter.

Commonly used related units of length are:-

1000 millimeters (mm) = 1 meter (m)

100 centimeters (cm) = 1 meter (m)

1000 meters (m) = 1 kilometer (km)

Most biologists measure lengths with metric rulers.

1. Examine intervals marked on the metric rulers available in lab.

2. Make the following measurements. Be sure to include units for each measurement.

Length of this page _____________

Width of this page _____________

Your height _____________

Thickness of this manual _____________

Height of beaker _____________





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B. Volume:

Volume is the space an object occupies. The standard unit of volume is the liter (L), and

the most commonly used subunit is the milliliter (mL). There are 1000 mL in 1 liter.

1 cubic centimeter (cc) = 1mL

Scientists often measure volumes with beakers and graduated cylinders. To appreciate how to

make these measurements accurately, pour 40-50 ml of water into a 100 ml graduated cylinder and

observe the interface between the water and air. This interface, which is called the meniscus, is

curved because of surface tension and the adhesion of water to the sides of the cylinder. When

measuring the liquid in a cylinder such as a graduated cylinder, always position your eyes level

with the meniscus and read the volume at the lowest level.

1. Measure the milliliters needed to fill a named container (e.g. Can, mug)

C. Mass:

Mass is the quantity of matter in a given object. The standard unit is the kilogram (kg), and

other commonly used units are the milligram (mg) and gram (g). There are 1,000,000 mg in 1 kg

and 1000 g in 1 kg. A chicken egg has a mass of about 60 g. Note that the following discussion

avoids the term weight. This is because weight depends on the gravitational field in which the

matter is located. For example, you weigh less on the moon but your mass remains the same as it

is on earth. Although it is technically incorrect, mass and weight are often used interchangeably.

1. How many milligrams are there in 1 g?

mg

2. Convert 1.7 g to milligrams and kilograms.

___mg ___kg

Make metric measurements of mass

Biologists often use a triple-beam balance to measure the mass.

1. Locate the triple-beam balances in the lab.

2. Each of three beams of the balance is marked with graduations: the closest beam has 0.1-g

graduations, the middle beam has 100-g graduations, and the farthest beam has 10-g

graduations.

3. Before making any measurements, clean the weighing pan and move all of the suspended

weight to the far left.

4. The balance marks should line up to indicate zero grams

5. Measure the mass of an object by placing it in the center of the weighing pan and moving the

suspended masses until the beams balance.

6. The mass of the object is the sum of the masses indicated by the weights on the three beams.

7. Measure the masses of the following items. Be sure to include units for each measurement.

Paper clip, Pencil, Rock, 100-ml beaker (empty) and 100-ml beaker containing 50 ml of water.





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D. Density:

Density is mass per unit volume. Use data that you’ve gathered from part C (question

7) to determine the density of water.

Density of water = (mass/volume) = ___________

E. Temperature:

The degree of hot or cold of an object is termed temperature. More specifically, it is the

average kinetic energy of molecules. Heat always flows from high to low temperatures. This is

why hot objects left at room temperature always cool to the surrounding or ambient temperature,

while cold objects warm up. Consequently, to keep a heater hot and the inside of a refrigerator

cold requires energy. Thermometers are instruments used to measure temperature.

To convert Celsius degrees to Fahrenheit degrees, multiply by 9/5 and add 32. Is 4°C the

temperature of a hot or cool day? ____

What temperature is this in degrees Fahrenheit? ____ °F

To convert Fahrenheit degrees to Celsius degrees, subtract 32 and multiply by 5/9.

What is body temperature, 98.6°F, in degrees Celsius? ____ °C

Location 0c °F

Room

Cold running tap water

Hot running tap water

Ice water

Boiling water

Table 1.2 - Comparison of Celsius and Fahrenheit Temperatures

In summary, the formulas for these temperature conversions are:

°F = 9/5 °C + 32

°C= 5/9 (°F -32)

Question

What are the relationships of each of the following mass units with the base gram unit? Write each

of them down.

centigram (cg)

milligram (mg)

microgram (µg)

nanogram (ng)





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Part 2: The Compound Microscope

Objectives:

After completing the exercise, you will be able to:

1. Describe how to care for a compound light microscope;

2. Recognize the function of the parts of a compound light microscope;

3. Correctly use a compound light microscope;

4. Make a wet mount;

5. Use your skills to explore the microscopic world.

Introduction:

All individual cell parts, most cells, and even some entire plants are so small that they

cannot be seen with the unaided eye. The study of such small plants and plant parts may be

facilitated by the use of a hand lens, but in order to observe clearly the minute structure of such

things, the compound microscope is most useful.

The invention of the compound microscope generally is attributed to Hans and Zacharias

Jansen, Dutch spectacle makers, who in 1590 developed the crude forerunner of today's fine

precision instruments. For many years, however, the grinding of lenses was a hit-or- miss affair; and

it was not until about 1872 that Ernst Abbe derived the mathematical formula which enabled lens

grinders to make their lenses with extreme precision and to duplicate any previously made lens.

Since that time both the optics and the mechanics of the microscope have been improved until they

have reached a very high degree of perfection.

The compound microscope consists essentially of an optical system composed of a series of

lenses arranged in such a manner as to give a clear image, much enlarged, of small objects, and a

mechanical system which permits the lenses to be adjusted in such a way as to bring them into

critical focus on the object being examined.

The optical system consists fundamentally of an ocular lens or eyepiece which fits snugly

into the upper end of a light-tight body tube, and objective lens fitted to the opposite end of the

body tube, and a mirror or other source of light below the objective lens. In addition, most

microscopes now have an Abbe condenser between the mirror and the objective, the purpose of

which is to concentrate the light into a small beam. In practically all microscopes several objective

lenses with different powers of magnification are fitted into a revolving nosepiece attached to the

lower end of the body tube. Thus several degrees of magnification may be done by simply rotating

the nosepiece so as to bring different objectives into the optical axis.

When the revolving nosepiece is used, the objectives are adjusted so as to be parfocal. That

is, an object seen in clear focus under one objective will also be in focus under other objectives.

The magnifying power of a microscope is obtained by multiplying the power of the

objective by the power of the ocular; thus a 10 x ocular used with a 10 x objective gives a





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magnification of 100 diameters. The highest magnification which can be obtained with an optical

microscope using visible light is about 3,000.

The electron microscope uses a beam of electrons instead of light and a series of magnetic

fields instead of glass lenses. A clear image at 10,000 magnification or more may be obtained.

The instructor will discuss the parts and the use and care of the microscope, but each student

should familiarize themselves with the names and functions of all parts of the instruments. Label

each of the parts shown in the illustration.

Above all, remember that your microscope is an expensive precision instrument and should

be treated accordingly.

Materials:

For each student

Laboratory kit.

Laboratory manual.

Microscope.

Prepared slide of a single letter or a small piece of printed matter.

Prepared slide of several kinds or colours of fibers.

For each student table

A piece of newspaper with small newsprint.

Several small pieces of fabric of different kinds and colours.

A dish containing Spirogyra or other filamentous algal.

Dropping bottles of water, 50 per cent alcohol, eosin, and IKI solution.

For supply-demonstration table

Optional but informative:

1. Display of different makes, ages, and models of microscopes, oculars, and objectives.

2. Display of microscopes accessories such as lamps and micro-manipulators.

3. Wall charts of microscopes showing the various parts.

Methods:

I. Microscope Preparations

Materials which are to be examined microscopically are handled as one of two types of

preparation, the temporary mount or the permanent mount. Since the microscope examination

of most materials involves transmitted light, the preparation must be thin enough for making

temporary mounts for laboratory use, take care to use only a little material and see that the material

is spread out in a thin and uniform layer.





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A. The temporary mount:

A temporary mount, as the name implies, is one intended for a brief period of examination,

after which it may be discarded. Prepare such a mount in the following way:

1. Take a clean glass slide and place, near the center, a drop of clean water. For some preparations

you may need to use alcohol, glycerine, iodine solution, or other liquid.

2. Into the water (or other liquid) introduce a small amount of material to be examined, making

sure that the material is covered with liquid.

3. Cover the material with a plastic cover slip. The cover slip should be leaned over the material

rather than dropped flat, for dropping it flat on the material tends to trap many small air bubbles

which cause considerable annoyance to the observer. Mounts which may need to be kept from

one laboratory period to the next should be made with glycerine or with paraffin oil rather than

with water.

B. The permanent mount:

The preparation of permanent microscope slides is a long and tedious process. The principal

steps in the preparation of such slides are as follows:

1. The material to be made into slides is killed by being placed in one or another of several killing

solutions. One of the most commonly used killing solutions is referred to as F.A.A. and contains

formalin, acetic acid, and alcohol.

2. The killed material is dehydrated by being passed through a number of grades of alcohol -

50,70,85,90 and 100 percent.

3. The dehydrated material is then infiltrated with and imbedded in a block of paraffin wax.

4. Using an instrument called the microtome, very thin sections of the imbedded material are cut

off, and these sections are affixed to clean glass slides with an adhesive.

5. The paraffin is dissolved away with some organic solvent, such as xylene or toluene, and the

slides are passed back down through the graded alcohols to 50 per cent alcohol or even to water.

6. The slides are then passed through certain dye solutions and are again dehydrated and passed

into xylene or toluene, which "clear" the material and makes it more nearly transparent.

7. Finally the cover slip is added and is sealed to the slide with a resin.

This process may require anywhere from 2 or 3 days to several weeks, depending on the

specific procedure followed by the technician. The method of preparation of permanent slide is

outlined here for one specific reason: As you use prepared slides in this course, remember that

someone contributed a great deal of time, of patience, and of technical skill to their preparation.

Handle them with care.

II. Preparing to Use the Microscope

Properly used, the microscope is one of the most important tools of biologist. From the very

beginning, try to learn to use it properly. The following steps will help you to do this.

1. Place the microscope on the table in front of you with the heel of the base toward you and about

an inch from the edge of the table. If you are tall enough to look through the upright

microscope, do so. If you are not, incline the instrument toward you only as far as you need to in





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order to look through it comfortably. Do not set the microscope obliquely in front of you, and

do not incline it more than absolutely necessary.

2. Clean the lenses with lens paper, first the high-power objective, then the low-power objective,

and finally the ocular. (CAUTION: Never touch the lenses with your fingers, and never clean

them with anything else but use only lens paper.) The mirror can be cleaned with a handkerchief

or with a towel. Snap the low-power objective into position beneath the body tube and lower the

body tube until the lens is about 1/4 above the stage (or as far as it will go if your microscope

has a stop to keep the lens from striking the stage ).

3. See that the iris diaphragm is opened wide, and then turn the concave surface of the mirror up.

Watching the objective from one side, turn the mirror toward the source of light and adjust it

until the tip of the objective is brightly and evenly illuminated. Now look through the ocular.

You should see a clear circle of light, the microscope field. How large does this field appear to

be?

Actually it is approximately 1.6 mm in diameter, or about the size of this letter "o".

4. Now place the slide to be examined on the stage of the microscope, making sure that the

material to be viewed is centered over the opening in the stage, and clip the slide in place with

the stage clips.

5. While looking through the ocular, raise the body tube slowly by turning the large coarse

adjustment knobs slowly backward. Soon the object should come into view. If it does not, again

observe the objective from the side and lower it until it almost touches the slide; and then look

for the object again. (CAUTION: Never lower the body tube with the coarse adjustment

knobs while you are looking through the microscope.) When the object comes into view use

the small fine adjustment knobs in order to get the best image possible. If the light is too bright

for your eyes, reduce the intensity by partially closing the iris diaphragm. Do not attempt to

reduce the light intensity by turning the mirror aside, for this will invariably cause distortion.

Use whichever eye seems most natural to you, but learn to keep both eyes open.

6. To locate the object under high power, first make sure it is in the exact center of the field, and

then switch the high-power field which has a diameter of only 0.4 mm., or about one-fourth that

of the low-power field. Any object which is not at or near the center of the low-power field will

be out of the high-power objective view. Adjust the focus as required, using the fine

adjustment knobs. (CAUTION: Never use the coarse adjustment with the high power.)

7. To remove a slide from the microscope, first switch the low-power objective into position so as

to avoid any possibility of damage to the high-power lens.

III. Exercises in the Use of the Microscope

1. Knowing that the low-power field has a diameter of 1.6 mm, calculate the area of the field by

using the formula Area = Π r2. The area of the low-power field of my microscope (Number

__________) is ________ mm2. Do the same for the high-power field. The area of the high-

power field of my microscope is __________ mm2.

2. Using some small newsprint, tear or cut out a bit of paper containing a few letters and make a

temporary mount in water, following the procedure outlined under Experiment I- A. Study this

mount microscopically. What does the microscope do to the size of the letters?

__________________________________________________________________





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What does it do to the position of the letters?

__________________________________________________________________

Move the slide gently to the right and note in which direction the image appears to move. Move

it to the left, toward you, and away from you, noting each time the apparent direction of

movement

3. Using a few fibers of cotton, silk, or other material which have been dyed different colours,

make a temporary mount in which a fiber of one colour crosses a fiber of another colour.

Can you focus the microscope so as to tell which fiber is on top?

__________________________________________________________________

Open the diaphragm wide. Can both fibers be seen clearly?

__________________________________________________________________

Close the diaphragm until the light is almost cut off and examine the fibers again. Can you see

both of them now?

__________________________________________________________________

4. Mount a small amount of a filamentous alga, such as Spirogyra, in water and examine

microscopically. Knowing the diameter of the field, estimate the length of one of the structural

units or cells. Study the scale engraved on the fine adjustment knob on the right side of your

microscope. Moving this knob a single space changes the elevation of the objective by 0.002

mm., or 2 μm. Experiment with this until you can measure the diameter (thickness) of the

Spirogyra cell. To do this, raise the fine adjustment until you can just make out the upper

surface of the cell as you view it under high power. Make a record of the position of the

adjustment knob. Now lower the lens until you can just make out the lower limit of the cell, and

again record the position of the knob. How many spaces did you move the knob in changing the

focus from the top of the cell to the bottom?

__________________________________________________________________

How thick is the cell?

__________________________________________________________________





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Experiment 2

Title: Cell Structure I

Objectives:

After completing the exercise you will be able to:

1. Prepare specimens for staining

2. Identify unique and standard structures of the microscopic world

3. Identify and use different stains for the different types of organelles

Introduction:

Cells were first described in 1665 by Robert Hooke and are now known to be of almost

universal occurrence in organisms.

The Cell Theory states that the cell is the basic unit of an organism, the whole organism

being little more than a collection of independent cells. Though basically similar, cells show

considerable diversity in their contents, shape and function. In all cases, there is a close relationship

between cell structure and function.

To see a particular structure it may be necessary to stain the cell. The choice of stains is

important because certain stains are specific to certain structures; thus acetocarmine stains the

nucleus and its contents; iodine solution stains starch grains.

Materials:

x.s. pine needle

Rheo discolour (leaf)

Yeast (in 1% Sucrose)

Blood cells (Prepared Slides)

Spirogyra (Prepared Slides)

Euglena (Prepared Slides)

Celery

Eosin

5 % Sucrose

Lactophenol

Iodine

Janus Green

Acetocarmine

Methylene Blue

Methods:

I. Epidermal Cells of Plants

1. Remove the surface layer of cells and the epidermis from the upper surface of the Rheo

discolours leaf.

2. Mount the transparent tissue, torn side down, in water on a clean slide and cover with a cover

glass.

3. Study this tissue under low power to determine the shape and the arrangement of the cells and

then under high power to observe the details of cell structure.

4. In which of these cell types are the nuclei most readily seen?

5. If details of cell structure are not readily seen, mount the material in eosin.

6. Label completely your drawing of the epidermal peel.

7. Do these cells differ from one another in any particular respect?

8. If so what differences do you observe?





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9. Repeat your observations using a piece of epidermis from the lower side of the leaf. Has this

tissue the same uniform structure as the upper epidermis?

Do you find any cells which contain green plastids?

What is the shape of such cells?

10. Epidermal peels from leaves of other plants such as Zebrina and corn can be compared.

II. Cell Wall and Middle Lamella.

1. Examine the large cells at the corners of transverse section of a pine needle.

2. Notice the thick cellulose walls which have been laid down in layers.

3. The thin line separating the cellulose walls of adjacent cells, the middle lamella, is clearly

visible. What does it represent?

4. Fine channels in the cellulose walls connect adjacent cells, these are the

____________________________?

III. Yeast Cells (Saccharomyces)

Yeast is a unicellular fungus which grows naturally on the surface of fruits.

1. Pipette a small drop of yeast suspension onto a slide and add a drop of iodine solution. Cover

with a cover slip.

2. On a second slide mount a drop of yeast in lactophenol. Observe the slide under high power.

What do you see?

How does the stain help you to make out details of the cells?

Yeast reproduces by budding: one or more buds grow out from the parent cell and eventually

break away. Can you see yeast cells budding?

What is the significance of budding?

In what way does the yeast cell resemble:-

a) an animal cell ?

b) a plant cell?

What conclusion do you draw from the comparison?

IV. Blood Cells.

1. Examine white blood cells in a prepared smear of human blood. Observe the granulocytes. They

move by amoeboid locomotion and engulf bacteria by phagocytosis.

What other unusual feature is apparent in these cells?

While looking at the blood smear notice the red blood cell. How do they differ from typical

animal cells?

2. Draw and label.

V. Spirogyra Cells

1. Mount a filament of the fresh water algae, Spirogyra and observe under high powers.

2. The filament consists of a chain of elongated cells joined end to end.





18

3. Observe one of its cells in detail.

4. Draw and label.

Can you identify the cell wall?

Study the shape of the chloroplast with the pyrenoids, cytoplasm, nucleus suspended in the

centre of vacuoles by cytoplasmic bridles. What is the 3-dimensional shape of the cells?

Investigate the detailed structure of the cells by staining 3 separate slides with

a) acetocarmine for the nucleus

b) methylene blue for cell wall

c) iodine solution for starch grains near the pyrenoids

5. The cell wall contains mucilage (slimy). Why is this useful?

VI. Flagellum

1. Examine Euglena or some other comparable unicellular flagellate, under high power, using low

illumination and watch the flagella in action.

2. Irrigate with Noland's solution which fixes flagella and stains them blue. Draw and label.





19

Experiment 3

Title: Cell structure II

Objectives:

To examine different types of plant cells and to identify their special organelles

Introduction:

Just as there is diversity of form in life, so there is in the form and function of cells that

make up living organisms. Single cells such as Euglena, Chlamydomonas or other unicellular

organisms can be free-living, capable of carrying on an independent existence. Some cells live as

part of a loosely organized colony of cells that move from place to place.

Cells vary in size, shape and functions whatever its form or function. The cell is recognized

as the basic unit of living matter, containing all those properties and processes that are collectively

called life.

The living plant contains many kinds of cells, each specialized for or adapted to a certain

function. Though the plants are considered to be living organisms, they contain many non-living

cells; and though they maybe chlorophyll-bearing, many of them lack chlorophyll. The purpose of

this study is to examine different types of plant cells.

Materials:

Microscope

Filter paper

Navashin’s solution or FAA

Eosin

Moss

Ripe tomato

Slides and cover slip

Iodine solution

Saline solution (concentrated)

Elodea

Potato

Carrot

Methods:

I. Elodea

1. Mount an entire leaf of Elodea on a slide and observe it under the low power microscope. Note the

thorn-like spur cells projecting from the leaf margin.

2. Choose a place about half way from the midrib to the margin of the leaf, using high-power if

necessary, try to determine by focusing up and down the number of layers of cells in the leaf.

i) How many layers of cells do you find?

ii) Examine the margin of the leaf. How thick is the leaf at the margin?

iii) Returning to former position, study a single cell of this leaf under high power. Note the light

coloured lines and the transparent cell walls. Each light coloured line actually is the combined

walls of 2 adjacent cells cemented together by the middle lamella, which consists largely of

calcium pectate.

3. Study carefully the contents of a single cell. The organized, living material enclosed by the cell

wall is the protoplast and the living substance itself is protoplasm.

i) Can you locate the spherical or oval gray granular nucleus?





20

ii) Look carefully, for it may be hidden by other parts of the cell. Look for the cytoplasm, a gray

or colourless fluid and materials located just inside the cell wall. Do you find the green

chloroplasts?

iii) The nucleus is surrounded by a nuclear membrane. Inside the nucleus are the granular

chromatin, one or more spherical nucleoli, and the jelly-like nuclear sap. Try to locate and

identify these parts.

iv) The central part of the cell is occupied by the vacuole, a cavity in the cytoplasm which is filled

with cell sap, water in which sugars, salts, and other substances are dissolved. The vacuolar

membrane or tonoplast separates the vacuole from the cytoplasm and a similar membrane, the

plasma membrane, lies between the cytoplasm and the cell wall. These cell membranes are

difficult to distinguish in the living cell.

4. Study carefully one of the spur cells found at the margin of the leaf.

i) How does the number of chloroplast in this cell compare with the number in the regular leaf

cell?

ii) What cell part or parts can you find here which you could not find in the regular leaf cell?

iii) Draw the details of the cell structure in the spur cell.

Note: if you cannot find the different part of the cell in the leaf mounted in water, lift the cover

glass, drain off the water, and add a drop of iodine solution. Replace the cover glass and observe

again. Or remove the water from under the cover glass with a piece of filter paper, and then add a

drop of Navashin’s solution or a drop of F.A.A. under the cover glass. Any of these solutions will

kill the protoplasts and stop all movement, but a number of cell structures will be much more easily

observed after the treatment.

II. Moss

1. Mount a small leaf of the moss in water and examine its cells under low power, then high power.

i) How many layers of the cells do you find?

ii) Can you see the chloroplast?

iii) What is the green colour of the chloroplast caused by?

III. Other types of plastids

In many plant cells, there are plastids which do not contain chlorophyll. Such plastids may be

colourless (leucoplasts) or they may contain pigments other than chlorophyll (chromoplasts).

Under certain conditions, one type of plastid may be changed into another type.

a. Leucoplasts

1. Scrape off some material from the potato and mount it in dilute iodine solution.

i. What do you observe?

ii. Dilute the iodine solution further and scrape off more material and mount. Can you see the

structure of the starch grains? Draw.

iii. Observe these grains mounted in water. They should be colourless. Such colourless plastids are

leucoplasts.





21

b. Chromoplasts

1. Make a temporary mount of a bit of pulp from a ripe tomato or a very thin section of carrot.

2. Observe under the microscope.

3. Look for coloured bodies, the shape and number of which will vary from one plant to another.

Draw a cell showing the chromoplasts.





22

Experiment 4

Title: Membrane Permeability

Objectives:

After completing this exercise, you will be able to:

1. Define solvent, solute, solution, selectively permeable, diffusion, osmosis, concentration gradient,

equilibrium, turgid, plasmolyzed, plasmolysis, turgor pressure, tonicity, hypertonic, isotonic,

hypotonic;

2. Describe the effects of hypertonic, isotonic, and hypotonic solutions on Elodea leaf cells and onion

scale leafs.

Introduction:

Living cells are made up of 75% - 85% water. Virtually all substances entering and leaving

cells are dissolved in water, making it the solvent most important for life processes. The substances

dissolved in water are called solutes and includes such substances as salts and sugars. The combination

of a solvent and dissolved solute is a solution. The cytoplasm of living cells contains numerous solutes,

like sugars and salts, in solution.

All cells possess membranes composed of a phospholipid bilayer that contains different kinds

of embedded and surface proteins. Membranes are boundaries that solutes must cross to reach the

cellular site where they will be utilized in the process of life. These membranes regulate the passage of

substances into and out of the cells. They are selectively permeable, allowing some substances to

move easily while completely excluding others.

The simplest means by which solutes enter the cell is diffusion, the movement of solute

molecules from a region of high concentration to one of lower concentration. Diffusion occurs without

the expenditure of cellular energy. Once inside the cell, solutes move through the cytoplasm by

diffusion, sometimes assisted by cytoplasmic streaming.

Water (the solvent) also moves across the membrane. Osmosis is the movement of water

across selectively permeable membranes. Think of osmosis as a special form of diffusion, one

occurring from a region of higher water concentration to one of lower water concentration.

The difference of concentration of like molecules in two regions is called a concentration

gradient. Diffusion and osmosis take place down concentration gradients. Over time, the

concentration of solvent and solute molecules becomes equally distributed, the gradient ceasing to

exist. At this point, the system is said to be at equilibrium.

Molecules are always in motion, even at equilibrium. Thus, solvent and solute molecules

continue to move because of randomly colliding molecules. However, at equilibrium there is no net

change in their concentrations.





23

Plasmolysis in Plant Cells:

Plant cells are surrounded by a rigid cell wall composed primarily of the glucose polymer,

cellulose. Many plant cells have a large central vacuole surrounded by the vacuolar membrane. The

vacuolar membrane is selectively permeable. Normally, the solute concentration within the cell’s

central vacuole is greater than that of the external environment. Consequently, water moves into the

cell, creating turgor pressure, which presses the cytoplasm against the cell wall. Such cells are said to

be turgid. Many nonwoody plants (like beans and peas) rely on turgor pressure to maintain their

rigidity and erect stance.

In this experiment, you will discover the effect of external solute concentration on the structure

of plant cells.

Materials:

For each student

Forceps

2 microscope slides

2 coverslips

Compound microscope

For each table

Elodea in tap water

2 dropping bottles of dH2O

2 dropping bottles of 20% sodium

chloride (NaCl)

Methods:

1. With a forceps, remove two young leaves from the tip of an Elodea plant.

2. Mount one leaf in a drop of distilled water on a microscope slide and the other in 20% NaCl

solution on a second microscope slide

3. Place coverslips over both leaves.

4. Observe the leaf in distilled water with the compound microscope. Focus first with the medium–

power objective and then switch to the high-dry objective.

5. Now observe the leaf mounted in 20% NaCl solution. After several minutes, the cell will have lost

water, causing it to become plasmolyzed. (This process is called plasmolysis.)

6. To observe deplasmolysis, slowly and gently remove the coverslip from a preparation exhibiting

plasmolysis, drain the salt solution and add two drops of distilled water. After 1 minute, place a

coverslip on the preparation. Examine under high-dry for 5 minutes and then give a see what

happens during deplasmolysis.

Tonicity describes a solutions solute concentration compared to that of another solution. The solution

containing the lower concentration of solute molecules than another is hypotonic relative to the second

solution. Solutions containing equal concentrations of solute are isotonic to each other, while one

containing a greater concentration of solute relative to a second one is hypertonic.

Question:

Were the contents of the vacuole in the Elodea leaf in distilled water hypotonic, isotonic, or hypertonic

compared to the water?





24

Was the 20% NaCl solution hypertonic, isotonic or hypotonic relative to the cytoplasm?

If a hypotonic and a hypertonic solution are separated by a selectively permeable membrane, which

direction will the water move?

Name two selectively permeable membranes that are present within the Elodea cells and that were

involved in the plasmolysis process.

1.________________________________________________________________

2.________________________________________________________________





25

Experiment 5

Title: Macromolecules

Objectives:

After completing this exercise, you should be able to:

1. Define monosaccharide, disaccharide, and polysaccharide and give examples of each.

2. Name the monosaccharide components of sucrose and starch.

3. Describe the test that indicates the presence of most small sugars.

4. Describe the test that indicates the presence of starch.

5. Define hydrolysis and give an example of the hydrolysis of carbohydrates.

Introduction:

Living organisms are composed of molecules that come in diverse shapes and sizes and serve a

variety of purposes. Some molecules form the structure of an organism’s body, for example, the

cellulose that makes up the cell walls in plants, the proteins and phospholipids that comprise cell

membranes, and the fibers that make up animal muscles.

There is also a wide array of molecules that perform all the functions of life. For example,

enzymes catalyze the chemical reactions necessary for biological processes, neurotransmitters convey

information from one neuron to another, and visual pigments absorb light so that you can see with your

eyes.

In this laboratory, you will study three classes of the largest biological molecules, called

macromolecules: carbohydrates, lipids, and proteins.

Part 1: Carbohydrates

Most carbohydrates contain only carbon, oxygen, and hydrogen. The simplest forms of

carbohydrate molecules are the monosaccharides (single sugar). One of the most important

monosaccharides is glucose, the end product of photosynthesis in plants. It is also the molecule that is

metabolized to produce another molecule, ATP, whose energy can be used for cellular work. There are

many other common monosaccharides, including fructose, galactose, and ribose.

Some disaccharides (double sugars) are also common. A disaccharide is simply two

monosaccharides linked together. For example, maltose consists of two glucose molecules, lactose

(milk sugar) consists of glucose and galactose, and sucrose (table sugar) consists of glucose and

fructose. Can you discern a rule used in naming sugars?

Carbohydrates are also found in the form of polysaccharides (many sugars), which are long

chains of monosaccharide subunits linked together. Starch, a polysaccharide composed of only glucose

subunits, is an especially abundant component of plants. Most of the carbohydrates we eat are derived

from plants.

Starch is the plant’s way of storing the glucose it makes during photosynthesis. When you eat starch,

you are consuming food reserves that the plant has stored for its own use. The starch of potatoes and





26

root vegetables, for example, would be used the next spring for the plant’s renewed growth after the

winter die-back.

Animals store glucose in glycogen, which is another form of polysaccharide. Although starch

and glycogen are both composed of glucose subunits, the glucose molecules are bonded together in

different ways, so these polysaccharides are not identical. Glucose subunits are bonded together in a

third way in the polysaccharide cellulose. While starch and glycogen are meant to be metabolized for

energy, cellulose, which is the most abundant carbohydrate in the world, is a structural molecule that is

designed not to be metabolized. Cellulose makes up the cell walls of plants and is a primary

component of dietary fiber. For most animals it is completely indigestible. Those that can digest it,

such as termites and cows, do so only with the assistance of organisms such as bacteria, fungi, or

protistans.

Most disaccharides and polysaccharides can be broken down into their component

monosaccharides by a process called hydrolysis, which is accomplished in organisms by digestive

enzymes. This process is important in seeds. If the seed’s food resource is starch, it must be able to

convert the starch to glucose. The glucose is then used to generate ATP, which in turn is used to

provide the growing plant embryo with energy for metabolic work. Hydrolysis of starch begins when

the seed takes up water and begins to germinate.

A chemical hydrolysis can be done in the laboratory by heating the molecules with acid in the

presence of water. You will perform a chemical hydrolysis in this exercise.

Investigation 1: Monosaccharides and Disaccharides

You will use Benedict’s reagent as a general test for small sugars. When this reagent is mixed

with a solution containing single or double sugars and then heated, a coloured precipitate (solid

material) forms. The precipitate may be yellow, green, orange, or red. If no monosaccharide or

disaccharide is present, the reaction mixture remains clear. However, Benedict’s reagent does not react

with all small sugars. For example, sucrose gives a negative Benedict’s reaction.

Glucose will be used to demonstrate a positive Benedict’s test. What should be used as a negative

control for this test?

Materials:

Benedict’s reagent

1% solutions of glucose, fructose, lactose, sucrose, and starch.

Test tube

Beaker

Hot plate

Methods:

1. Make a boiling water bath by filling a beaker about half full of water and heating it on a hot plate.

You will need to use this water bath in several activities.





27

2. Place in a test tube 1 ml of a 1% glucose solution and 5 ml of Benedict’s solution.

3. Prepare a control.

4. Place the two tubes in boiling water for 2-3 minutes.

5. Observe the colour of the solution and note whether a precipitate has formed.

Note: a change in colour of the solution is not indicative of a positive reaction. A precipitate must

appear.

6. Repeat the test with 1% solutions of fructose, lactose, sucrose and starch.

7. Record all of your results in a table.

Investigation 2: Starch

Starch is tested by using iodine reagent. A dark blue colour indicates the presence of starch.

You will use a solution of potato starch to demonstrate a positive test. What negative control should be

used for this test?

Materials:

Starch solution

Iodine reagent

Test tubes

Methods:

1. Prepare a starch solution by mixing thoroughly 2g of starch with 10 ml of water and then pouring

this mixture into 200 ml of boiling water.

2. Get two test tubes and label them 1 and 2.

3. Put a few ml of the starch solution in Tube 1. This is the positive control.

4. Tube 2 is the negative control. What substance goes in it? How much should be used?

5. Put a few drops of iodine reagent into each tube.

6. Record the observations.

Investigation 3: Hydrolysis of Carbohydrates

As discussed earlier, disaccharides are composed of two monosaccharides linked together.

Polysaccharides are long chains of monosaccharides. The bonds joining these subunits can be broken

in a process called hydrolysis. In this procedure, you will hydrolyze sucrose and starch by heating

them with acid.

What monosaccharides will result from the hydrolysis of sucrose?

What monosaccharide will result from the hydrolysis of starch?

The hydrolysis reactions will be carried out in two large test tubes. One contains sucrose and

hydrochloric acid (HCl) and the other contains starch and HCl. You will sample the sucrose tube

twice: once before the hydrolysis has begun and again after 3 minutes. You will take 6 samples from





28

the starch tube: 2 before the hydrolysis has been done, 2 after 5 minutes of hydrolysis, and 2 after 15

minutes. Two samples are needed at each time so that one can be tested for small sugars (Benedict’s

test) and one can be tested for starch (iodine test).

Materials:

Starch solution

Sucrose solution

2N HCL solution

Benedict’s reagent

Iodine reagent

Test tubes

Pipettes

Water bath

Methods:

1. Get eight test tubes and label them 1 through 8. Line up the test tubes in order in a test tube rack.

2. Get two large test tubes and label them starch and sucrose. Use an empty beaker as a test tube

holder if the test tubes don’t fit in the rack.

3. Pipette 6 ml starch solution and 3 ml 2N HCl into the tube labeled starch.

Caution: HCl is a strong acid. Handle it with caution.

4. Pipette 5 ml sucrose solution and 1 ml 2N HCl into the tube labeled sucrose.

5. Swirl each tube gently to mix the contents.

Sampling

6. Draw 1 ml of solution from the sucrose tube and put it in Tube 1.

7. Using a different pipette, draw 1 ml of solution from the starch tube and put it in Tube 3. (Skip

Tube 2 for now.)

8. Draw an additional ml of solution from the starch tube and put it in Tube 4.

9. Place the extra-large starch and sucrose tubes in your boiling water bath. Note the time.

10. After 2 or 3 minutes, draw 1 ml of solution from the sucrose tube and put it in Tube 2. You are

now finished with the sucrose solution. You may remove it from the water bath.

11. After 5 minutes, draw 1 ml of solution from the starch tube and put it in Tube 5.

12. Put a second ml of starch solution in Tube 6.

13. Wait 10 more minutes and then repeat steps 11 and 12, putting the solution in Tube 7 and 8.

Testing for Starch and Sugar

14. Add 5 ml of Benedict’s reagent to Tubes 1, 2, 3, 5, and 7. Place these tubes in the boiling water

bath for 5 minutes.

15. Add 3 or 4 drops of iodine reagent to Tubes 4, 6, and 8.

16. Remove the tubes from the water bath and wait 5 minutes for them to cool. Record the results in

the following table:





29

Tube Number

Sucrose Starch

1 2 3 4 5 6 7 8

Time

(min) 0 2-3 0 0 5 5 15 15

Benedict’s

reagent

Iodine

reagent

Interpretation of Results:

Explain the results you obtained using the Benedict’s test on the sucrose solution.

Explain the results you obtained using the iodine reagent test with starch.

Explain the results you obtained using the Benedict’s test with starch.

Why does hydrolysis of starch take longer than hydrolysis of sucrose.

Part 2: Proteins

A protein’s structure is determined by the amino acid subunits that make up the molecule.

Although there are only 20 different naturally occurring amino acids, each protein molecule has a

unique sequence. The amino acids are linked by fairly tight bonds, and the side groups that are part of

the amino acids also interact with each other to help shape the molecule.

The bond between amino acids in a protein is a peptide bond and is identified by a Biuret test.

Specifically, peptide bond in proteins complex with Cu2+

in Biuret reagent and produce a violet colour.

A Cu2+

must complex with four to six peptide bonds to produce a colour; therefore, free amino acids

do not react positively.

Biuret reagent is a 1% solution of CuSO4 (copper sulfate). A violet colour is a positive test for

the presence of protein; the intensity of colour relates to the number of peptide bonds that react.

You will use a solution of egg albumin (a protein extracted from egg whites) to demonstrate a

positive Biuret test. What negative control should be used for this test?

Materials:

1% egg albumin

Concentrated KOH

0.5% CuSO4

Test tubes

Methods:

1. Get two test tubes and label them 1 and 2.

2. Put 3 ml of 1 % egg albumin into Tube 1.

3. Tube 2 is the control. What substance goes in it? How much should be used?





30

4. Add an equal volume of concentrated KOH (~ 20%) to both tubes. Mix thoroughly.

5. Slowly add 1 ml of 0.5% CuSO4. Mix.

6. After 2 minutes, record the colour in each tube.

Part 3: Lipids Lipids are oily or greasy compounds insoluble in water, but dissolvable in nonpolar solvents

such as ether or chloroform. The lipids we will consider in this laboratory are fats and oils, which are

generally used as storage molecules in both plants and animals.

Lipids provide long-term energy storage in cells and are very diverse. Lipid digestion occurs

primarily in the small intestine where bile produced by the liver breaks lipid globules into smaller

droplets, and then pancreatic enzymes break large lipid molecules into smaller components for

absorption.

You will use the paper test to indicate the presence of lipids in various foods. Although this test

is not sophisticated, it is quick and convenient.

Materials:

Brown paper

Vegetable oil

Water

Methods:

1. Get a small square of brown paper. Write “oil” on one half and “water” on the other.

2. Put a tiny drop of vegetable oil on the half of the paper labeled oil. Rub it gently with your

fingertip.

3. As a negative control, put a tiny drop of water on the half of the paper labeled water. Rub it gently

with a different fingertip to avoid contamination.

4. Allow the spots to dry. This may take a while.

5. When the spots are dry, hold the paper up to the light.

6. Record your observations.





31

Experiment 6

Title: Macromolecules in Foods

You have learned the methods to test the presence of carbohydrates, starch, protein and lipid in a

sample. Now it is time to put them into good use.

You will be given a few samples. Your job is to test the presence of carbohydrate, starch, protein and

lipid in each sample.

Record your observations in a table.

Samples:

Apple juice

Potatoes

Soft drink

Onions

Instant Noodles

Soya Beans





32

Experiment 7

Title: DNA Extraction

Objectives:

To extract the DNA from various plant samples.

Introduction:

DNA is found in almost every cell of every organism. You’ve heard about it, you’ve seen diagrams of

its structure, but have you ever actually seen DNA molecules? In this activity you will extract DNA

from various cells. After breaking up membranes of the cells in detergent, the DNA is then separated

from the rest of the cell contents.

Materials:

Onion

Detergent

Salt solution

Alcohol

Test tubes

Glass rods

Filter paper

Methods:

1. Grind up a piece of onion (and other plant or animal samples provided) in small amount of water.

2. Add some detergent and salt solution. Stir gently for 5 minutes.

3. Decant the liquid from the mixture into a clean test tube (use filter paper to get clear supernatant).

4. Add in cold alcohol by tilting the test tube at a 45-degree angle and very slowly pour the alcohol

down the side of the tube. The alcohol should just trickle down the side and come to rest on the top

of the water so that it forms a separate layer (don’t let the layers mix).

5. Place the test tube in its rack and do not move it for at least 15 minutes. The DNA will begin

precipitating out immediately between the two layers of liquid.

6. After 15 minutes, the DNA should be floating on the top of the test tube. Use a Pasteur

pipette/glass rod/wooden stick to spool it like cotton candy.

7. Describe what the DNA looks like.

The precipitated DNA in the alcohol layer is only part of the total DNA from the onion cells. Much of

the DNA is still in the water below. You can bring this DNA into the alcohol, where it will precipitate.

Tilt the test tube slightly and insert the Pasteur pipette into the yellowish water fraction, then pull the

pipette up into the alcohol. You should see more DNA come up with it. Be careful not to stir the layers

together. When it reaches the alcohol, the DNA will precipitate in its stringy form and you can spool it,

too.

Use the same procedure for the other samples provided. From which sample can you extract the most

DNA? Can you tell which sample has the smallest cells, the biggest cells?





33

Alternative method:

1. Buffer: add ¼ teaspoon of salt, 1 teaspoon of baking soda and 1 teaspoon of detergent/shampoo

into minimum amount of distilled water. Stir gently to dissolve and mix the ingredients. Add in

enough chilled distilled water to make up 120 ml.

2. Dice and grind onion or other sample provided, add in a little water to the mixture

3. Place 5ml of the mixture into a clean beaker or test tube and add in 10 ml chilled buffer prepared in

step 1. Stir vigorously for at least 2 minutes and then filter away the debris by passing through a

cotton filter.

4. Save at least 5 ml of the supernatant. Slowly add in 10 ml cold 95% ethanol at an angle down the

side of the test tube. Do not mix the ethanol and buffer layers together.

5. Insert a Pasteur pipette/glass rod through the layer of alcohol and twirl back and forth with the tip

of the rod suspended just below the boundary between the alcohol and the buffer solution. Longer

pieces of DNA will spool onto the rod.

Questions:

1. What does the DNA look like?

2. What do you think was the specific purpose of adding each of the following: (i) detergent,

(ii) salt, (iii) alcohol?

3. Why onion is suitable for DNA extraction?

4. What is the component in the detergent that helps in the DNA extraction?





34

Experiment 8

Title: Cell Division: Mitosis

Objective:


1. Identify and describe the stages of the cell cycle

2. Distinguish between mitosis and cytokinesis

3. Stain and examine chromosomes in mitotic cells

Introduction:

All cells come from preexisting cells. The complex series of events whereby the nucleus and

cytoplasm are divided, into two parts, which are usually equal and alike, is called cell division or

mitosis. There are certain structural differences in plant and animal cells, and the two vary to some

extent also in the process of cell division. However, in all organisms the process of cell division is

essentially the same.

Cell Division in Plant Cells

The onion root tip is one of the most widely used materials for the study of mitosis. The onion

root is available in quantity and preparations of the dividing cells are easily made. The chromosomes

are relatively large and few in number and hence easier to study than the cells of many other

organisms. There are regions of rapid cell division in root tips; therefore, the chances are good that

within such tissues one can identify every stage in mitosis.

There are several reasonably distinct stages in cell division, although the process is continuous

and there is some gradation between the various steps. These steps in sequence are prophase,

metaphase, anaphase, and telophase. These stages of mitosis can be observed in onion root tip squash

or in longitudinal sections of the onion root tip on prepared slides.

a) During prophase, the chromosomes become distinguishable in the nucleus, the nuclear membrane

breaks down, and the chromosomes then become distributed haphazardly through the cytoplasm.

At this stage in the onion root tip the chromosomes often appear as a coiled mass and in some

cases the nuclear membrane may still be intact. These elongated chromosomes later become

condensed into shorter chromosomes and. the nuclear membrane disappears. Even at this early

stage each chromosome has probably doubled, although this will be difficult to see on the slides.

b) During metaphase, the chromosomes arrange themselves near the center of the cell. In tile onion,

the ends of the chromosomes will protrude into the cytoplasm on each side of the cell. The

metaphase stage is apparently a preparation for the equal division of chromosomes between the

daughter cells. During or somewhat before metaphase, a small threadlike structures called, spindle

fibers form in the dividing cell.

c) At the beginning of anaphase the two members of the previously doubled chromosomes separate,

one moving toward one side of the cell, the other toward the opposite side. This stage can be

recognized in the onion because there will be two groups of V-shaped chromosomes on opposite





35

sides of the cell. The sharp end of the V is pointed toward the center of the cell. The onion has 16

chromosomes; hence it is seldom possible to see all of them at one time. Out down the light on the

microscope, and see if you can find any spindle fibers near the center of the cell.

d) Cell division is completed during telophase, and reorganization of the cell contents of the two

daughter cells begins. It is often difficult to distinguish between late anaphase and early telophase

in the cells of the onion root tip. During telophase, however, a cell plate starts to form across the

center of the cell, which when complete will divide the original cell into two daughter cells. This

cell plate will appear as a fine line that passes across the dividing cell. Another distinction between

telophase and anaphase is that during telophase the individual chromosomes are not as distinct as

in anaphase as telophase progresses, the nuclei begin to reorganize and the chromosomes become

distinct in the chromatin throughout the nuclear material.

In both plant and animal cells, the daughter cells resulting from mitotic division have the same number

and kinds of chromosomes as the original cell from which they came. Thus, in the onion each daughter

all has l6 chromosomes, just as the original cell did.

Figure 2: Method of supporting an anion to promote growth of roots

Materials:

Prepared slide of onion, Allium, root tip mitosis

Compound microscope

Methods:

1. Examine a prepared slide of a longitudinal section through an onion root tip

2. Focus first with the low-power objective of your compound microscope to get an overall

impression of the root’s morphology





36

3. Concentrate your study in the region about 1 mm behind the actual tip. This region is the apical

meristems of the root. Search for examples of all stages of mitosis. Draw and name in the correct

sequence the different stages of mitosis as you can see at the root tip. 4. Search for signs of cell plate formation.

Preparing and staining chromosomes

Making the squash preparation:

Squash the softened, stained, root tips by lightly tapping on the cover-slip with a pencil: hold

the pencil vertically and let it slip through the fingers to strike the cover-slip. The root tip will spread

out as a pink mass on the slide; the cells will separate and the nuclei, many of them with chromosomes

in various stages of mitosis (because the root tip is a region of rapid cell division) can be seen under

high power of the microscope lens.

Study the section of the onion root tip and the root tip squash that you have prepared. Keep in mind the

sequence in which the different stages occur. Chances are that all stages will not be present in any one

section. You will notice that most of the cells will be in interphase. The next largest number will be in

prophase, only a few will be seen in metaphase, anaphase, and telophase. The reason is that these cells

remain in interphase and prophase longer than in other stages. Why?

Materials:

Onion root tip.

Compound microscope

Support onions over beakers or jars of water using tooth-picks as shown in Figure 2. Keep the onions

in darkness for several days until the roots growing into the water are 2-3 cm long.

Methods:

If you do not intent to use it immediately, follow steps (1) to (3):

1. Fix (i.e. coagulate, harden and preserve the protoplasm) the root tips in Carnoy's fluid for 24 hours.

2. Wash in water for a few minutes.

3. If the material is not to be used at once, store it in 70% alcohol; otherwise proceed to (4).

For immediate use:

4. Place the root tips in a test-tube containing some normal hydrochloric acid for a few minutes (6

min) or until the root tip feel soft. This treatment affects the DNA of the chromosomes so that it

reacts with the stain later on. It also dissolves the middle lamella so that the cells are easily

separated during the squashing process.

5. Pour the contents of the tube into a petri dish, pick out the root tips with forceps or a point brush

and cut off about 3 to 4 mm of the 2-3 cm root tips.

Place them into any of the following-

i. a specimen tube containing leucobasic fuohsin (Schiff's reagent). Leave the root tips in the

corked tube for 23 hours in darkness, during which time they will take on a magenta





37

colouration. Place the root tips on a clean glass slide in a drop of 45 per cent acetic acid and

cover with a cover-slip. View the sample under the microscope.

ii. into a glass slide containing a drop of acetocarmine stain or 0.5% toluidine blue

6. Using a razor blade cut off and retains the tip-most 1mm of the root. Chop the remaining root tip

into many pieces with the razor blade and crush the material with a glass rod

7. Upon completing this procedure, apply a clean cover glass slip to the slide and heat it gently over

an alcohol lamp (Do not boil). Then invert the slide on a tissue and push downward firmly,

applying pressure with your thumb over the cover glass. This should flatten the cells and disperse

them so they can be observed under the microscope.

Questions:

1. Interphase has some times been called a “resting stage.” Why is this inaccurate?

2. Some specialized cells such as neurons and red blood cells lose their ability to replicate when they

mature. Which phase of the cells cycle do you suspect is terminal for these cells? Why?

3. Why do you suppose cytokinesis generally occurs in the cell’s midplane?

4. What would happen if a cell underwent mitosis but not cytokinesis?





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Experiment 9

Title: Cell Division: Meiosis

Objective:


1. Highlight the unique features of meiosis which are different from mitosis

2. Compare and contrast spermatogenesis and oogenesis by identifying the unique features of each

process

3. Identify the basic features of meiosis in microsporogenesis and megasporogenesis

4. Compare and contrast the microsporogenesis and megasporogenesis in angiosperms

Introduction:

In all organisms which reproduce sexually, there is a doubling of chromosome number at the

time of fertilization when the nuclei of the two sex cells or gametes fuse. Consequently there must also

be in the life cycle a compensatory mechanism, before the next sex cells are produced, in which the

chromosome number is halved again. This halving is achieved in the process of meiosis. Thus meiosis

is an invariable component of the life cycle of sexually reproducing organisms, alternating with

fertilization in the life cycle.

The beginning student sometimes confuses the events and significance of the two distinctly

different processes, mitosis and meiosis. Mitosis is a division of the nucleus which, with the cell

division which usually immediately follows, results in new cells with kinds and numbers of

chromosomes the same as are in the parent cells. Meiosis, on the other hand, is sharply restricted to a

particular part of the life cycle, and to a particular region of the organism. In all plants except some of

the lower forms, it occurs in specialized regions of the plant body, and results in the production of

spores which initiate the alternate generation in the life cycle. This alternate generation was half the

chromosome number of the generation which results from fertilization.

The halving occurs in the meiotic process because there are two nuclear divisions and

separation of each chromosome occurs in only one of them. Usually in the first division homologous

chromosomes pair and then separate, while in the second division the chromatids of each chromosome

separate. Thus from a parental cell with 2 members of each kind of chromosome, 4 cells are produced,

each with 1 member of each kind.

Certain events in meiosis are of the utmost genetic significance. In the prophase of the first

division there is a pairing of homologous chromosomes, and an interchange of parts between

homologous chromatids. This and the ensuing random assortment of nonhomologous chromosomes

create enormous possibilities of variation through gene recombination.

Meiosis in Lily

In this exercise, meiosis will be studied as it occurs in the development of mature pollen grains

of the flowering plants. These pollen grains give rise to the male gametes, which fuse with the egg to





39

produce a seed; the seed ultimately germinates and produces another plant. The first meiotic division is

meiosis I. This is followed by a second division, i.e., meiosis II.

Meiosis I:

Prophase of meiosis I involves the separation of chromosomes that had, in prophase, pained

with each other. One chromosome of each pair is of maternal, and the other of paternal, origin, and

their separation leads to the formation of haploid nuclei.

Meiosis I is divided into four stages: prophase, metaphase, anaphase and telophase. It involves the

separation of chromosomes that had, in prophase, pained with each other. One chromosome of each

pair is of maternal, and the other of paternal, origin, and their separation leads to the formation of

haploid nuclei.

Prophase I Prophase I is the stage in which the most profound, and genetically the most significant, modifications

take place, is subdivided into 5 separate stages.

Leptonema

This stage does not differ appreciably from the earliest prophase stages in mitosis except that the

cells and nuclei of meiotic tissues are generally larger than those surrounding somatic tissues. The

chromosomes, too, one longer and more slender. Unlike mitotic chromosomes, along their length

may be seen a series of bead-like structures called chromomeres (these are constant in number,

size, and position).

Zygonema

The pairing or synapsis of chromosomes in intimate association begins in zygonema. Since, to a

diploid offspring, each parent has contributed a haploid set of chromosomes, and since the

chromosomes contributed by the sperm are, with certain exceptions such as the sex chromosomes,

identical with those contributed by the egg, all diploid cells possess pairs of similar or homologous

chromosomes. These chromosomes pair lengthwise with each other in a pattern characteristic of

the species.

Syriapsis, when once initiated, proceeds in zipper-like fashion to bring the homologous

chromosomes together along their entire length.

Pachynema

If zygonema is considered to be the period of active pairing, pachynema is the stable stage. The

chromosomes, which are visibly thicker, appear to be present in the haploid number, but each

thread can be recognized as 2 chromosomes closely oppressed. These pairs are referred to as

bivalents. The nucleolus is particularly evident at this stage, and certain of the chromosomes are

attached to it.

Diplonema

Longitudinal separation of the paired chromosomes initiates diplonema. At the same time the

longitudinal duality of each chromosome becomes clearly evident, revealing that each bivalent

consists of 4 chromatids. As the pairing relationships of pachynema lapse, the homologues move





40

apart from each other. The separation is generally not complete. However, for the paired

chromosomes are held together at one or more points along their length. The bivalents, as a

consequence, take on the appearance of a cross if there is one point of contact, a loop if there are 2

points of contact or a series of loops if there are 3 or points of contact. Each point of contact is a

chiasma (pl. chiasmata). Since the 2 sister chromatids of any chromosome do not separate laterally

from each other, the chiasma is a point of exchange that preserves the bivalent structure.

During diplonema the chromosomes are actively shortening, and soon their coiled nature is

apparent. During this time the nucleolus may become diminished greatly in size although it

continues to remain attached to its particular chromosome or chromosomes.

Diakinesis

The distinction between diakinesis and diplonema is not a sharp one although diakinesis is

characterized by a more contracted state of the chromosomes, by the disappearance or detachment

of the nucleolus from its associated chromosomes, and by the even distribution of the bivalents

throughout the nucleus. The chromosomes continue to shorten by coiling more tightly. The

bivalents consequently assume a more rounded shape, with the homologues joined to each other

largely at their terminal ends. This union comes about by the terminalization of chiasmata as the

chromosomes shorten.

Metaphase I

Metaphase is characterized by the complete disappearance of the nuclear membrane and the formation

of the spindle. Similarly, as in mitosis, the bivalents congress onto the metaphase plate where they

subsequently become properly orientated.

A difference between mitotic and meiotic chromosomes should be noted. In mitosis, the metaphase

chromosome possesses a functionally undivided centromere, which, together with the centromeres of

the other chromosomes, lies on the metaphase plate. In meiosis, the bivalent has 2 functionally

undivided centromeres, which instead of lying on the plate are simply orientated in the long axis of the

spindle, the distance between them being regulated by the proximal position of the chiasmata. At this

time, and prior to the separation of the chromosomes, an active repulsion appears to exist between

homologous centromere.

Anaphase I

The movement of chromosomes from the metaphase plate do the poles constitute anaphase. Unlike

mitosis, in which the centromere divides and sister chromatids pass to the opposite poles, the

centromeres of each bivalent in meiosis one undivided as they move poleword with the result that

whole chromosomes instead of chromatids segregate. Each anaphase group, therefore, is made up of a

haploid number of chromosomes instead of a diploid member of chromatids. In this manner a

reduction in chromosome number results from the first meiotic division.





41

Telophase I

Telophase and interphase are not necessarily component stages in the full meiotic cycle. In most

organisms a nuclear membrane is formed in telophase, and a regrouping of the chromosomes occurs

with a relaxing of the coiled structure of the chromosome, e.g. grasshoppers, corn, and Tradescantia.

In Trillium, the anaphase chromosomes of the first division in the pollen mother cells orient

themselves at the pole following the disappearance of the spindle, and pass directly to metaphase of

the second division. The coiling of the chromosomes is retained, and persists in fact through to the

interphase which terminates the meiotic divisions.

Second Meiotic Division:

The meiotic process is completed, when each of the two haploid nuclei divide by a process that

is essentially mitotic. (Referred to as MEIOTIC MITOSIS) Four haploid nuclei result. Whether the

usual mitotic prophase is present depends upon whether or not there was an interphase period.

In any event, 3 differences serve to distinguish the second meiotic division from a mitotic division.

First, the chromosomes are present; a haploid number; second, the chromatids in general are widely

separated from each other and exhibit no relational coiling; and third each chromatid might be quite

different generically from its condition at the initiation of the meiotic process.

Cytokinesis in Meiotic Cells:

Segmentation of the original meiotic cell by walls or membranes may or may not take place.

Higher plants - the meiotic cells (microsporocytes) of the anther usually develop a cross well at the end

of the first meiotic division and in a plane at right angles to the axis of division. A second wall at right

angles to the first divides the cells at the end of the second meiotic division. Each of the form cells

(microspores) has its own wall, and as they enlarge they burst through, freeing themselves from the

original wall of the meiotic cell.

The spermatocytes of animals behave like the microsporocytes of the higher plants in that 4 cells are

formed from a single meiotic cell, Cytokinesis, however, is by a process of furrowing, as it is in

somatic cell division of animals.

Materials:

Set of prepared slides for microsporogenesis and megasporogenesis of angiosperms and

spermatogenesis and oogenesis of animals.

Preparing squash slides of Microsporogenesis from plant anthers.

A. Collecting the Material

Materials:

Flower buds

Carnoy’s solution (6 parts absolute ethyl alcohol : 3 parts chloroform : 1 part glacial acetic acid)

Farmer’s fixative (3 parts absolute ethyl alcohol : 1 part glacial acetic acid

70% ethyl alcohol





42

Methods:

1. Collecting, killing, and fixing developing flowers of various sizes will provide a source of anthers

to be used for the study of meiosis by the “squash” technique. Both cultivated and native plant

material may be used for this purpose. Among the especially satisfactory or convenient species are

maize, rye, Rhoeo spathacea, Tradescantia spp., wild and cultivated lily species, and Tragopogon

spp. The usefulness of Ornithogalum and chives as angiosperms in which microsporogenesis can

be readily studied is also documented in the literature. All of these plants have relatively large

chromosomes in comparatively low numbers. Another advantage of plant such as maize,

Tradescantia, Rhoeo, Ornithogalum, and Tragopogon is that one can collect, kill and fix entire

inflorescences (flower clusters), each consisting of numerous lower buds in various stages of

meiosis. The plant material must be collected early enough to catch the meiotic divisions while

they are occurring. Often the novice will collect too late in the season, after meiosis has been

completed and the anthers are filled with mature pollen grains. Experience is the best teacher in

this matter.

2. The collected flower buds must be preserved (fixed) in appropriate chemicals that stop the meiotic

process and preserve the chromosomes in their normal form and position. Two of the most widely

used fixatives are Carnoy’s solution (6 parts absolute ethyl alcohol: 3 parts chloroform: 1 part

glacial acetic acid) and Farmer’s fixative (3 parts absolute ethyl alcohol: 1 part glacial acetic acid).

For best results, these fixatives should always be prepared fresh just before use. Both fixatives

produce acid-fixation images; they preserve the chromosomes, nucleoli, and spindle apparatus

particularly well.

3. Materials are placed in vials and completely covered with the appropriate killing-fixing solution,

using 50 parts of fixative to one volume of material. After material is fixed for 18-24 hours, it may

be stored in 70% ethyl alcohol in a refrigerator or freezer. Materials treated in this fashion are

effectively preserved for use over a period of years.

B. Preparing a Squash

Materials:

Flower buds

70% ethyl alcohol

Acetocarmine stain

Watch glass

Microscope slides

Cover glass

Alcohol lamp

Scalpel

Paper towels

Stereomicroscope

Compound microscope

Methods:

1. Remove an entire inflorescence or an individual flower bud from the storage

container and place it in a watch glass. Add a few drops of 70% ethyl alcohol to keep

the plant material moist. If the flower bud and its anthers are small, you may wish to

place the watch glass containing the flower bud on the stage of a stereomicroscope to

assist in differentiating the anthers from other flower parts. It is helpful to examine

the flower bud against a dark background, because the killed and fixed plant material





43

has been bleached white by the fixative and alcohol. A black card placed on the

microscope stage will help you to see well. Continue to keep the inflorescence and

the individual flower buds moist in alcohol as you dissect the anthers from the bud.

2. Now transfer one or two anthers to a clean microscope slide and place them in a drop

of acetocarmine stain. Using a teasing needle, scalpel, or razor blade, macerates the

anthers, freeing the microsporocytes from the anther walls. If possible, remove the

anther walls from the drop of stain. Apply a cover glass and heat the slide over an

alcohol lamp, being careful not to allow the stain to boil. Add stain if necessary to

prevent drying.

3. After heating, cover the slide with paper toweling and press down firmly with your

thumb. This pressure will squash or flatten the microsporocytes, making it possible to

observe the chromosomes in the various meiotic stages. Most of the microsporocytes

from any one flower tend to be in about the same stage of meiosis. If anthers from

more than one flower are crushed on a slide, different stages are likely to be observed.

Examine the slide under the low and high-power objectives of a compound

microscope.

C. Study of Meiosis in the Course of Microsporogenesis

Study your slides, looking for various meiotic stages. In microspore mother cells

you may expect to see all of the substages of prophase I- leptonema, zygonema,

pachynema, diplonema, and diakinesis. Some materials are better for certain stages. For

example, maize is especially good for pachytene chromosomes, whereas lily

chromosomes effectively reveal the chromomeres that are characteristic of leptonema and

zygonema. Metaphase I and anaphase I will also occur in the pollen mother cell.

Depending on the species, cytokinesis may or may not occur at telophase I. For example,

cytokinesis does occur after telophase I in lily, rye, and Tradescantia, but it does not take

place in the common garden pepper until after telophase II, when it occurs in two planes,

thus producing the four microspores.

Meiosis II takes place in the nuclei produced as a result of the first meiotic

division. Prophase II, metaphase II, anaphase II, and telophase II occur and result in the

production of four haploid nuclei, each of which is isolated in a separate microspore by

means of cytokinesis. The four microspores separate and become covered by a pollen

wall that is characteristic of the species and, thus, constitute four individual pollen grains.

Record your observations in Table 6.1 about meiotic cells that occur on the slides that

you have made.

If a slide is made of an anther in which meiosis has already been completed, you

might find microspores or mature pollen grains. Although not revealing meiotic stages,

such a slide can be useful to demonstrate the first postmeiotic mitosis in which the

haploid microspore nucleus divides to form the spherical tube nucleus and the elongate

generative nucleus. This mitotic division can reveal both chromosome number and

chromosome morphology.





44

Record each meiotic stage you have been able to detect in the plants for which you have prepared squashes

of anthers. Indicate the name of the plant used in each case.

Meiotic stage Name of plant: _______________ Name of plant: _______________

Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3

Prophase I

Metaphase I

Anaphase I

Telophase I

Prophase II

Metaphase II

Anaphase II

Telophase II

Mature

pollen grains

Table 10.1 – Observations of Microsporogenesis in Angiosperms

An occasional anther may produce microsporocytes in which the consequences of

chromosome aberrations are evident. Depending on the stages of meiosis present, you

might encounter chromosome bridges, acentric fragments, lagging chromosomes,

micronuclei, and various combinations of these abnormalities. Show your instructor any

slide that you make that reveals such abnormalities.

uddd1104 cell biology lab manual 2013

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