Z

UTAR
Centre for Foundation Studies

FHSC1214
Fundamentals of Cell Biology
UNIVERSITI TUNKU ABDUL RAHMAN

CENTRE FOR FOUNDATION STUDIES
FHSC 1214 Fundamentals of Cell Biology
Laboratory Manual
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UNIVERSITI TUNKU ABDUL RAHMAN

CENTRE FOR FOUNDATION STUDIES
LABORATORY SAFETY RULES
The following rules must be obeyed by all students in the science laboratory of the faculty.
Wilful or repeated inadvertent noncompliance may result in dismissal or suspension from
the laboratories.
I. No entry without permission:
i)
Outsiders are not allowed to enter the laboratory without permission.
ii) Visitor must request for a lab coat from the laboratory officer before enter to the laboratory.
iii) No student is allowed to enter the laboratory unless permission has been given by
laboratory officer or lecturer.
II. At work in the laboratory:
i)
No experiment may be attempted without the knowledge and permission of lecturer or lab
officer.
ii) Laboratory coat must be worn at all times in the laboratory.
iii) Students must wear covered shoes in the laboratory. Students wearing open toes shoes
such as slippers or sandals are not allowed to work in the laboratory.
iv) Safety glasses must be worn when necessary.
v) Mobile phones are to be switched off at all times in the laboratory.
vi) Do not smoke, drink, eat, bite nails or pencils, or apply cosmetics in the laboratory.
vii) Do not pipette chemicals with mouth.
viii) Do not taste any chemicals, including diluted solutions. If any acid or alkali accidentally
enters your eyes or mouth, wash immediately with plenty of water. Inform your lecturer or
laboratory staff, and seek medical attention if necessary.
ix) Any accident must report to the lecturer or lab officer immediately.
x) Paper should never be used to light up the Bunsen burners.
xi) Used match sticks, filter papers, and other solid waste must never be thrown into the
sinks. They must be thrown into the dustbins provided. Lighted match sticks and
smoldering materials must be extinguished with tap water before thrown into the
dustbins.
xii) Students must take responsibility for apparatus and equipment under their charge in the
laboratory.
xiii) Any glassware breakages, apparatus lost and equipment damages or malfunctioning
must be reported to the laboratory officer.
III. Before leaving the laboratory:
i)
Ensure all the equipment and working benches used are thoroughly cleaned and dried.
ii) Wash hands and arms with soap and water before leaving the laboratory.
iii) All stools must be kept under the benches.
iv) No student is allowed to take away any chemicals, equipment or other property of the
laboratory without permission.
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Table of Contents:
Practical
Practical 1
Biological molecules I
Practical 2
Biological molecules II
Practical 3
Enzyme studies I
Practical 4
Enzyme studies II
Practical 5
Cell studies I
Practical 6
Cell studies II
Practical 7
Cell studies III
Practical 8
Cell studies IV
Practical 9
Energetics I
Practical 10
Energetics II
Experiment Description
Identification of Biochemicals in Their Pure
Form
Investigation of Action of Saliva and 3 M
Hydrochloric Acid in Two Carbohydrate
Solutions
Investigation of the effects of catalase
concentration on hydrogen peroxide
Investigation of the Enzymatic Effects of
Materials on Hydrogen Peroxide Solution
The Microscope and Its Uses
Page
1
Extraction of Cell Organelles by Differential

Centrifugation
Determination of the Isotonic Concentration of
the Potato Cell Sap
Effects of various treatments on pieces of
stained potato tissues
Respiration of Germinating Beans
17
Respiration of Yeast
33
7
9
11
21
26
30
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Practical 1
Identification of Biochemicals in Their Pure Form Identification of

Biochemicals in Their Pure Form
Objective:
To identify the components of the solution in its pure form with various food tests
Apparatus & Equipments:
Boiling tubes
Boiling tube rack
o
Water bath, 95 C
Boiling tube holder
Materials:
Iodine
1 M hydrochloric acid
Sudan III
Starch solution
Corn oil
Egg albumin
1% copper sulphate solution
DCPIP (dichlorophenolindophenol) solution
1% carbohydrate solutions
Sodium hydroxide
Absolute ethanol
Introduction:
The nutrients in the food you eat supply your body with energy for growth and repair. These
principle substances include carbohydrates, proteins, fats, minerals and vitamins. We can test for
the presence of these important compounds in food by using chemical reagents that react in
predictable ways in the presence of these nutrients.
Part 1: Identification of Carbohydrates

Test for reducing sugars
The reducing sugars include all monosaccharides, such as glucose and fructose, and some
disaccharides, such as maltose and lactose.
See basis of test below for explanation of the following reaction:
-C-C=O + 2 CuSO4 + 4 NaOH

Na2SO4
(blue)
RCOOH + Cu2O + 2 H2O + 2

red ppt.
Benedicts solution
Benedicts Test for reducing sugars
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Procedure*
Reducing sugar test
3
Add 2 cm of any one
solution of the reducing
sugar to boiling tube.
Add an equal volume of
Benedicts solution.
Using a test-tube holder,
shake and heat at a high
temperature for 2 minutes.
Basis of test
Observation
Benedicts solution contains copper

sulphate. Reducing sugars reduce
soluble alkaline blue copper
sulphate containing copper (II) ions,
2+
Cu to insoluble red-brown copper
oxide containing copper (I). The
latter is seen as a precipitate.
Test for non-reducing sugars

The most common non-reducing sugar is sucrose, a disaccharide. If reducing sugars have been
shown to be absent (negative result in above test), a brick-red precipitate in Benedicts test on the
acid-hydrolysed unknown sample in the test below indicates the presence of a non-reducing
sugar.
Procedure*
Non-reducing sugar test
3
Add 2 cm of sucrose
solution to a boiling tube.
3
Add 1 cm 1 M hydrochloric
acid and heat at a high
Carefully neutralize with
3
equal volume (1 cm ) of 1 M
sodium hydroxide.
Basis of test
Observation
A polysaccharide or disaccharide
can be hydrolyzed to smaller
component constituents by boiling
with 1 M Hydrochloric acid .
Sucrose is hydrolyzed to glucose
and fructose, both of which are
reducing sugars and give the
reducing sugar result with the
Benedicts test.
Finally, add an equal volume

3
(4 cm ) of Benedicts
solution to the acidhydrolysed sugar solution
and heat at a high
Additional Information
The test is semi-quantitative, that is, a rough estimation of the amount of reducing sugar
present will be possible.
The final precipitate will appear green to yellow to orange to red-brown with increasing
amounts to reducing sugar. The initial yellow colour blends with the blue of the copper
sulphate solution to give the green colouration.
Test for starch

Starch is only slightly soluble in water, in which it forms a colloidal suspension.
Procedure*
Basis of test
Observation
Iodine test
Add a few drops of 1%
A polyiodide complex is formed
starch solution on a white
with starch.
tile.
Add a few drops of iodine to
the starch solution
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Part 2: Identification of Lipids

Lipids include oils (such as corn oil and olive oil), fats and waxes.
Procedure*
Sudan III
3
3
Add 2 cm of oil to 2 cm of
distilled water in a boiling tube.
Add a few drops of Sudan III
and shake.
Emulsion test
3
Add 2 cm of oil to a boiling
3
tube containing 2 cm of
absolute ethanol. Dissolve the
lipid by shaking vigorously. Add
an equal volume of cold water.
Basis of test
Observation
Fat globules are stained red and

are less dense than water.
Lipids are immiscible with water.

Adding water to a solution of the
lipid in alcohol results in an
emulsion if tiny lipid droplets in the
water which reflect light and give a
white, opalescent appearance.
Part 3: Identification of Proteins

A suitable protein for these tests is egg albumen.
Procedure*
Biuret Test
3
Add 2 cm protein solutions to a
boiling tube. Add an equal
volume of 5% sodium hydroxide
solution and mix. Add 2 drops of
1% copper sulphate.
Basis of test
Observation
A test for peptide bonds. In the

presence of diluted copper
sulphate in alkaline solution,
nitrogen atoms in the peptide chain
form a purple complex with copper
2+
(II) ions, Cu .
Tabulation of qualitative data

Tabulate your observations above for each biochemical food test executed:
o
o
o
Liquid
mixture, solution, suspension, emulsion?
transparent, translucent, opaque?
Colour
state initial and final colours?
Precipitate (if any)
colour of precipitate?
amount of precipitate?
For tests involving carbohydrates, observe and report characteristics of tube contents before and
after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate as above.
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Practical 2
Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two

Carbohydrate Solutions
Objective:
(Students are expected to state the objective of this experiment.)
Apparatus & Equipments:
Boiling tubes
Beaker
Graduated plastic dropper
o
Water bath, ~ 37 C
o
Water bath, ~ 95 C
Materials:
Carbohydrate solution A
Carbohydrate solution B
Benedicts solution
3 M Hydrochloric acid
3 M Sodium hydroxide
Procedures:
Part 1
1.
Prepare two boiling tubes containing 1 ml solution A and 1 ml solution B respectively. Add 1
o
ml Benedicts solution to each tube. Heat both tubes together in the (~95 C) water bath for
two minutes. Record the results in table 1.
2.
Add a few drops of solution A and B separately on a white tile. On each solution, add 1-2
drops of iodine solution. Record your observations in the table 1.
Part 2
3.
Label the boiling tubes 1, 2, 3 and 4. Pipette 2 ml of solution B into each of four boiling
tubes.
o
4.
Place tubes 1 and 2 in a water bath of ~37 C to heat up the solution.
5.
Salivate into a small beaker till it reaches about 5 ml.
6.
This step is to be done approximately at the same time. Pipette 2 ml of saliva prepared into
tubes 1 and 4. Shake the contents of the tubes well to ensure thorough mixing. Pipette 2 ml
of HCl into tubes 2 and 3.
7.
Let tubes 1, 2, 3 & 4 incubate at their respective temperatures (see Table 2) for 30 minutes
from this moment.
8.
Label 4 more new boiling tubes as follows: 1, 2, 3 and 4.
9.
After 5 minutes of incubation of tubes 1 to 4, pour out 2 ml of the contents from all these
tubes into the respective newly labelled tubes (e.g., 1 into 1, 2 into 2 etc.). Place back the
original tubes back into the respective temperatures of incubation.
10.
Neutralize the acid in each of tube labelled 2 and 3 with 1 ml of sodium hydroxide (each).
Shake each tube (2 and 3) to ensure uniform mixing.
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11. Perform Benedict test on the contents of tubes 1 to 4 by pipetting 2 ml of Benedict solution
o
into each tubes and heating them in 95 C water bath for 2 minutes. Record your
observations in Table 2.
12.
After 30 minutes of incubating tubes 1 to 4, neutralize the acid in each test tube labelled 2
and 3 with 1ml of sodium hydroxide.
13.
Carry out Benedicts test with an equal volume of Benedicts solution (2 ml) for each tube.
Remember to heat your sample. Record your observations in table 2.
Table 1:
Observations
Conclusions
Benedicts test:
Solution A
Solution B
Iodine test:
Benedicts test:
Iodine test:
Table 2:
Benedicts TestColour Observation
Tube
Contents
2 ml solution B
2 ml saliva
2 ml solution B
2 ml HCl
2 ml solution B
2 ml HCl
2 ml solution B
2 ml saliva
2
3
4
Temp.
(C)
After 5 min of incubation

(1 4)
After 30 min of incubation

(1 - 4)
37
37
95
95
Discussions:
Discussions should contain:
1) Name of enzyme involved.
2) Specific action(s) of enzyme involved.
3) Effect of temperature on enzyme structure (bonds, active site etc.)
4) Effect of HCl on solution B.
5) Product identification : make suggestion(s).
6) Product structure and chemical classification.
7) Chemical bases of tests used.
8) Which carbohydrate is more complex, A or B? Give a reason.
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Flow chart for Part 2:
Incubate at
Mix with
Incubate at
After 5 mins
Pour out for
Benedicts test
Pour to
Solution B
2 ml
Solution B
2 ml
Solution B
2 ml
Solution B
2 ml
37C
2 ml saliva
37C
37C
2 ml HCL
2 ml HCL
37C
95C
Remove tube 1, 2, 3, 4 from water bath.
2 ml saliva
95C
2 ml
2 ml
2 ml
2 ml
Place tube 1,2,3,4 back into water bath for continuous incubation
NaOH
Benedicts
solution
2 ml
1 ml
2 ml
1 ml
2 ml
2 ml
Heat for 1 min

Record observation at Table 2 (After 5th min)
After 35 mins
Remaining
content to
perform
Benedicts test
NaOH
Benedicts
solution
Remove tube 1, 2, 3, 4 from water bath.

2 ml
2 ml
2 ml
2 ml
2 ml
1 ml
2 ml
1 ml
2 ml
2 ml
Heat for 1 min

Record observation at Table 2 (After 35th min)
Results
(After 5 mins)
Results
(After 35 mins)
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Practical 3
Investigation of the effects of catalase concentration on hydrogen peroxide
Objective:
To investigate the effects of different catalase concentration on the decomposition of hydrogen
peroxide.
Apparatus
Boiling tubes
Scalpel/ pen knife
White tile
Mortar and Pestle
Weighing boat
1 rubber bung with delivery tube
Potato
Pipette
Materials:
1% hydrogen peroxide solution
Introduction:
Enzymes are proteinaceous molecules that speed up chemical reactions within living systems. In
this experiment, the effect of catalase concentration on hydrogen peroxide is investigated.
Catalase is an enzyme present in the cells of plants, animals and aerobic (oxygen requiring)
bacteria. It promotes the conversion of hydrogen peroxide, a powerful and potentially harmful
oxidizing agent, to water and molecular oxygen.
2H2O2 + catalase 2H2O + O2
Warning: H2O2 is corrosive
Procedures:
1. Cut and weigh 5g of potato using a weighing boat.
2. Cut the potato samples into smaller pieces and mash the potato sample using the mortar and
3
pestle. Add 6 cm of distilled water to the potato samples after the mashing process.
3. Collect the potato juice.
4. You are going to set up the apparatus as shown below.
5. Fill an empty test tube with tap water.
3
6. Add 5cm of hydrogen peroxide into a boiling tube.

3
7. Draw 1cm of the juice from the mashed potato samples and add to the boiling tube.
Immediately close the boiling tube with the rubber bung.
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8. Immediately immerse the tube in the water bath. Count the number of gas bubbles produced
for 2 minutes and record it.
9. To get a 2
nd
rd
and 3 measurement, dispose the contents of tube A. Repeat step 5 to 8.
10. Repeat Step 2 to 9 but with 10g of potato, then 15g and finally 20g.
11. Record the data in table 1.
Table 1:
Number of Attempt
Number of gas
bubbles produced
st
5g
nd
2
(What heading should you write here?)

10g
15g
rd
st
nd
rd
st
nd
rd
st
3
1
2
3
1
2
3
1
20g
nd
2
rd
Results and Discussions:

1. From the data you have collected in the practical, account fully for the results which you have
obtained. Discuss any anomalous data/ results that you might have.
2. Plot a graph of average number of bubbles produced against potato samples used. Explain
the trend or pattern of the graph.
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Practical 4
Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide

Solution
Objective:
To investigate the enzymatic effect of various materials in the hydrogen peroxide solution.
Apparatus & Equipment:
Beaker
Boiling tubes
o
Water bath (95 C)
Wood splints
Glass rod
Materials:
Fresh Liver
Manganese dioxide
Potato cubes
Hydrogen peroxide
Warning: H2O2 is corrosive

Procedures:
1. Label six fresh empty boiling tubes 1, 2, 3, 4, 5, 6.
2. Using a blade, cut the provided liver into 3 pieces of roughly 0.8 cm x 0.8 cm x 0.5 cm.
3. Place one piece of liver into tube 1.
4. Place the second piece of liver into the bottom of tube 2. Place tube 2 in the boiling or water
o
bath (95 C) until the liver is cooked.
5. Now put the third piece of liver into tube 3. Mash it gently into a pulp using a glass rod.
6. Using the weighing balance, measure out two 0.5 g portions of manganese dioxide powder
each onto a weighing boat. Pour each portion into tube 5 and tube 6.
o
7. Put tube 6 in the water bath (95 C) for five minutes.

8. After five minutes let cool tube 2 and 6.
9. Cut potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into a tube 4.
3
10. Prepare another six fresh empty test tubes. Put 5 cm of hydrogen peroxide into each of
them.
11. Next, quickly add hydrogen peroxide into the test tubes 1, 2, 3, 4, 5, and 6.
12. Using the parafilm provided, stretch it quickly seal the mouth of the test tubes by stretching
the film over it and leave for 20 minutes
13. Insert a glowing splint into each tube by just penetrating the parafilm with it.
14 Record your observations on each tube immediately after the reaction has started.
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Table 1:
Tube
1
2
3
Contents with 5 cm
hydrogen peroxide
Before using wood splint
After using wood splint
Fresh liver
Boiled liver
(cooled)
Pulped liver
Potato cube
Manganese dioxide
(untreated)
Observations
Boiled manganese
dioxide
(cooled after heating)
Results and discussion:

In your report, be sure to address the following:
1. What is the equation of the reaction observed?
2. What plant or animal organelle is involved?
3. What effect does pulping the liver have upon the reaction? Account for this.
4. What effect does boiling the liver have upon the reaction? Account for this.
5. What were the differences between the reactions with fresh liver and with fresh potato
cubes? Account for these differences.
6. What were the differences between the effects on the reaction of boiling the liver and heating
the manganese dioxide? Account for these differences.
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Practical 5
The Microscope and Its Uses
Objective:
1. To study the uses of microscope and its parts.
2. To learn microscopic techniques such as focus the object with correct illumination under different
power of magnifications.
3. To study the microscopic structure of biological samples and to learn the preparation of
biological samples for microscopic study purposes.
Introduction:
The microscope is a basic tool of the biologist. It is a valuable precision optical instrument easily
damaged by careless usage. It is very important for the student to become familiar with
the parts of the microscope and the procedures in the handling of it.
Apparatus:
Binocular Microscope
Cover slips
Microscope slide
Newspaper
Materials
Potato
Onion
Hair
Iodine
Safranin
Parts of the Microscope:
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Component
Arm
Base
Body tube
Eyepiece or ocular
lenses
Revolving nosepiece
Objective lenses
Focusing adjustments
Stage
Mechanical stage
Specimen holder
Vertical feed knob
Horizontal feed knob
Condenser
Built-in light source
Brightness
adjustment knob
Main switch
Function
For lifting and carrying the microscope.
To provide stability.
To house the lenses.
The magnification of the eyepiece (given as 10X) is printed on the metal
part of the ocular.
Located at the lower end of the body tube, it carries 3 objectives of
different lengths. Rotating this part changes the magnification of the
objectives.
They are of different magnifications with the following visible properties:
Objectives
Magnification
Length
Lens opening
Scanning lens
4x
Shortest
Widest
Low power lens
10x
short
wide
High power lens
60x
longest
narrowest
These comprise two knobs located on either side of the microscope
which are used to change the distance between the object being viewed
and the objective lens. Changing the distance determines the focus.
For the object to be viewed in focus under high magnification, the lens
must be much closer to the object than when it is under low
magnification.
Coarse adjustment
Made by the large knob beside the body tube for
focusing under low power magnification.
Fine adjustment
Made by the small knob, which is for focusing
under high power magnification and accurate
focusing.
This is the platform for slides and specimens to be viewed under the
microscope.
This movable portion of the stage is attached to the specimen holder and
allows the slide to be moved in different directions to facilitate viewing.
This holds the glass slide in place.
Rotating this moves the glass slide in the vertical direction.
This moves the glass slide in the horizontal direction.
Located just beneath the stage of the microscope, it incorporates a lens
which collects light on the stage to bear on the object.
This is situated below the iris-diaphragm to provide light for illuminating
the object. It can be switched on or off.
This provides adjustment to the illumination brightness.
This ensures that power is turned on or off.
Preliminaries before Use:

1. Use the coarse adjustment to raise the body tube so that the objective can clear the stage
when the revolving nosepiece is turned.
2. Turn the nosepiece until the scanning objective is in-line with the eyepiece. You should
hear a soft click or else feel a distinct falling into place as the objective moves into
position.
3. Turn the diaphragm to its largest opening.
4. Look into the eyepiece and make a final adjustment to the light adjustment knob so that
the field of view (i.e., the lit circle which you see) is evenly illuminated. Any glare should
be removed by adjusting the diaphragm.
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5. The microscope is now ready for use.
6. Ensure that the microscope stage is at its lowest position. This will prevent breaking of slides
and lenses by mistake when adjusting the objectives by moving the stage with the coarse
adjustment knob.
REMEMBER
Always handle glass slides and cover slips by their
edges, never by their flat surfaces.
Viewing the slides:
1. Place the slide carefully on the stage and position such that the specimen is in the centre of
the hole in the stage and also in the middle of the circle of light emanating from the lamp
through the stage hole.
2. Ensure that the scanning objective is in place.
3. Place your head just above the eyepieces. Slowly, slide the eyepieces towards each other
horizontally so that they fit the position of the eyes on your head. If the eyepieces are in
correct position, you should be able to observe only one illuminated circular field of view.
4. Adjust the brightness adjustment knob to give the right amount of light for viewing the object
clearly.
5. Looking down the eyepiece, slowly adjust the position of stage with the coarse adjustment
knob until the object comes into focus. Focus accurately by using the fine adjustment knob.
6. If the details of the specimen are not clear, adjust the brightness adjustment knob and/or
condenser.
7. Once the object is in sharp focus, its time to view it at higher magnification (i.e., the 10X
objective).
Using a higher power objective:
1. Most microscopes have parfocal objectives. This means that if one switches from viewing a
specimen in sharp focus under a lower power objective to a higher one, the object should
automatically come approximately into focus. Only some slight further focussing with the fine
adjustment knob is required to see the specimen clearly. Therefore, if youre using the
higher power objectives, do not use the coarse knob to refine focus or youll risk
breaking the slide and lenses.
2. When changing from one objective to another, you will hear a click when the objective is set
in position.
3. You are now ready to switch from the scanning objective to a higher power objective after
obtaining a sharp focus of the object.
4. Adjust the fine adjustment knob to see the specimen clearly.
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5. If youre going to switch to the next higher power objective, look from the side of the
microscope and move the revolving nosepiece slowly till that higher power objective clicks
into position.
6. Take care that the lower end of the high power objective does not touch the cover slip. If this
happens, you must repeat the whole procedure focusing again, starting with the scanning
objective.
Trouble-shooting
Below are some common problems associated with not being able to find and focus on an object
under high-power magnification.
Is the objective lens in position?

Is the cover slip on the slide facing upwards?
Is the object in the centre of the stage?
Are the lenses clean and free from dirt and moisture?
Is the condenser adjusted and focused?
Exercise 1
Focusing the Microscope - e slide
1. Prepare a microscope slide to view the letter e. Using a pair of scissors, cut a
piece of newspaper that includes a tiny sharp-lined letter (an R or "e" is best).
2. Place this square piece of newspaper in the centre of the slide with the printed
side up.
3. Add one or two drops of water onto the newspaper using a dropper.
4. P lac e t he cover slip carefully over the newspaper. This is to be done by holding the
cover slip about 45 to the slide, let it slip down the slide till the lower edge touches
the water, and then slowly lower the cover slip down onto the slide.
How to Mount an Object for Microscopic Examination

Carry out the observations as follows:
1. Compare the position of image as seen through the eyepiece with that of the printed letter as
seen with the unaided eye. Does the image appear to be reversed, i.e. as it would appear if
seen in a mirror?
2. Slowly move the slide from left to right, observe and describe the way the image moves.
Repeat right to left.
3. Move the slide away from yourself and describe observe the movement of the image again.
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Exercise 2
Observation of Onion Cells:
1. Bend the onion scale leaf towards the outer epidermis until it breaks on the upper surface.
2. With your fingers or forceps, pull the inner epidermis gently away from the scale leaf.
3. Using a dropper, place 1-2 drops of water on the slide and place the epidermis on the water.
Make sure that there is some water covering the specimen and surrounding it.
4. Hold a cover slip at about 45 to the slide and lower it so that one edge touches the water
droplet.
5. Slowly lower the cover slip onto the slide using the points of a pair of fine forceps.
6. The mounting of a specimen on a slide with solution is called a wet mount.
7. Prepare another slide of the onion epidermis. This time add a drop of safranin or iodine onto
the epidermis instead of distilled water. Allow the stain to take for 10 minutes before drawing
it off with tissue paper. Finally put on a cover slip.
8. Examine first under low power (begin with the scanning objective first) and then under high
power.
Answer these questions:
1. Are all the cells identical in shape and size?
2. Is the nucleus located in the same position in all the cells?
3. Suggest reasons to explain any apparent differences in the shape and size of the cells as well
as the location of the nucleus.
4. What are the effects of the iodine or safranin stain on the cells?
5. Can you observe any changes in the cells? If so, describe them.
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Exercise 3
Observation of Starch Grains
1. Place a clean glass slide on the white tile. Place a small piece of potato in the centre of the
slide and rub the piece of the potato to distribute the potato juice in an even layer.
2. Add a drop of water and then a clean cover slip to the slide.
3. Examine the preparation under low power.
4. You can prepare another slide but this time stain the potato juice with iodine.
5. Examine the iodine-stained mount first under the scanning objective and then under low and
high power.
Exercise 4
Observation of Hair
1. Mount a small portion of your own hair in a drop of water on a slide. Add a cover slip.
2. Adjust the diaphragm of the microscope to its largest opening and bring the hair into sharp
focus under low power (begin with the scanning objective first). Reduce light gradually by
progressively closing the diaphragm. In this way, determine the diaphragm setting that
provides the clearest image of the hair. As you further examine the hair, shift the focus by
slowly turning the fine adjustment back and forth.
3. Move the hair to the centre of the scanning field and shift to higher power magnifications.
Note any changes in the brightness of the field of view. Bring the hair image into the sharpest
possible focus and examine carefully.
4. As an interesting corollary of this exercise you could examine hair from different members of
the class and try to determine differences between fine and coarse hair, curly and straight
hair, and between hair of different shades or colours.
5. The exercise in step 4 above can be further extended by using hair from different animals,
e.g. dog, cat, mouse, rabbit, guinea pig, etc.
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Practical 6
Extraction of Cell Organelles by Differential Centrifugation
Objective:
To show how organelles can be purified from homogenated liver tissue by differential
centrifugation.
Apparatus and equipment:
Centrifuge
Centrifuge tubes
Mortar and pestle
Materials:
Liver
Isotonic solution
Introduction: Cell fractionation
A. Homogenization
Cells or tissues are ground up/ blended in such a way that its consistency is even. This is to
destroy the cell membrane so that the cytoplasmic components flow out.
B. Centrifugation
Principle: Different cell components are of a certain size and density, and descend to the bottom
of the centrifuge tube at different speeds. The faster the rotations of the centrifuge, the smaller
the particles are sedimented. Components can be separated from larger to smaller ones based
by using a series of increasing speeds. This is called differential centrifugation.
The process of differential centrifugation is based on the fact that organelles have differences in
size, shape and density. As a result, the effect of gravity on each is different. We can use this
principle to separate an organelle from a homogenous solution of particles by artificially
controlling the gravity of a solution. This is done by putting the solution in a variable speed
centrifuge and rotating them at a high rate of speed. This creates a force that can be much
greater than the force of gravity, and particles that would normally stay in solution will fall out and
form a pellet at the bottom of the tube.
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A piece of tissue is homogenized

by physically grinding it.
The cell homogenate contains

large and small organelles.
A centrifuge is used to separate

the organelles based on size
and density.
Golgi
Mitochondria
The heaviest organelles can be

removed and the remaining
suspension re-centrifuged until
the next heaviest organelles
reach the bottom of the tube.
Nuclei
Cell Fractionation The organelles can be separated from one another after cells are broken open and
centrifuged.
th
Diagram: Life, the science of biology (6 Ed.). William K. Purves,

David Sadava, Gordan H. Orians, and H. Craig Heller (2001).
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Centrifugation Scheme Homogenized liver tissue is subjected to low centrifugation force to

separate larger cell structures and then higher centrifugation force to separate smaller organelles.
Procedures:
1. Obtain an uncut piece of liver (~3cmx3cm).
2. Using a mortar and pestle, grind the liver piece into a pulp. This is called homogenization and
the product the homogenate.
3. Add 10 ml or more isotonic solution as you grind
4. If the pulp is very thick, pour about 10-15 ml into the 50 ml graduated plastic tube and top up
to 20 ml of the tube with isotonic solution.
Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as
possible, so that the centrifuge will not be damaged during spinning.
5. Spin the tube for 5 minutes at 600g.
6. After 5 minutes, collect your centrifuged sample. Note: Do NOT shake the tube.
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7. Slowly insert the pipette tip into the supernatant, being careful not to mix up the contents.
8. Aspirate out the supernatant into 15 ml graduated plastic tubes.
9. Unless there is enough supernatant, top up the 15 ml graduated plastic tubes to 10 ml with
distilled H20.
Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as
possible, so that the centrifuge will not be damaged during spinning.
10. Centrifuge the graduated plastic tube for about 20 minutes at 3000g.
11. After 20 minutes, collect your centrifuged sample. Note: Do NOT shake the tube.
12. You can repeat the procedure according to the Centrifugation Scheme above.
13. Dispose of the excess liver pulp or pieces into the given plastic bag (not the dustbin).
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Practical 7
Determination of the Isotonic Concentration of the Potato Cell Sap
Objective:
To prepare NaCl solutions of various concentrations from a stock solution of 1.0 M by dilution
technique
To find out the isotonic concentration of potato cell sap as a function of changes in the
concentration of sodium chloride (NaCl) solution and in the weight of potato strips.
Introduction:
Water potential,
Water potential (denoted by the Greek letter , psi) is used to describe combined effects of
concentration and pressure in a solution on the tendency of water molecules to diffuse across
a membrane.
Water potential is defined as the net tendency of water to diffuse out of a solution by
osmosis
Water potential of a solution = effect of the solute concentration of that solution + effect of
pressure on that solution
= s + p
where = water potential of the cell
(its a -ve pressure, i.e., inward force like that created on
solutions or water when the plunger of a syringe is pulled
to draw liquid up1; see picture arrows)
s = solute potential of the cell

(its a -ve pressure also, i.e., inward force; see picture arrows)
p =
pressure potential, due to wall pressure (its a +ve pressure;

outward force like that created on solutions or water when the
plunger of a syringe is pressed to expel liquid; see picture
arrows)
For the purposes of comparison, pure water at atmospheric pressure has a water
potential of 0. If a solute, such as sugar, is added to this water, its water potential is
effectively decreased or lowered. This is because
water concentration in a given space is decreased
water molecules are attracted to the solute molecules and so move less freely
Water potential of a solution is always negative owing to the presence of solutes.
The more concentrated the solution, the lower its water potential i.e., the more
negative is its water potential.
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Essential knowledge for practical: Osmosis in living plant cells
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At full turgor
The plant cell is fully turgid
The cell wall is stretched fully
The pressure of the cell contents resists the uptake of water
Turgor pressure (p) is at a maximum
There is no net tendency of water to move in or out of the cell, i.e., water potential () = 0,
because the rate of water movement into the cell equals that of water out of the cell.
Incipient plasmolysis
Is when the shrinkage of the protoplasm reaches the point where the cell surface membrane
is just about to pull away from the cell wall
No pressure is exerted by the protoplast (= cytoplasmic contents + membrane) against the
cell wall, i.e. (p) = 0, i.e. ()= (s).
The cell is flaccid.
Full plasmolysis
Occurs when the cell surface membrane is pulled away from the cell wall with the cytoplasm
contracted.
Apparatus:
Boiling tubes
Boiling tube rack
Knife/ food slicer
Forceps
Graduated glass pipette
Materials:
NaCl solution, 1.0 M
Distilled water
Potato
Procedures:
3
1. In labelled boiling tubes, prepare a series of 40 cm of NaCl solutions at the concentrations of
0.0 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M from the stock solution using dilution technique.
Record the volumes of solution and of distilled water used in the table below.
The method:
M1V1=M2V2
Existing concentration: 1 M (M1) of NaCl
3
Desired Concentration: 0.1M (M2) of 40cm Nacl
Final volume of 1 M NaCl:
3
(1M) (V1) =(0.1M) (40 cm )
V1 = ?
Molarity
Volume of 1.0 M
3
NaCl solution (cm )
Volume of distilled
3
water (cm )
0.0 M
0.1 M
0.2 M
0.3 M
0.4 M
0.5 M
2. Prepare 15 cubes of potato tissue of about equal size (1 cm x 1 cm x 1 cm) using knife or you
can choose to cut using food slicer.
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3. Record the weight or each cube and place 3 cubes into the boiling tubes.
4. After 30 minutes, remove the cubes with the forceps provided and record the final weight and
calculate the change in weight of potato cubes in your table.
5. Record any change to the physical condition of the potato cubes.
6. Plot a graph of the change in average weight of potato cubes against the concentration of the
NaCl solution. Use a best fit curve.
Results:
weight of potato strips
(g)
Initial weight
Average initial weight
Final weight
Average final weight
Change in average
weight
Physical condition
0.0M
0.1M
Sucrose solution (M)

0.2M
0.3M
0.4M
0.5M
Discussions:
General
Positive change in
Weight
Zero change in weight
Negative change in
weight
Describe and explain:

a. Osmosis
b. Water potential
c. Solute potential
d. Turgor pressure
Comment on:
a. Change in weight
b. Tonicity (e.g., is solution hypertonic? etc.)
c. Condition of cells (e.g., turgid, etc.)?
d. (Net) movement of water?
e. Relationships among w plant cell , s plant cell , p plant cell , w NaCl, p
NaCl, s NaCl
Comment on:
a. Change in Weight
e. Relationships among w plant cell , s plant cell , p plant cell , w NaCl, p
NaCl, s NaCl
Comment on:
a. Change in Weight
f. Relationships among w plant cell , s plant cell , p plant cell , w NaCl, p
NaCl, s NaCl
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Conclusion
State the:
a. The concentration of NaCl solution at which the water potential of

the potato tissue is equal to the water potential of NaCl solution
(determine from graph).
b. Relationship between change in weight and NaCl concentration
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Practical 8
Effects of various treatments on pieces of stained potato tissues
Objective:
To investigate the effects of various treatments on pieces of stained potato tissues
Apparatus:
Knife/ food slicer
Forceps
Boiling tubes
Petri dish
Beaker
Materials:
Potato
Methylene blue solution
50% ethanol
Procedures:
1. Using knife/food slicer, cut five cubes from the potato provided, each approximately 10 mm x
10 mm x 5 mm. Trim off any peel which is still attached.
2. Place the potato cubes in a small beaker, immerse them in methylene blue solution for 10
minutes.
3. After 10 minutes, pour off the methylene blue solution and wash the cubes with tap water
until the water contains little or no stain.
3
4. Label four test tubes A, B, C and D. To each of the tubes A, B and C, add 5cm of distilled
3
water, to tube D add 5 cm of 50% ethanol.
o
5. Place tube A in boiling water, tube B in a water bath of 45 C and tube C and D at room
temperature.
6. After 5 minutes, add one stained potato cube to each of the four test tubes. Start the stop
watch immediately.
7. After 2 minutes, remove tubes A and B from the water baths and place them in the rack with
tubes C and D. Shake the tubes.
8. Separate the tissues from solutions using forceps and place the tissues on petri dish.
9. Record your observations of each test tube.
What is the texture and colour of the tissue?
What is the colour of the liquid?
How will you record the difference in intensity of colouration of the liquid?
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Results:
Table 1:
Tube
Intensity of blue colouration in liquid

(arbitrary unit)
Observation of potato tissue
A
B
C
D
Arbitrary units: 4 most intense, 1 least intense
Discussions:
From the data you have collected, account fully for the observations you have made and draw
clear conclusions, using your knowledge and understanding. Use the following questions as
guidelines.
1. Why should the surface area be kept constant for each piece of potato tissue?
2. Why are the potato cubes stained with methylene blue (why is a colour stain chosen)?
3. What is the purpose of having tube C placed in water at room temperature?
Why isnt tube D placed at high temperature?
4. What happen to the stained potato cubes when they are placed in water at room temperature?
5. What are the effects of temperature on potato cubes in tubes A, B and C?
6. What are the effects of the ethanol on the potato cubes?
7. State the reason for using equal volumes of liquids in all tubes.
8. Explain the significance of the 5 minutes incubation before adding the potato cubes.
9. Explain the significance of the 2 minutes incubation after adding the potato cubes.
10. Explain the significance of separating the tissue from solutions after the 2 minute incubation.
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Name : .........
Group :
Partial Report
Practical 8
Effects of various treatments on pieces of stained potato tissues
Results:
Table 1
Tube
Intensity of blue colouration in liquid

(arbitrary unit)
Observation of potato tissue
A
B
C
D
Arbitrary units: 4 most intense, 1 least intense
Discussions:
1. Why should the surface area be kept constant for each piece of potato tissue?
2. Why are the potato cubes stained with methylene blue (why is a colour stain chosen)?
3. What is the purpose of having tube C placed in water at room temperature?

Why isnt tube D placed at high temperature?
4. What happen to the stained potato cubes when they are placed in water at room temperature?
5. What are the effects of temperature on potato cubes in tubes A, B and C?
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6. What are the effects of the ethanol on the potato cubes?
7. State the reason for using equal volumes of liquids in all tubes.
8. Explain the significance of the 5 minutes incubation before adding the potato cubes.
9. Explain the significance of the 2 minutes incubation after adding the potato cubes.
10. Explain the significance of separating the tissue from solutions after the 2 minute incubation.
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Practical 9
Respiration of Germinating Beans
Objective:
To investigate the aspects of respiration in germinating bean seeds
To understand the concept of respiratory quotient
Apparatus:
Syringe
Capillary tube
Gauze
Rubber tube
Materials:
Colour dye
Beans
Introduction:
Respiratory quotient
When a respiratory substrate (eg. glucose) is oxidized for energy, carbon dioxide is produced.
The volume (or moles) of carbon dioxide produced with reference to the volume (or moles) of
oxygen consumed during oxidation of a respiratory substrate for a fixed period of time is termed
as the respiratory quotient (RQ).
volume of CO2 produced
RQ = volume of O2 consumed
RQ gives indication of the
type of respiration,
nature of respiratory substrate, and hence
amount of metabolic energy that can be produced.
For example, the complete oxidation of glucose is represented by the following equation:
C6H12O6 + 6O2
6CO2 + 6H2O + energy
RQ for glucose = 6/6 = 1.0

In general, the lower the respiratory quotient, the more oxygen is required for complete oxidation
of a respiratory substrate, and hence the greater the potential for generating ATP from that
respiratory substrate.
The table given illustrates some common values and their significance.
When RQ is
>1.0
Carbohydrates are used as a respiratory substrate with some anaerobic

respiration occurring simultaneously.
1.0
Carbohydrates are used as the respiratory substrate.
0.7
Mainly fats are being used as the respiratory substrate.
0.8 0.9
A mixture of carbohydrates, lipids and proteins are used as respiratory substrate
0.85
A mixture of carbohydrates and lipids are used as respiratory substrates.
0.9
Proteins are the respiratory substrates. Note that the composition of proteins is
too varied for them to give the same RQ. However, most of them have a value
around 0.9
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Warning: Do not have direct contact with soda lime as it will burn your skin
Procedures:
1. You are provided with a syringe with soda lime has been placed.
2. Place four or five bean seeds into the barrel of the syringe and carefully replace the plunger.
3. Attach the length of glass capillary tube to the syringe, using the rubber tubing provided.
4. Drop of coloured liquid into the capillary tube.
5. Place the apparatus on a sheet of white paper alongside a milimeter ruler. Your assembled
apparatus should now look like the one shown in Fig. 1.
6. Wait until the drop of coloured liquid starts to move.
7. Mark the position of the coloured liquid on the capillary tube with a marker pen.
8. Measure how far the liquid moves in one minute. Repeat the measurement every minute for
the next nine minutes.
9. If you do not get any liquid movement after 3 minutes, adjust the apparatus or reset-up.
10. If youve obtained reasonable readings, you may dismantle the apparatus and dispose the
used soda lime (without touching it) in the syringes into the beakers provided.
Fig. 1
Results and Discussions:
1. Construct a table and record your results of how far the liquid moves in one minute over ten
minutes.
2. Plot your results on a piece of graph paper. Use a best fit line.
-1
3. From your table, calculate the mean distance travelled in mm min . Show your working.
4. Assume the diameter of the capillary tube hollow is 0.2 mm. The area of the end of the
2
capillary tube can be calculated by using the formula r , where = 22/7. Calculate the
volume of gas that is absorbed by the seeds in one hour. Show your working.
5. With reference to respiration of the green bean seeds, explain why the drop of liquid moves
along the capillary tube.
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6. The formula used for calculating the RQ (respiratory quotient)is given as follows:
RQ = Vol. of carbon dioxide evolved during respiration
Vol. of oxygen absorbed during respiration
Explain how you would use or modify the apparatus in our experiment to calculate the RQ of
the seeds.
7. Experiments of this kind are very susceptible to changes in temperature. Explain how you
could compensate for any temperature changes during the experiment.
8. Discuss the sources of errors and ways to improve the experiment.
Sources of
error
Explanation
Improvement
Explanation
This will ensure that

the change in
volume of air in the
syringe is due to
oxygen absorbed by
the green bean
during the
experiment.
This will ensure
constant rate of
respiration as
oxygen is not a
limiting factor.
This will ensure a
more accurate
measurement of the
rate of respiration of
the green beans in
the specified
environment.
Movement in liquid
will be more
sensitively detected.
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Practical 10
Respiration of Yeast
Objective:
To investigate the effect of different nutrients on anaerobic respiration of yeast.
Apparatus and Equipment:
Incubator
Test-tubes (5 units)
Test tube rack
Fermentation tubes (Durham tube)
Materials:
20% sucrose solution
20% glucose solution
20% lactose solution
10% starch suspension
10% yeast suspension
Distilled water
Procedures:
1. Label five test tubes used for fermentation from A to E.
2. Using pipettes, place the following solutions into each fermentation glass tube.
Fermentation Tube
A
B
C
D
E
Solution
5 drops distilled water
5 drops glucose solution
5 drops sucrose solution
5 drops lactose solution
5 drops starch solution
3. Add 5 drops of yeast suspension into each fermentation tube. Add distilled water to fill up the
fermentation tubes.
4. Using pencil, support the fermentation tube as shown in Diagram I. Without removing the
pencil, inverse the fermentation tube. Take care not to spill out the fluid in the fermentation
tube.
5. Record the height of fluid in fermentation tubes A to E, as shown in diagram II.
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o
6. Place all the tubes in the incubator at 37 C 40 C for 30 minutes.

7. Remove the tubes from the incubator and measure the final height of the fluid in each
fermentation tube.
8. Record the difference in height between the initial and final readings for each of the tubes.
Results:
Tube
A
Nutrient
Final height of fluid,

y/mm
Difference
(x-y)/mm
Distilled water
Glucose
Sucrose
D
E
Initial height of fluid,

x/mm
Lactose
Starch solution
Discussions:
1. Name the gas collected in the fermentation tubes.
2. Which tube/tubes did not release any gas?
3. State the reasons for your answer in 2.
4. Based on experimental results, suggest the most suitable nutrient for the yeast to carry
out its activity.
5. Write an equation representing anaerobic respiration in yeast.
34

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UTAR

Centre for Foundation Studies

UNIVERSITI TUNKU ABDUL RAHMAN

FHSC 1214 Fundamentals of Cell Biology

UNIVERSITI TUNKU ABDUL RAHMAN

Extraction of Cell Organelles by Differential

Identification of Biochemicals in Their Pure Form Identification of

Part 1: Identification of Carbohydrates

-C-C=O + 2 CuSO4 + 4 NaOH

RCOOH + Cu2O + 2 H2O + 2

Benedicts solution contains copper

Test for non-reducing sugars

Finally, add an equal volume

Test for starch

Part 2: Identification of Lipids

Fat globules are stained red and

Lipids are immiscible with water.

Part 3: Identification of Proteins

A test for peptide bonds. In the

Tabulation of qualitative data

Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two

Place tubes 1 and 2 in a water bath of ~37 C to heat up the solution.

Salivate into a small beaker till it reaches about 5 ml.

Label 4 more new boiling tubes as follows: 1, 2, 3 and 4.

After 5 min of incubation

After 30 min of incubation

Heat for 1 min

Remove tube 1, 2, 3, 4 from water bath.

Heat for 1 min

Investigation of the effects of catalase concentration on hydrogen peroxide

6. Add 5cm of hydrogen peroxide into a boiling tube.

and 3 measurement, dispose the contents of tube A. Repeat step 5 to 8.

(What heading should you write here?)

Results and Discussions:

Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide

Warning: H2O2 is corrosive

7. Put tube 6 in the water bath (95 C) for five minutes.

Before using wood splint

After using wood splint

Results and discussion:

The Microscope and Its Uses

Preliminaries before Use:

Is the objective lens in position?

Focusing the Microscope - e slide

How to Mount an Object for Microscopic Examination

Observation of Onion Cells:

Observation of Starch Grains

Extraction of Cell Organelles by Differential Centrifugation

A piece of tissue is homogenized

The cell homogenate contains

A centrifuge is used to separate

The heaviest organelles can be

Diagram: Life, the science of biology (6 Ed.). William K. Purves,

Centrifugation Scheme Homogenized liver tissue is subjected to low centrifugation force to

Determination of the Isotonic Concentration of the Potato Cell Sap

s = solute potential of the cell

pressure potential, due to wall pressure (its a +ve pressure;

Water potential of a solution is always negative owing to the presence of solutes.

Sucrose solution (M)

Zero change in weight

Describe and explain:

a. The concentration of NaCl solution at which the water potential of

b. Relationship between change in weight and NaCl concentration