BioI Cell Bio LabManual SL Version 6 201505

Table of Contents:
FHSB 1214
FHSC 1214
Biology I
Cell Biology
Introduction
Practical 1
Practical 1
Cell Biology
Biological
Studies I
molecules I
Practical 2
Practical 2
Cell Biology
Biological
Studies II
molecules II
Practical 3
Cell Biology
Studies III
Practical 4
Cell Biology
Studies IV
Practical 8
Cell Biology
Studies VIII
Practical 5
Cell Biology
Studies V
Practical 6
Cell Biology
Studies VI
Practical 7
Cell Biology
Studies VII
Practical 9
Cell Biology
Studies IX
Practical 10
Cell Biology
Studies X
-
Experiment Description
Page
Writing of Lab Reports

Identification of Biomolecules
5
13
Identification of Unknown Carbohydrate

Solutions and Investigation of Action of
Saliva and HCl in Carbohydrate Solution
at Two Different Temperatures
Investigation of the Effects of Catalase
Concentration on Hydrogen Peroxide
Decomposition
20
Synthesis of Starch Using an Enzyme

Extracted from Potato Tuber
Investigation of the Effects of Different
Catalytic Conditions on Hydrogen
Peroxide Decomposition
Microscopy
27
Practical 6
Cell studies II
Practical 7
Cell studies III
Extraction of Cell Organelles by Cell

Fractionation
Determination of Solute Potential of
Potato Cell Sap
47
Practical 8
Cell studies IV
Effects of Different Treatments on

Stained Potato Cells
64
Practical 9
Energetics I
Respiration of Germinating Beans
67
Microscopic Examination of Cells at

Various Stages of Plant Mitosis and
Meiosis
DNA, Mitosis and Meiosis Modelling
71
Respiration of Yeast
93
Practical 3
Enzyme studies
I (Experiment 1)
Optional:
Practical 3
Enzyme studies
I (Experiment 2)
Practical 4
Enzyme studies
II
Practical 5
Cell studies I
Practical 10
Energetics II
Lab manual version 6_201505

FHSB1214 Biology I & FHSC1214 Fundamentals of Cell Biology
24
29
32
54
89
Important rules on tests and lab assignments
Details:
If a student fails to submit an assignment or misses a test, the lecturer will NOT remind
you to submit a new assignment nor to sit for a replacement test. The replacement
test will be announced to everyone in general and not to individual absentees. Those
who are supposed to attend must turn up and will not be reminded. It will be conducted
at the end of the semester on a different topic (usually more difficult) when all students
are so busy with tests and assignments.
It is the responsibility of the student who misses a graded/full report (with valid
reasons) to submit a replacement report, which is based on a different experiment than
the one carried out for the graded/full report.
If a submission is done online, a minimum of 7 days are given to submit your
assignment. As such, no excuses will be entertained if theres a server/ IT failure or
technical problems with your UTAR account. Hence, you have an option to submit your
assignment on day 1 to be safe, or on day 7 to be stupid. You may submit a
replacement report upon the approval of the practical lecturer. However, it is NOT the
responsibility of the lecturer to remind you about it.
I acknowledge reading the above & agree to be bound by terms therein.
Your signature:
___________________
Name:
Student ID:
Date:

How YOU can do well in BIOLOGY

Follow the 4As and you can expect As.
ttitude
Attend ALL lectures, tutorials and practicals on
time without fail.
Be attentive in class and revise your notes after
class while the topic is still fresh in your mind.
Why waste time re-reading 2-3 months later?
Do your assignments faithfully as they carry
marks for the finals.
Come prepared for lessons (i.e. read up
beforehand).
Read up beforehand before attending lectures
so that you wont be lost and wasted hours of
your life week after week.
Why stress yourself out if you can avoid it? Do
NOT count on last minute revision for tests and
examinations, as it will be too late to catch up
and seek help in areas where you may find
confusing or unclear of.
Why panic before exams because you cant find
this or that? Keep separate files for lecture,
tutorial and practical. File up the respective
notes systematically so that you do not lose
them along the semester.
Do you expect the lecturer/ tutor to be available
all the time to answer your questions? It is YOUR
responsibility to take the initiative to clear your
doubts or satisfy your curiosity to understand
certain scientific phenomena by reading up on
the relevant topics.
Based on a true story

A professor at the National University of
Singapore recounts how on one
occasion a student consulted him days
before the exam.
Student: Prof, could you explain this
page to me please?
Professor: What dont you understand
about this page?
Student: EVERYTHING.
Professor: But I already went through
this during lecture.
Student: Oh, I didnt attend most of the
lectures actually. As for the next page,
could you explain this page to me
please? ... and this page too and that
too
Prof: Im sorry, I cant help you.
Student: (Hmmmph, HES so selfish.
Hey, I paid to study here!)
What do YOU think?
If the student failed, whose fault was
it?
Was this student clever in skipping
lectures?
Was it fair for the student to make
demands on the lecturers precious
time to answer his questions?
How would the student have
benefited himself if he looked up
books and other sources of
information for himself first?
ttendance for lectures, tutorials and practicals

Lectures, tutorials and practicals carry marks
that count towards your finals.
You are expected to be present at ALL lectures,
tutorials and practicals.
Absence from any lesson must be accompanied
by a photocopy of your medical certificate
presented to your lecturer/ tutor at your next
meeting.
If you know in advance that you will not be able to attend the practical for a particular
week, you are expected to inform your tutor latest by the Friday before the affected
week.

ssignments
Use proper A4 foolscap for all handwritten assignments.
Write neatly and legibly in blue or black ink. Your tutor reserves the absolute right to
reject your assignment and ask you to re-do the assignment should he/she consider it
to be below the expected quality.
Submit your assignment on time. Late submissions may entail mark deduction or not
be graded at all.
ssessments
ALL academic tests and examinations help prepare you better for the finals.
As such, to sit for them all is not only compulsory, but beneficial. After sitting for one,
youll just want to sit for another, and another, and another
Absence from tests and examinations MUST be covered by a medical certificate, or
will be considered to have failed the tests.

Introduction
Writing of Lab Reports
hy should I bother writing lab reports in the correct way? The Foundation
Programme is designed to prepare you for undergraduate studies at UTAR which
will require the writing of lab reports all years generally. At the end of your third
year, you may have an opportunity to work on scientific projects which will culminate in an
official scientific report. Depending on the quality of your report, the golden chance
remains of publishing your report in a scientific journal. Such recognition may open doors
of opportunity (e.g., strengthen application for scholarships and further studies etc.).
Science professors are evaluated in most parts of the world by the papers they write.
Format of a lab report

Your lab report should be preceded by a cover page which contains the following:
Name
Partners name
Group
Date
Program
Unit code
Unit description
Year and semester of study
Title of lab report
Lecturers name
Example:

Your lab report should contain the following sections:
Title
Objective
Apparatus
Materials
Procedure
Results/ observations
Discussion with citations
Conclusion
References
The following guidelines on report writing are those required by the actual internationallyrecognized scientific community. The text in quotation marks in the following section is
taken from Warren D. Dolphin of Iowa State University. Credit has been given to the author
by citing the source. This is good practice as opposed to plagiarism, in which copied
material is claimed as the possession of the copyist.
1 Apparatus, materials and procedure

As the name implies, the materials and procedure used in the experiments should be reported in
this section. The importance in writing this section is to provide enough detail for the reader to
understand the experiment without overwhelming him or her. When procedures from a lab book or
another report are followed exactly, simply cite the work, noting that details can be found in that
particular source. However, it is still necessary to describe special pieces of equipment and the
general theory of the assays used. This can usually be done in a short paragraph, possibly along
with a drawing of the experimental apparatus. Generally, this section attempts to answer the
following questions:
1. What materials were used?
2. How were they used?
3. Where and when was the work done? (This question is most important in field studies.)
2 Results and observations

Results
The results section should summarize the data from the experiments without discussing their
implications. The data should be organized into tables, figures, graphs, photographs, and so on.
But data included in a table should not be duplicated in a figure or graph.
All figures and tables should have descriptive titles and should include a legend explaining any
symbols, abbreviations, or special methods used. Figures and tables should be numbered
separately and should be referred to in the discussion by number, for example:
Figure 1 shows that the activity decreased after five minutes.
The activity decreased after five minutes (fig. 1).
Figures and tables should be self-explanatory; that is, the reader should be able to understand
them without referring to the text. All columns and rows in tables and axes in figures should be
labelled.
This section of your report should concentrate on general trends and differences and not on trivial
details. Many authors organize and write the results section before the rest of the report.

2.1 Recording Qualitative Data

Qualitative experiments include those that require observations of non-quantifiable data such as
observations of colour, slides and whole specimens. Below are guidelines on reporting a segment
of qualitative experiments.
Liquid in container:
Be careful to distinguish accurately among solution, suspension and emulsion. It is your
responsibility to look up the definitions as studied in secondary school.
KI solution was added to the starch suspension

emulsion of lipid droplets in water
Amount of light penetrating solution

Be careful to distinguish accurately among clear, cloudy/murky and milky. It is your responsibility
to look up the definitions as studied in secondary school.
Colour
Some descriptions of colour are unacceptable as they are ambiguous.
Light/pale brown, instead of beige
Murky/ cloudy white, instead of milky
If theres a change in colouration, you may choose to report as follows.
The initial blue colouration of the solution turns green, then yellow and may finally appear
brick red.
If the transition cannot be easily seen, at least state the initial and final colours.
If there is no change, one must state the colour (e.g., it remained blue). It is incomplete to only
report there was no colour change without at least recording the initial colour.
Precipitate
One should comment on the precipitate colour and relative quantity. To do so, the mixture must be
left to settle.
Colour of precipitate - green, yellow, brick red precipitate

Amount of precipitate - a little, moderate amount, abundant
Example:
When describing observations involving Benedicts test, one should report that when one shakes
the test tube containing Benedicts solution and precipitate, the entire mixture will take the colour
of the precipitate. This colour upon shaking is recorded and also the amount of light penetrating
solution (transparent/ translucent/ opaque).
Moderate amount of brick red precipitate suspended in solution, which bore a tinge of blue.
Solution was milky.
Note: Particles cannot be regarded as precipitate. (e.g. groundnut particles in water.)

2.2 Recording Quantitative Data

Quantitative experiments include those that require observations of quantifiable data such as time,
quantity, weight, etc.
Tabulation and graphing
There are two categories of data normally used in reporting quantitative results raw data and
processed data. Raw data refers to the readings obtained from measurements (e.g., length, weight,
height, quantity, etc.).
The table must be accompanied by the following features:
Informative table title
Gridlines
Columns/ rows with appropriate headings and units (units and calculations should not be
in the table body)
All processed data related to and required for plotting graph must be shown in the table.
E.g. Averages, rate of yeast respiration in terms of no. of bubbles formed per minute.
Precision and decimal places:
One must express data according to the precision afforded by the instrument. E.g., if the instrument
can weigh an item as light as 0.1 g, then do not record it as 0.10 g, so as to correctly reflect the
precision of the instrument.
Note that the decimal places in the table must be the same for the same unit of measurement, and
reflect the precision of the instrument. If a measurement unit is converted to percentage or any
other unit, one is not bound by the precision of the instrument.
However, the recording should maintain a consistent and reasonable use of the number of decimals
(e.g., avoid too many decimals 88.8888888 %). Note that the table and graph below feature such
consistency of decimal places.
Precision of processed data can be presented in the following manner:
Averages calculated should follow the decimal places of the raw data.
Processed data involving summation and/ or subtraction should follow decimal places of
the raw data.
Decimals arising from processed data involving multiplication and/ or division should be
reasonable (e.g., not unnecessarily long).
Sample table:
Title: Mass of precipitate of standards at various concentrations of glucose solutions.
Glucose
concentration (%)
4
2
1
0.5
0.1
Reading 1
0.1
8.2
5.2
2.3
0.4
Precipitate mass (g)

Reading 2
Reading 3
18.6
9.3
4.5
1.8
0.3

18.4
9.0
4.8
2.1
0.4
Ave.
18.7
8.8
4.8
2.1
0.4
Graph
Plot a graph that will show the trend of the investigation. Include the following in the plotting of
graph:
Informative title
x-axis : labelled, including units (independent variables)
y-axis : labelled, including units (dependent variables)
appropriate scale used
points plotted
centroid point
Shape of graph can only be drawn using pencil, blue and black ink pen
points plotted according to table of data
best fit line/ curve
Sample graph:
Average mass of precipitate of standards at various

concentrations of glucose solutions
20
Ave. precipitate mass (g)
18
16
14
12
10
8
6
4
2
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Concentration of glucose solution (%)
Note:
The line of the plot does not go beyond the concentrations used (no extrapolation of points).
Hence, one should not extrapolate otherwise it is a claim that a certain y value is predicted
for a certain concentration.
Avoid clashing headings with clashing units (e.g., headings with two different units gram eggs vs.
gram nutrients per gram plain feed)
Amount of nutrients
(g/ g plain feed)
Mean
Mass of eggs laid in a week (g)

0.30
0.25
0.20
0.15
0.10
0.00
78.0
62.7
59. 7
58.0
74.0

69.3
2.3 What if I do not obtain desired results?

For the purpose of your UTAR lab report, if you dont obtain the desired results, just record them
as they are. By right, you should repeat it however, you may be constrained by a limited amount
of supplied solutions in the UTAR lab and time.
Hence, if your repeats involve consuming more solutions, please ask your tutor first. You may put
a footnote concerning the expected results. In your discussion, be sure to explain the possible
reasons for the anomaly.
3 Discussion with citations

This section should not just be a restatement of the results but should emphasize interpretation of
the data, relating them to existing theory and knowledge. Speculation is appropriate, if it is so
identified.
Start your discussion with a brief summary of the experiment. Be careful not to repeat the
procedure here. This summary should briefly introduce the readers about the experiment.
This brief summary should be then followed by a brief introduction on the biological theory
behind the experiment.
Explain how the independent variables affected the dependent variables, you may use
equations provided and show the dependent/independent variables.
Do not include all the list of each and every number on the data sheet.
Suggestions for the improvement of techniques or experimental design may also be included
here.
In writing this section, you should explain the logic that allows you to accept or reject your original
hypotheses. You should also be able to suggest future experiments that might clarify areas of doubt
in your results.
When citing references in the text, do not use footnotes; instead, refer to articles by the author's
name and the date the paper was published.
Fox in 1988 investigated the hormones on the nest-building behavior of catbirds.

Hormones are known to influence the nest-building behavior of catbirds (Fox, 1988).
When citing papers that have two authors, both names must be listed. When three or more authors
are involved, the Latin et al. (et alia) meaning "and others" may be used. A paper by Smith, Lynch,
Merrill, and Beam published in 1989 would be cited in the text as:
Smith et al. (1989) have shown that...
This short form is for text use only. In the References, all names would be listed, usually last name
preceding initials.
3.1 General Comments on Style

1.
All scientific names (genus and species) must be italicized. Underlining indicates italics in a
typed paper.
2.
Use the metric system of measurements. Abbreviations of units are used without a following
period.

10
3.
Be aware that the word data is plural while datum is singular. This affects the choice of a
correct verb. The word species is used both as a singular and as a plural.
4.
Numbers should be written as numerals when they are greater than ten or when they are
associated with measurements
6 mm or 2 g
two explanations of six factors.
When one list includes numbers over and under ten, all numbers in the list may be expressed
as numerals; for example,
17 sunfish, 13 bass, and 2 trout.
Never start a sentence with numerals. Spell all numbers beginning sentences.
5.
Be sure to divide paragraphs correctly and to use starting and ending sentences that indicate
the purpose of the paragraph. A report or a section of a report should not be one long
paragraph.
6.
Every sentence must have a subject and a verb.
7.
Avoid using the first person, I or we, in writing. Keep your writing impersonal, in the third
person. Instead of saying, "We weighed the frogs and put them in a glass jar," write, "The
frogs were weighed and put in a glass jar."
8.
Avoid the use of slang and the overuse of contractions.
9.
Be consistent in the use of tense throughout a paragraph--do not switch between past and
present. It is best to use past tense.
10.
Be sure that pronouns refer to antecedents. For example, in the statement, "Sometimes
cecropia caterpillars are in cherry trees but they are hard to find." Does "they" refer to
caterpillars or trees?
After writing a report, read it over, watching especially for lack of precision and for ambiguity. Each
sentence should present a clear message. The following examples illustrate lack of precision:
"The sample was incubated in mixture A minus B plus C."
Does the mixture lack both B and C or lack B and contain C?
"Protection against Carcinogenesis by Antioxidants"
The title leaves the reader wondering whether antioxidants protect from or cause cancer.
The only way to prevent such errors is to read and think about what you write. Learn to reread and
edit your work.
Identify trends/ patterns by in words the trend shown in the graph. Remember to make reference
to the values shown on the graph. Explain all the observations or trend obtained during the
investigation.
As temperature increases from 25 oC to 50OC, rate of yeast respiration/ mean number of

bubbles formed per 3 mins. increases proportionately/ linearly from 7 to 28.

11
In summary, the discussion should be correctly applying the theoretical concept involved in the
experiment.
4 Conclusion
State the general trend obtained through the investigation and provides a concise conclusion about
the investigation. Conclusion should be an attempt to answer the experimental objective.
5 References
This section lists all articles or books cited in your report. It is not the same as a bibliography, which
simply lists references regardless of whether they were cited in the paper. The listing should be
alphabetized by the last names of the authors. Different journals require different formats for citing
literature.
For articles:
Fox, J.W. 1988. Nest-building behavior of the catbird, Dumetella carolinensis. Journal of Ecology
47: 113-17.
For Books:
Bird, W.Z. 1990. Ecological aspects of fox reproduction. Berlin: Guttenberg Press.
For chapters in books:
Smith, C.J. 1989. Basal cell carcinomas. In Histological aspects of cancer, ed. C.D. Wilfred, pp.
278-91. Boston: Medical Press.
For electronic resources:
For web page with personal author
Irving, I. (2009, August 25). Crime, punishment and poverty in the United States. Retrieved from
http://ideas.repec.org/p/dal/wparch/uspov.html/
For web page with corporate author
U.S. Food and Drug Administration. (2009). Smoking cessation products to help you quit. Retrieved
from http://www.fda.gov/hearthealth/riskfactors.html/
For web page without author & without date
Perceptions of university student leadership and achievement
http:/www.cc.edu/user_surveys/1998-10/

(n.d.). Retrieved from
12
Practical 1 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)

Identification of Biomolecules
______________________________________________________________________
Objective:
To identify the biomolecules in a solution using various food tests and state the
justifications.
Introductory instructions:
You may perform this experiment in groups of 2-3.
Important notice:
Any heating that has to be done in the following tests should be carried out in a water bath
at 95oC. Direct heating of test-tubes should not be taking place.
Apparatus & Equipments:
Test tubes
Boling tubes
Water bath, 95oC
Materials:
Iodine
0.1 M hydrochloric acid
Sudan III
Starch solution
Corn oil
Egg albumin
1% copper sulphate solution
Test tube rack

Wooden holder
Spatula
1% sucrose solution
0.1 M Sodium hydroxide
1% glucose/fructose/lactose solution
Absolute ethanol
DCPIP (dichlorophenolindophenol)
solution
Ascorbic acid
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.
Please refer to the notes given above on:
How to record qualitative data.
(Marks will be awarded based on proper recording.)
What to do if you dont obtain the desired results.
Flowchart
Students will be allowed to proceed with the experiment only if they have come into the
laboratory with a flowchart of the days experiment.

13
Procedures:
Part 1: Identification of Carbohydrates
(A)
Test for reducing sugars
The reducing sugars include all monosaccharide, such as glucose and fructose, and some
disaccharides, such as maltose and lactose, using 0.1 1% sugar solutions. Common
tests for reducing sugars include Benedicts test (described below) and Fehlings test (not
described here).
See basis of test below for explanation of the following reaction:
Benedicts test for reducing sugars:

Procedure*
Basis of test
Observation
Benedicts solution
contains copper sulphate.
Reducing sugars reduce
soluble alkaline blue
copper sulphate
containing copper (II)
ions, Cu2+ to insoluble
red-brown copper oxide
containing copper (I). The
latter is seen as a
precipitate.
[Note: report after

shaking and after
contents settle down; see
introduction pg. 7]
Reducing sugar test

Add 2 cm3 of any one
solution of the reducing
sugar(s) provided into a
boiling tube. Add an equal
volume (2 cm3) of
Benedicts solution into
the same boiling tube.
Using a wooden holder,
shake and heat the
mixture in 95C water
bath for one minute,
shaking continuously to
minimize spitting.
*: Please do NOT remove measuring cylinder or any other item from the stations
provided.
Observe and report characteristics of tube contents before and after precipitate settles to
bottom of tube, taking note of liquid, colour and precipitate.
(B)
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 for test (A)), a brick-red precipitate in the
test below indicates the presence of a non-reducing sugar. If reducing sugars have been
shown to be present, a heavier precipitate will be observed in the following test than with
the reducing test if non-reducing sugar is also present.
The proper procedure to test for the presence of an unknown carbohydrate sample
containing non-reducing sugars involves:
14
First test for reducing sugars: Benedicts test on the unknown fresh sample
Why is this step necessary?
What results will one get which will cause this step to be called a negative test?
Second test for reducing sugars: Benedicts test on the acid-hydrolysed unknown
sample
What results will one get which will cause this step to be called a positive test?
Procedure*
Basis of test
Observation
Non-reducing sugar test

Add 2 cm3 of fresh
sucrose solution into a
boiling tube. Add 1 cm3 of
0.1 M hydrochloric acid.
heat the mixture at 95C
for one minute.
Carefully neutralize the
mixture with equal volume
(1 cm3) of 1 M sodium
hydroxide.
A polysaccharide or
disaccharide can be
hydrolyzed to smaller
component constituents
by boiling with 0.1 M
hydrochloric acid.
[Note: report after

shaking and after
contents settle down; see
introduction pg. 7]
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 (4 cm3) of
Benedicts solution to the
acid-hydrolysed sugar
solution.
shake the mixture
continuously to minimize
spitting while heating at
95C for one minute.
Additional Information
The mixture is likely to bump violently during heating and extra care should
therefore be taken. 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.
Is the precipitate that of reducing sugar or copper oxide?
*: Please do NOT remove measuring cylinder or any other item from the stations provided.
Observe and report characteristics of tube contents before and after precipitate settles to
bottom of tube, taking note of liquid, colour and precipitate.
15
(C)
Test for starch
Starch is only slightly soluble in water, in which it forms a colloidal suspension. It can be
tested as a mainly solid in suspension.
Procedure*
Basis of test
Observation
Iodine test
***Note: The starch
prepared for you is already
cooked starch.
A polyiodide complex is
formed with starch.
Add a few drops of 1%

cooked starch solution on
a white tile.
Add a few drops of I2/ KI
solution (iodine). Be sure
to mix them together on
the tile with a glass rod.
provided.
Part 2: Identification of Lipids
Lipids include oils (such as corn oil and olive oil), fats and waxes.
Procedure*
Basis of test
Observation
Sudan III
Sudan lll is a red dye. Add
2 cm3 of oil to 2 cm3 of
distilled water in a test
tube. Add a few drops of
Sudan III and shake.
Fat globules are stained

red and are less dense
than water.
[***Note: report after

shaking and after contents
settle down]
Lipids are immiscible with

water. Adding water to a
solution of the lipid in
alcohol results in an
emulsion of tiny lipid
droplets in the water which
reflect light and give a
white, cloudy/milky
appearance.
[***Note: report after

shaking and after contents
settle down]
Emulsion test
Add 2 cm3 fat or oil to a
test tube containing 2 cm3
of absolute ethanol.
Dissolve the lipid by
shaking vigorously. Add 4
cm3 volume of distilled
water.
provided.
16
Part 3: Identification of Proteins

A suitable protein for these tests is egg albumin.
Procedure*
Basis of test
Observation
Millons Test
Add 2 cm3 of protein
(albumin) solution into a
boiling. Add 1 cm3 of
Millons reagent.
heat the mixture at 95C
for one minute.
Millons reagent is
poisonous: be extremely
careful!
Millons reagent contains

mercury acidified with
nitric acid, giving mercury
(II) nitrate and nitrite. The
amino acid tyrosine
contains a phenol group
which reacts to give a red
mercury (II) complex.
This is a reaction given
by all phenolics and is not
specific for proteins.
Protein usually
coagulates on boiling.
Thus appearing solid.
The only common protein
lacking tyrosine likely to
be used is gelatin.
Biuret Test
Add 2 cm3 (albumin)
protein solution into a test
tube. Add an equal
volume (2 cm3) of 5%
sodium hydroxide solution
and mix. Add 2 drops of
1% copper sulphate
solution and mix. No
heating is required.
A test for peptide bonds.

In presence of dilute
copper sulphate in
alkaline solution, nitrogen
atoms in the peptide
chain form a purple
complex with copper (II)
ions, Cu2+.
Biuret is a compound
derived from urea which
also contains the
CONH group and gives
a positive result.
provided.

17
Part 4: Identification of Vitamin C (ascorbic acid)

***Note: If more than 5 drops of ascorbic acid are required to turn DCPIP to colourless,
please dilute the latter significantly.
This test can be conducted on a quantitative basis if required, in which case the volumes
given below must be measured accurately. A suitable source of vitamin C is a 50/50 mix
of fresh orange or lemon juice with distilled water. Vitamin C tablets may also be
purchased.
Procedure*
Basis of test
Observation
DCPIP test
Using 0.1% ascorbic acid
solution as a standard.
Add 1 cm3 of DCPIP
solution to a test-tube.
DCPIP is a blue dye

which is reduced to a
colourless compound by
ascorbic acid, a strong
reducing agent.
***Add the 0.1% ascorbic

acid to the DCPIP drop by
drop until it becomes
approximately colourless
(or by stirring gently if
youre provided with a
syringe needle/ glass
rod).
Note the no. of drop(s) of
ascorbic acid solution
used.
Additional Information
Shaking the solution would result in oxidation of the ascorbic acid by oxygen in the
air. The effects of shaking and of boiling could be investigated.
provided.

18
REPORT GUIDELINES
Results/ observation: Tabulation of qualitative data
1. Tabulate your observations above for each biochemical food test executed,
according to the guidelines provide in the introduction on writing lab reports. Note:
The table in the lab manual for this task is not presented correctly.
2. Wrong results are alright for this experiment.
3. No need to write procedure, basis of test, discussion or conclusion.
4. You may choose to construct one or more tables.
5. 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.
o Liquid
mixture, solution, suspension, emulsion?
clear, cloudy/murky, milky?
o Colour
state initial and final colours?
o Precipitate (if any)
colour of precipitate?
amount of precipitate?
Discussion:
1. How could you determine the concentration of ascorbic acid in an unknown sample?
2. You are provided with three sugar solutions. First one contains glucose, second one
is a mixture of glucose and sucrose, and lastly is sucrose solution.
(a) How could you identify each solution?
(b) Supposing that the apparatus were available, and time permitted, briefly discuss
any further experiments you could perform to confirm your results.
3. After carrying out Benedicts test, a student concludes that the obtained positive
results prove that glucose is present. True or false? Provide a reason.
4. After carrying out Benedicts test, a student identifies the coloured precipitate as
reducing sugar. True or false? Provide a reason.
5. A student pours Benedicts solution into a tube containing a carbohydrate. No colour
change is obtained. The student concludes that the carbohydrate is not a reducing
sugar. True or false? Provide a reason.
6. A student adds acid to a solution of sucrose followed by neutralization and, finally,
Benedicts test. A negative (instead of positive) result is obtained. Explain why.
7. Why does sucrose yield positive results after carrying out the non-reducing sugar
test? What are the components of sucrose?

19

Identification of Unknown Carbohydrate Solution and Investigation of Action of
Saliva and Hydrochloric Acid in Carbohydrate Solution at Two Different
Temperatures
______________________________________________________________________
Objective:
Students are expected to state the objective of this experiment.
Apparatus & Equipments:
Boiling tubes
Water bath, ~37-40oC
Beaker
Wooden holder
Materials:
Carbohydrate solution A
Benedicts solution
3 M Sodium hydroxide
Metal test tube racks

Water bath, ~90-95oC
Dropper
Carbohydrate solution B
3 M Hydrochloric acid
Flowchart
Procedures:
This experiment is to be done in pairs. To avoid congestion, each pair should collect the
following before beginning the experiment:
4 ml NaOH
18 ml Benedicts Solution
2 ml Solution A
10 ml Solution B
8 ml HCl
1 dropper
5 ml measuring cylinder (to be washed with distilled water each time before reuse)
Metal test tube racks
Overview
Please see tables 1 & 2 on the next page to get a rough idea of what is required in the
experiment. Can you identify in the instructions that follow, how the tubes are to be placed
under various temperatures and time periods?
Carry out your investigation as follows.
1.
Prepare two boiling tubes containing 1 ml solution A and 1 ml solution B respectively.

Add 1 ml Benedicts solution into each test tube. Heat both tubes together in the
hotter (~90-95oC) water bath for two minutes. Record the results in table 1.

20
2.
Add a few drops of fresh solution A and B separately spaced on a white tile. On each
solution, add 1-2 drops of I2/KI solution (iodine). Be sure to mix them together on the
tile with a glass rod. Record your observations in the Table 1.
3.
Pipette 2 ml solution B into each of four test-tubes and, label the tubes 1, 2, 3 and 4
respectively with labelling paper (or masking tape) near mouth of tube. Write the
initials of your group name or individuals.
4.
Place tubes 1 and 2 in a water bath of ~37o (it doesnt matter how long you put it in
at this stage as no saliva or HCl have been added yet).
5.
Salivate into a measuring cylinder till it reaches 5 ml.
6.
Ensure that the following two steps (6 and 7) adding of saliva or HCl into the
respective tubes (mentioned in the next sentence and below) is done approximately
at the same time. (Why is this necessary?)
7.
Pipette 2ml of saliva each into 1 and 4. Shake the contents of the tubes well to ensure
thorough mixing.
8.
Measure 4 ml HCl and pipette 2 ml each into tubes 2 (already in water bath of ~37oC)
and 3. Place tubes 3 and 4 in a water bath set at 95 oC. Let tubes 1, 2 (already in
water bath of ~37oC), 3 & 4 (recently in water bath of ~95oC) incubate at their
respective temperatures (see Table 2) for 35 minutes from this moment.
9.
Label 4 more new tubes (either test tubes or boiling tubes) as follows: 1, 2, 3 and
4. After 5 minutes of incubation of tubes labelled 1 to 4 prepared previously, pour
out about half of the total volume of the contents from all these tubes into the
respective newly labelled test tubes (e.g., 1 into 1, 2 into 2 etc.). Straightaway, place
back the original tubes (labelled 1-4) back into the respective temperatures of
incubation.
10.
Neutralize the acid in each of tube labelled 2 and 3 with 1ml of sodium hydroxide
(each). Shake each tube (2 and 3) to ensure uniform mixing.
11.
To carry out Benedicts test, add an equal volume of Benedicts solution (2 ml) for
each tube. Using a wooden holder, shake and heat at 95C for one minute, shaking
continuously to minimize spitting. Record your observations in Table 2.
12.
After 35 minutes of incubating tubes 1 to 4, neutralize the acid in each test tube
labelled 2 and 3 with 1ml of sodium hydroxide. (Why is neutralization necessary?)
Remove 2ml of solution from each tube 1 to 4 and carry out Benedicts test with an
equal volume of Benedicts solution (2 ml) for each tube. Remember to heat your
sample (please see previous steps). Record your observations in Table 2.

21
REPORT GUIDELINES
Results/ observation:
Table 1: (title)
Observations
Conclusions
Benedicts test:
Absence/presence of what type

of carbohydrate?
Solution A
Iodine test:
Benedicts test:
Absence/presence of what type

of carbohydrate?
Solution B
Iodine test:
Table 2: (title)
Benedicts TestColour Observation
Tube
Contents
Temp
(C)
2 ml solution B
2 ml saliva
37
2 ml solution B
2 ml 3 M HCl
37
2 ml solution B
2 ml 3 M HCl
95
2 ml solution B
2 ml saliva
95
After 5th min

(from tubes 1 4
into 1 4)
After 35th min

(tubes 1 4)
For Benedicts test and Iodine tests, please follow lab manual guidelines for students on
writing lab report on the following:
o Liquid
Solution, suspension, emulsion?
Clear, cloudy/murky, milky?
o Colour
State initial and final colours?
o Precipitate
Colour of precipitate?
Amount of precipitate?

22
Discussions:
1. Discussion should contain:
1)
2)
3)
4)
5)
Name of the enzyme involved

Specific action(s) of enzyme involved
Effect of HCl on Solution B
Effect of temperature on saliva and/ or solution B
Product:
a. Identification (make suggestion(s)/ educated guesses)
b. Structure (e.g., chemical classification etc.)
6) Bases of chemical test(s) used
7) Which carbohydrate is more complex, A or B? Give a reason.
Conclusion:
How do saliva, HCl and temperature affect the carbohydrate?

23

Investigation of the Effects of Catalase Concentration on Hydrogen Peroxide
Decomposition
Objective:
To investigate the effects of different catalase concentration on the decomposition of
hydrogen peroxide.
Apparatus and Materials:
5 test or boiling tubes
1 beaker (500cm3)
1 beaker (250cm3)
4 test tubes
1 rubber bung with delivery tube
1 Retort Stand (optional)
1% hydrogen peroxide solution **
Water bath 37C
Scalpel/ pen knife

White tile
Mortar and Pestle
Weighing boat
Measuring cylinder
4 filter funnel and filter paper (optional)
Potato
**Caution:
Hydrogen peroxide is formed continuously as a by-product of
chemical reactions in living cells; it is a very toxic (poisonous) substance.
Note to lecturer:
This experiment may be done together with Experiment 2 if the lab session is 3 h long.
Introduction:
Enzymes are proteinaceous molecules that speed up chemical reactions within living
systems. In this experiment, the effect of catalase 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. For the person handling, please wear gloves.
Flowchart
Procedures:
1. Depending on the size of the rubber bung holding the delivery tube, select either one
boiling or test tube and label it as tube A.
2.
Cut the potato and weigh 5g of potato using a weighing boat.
3.
Cut the 5g potato samples into smaller pieces (the smaller the easier for you to mash)
and mash the potato sample using the mortar and pestle.
Note: dont spend too much time on this.

24
4.
Add 6 cm3 of distilled water to the potato samples during the mashing process.
5.
Separate the solid mashed potato from the liquid either (i) pouring the liquid into a test
tube; or (ii) by filtering the mashed potato sample (with filter paper and funnel) and
collect the filtrate in a test tube.
Note: using filter paper and funnel is more time-consuming.
6.
Fill an empty test tube with tap water (see Figure 1).
7.
Add 5cm3 of hydrogen peroxide into tube A.
8.
Seal the end of the delivery tube furthest away from the rubber bung with a piece of
parafilm.
9.
Add 1cm3 of the filtrate from the mashed potato samples into tube A.
10. Immediately close the test tube with a rubber bung that has been connected with a
delivery tube.
11. Set up the apparatus as shown in Figure 1. (if retort stand is provided; if not just use
each others hands).
Figure 1 Experimental Setup

12. Remove the parafilm and immediately immerse the tube containing the mixture in a
beaker containing 37C water.
13. Start your watch and count the number of gas bubbles produced for 2 minutes and
record it. After you finish, return the water you took back to the electric water bath.
[Note: water can maintain the heat in it for quite some time.]
14. Get a 2nd measurement by disposing the contents of tube A to repeat step 7 to 13.
After you finish, return the water you took back to the electric water bath.

25
15. Repeat the experiment with 10g of potato, then 15g and finally 20g (optional, depends
on time available).
16. Record the data in Table 1. Calculate the averages in order to plot graphs.
REPORT GUIDELINES
1) Tabulation of quantitative data
Table 1: (title)
(What heading should you write here?)
10g
15g
*20g
*3rd 1st 2nd *3rd 1st 2nd *3rd *1st *2nd *3rd
5g
Number of
1st 2nd
Attempt
Number of gas
bubbles produced
*Optional, depends on time available.
2) Graphing of quantitative data

Present your graph (pasted from Excel) of the average number of bubbles produced
against potato samples used. Use a best fit curve.
Discussion:
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. Explain
the trend or pattern of the graph.

26
Practical 3 Experiment 2 (FHSC 1214 Cell Biology ONLY)

Synthesis of Starch Using an Enzyme Extracted from a Potato Tuber
______________________________________________________________________
Objective:
To investigate which compounds the enzyme in the potato can act on to produce starch
(investigate three possible substrates: glucose, maltose and glucose-1-phosphate).
Centrifuge and centrifuge tubes
Test tube rack
Pestle and mortar
Knife
Labelling paper (or masking tape)
Test tubes
White tile
Glucose-1-phosphate (2%)
Glucose solution (2%)
Maltose solution (2%)
Iodine solution
Potato tuber
Measuring cylinder
Procedures:
Create a flowchart before you enter the lab in order to understand the steps in this
experiment. Show this to your tutor before starting the experiment.
Perform this experiment in pairs.
Take 5 ml iodine only when ready to begin the experiment.
Groups may have to take turns to centrifuge, depending on the number of groups
and holders in the centrifuge.
NOTE: After carrying out steps 1 to 2, proceed to Experiment 2. Return to
Experiment 1 only during the waiting periods of Experiment 2.
A. Extracting the enzyme from potato tissue
1. Peel a medium-sized half potato. Cut half of it into small cubes on a white tile (the
smaller the easier for you to grind). Grind a few pieces of potato cubes in a pestle and
mortar with 20cm3 of water.
2. Separate the aqueous part of the extract from the solid as best as possible. You can
do this by pouring it out while restraining the solids with your fingers or an appropriate
instrument. Divide the aqueous part of the extract into two equal portions and pour
them into two centrifuge tubes. As far as possible, avoid letting sand and solid matter
to get into the tubes.
3. Spin the extracts in a centrifuge for ten minutes at 5000 rpm so that the starch, cell
walls and other solid matter will settle at the bottom of the centrifuge tubes. The starchfree liquid above the deposit, or supernatant, should contain the enzyme.
4. Using a teat pipette, carefully, without disturbing the deposit beneath, withdraw as
much the clear enzyme solution as possible from the centrifuge tube.
5. To check whether this enzyme solution is starch-free, transfer a few drops of it into a
test tube and add 2 drops of iodine solution onto it. If a blue colour appears, then the
potato extract would need to be centrifuged again.
27
B. Attempting starch synthesis

6. Label three clean test tubes G, M and G1P, respectively. Use a separate teat pipette
and measuring cylinder in each case to place 3 cm3 of glucose solution in the G tube,
3 cm3 of maltose solution in the M tube, and 3cm3 of glucose-1-phosphate solution in
the G1P tube.
7. To synthesise starch, pour 2 cm3 of the enzyme solution (the liquid or supernatant you
obtained after centrifuging above) into the substrate tube (G, M and G1P), mix well
and note the time.
8. For each substrate, place 15 discrete drops of iodine solutions on clearly labelled piece
of white tile.
9. After one minute of the reaction use a teat pipette to place one drop of enzymesubstrate solution onto one existing drop of iodine solution on the white tile. Stir with a
suitable object (e.g. woodsplint or tooth pick) and record the colour produced. Repeat
at intervals of 1 minute over 15 minutes, all the three tubes simultaneously.
REPORT GUIDELINES
Discussion
Discuss the following questions:
1. Draw the structural formula of the substrates. What features of the starch-synthesizing
substrate molecule might have been recognized by the starch-synthesizing enzyme?
2. The synthesis of polymers such as starch requires metabolic energy. What was the
energy source in the successful reaction?
3. The enzyme isolated from potatoes is known as starch phosphorylase. In the intact
potato tuber it is also used to break down starch. How did conditions in the test tube
favor starch synthesis? In what circumstances does the enzyme bring about starch
synthesis in a potato?
4. In plant leaves, starch accumulates in chloroplasts. The synthesis of starch requires
ATP. Where do you think this ATP comes from?

28

Investigation of the Effects of Different Catalytic Conditions on Hydrogen Peroxide
Decomposition
Objective:
To investigate the catalytic effect at different conditions on the hydrogen peroxide
decomposition.
Apparatus & Equipment:
Beaker
Water bath (95oC)
Glass rod
Materials:
Fresh Liver
Manganese dioxide
Wooden splinters
Boiling tubes & test tubes

Pen knife/ blade
Parafilm
Potato cubes
10% hydrogen peroxide**
**Caution:
Hydrogen peroxide is formed continuously as a by-product of
chemical reactions in living cells; it is a very toxic (poisonous) substance.
Flowchart
Procedures:
1. Label six clean empty boiling tubes as 1, 2, 3, 4, 5 and 6.
2. Cut the provided liver into three 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 tube 2. Place tube 2 in water bath (95oC) for about
five minutes. Remove it from the water bath and let it cool.
5. Put the third piece of liver into tube 3. Mash it gently into a pulp with a glass rod.
6. Cut the potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into tube 4.
7. Measure two portions of 0.5 g manganese dioxide powder. Pour each portion into tube
5 and tube 6.
8. Put tube 6 in the water bath (95oC) for five minutes. Remove it from water bath and let
it cool.
9. Prepare another six clean empty test or boiling tubes. Put 5 cm3 of hydrogen peroxide
into each of them.
29
10. Quickly add hydrogen peroxide into tubes 1, 2, 3, 4, 5, and 6 respectively.

**Step 9 and 10 are to be done quickly.
11. Stretch the parafilm and seal the mouth of the boiling tubes quickly.
In order to prevent the parafilm from being displaced if a lot of gas is produced, secure
the parafilm covering the side of the test or boiling tube with another piece of parafilm.
12. Record your observations in Table 1.
13. Leave the tubes for 20 minutes or till when you see quite a lot of gas being produced
in some boiling tubes as seen by the bulging of parafilm from the test or boiling tube
mouths.
14. Insert a glowing wooden splinter (flame extinguished but glow remains) into each tube
one at a time by just penetrating the parafilm with it.
Why is it important to test each boiling tube at least without too much difference in the
duration of sealing among the tubes?
15. Again, record all your observations in Table 1.
REPORT GUIDELINES
Results/ observations:
Table 1: (title)
Test
Tube
1
2
3
Contents with 5 cm3

hydrogen peroxide
Fresh liver
Boiled liver (cooled)
Pulped liver
Potato cubes
Manganese dioxide
(untreated)
Observations
Before inserting glowing
After inserting glowing
splinter
splinter
Boiled manganese
dioxide (cooled after
heating)
[Note: be sure to use the following terms correctly: glowing splinter glowed brighter, flame
rekindled, effervescence (bubbles) observed, reference to sound, etc.]
30
Discussion:
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 (include
reference to enzyme structure (bonds, molecular motion, shape, active site).
5. What were the differences between the reactions with fresh liver and with fresh potato
cubes? Account for these differences (include reference to enzyme structure (bonds,
molecular motion, shape, active site)
6. What were the differences between the effects on the reaction of boiling the liver and
heating the manganese dioxide? Account for these differences (include reference to
susceptibility (sensitivity) to heat, enzyme shape, bonds etc).

31
Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I)

Microscopy
Experiment 1 Microscope and Its Uses

Individual experiment
Objective:
To study the uses of microscope and its maintenances.
To learn microscopic techniques such as focus the object with correct illumination under
different power of magnifications.
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. Treat
your microscope well and it will serve you well.
Binocular Microscope
Microscope slide
Plastic millimeter ruler
Cover slips
Newspaper
Setting up the Microscope:

The microscope when not in use is usually kept in a case. Remove it by grasping the
handle arm while placing one hand under the base. Set it down gently on the
laboratory table and at a reasonable distance from the table edge. Always keep
the microscope upright in the vertical position and never touch any of the lens
surfaces with the fingers since it will deposit a thin film of oil on the glass.

32
Parts of the Microscope:
Component
Function
Arm
Base
Body tube
Eyepiece
or ocular lenses
For lifting and carrying the microscope.

To provide stability.
To house the lenses.
This is a set of lenses that rests loosely at the top end of the
body tube. It is obvious that if the microscope is tilted while being
carried, the lens may fall out and be ruined.
The magnification of the eyepiece (given as 10X) is printed on
the metal part of the ocular.
Revolving
nosepiece
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.
Objective lenses
They are of different magnifications with the following visible

properties:
Objectives
Magnification Length
Scanning lens
Low power lens
High power lens
4x
10x
40x

Shortest
short
longest
Lens
opening
Widest
wide
Narrowest
33
Focusing
adjustments
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
Fine adjustment
Made by the large knob beside the body

tube for focusing under low power
magnification.
Made by the small knob, which is for
focusing under high power magnification
and accurate focusing.
Precautions when using the focusing adjustments:

Turn both adjustment knobs at the same time.
Do not overturn the adjustment knobs (i.e. do not force them to
go
beyond their limits)
Do not use the coarse adjustment knobs when focussing under
the
40x objective lens.
Stage
Mechanical stage
Specimen holder
Vertical feed
knob
Horizontal feed
knob
Condenser
Iris diaphragm
Built-in light
source
Brightness
adjustment knob
Main switch
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.
A rotating disk under the stage. This diaphragm is used to vary
the amount light that is projected upward into the slide.
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.

34
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. If not, the field of view is totally dark or an illuminated crescent instead
of a complete circle.
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.
5. Should either of the lenses appear dirty, wipe it gently with a piece of special lens
paper. Use a circular motion with very light finger pressure.
6. The microscope is now ready for use.
7. Position it so that the stage faces you.
8. Connect the microscope to the power supply and turn on the built-in light.
9. 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.
Preparation of Wet Mount:

Materials for microscopic examination are usually placed on the glass slide of
standard size, the microscope slide. The materials are then covered by small thin
piece of glass, the cover slip. Both microscope slide and cover slip should be very
clean before use.
Cleaning microscope slides
Hold the microscope slide by the edges between the index flinger and the thumb
and dip in water. Then wipe dry using a soft tissue or a clean piece of cloth.
Cleaning cover slips
Cover slips are very fragile and need careful handling. Hold a cover slip by the
edges between the index finger and the thumb and then dip in water. To wipe dry
insert the cover slip into the fold of a piece of clean cloth or lens paper and apply
gentle pressure between the finger and thumb to both surfaces at the same time.
Use a gentle circular wiping motion for of effective cleaning.

35
REMEMBER
Always handle glass slides and cover slips by
their edges, never by their flat surfaces.
REMEMBER
Exercise 1
1.
Focusing theAlways
Microscope
- e slide
handle
glass
slides and cover

slips by their edges, never by their flat
Prepare a microscope slide to view the letter e. Cut out the letter e from a piece
surfaces.
of newspaper.
2. Place the tiny piece of newspaper in the centre of the slide with the printed side
up.
3. Add one drop of water onto the newspaper using a dropper.
4. Place the cover slip carefully over the newspaper.
Hold 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.
If this is done properly, the remaining water should spread out evenly with minimum
formation of air bubble between cover slip and slide.
Some air-bubbles may still be trapped even after the most careful preparation. If so,
gentle tapping of the cover slip with a pencil point may help remove them.
5. Make a drawing of the image under 4x magnification.
6. Carry out the observations as follows:
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)?
Slowly move the slide from left to right, observe and describe the way the image
moves. Repeat right to left.
Move the slide away from yourself and describe observe the movement of the
image again.

36
Exercise 2
Using a higher power objective
1. Great care must be taken when using higher power objectives. DO NOT focus the
high power objectives with the coarse adjustment knob or youll risk breaking the slide
and lenses.
2. Most microscopes have parfocal objectives. 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 slight further focussing with the
fine adjustment knob is required to see the specimen clearly.
3. When switching 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. Be careful that it does not touch the slide.
4. 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.
Exercise 3
Measurement with a Microscope
The unit of length used in nearly all microscopic measurement is the micrometer (um) which
equals 1/1000 mm. A simple way to gauge the size of an object viewed under the microscope
is to determine first the size of the circular field to view. We then use this measurement to
approximate the actual size of the object being viewed.
(A) Estimation of scanning field of view
1. Place a small plastic millimeter ruler on the stage.
2. Focus under the scanning objective so that a clear image of the millimeters divisions is
obtained.
3. Adjust the ruler so that the marked edge passes through centre of the field view.
4. Count the number of millimeter divisions seen within the field of view from one side to the
opposite side. Record of the diameter of the scanning field of view in both millimeters and
micrometers.
Diameter of the scanning field of view
= _______ mm
= _______ m
(B) Estimation of low power and high power field of view

We can find the low power field of view by a simple calculation. Divide the magnification
number of the low power objective being used by that of the scanning objective. Next,
divide the diameter of the scanning field (as estimated previously) by this quotient. This
gives the diameter of the low power field of view.
Example:
Scanning objective magnification
=4x

37
Low power objective magnification
= 10 x
Quotient
= 10 4
= 2.5
Diameter of scanning (4 x) field
= _____ m
Diameter of low power (10 x) field
= _____ 2.5
= _____ m
1. Using this simple method of calculation, determine the diameter of the high power field
of the microscope.
2. Replace the slide with the letter e onto the stage and re-examine the letter e.
Compare the height of the letter with diameter of the field of view.
3. Give an estimate of the actual height of the letter in both millimeters and micrometers.
Exercise 4
Magnification and Resolution
(A) Magnification Power:

The total magnification is the magnification of the eyepiece lens multiplied by the
magnification of the objective lens. By using different combinations of lenses, different
magnifications can be obtained. Do not use higher power than is necessary. More can be
made out under lower power with good illumination than under higher power with poor
illumination. Also, the larger the region of the object viewed, the easier it is to interpret
what you see.
(B) Resolving Power:
This following exercise illustrates to us the resolving power (or resolution) of a microscope
which is the ability to separate fine details to seen in the object. For most us, for example,
two dots separated by less than 0.1 mm will appear as a single dot. The microscope
therefore does two things for us it magnifies and it allows for finer resolution.
1. Prepare a wet mount using a piece of magazine photograph. Use the same procedure
as for e slide.
2. Examine the wet mount under low power (begin with scanning objective first) and
observe how the image compares with the photograph when seen with the unaided
eye.
Oil Immersion:
If you require a particularly high magnification, immersion oil may be used. Fluid with the
same refractive index as the objective lens is placed between a special objective lens and
the cover slip so that it touches both. The fluid permits a larger cone of light rays to enter
the objective from the specimen, and this increases the resolving power obtainable.
38
Note:
If your microscope comes with a 100 x objective, please DO NOT use it. Used the improper
way, it will break.
Microscope Care:
1. Turn the resolving nosepiece until the scanning objective is in position.
2. Adjust the boy tube so that the lower end of the objective is about 1 cm above the
stage.
3. Ensure that the stage surface is clean and dry.
4. Return the microscope in an upright position to its storage case.

39
Experiment 2
Preparation of Microscopic Slides
______________________________________________________________________
Objective:
To study the microscopic structure of biological samples and to learn the preparation of
biological samples for microscopic study purposes.
Introduction:
Examination of biological materials under the microscope will usually entail long periods
of looking into the eyepiece. It is useful to develop the habit of keeping both eyes open
and relaxed, as though you were looking at a distant object. This will cut out eye-strain
caused by continual forcing of one eye to remain closed.
Apparatus and Equipment:
Binocular Microscope
Microscope slide
Forceps
Cover slips
Soft tissue papers (lens cleaner)
Materials:
Onion
Potato (optional)
Safranin (optional)
Iodine
Hair (optional)
Observation of Onion Cells:

The onion scale leaf has generally two major surfaces an outer surface which faces the
exterior and an inner surface which faces the interior of the onion. The outer surface may
have pigmented portions of its outer epidermis while the inner surface may not.
Scale leaf
Toward interior
toward exterior
(Mackean, D. G., 1973. Introduction to biology, p. 25.)

40
Exercise 1
Preparation of microscopic slides
1. Cut an onion bulb into quarters. Remove one of its fleshy scale leaves.
2. Bend the onion scale leaf towards the outer epidermis until it breaks on the upper
surface.
3. Although broken, there is some thin tissue layer of the inner epidermis still intact. It
appears as a transparent paper-thin skin with a ragged edge along the broken edge
of the leaf.
4. With your fingers, pull the inner epidermis gently away from the scale leaf.
5. Using a dropper, place 1-2 drops of water on the slide and place the epidermis (~5mm
x 5mm) on the water.
6. Get rid of air bubble if there is any. Why are bubbles undesirable?
7. Slowly lower the cover slip onto the slide.
Some air-bubbles may still be trapped. If so, gentle tap
the cover slip with a pencil point to remove them.
8. Remove excess water from on top or around the cover slip with a piece of tissue paper.
9. The mounting of a specimen on a slide with solution is called a wet mount. Avoid tilting
the microscope when using a wet mount.

41
Exercise 2
Viewing the slides
1. Place the slide carefully on the stage. Position the specimen 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 by moving the revolving nosepiece.
(If not, the field of view is totally dark or an illuminated crescent instead of a complete
circle.)
3. Slide the eyepieces horizontally to the maximum length away from each other. 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. If not, youll see two overlapping illuminated circles.
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. Keep both eyes open when viewing through the eyepiece. Get accustomed to using
both eyes otherwise this will strain your eye or give you a headache over time.
7. Once the object is in sharp focus, its time to view it at higher magnification.
8. Never to lower the body tube while looking into the eyepiece and using the coarse
adjustment. If you miss the image, look up and repeat the whole procedure of focusing.
9. For viewing under every objective lens, use the fine adjustment to sharpen the focus
of the specimen.
10. Count the number of cells you see at 10X magnification.
11. Make a drawing of 4 6 cells, each 2 3 cm long. Include only the details you can
observe in your preparation. Label accordingly.
Are all the cells identical in shape and size?
Is the nucleus located in the same position in all the cells?
Suggest reasons to explain any apparent differences in the shape and size of the
cells as well as the location of the nucleus.
Notes:
The lines that form the network between individual cells are non-living cell walls made up
chiefly of cellulose. This cell wall is the outermost part of the cell and immediately
surrounds the cell membrane, also called plasma membrane, which in turn enclose the
cytoplasm. The central part of most plant cells is taken up by a vacuole filled with a fluid
made up mostly of water and various salts. The nucleus appears as a dense body in the
translucent cytoplasm.
42
12. Turn again to the scanning objective and remove the slide from the stage.
13. Stain the specimen by the technique of irrigation.
Place a drop of iodine at one edge of cover slip. A small piece of filter paper is brought
into contract with the water at the opposite edge of the cover slip. As water is absorbed
the iodine from the other side will be drawn under the cover slip. Continue this until the
iodine is drawn halfway across the space beneath the cover slip. The iodine will then
slowly spread throughout the mount.
Tissue
paper
The Technique of Irrigation
14. Examine first under low power (begin with the scanning objective first) and then under
high power.
What are the effects of the iodine stain on the cells?

Can you observe any changes in the cells? If so, describe them.
Are there starch grains in the cells?
How can you identify the starch grains if they are present?
15. Prepare another slide of the onion epidermis. This time add a drop of safranin onto the
epidermis instead of water. Allow the stain to take for 10 minutes before drawing it off
with tissue paper. Use the irrigation technique to dilute and wash off the excess free
stain. Finally put on a cover slip.
16. Examine first under low power (begin with the scanning objective first) and then under
high power.
What are the effects of the safranin stain on the cells?
How is this preparation different from the previous one observed in step 14?
Add to your drawing any additional details you may observe with this second
preparation.

43
[Additional practice tasks if time permits]

Exercise 3
Observation of Starch Grains
1. Place a small piece of potato in the centre of the slide and rub to distribute the potato
juice in an even layer. Discard the piece of potato.
2. Add a drop of water and then a clean cover slip to the slide. Take the usual precaution
of avoiding air-bubbles.
3. Examine the preparation under low power (begin with the scanning objective first).
The starch grains in the mount can be more readily observed if sized of the opening
in the iris diaphragm is decreased. This will increase the contrast between the starch
grains and the surrounding water.
4. Move the slide on the stage until you locate a field in which the grains are well
separated. Make a drawing of 4 6 starch grains to illustrate their typical shape.
5. After completing your drawings, turn again to the scanning objective and remove the
slide.
6. Stain the grains with iodine using the technique of irrigation.
7. Examine the iodine-stained mount first under the scanning objective and then under
low and high power. Draw 4 6 typical starch grains to illustrate their shape and
structure.
8. Prepare another slide of starch as outlined in step no. 1 but do not add the cover slip
yet. The grains are stained first by adding a drop of iodine onto them and the slide
gently rotated by tilting to-and- fro so that the whole area of grains is evenly covered
by iodine. Excess stain is drained off before a cover slip is added. Examine this
preparation carefully.
What observable changes may be seen in the starch grains exposed to relatively
high
iodine concentration?
What observable differences are there between these starch grains when
compared to
those exposed to lower iodine concentration?
Can the internal grain structure better observe in strained grains or unstained
ones?
9.
Biological materials are often stained before examination under a microscope. Based
on your experience in this exercise suggest reasons for such use of stains.

44
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,
taking the usual precautions not to trap air beneath it.
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. Estimate the width of the hair. State his measurement in millimeters as well as in
micrometers. (Refer to the procedure outlined in Experiment 1, Exercise 3)
While shifting the focus with the fine adjustment, what changes in the image can be
observed? Explain why these changes take place.
Does higher-power magnification allows greater detail to be seen?
Is the depth of focus as great with higher power as with low power?
Is the resolving power increased or decreased when magnification is increased?
5. 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.

45
GUIDELINES
On-site Assessment
Each student will be assessed on-the-spot identification of 3 structures within certain

minutes (10 marks) (The duration will be decided by the tutor).
This section may comprise 10 marks out of 20 marks. Any mistake will result in subtraction
of 1 mark.
Checklist for on-site slide structure identification
Observed
Yes
No
Skill: Manipulation
1. Position compound light microscope so that the stage faces you and
ensure that the microscope stage is at its lowest position.
2. Position the specimen holder such that it is roughly in the middle of
the stage and not at either left or right extremes.
3. Ensure that the scanning objective is first employed.
4. Ensure that the field of view is a complete circle and not totally dark
or an illuminated crescent.
5. Both eyes open and used to look through the eyepieces.
6. Adjust the brightness adjustment knob to give the right amount of light
for viewing the object details clearly (i.e., instead of either too dark or
too bright, obscuring the objects finer details).
7. When using the next higher power objective, look from the side of the
microscope to ensure that it does not touch the slide.
8. When using higher power objectives (e.g., 40 X onwards), only the
fine adjustment knob is used (i.e., not the coarse adjustment knob).
9. Focus on image accurately and sharply by using the coarse and fine
adjustment knobs.
Skill: Identification
10. Able to name the specimen from the slide or identify two - three
structures from the slide.
Total marks

46
Practical 6 (FHSC 1214 Cell Biology ONLY)

Extraction of Cell Organelles by Cell Fractionation
___________________________________________________________________
Objective:
To show how organelles can be purified from homogenated liver tissue by differential
centrifugation.
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 rotation of the centrifuge,
the smaller the particles is sediment. Components can be separated from larger to smaller
ones based by using a series of increasing speeds. This is called differential
centrifugation.
A cell component can be designated 70S. S is Svedberg unit or sedimentation coefficient.
It refers to how fast a substance /particle sediments in an ultracentrifuge, based on its size
and shape. The greater the S number, the greater the rate of sedimentation.
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. The relative centrifugal
force can be calculated by the following equation:
R.C.F. = 1.119 x 10 -5 (rpm2) r
Where rpm is the revolutions per minute of the rotor and r is the distance (in cm) of the
particle from the axis of rotation. The radius used is the distance from the center of the
axis of rotation to the middle of the centrifuge tube. The forces created at low speeds are
small (e.g. 600 X g) and only very large or dense particles will fall out of solution (nuclei,
whole cells and large cellular debris). At high speeds, the force created can be quite great
(e.g. as much as 300,000 X g). At these speeds, most particles will fall out of solution and
only very small, highly soluble molecules will remain in solution.

47
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
Nuclei
The heaviest organelles can

be removed and the
remaining suspension recentrifuged until the next
heaviest organelles reach the
bottom of the tube.
Figure 1 Cell Fractionation. The organelles can be separated from one another after
cells are broken open and centrifuged. Diagram: Life, the science of biology (6th Ed.).
William K. Purves, David Sadava, Gordan H. Orians, and H. Craig Heller (2001)

48

49
Differential centrifugation schemes (Figure 1) involve stepwise increases in the speed of

centrifugation. At each step, more dense particles are separated from less dense particles,
and the successive speed of centrifugation is increased until the target particle is pelleted
out. The final supernatant is removed, the pellet is re-suspended, and further study or
purification can be done on it. The fractionation of rat liver is an example of how this
process works.
An important thing to note is that there is cross contamination between the second and
third pellets. Mitochondria show up in Pellet 3 and lysosomes show up in Pellet 2. This
shows that the separations made by this technique aren't absolute purifications, but
relative enrichments of organelles.
In order to develop a differential centrifugation scheme to isolate a particular organelle, a
marker must be used to follow its isolation. The marker can be the activity of an enzyme
that is confined to that organelle. For example, enzymes of the electron transport chain
are membrane bound and confined to the inner membrane of the mitochondria. Therefore,
after a centrifugation to isolate mitochondria, both the pellet and supernatant can be
analyzed to see which part has more of the activity associated with these enzymes. The
fraction with more of the activity has been "enriched" with mitochondria. Purification of the
organelle is accomplished by following the enrichment through successive steps.
Another way to follow enrichment is by binding a radioactive label to protein on the
organelle. For example, cell surface membranes can be isolated by first binding a
radioactive drug that has its protein receptor in the cell membrane. The drug binds tightly
to the receptor and remains there, so the fractions that contain the most radioactivity are
enriched for cell surface membranes.

50
Figure 2 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 (~ 3 cm x 3 cm). Be sure to remove any associated fat
or surrounding foreign (non-liver) tissue.
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, to increase the volume and dilute
the pulp. [Use distilled or tap water if no isotonic solution if provided.]
(Why is isotonic solution needed?)
4. Remove and dispose any ungrounded tissue or fat.
5. Using labelling paper (or masking tape), label the 15 ml graduated plastic tube
provided with your groups name.

51
6. If the pulp is very thick, pour about 2-4 ml into the 15 ml graduated plastic tube and
top up to 14 ml of the tube with isotonic solution. The graduated tube has markings to
guide you.
7. If the pulp is not too thick, pour 5-8 ml of the pulp (homogenate) into the 15 ml
graduated plastic tube and top it up till 14 ml with tap water. The graduated tube has
markings to guide you.
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.
8. Wipe off any spillage on the tube exterior so as not to contaminate the centrifuge.
9. With the support of lab technical staff, spin the tube for 5 minutes.
While waiting, prepare for the next experiment.
10. After 5 minutes, collect your centrifuged sample.
Note: DO NOT shake the tube.
11. Use one hand to hold the 50 ml graduated plastic tube and another to hold a glass
pipette.
12. Press the air out of the rubber bulb of the pipette. Slowly insert the pipette tip into the
supernatant, being careful not to mix up the contents.
13. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one 15
ml graduated plastic tubes in roughly equal volumes.
14. Unless there is enough supernatant, top up just one of 15 ml graduated plastic tubes
to 10 ml with distilled H20.
15. Label the tube and wipe off any spillage on the tube exterior so as not to contaminate
the centrifuge.
16. Centrifuge the graduated plastic tube for about 20 minutes. While waiting carry on with
the next experiment.
17. After 20 minutes, collect your centrifuged sample.
Note: DO NOT shake the tube.
18. Use one hand to hold the 15 ml graduated plastic tube and another to hold a glass
pipette.
19. Press the air out of the rubber bulb of the pipette. Slowly insert the pipette tip into the
supernatant, being careful not to mix up the contents.
20. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one 15
ml graduated plastic tube.
52
21. Unless there is enough supernatant, top up just one of the 15 ml graduated plastic
tubes to 10 ml with distilled H20.
22. Label the tube and wipe off any spillage on the tube exterior so as not to contaminate
the centrifuge.
23. Centrifuge the labelled 15 ml graduated plastic tube for about 1 hour. While waiting
carry on with the next experiment.
24. After 1 hour, retrieve the tube and take a look at it. Due to shortage of time and
limitations of the centrifuge model used, this experiment will stop here.
25. Dispose of the excess liver pulp or pieces into the given plastic bag (not the dustbin).

53

Determination of the Solute potential of the Potato Cell Sap
______________________________________________________________________
Objectives:
To find out the tonicity of potato cell sap at different concentrations of sucrose solution
and its effect in the length of potato strips.
To find out the solute potential of potato cell sap based on the changes in concentration
of sucrose solution and length of potato strips.
Introduction
You are advised to read the following carefully before writing your lab report.
Water potential,
The tendency of water molecules to diffuse across a membrane is affected by:
the concentration of the solution on either side of the membrane
the resultant pressure in the solution on each side of the membrane
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
It is measured in pressure units such as kPa (kilopascals) and MPa (megapascals).
Water potential of a solution = effect of the solute concentration of that solution + effect of
pressure on that solution
where =
= s + p
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)
Campbell (2002). Chp 36. (6th Ed.).

54
Solute potential is the tendency of a solution to gain water

The potential of a solution to gain water is always negative
The more concentrated a solution, the more negative its solute potential
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.
(Pic. Hoh, 2003. A Level biology)

In pure water, water molecules are free to move about at random. In a solution, solute
particles attract water molecules and restrict their movement. Thus, water molecules
cannot leave a solution so easily, the presence of solute particles lowers the water
potential.
The movement of water molecules across a selectively-permeable membrane 2 from a
solution with a higher water potential to a solution with a lower water potential is known as
osmosis.
Never use the term semi-permeable membrane as it means that only water gets through and
no other solutes.
2

55
For example, if a plant cell is immersed in a solution with a higher water potential than the
cell, then osmotic uptake of water will cause the cell to swell.
By moving, water can perform work.
Therefore the potential in water potential refers to the potential energy that can be
released to do work when water moves from a region with higher psi to lower psi.
Plant biologists measure psi in MPa, where one MPa is equal to about 10 atmospheres of
pressure. 3
Campbell (2002). Chp 36. (6th Ed.).

56
For your understanding only: the concept of turgor pressure p
Any solution at atmospheric pressure has a negative water potential.
For instance, a 0.1-molar (M) solution of any solute has a water potential of -0.23 MPa.
In contrast to the inverse

relationship of psi to solute
concentration, water potential is
directly proportional to pressure.
Physical pressure - pressing the

plunger of a syringe filled with
water, for example - causes
water to escape via any
available exit.
If a solution is separated from

pure water by a selectively
permeable membrane, external
pressure on the solution can
counter its tendency to take up
the water due to the presence of
solutes or even forcing the water
from the solution to diffuse into
the compartment with pure
water.
It is also possible to create

negative pressure, or tension
when the plunger of a syringe is
pulled up.
If a 0.1 M solution is separated

from pure water by a selectively
permeable membrane, water will
move by osmosis into the
solution.
Water will move from the region of

higher psi (0 MPa) to the region of
lower psi (-0.23 MPa). (Fig. 36.3a)
If a 0.1 M solution (psi = -0.23 MPa) is separated from pure water (psi = 0 MPa) by a
selectively permeable membrane, then water will move from the pure water to the
solution. (Fig. 36.3d)
Application of physical pressure can balance or even reverse the water potential (Fig.
36.3).
A negative pressure can make water potential more negative (Fig. 36.3d).
Fig. 36.3. Values for & s in the left and right

arms of the U-tube respectively are given for
initial conditions, before any net movement of
water. (a) The addition of solutes makes water
potential more ve. (b,c) Application of physical
pressure increases water potential. (d) A ve
pressure (tension) decreases water potential

57
Essential knowledge for practical: Osmosis in living plant cells

58
The figure below shows the changes in water potential, pressure potential and solute
potential of a plant cell as it takes up or loses water and so changes in volume.
0 kPa
- ve kPa
p = s
= 0
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.
The curves for water potential () and solute potential (s ) are the same when the water
content of this cell falls below 50%.
As the cell contents shrink, there is no more force pushing on the cell contents. i.e., the
pressure potential (p) = 0. Hence, water potential () = solute potential (s) [Note: This
is a very important equation.]
59
In your lab report, be sure to use the proper terms (e.g., plasmolysis, the various
potentials etc.) when explaining the concepts herein.
Apparatus:
Boiling tubes
Ruler
Knife
Forceps
Glass rod
Boiling tube rack

Petri dish
White tile
Measuring cylinder
Materials:
Sucrose solution, 1.0 M
Distilled water
Potato
Tissue paper
Graph paper
Flowchart
Procedures:
1. Each group should have 35cm3 of sucrose. Label five boiling tubes as 0.1 M, 0.2 M,
0.3 M, 0.4 M, and 0.5 M.
2. Prepare a series of 20 cm3 sucrose solutions at respective concentrations from the
stock solution using dilution technique. Record the volumes of solution and distilled
water used in the table below.
M1V1 = M2V2
(M = molarity; V = volume)
Molarity
0.1 M
0.2 M
0.3 M
0.4 M
0.5 M
Volume of 1.0 M
sucrose solution (cm3)
Volume of distilled
water (cm3)
3. Prepare 15 potato strips of about 4.0 cm in length with a cross-section of about 0.5 cm
x 0.5 cm.
(Note: Dont spend too much time cutting dimensions can be approximated instead
of being identical. Ideally, it is important for each strip to be of equal dimensions. Why?)

60
4. Using a ruler, measure the initial level of all sucrose solutions in the boiling tube before
adding the potato strips. Record this in Table 2.
5. Take 3 potato strips, record the length of each potato strip in Table 1, and place the
strips into 0.1M boiling tube. Repeat these steps using 0.2 M, 0.3M, 0.4 M and 0.5 M
sucrose solutions.
6. After 30 minutes, remove the strips using forceps. Wipe them gently with tissue paper
and record the final length in Table 1.
7. Record the final level of the sucrose solution in each boiling tube and record any
changes to the physical condition of the potato strips in Table 2.
8. Calculate the average change in the length of the potato strips and record it.
9. Plot a standard graph of solute potential against the concentration of the sucrose
solution to determine the solute potential of the potato cell sap.
Concentration
(M)
Solute potential
(atm)
0.0
5
0.1
0
0.1
5
0.2
0
0.2
5
0.3
0
0.3
5
1.3
2.6
4.0
5.3
6.7
8.1
9.6
0.4
0
11.
1
0.4
5
12.
6
0.5
0
14.
3
0.5
5
16.
0
10. Plot a second graph of the change in average length of the potato strip against the
concentration of the sucrose solution. Use a best fit line.

61
REPORT GUIDELINES
Your table should have the following:
1. Initial, final and change in lengths of each strip and their averages
2. Initial, final and change in levels of sucrose solution in the boiling tubes before the
addition of potato strips
3. Physical condition of the potato strips.
Length of potato
Sucrose solution (M)
strips (mm)
0.1M
0.2M
0.3M
0.4M
0.5M
Initial length
Average initial length
Final length
Average final length
Change in average
length
The above table is just a suggested format. You may present in another way if suitable.
Present your graph (pasted from Excel) of the change in average length of the potato strip
against the concentration of the sucrose solution. Use a best fit curve. To get full marks,
please observe the guidelines given on pp8 and 9 as well.

62
Discussion:
Theory to apply: Refer to relevant information from lecture topics which may or may not
have been covered yet.
General
Note: Students must relate results with theory.

Definition of relevant terms:
a. Osmosis
b. Water potential
c. Solute potential
d. Turgor pressure
Positive change in
length
Zero change in
length
Negative change in
length (shortening of
strip)
Terms above used correctly below.

Comment on:
a. Change in length
b. Tonicity (e.g., is solution hypertonic? etc.)
c. Condition of cells (e.g., turgid, etc.)?
d. (Net) movement of water?
Comment on:
a. Change in length
e. Relationships among w plant cell , s plant cell , w sucrose, s
plant cell, s sucrose
Comment on:
a. Change in length
Conclusions
State the:
a. The concentration of sucrose solution at which the water potential of the potato tissue
is equal to the water potential of sucrose solution (determine from graph).
b. The solute potential in atm at which the water potential of the potato tissue is equal to
the water potential of sucrose solution (determine from graph).
c. Relationship between change in length and sucrose concentration.

63

Effects of Different Treatments on Stained Potato Cells
______________________________________________________________________
Objective:
To investigate the effects of various treatments on pieces of stained potato tissues
Pen knife or scalpel (if provided)
White tile
Forceps
Plastic ruler
8 test tubes
Stop watch
Potato
Methylene blue solution
50% ethanol
Petri dish
Beakers
Flowchart
Procedures:
1. Cut four cubes from the potato provided, each approximately 1 cm (breadth) x 1 cm
(length) x 2 to 5 mm (thick). Ensure all cubes are the same size and thickness. Trim
off any peel which is still attached.
Why should the surface area be kept constant for each piece of tissue?
2. Place the potato cubes in a 50mL size beaker, immerse them in methylene blue
solution for 10 minutes. Use only enough methylene blue (maximum 10mL) to cover
them (Swirl the contents to ensure that all surfaces of the cubes are exposed to the
stain).
Always read through your lab notes once before starting and look out for waiting
time. What can you do while waiting?
3. After 10 minutes, take out the stained potato cubes using forcep and wash the cubes
with running tap water until the water contains little or no stain. Then cover the cube
with tap water.
Why is a coloured stain chosen?
4. Label four test tubes A, B, C and D. To each of the tubes A, B and C, add 5cm3 of
distilled water, to tube D add 5 cm3 of 50% ethanol.
State the reason for using equal volumes of liquids in all tubes.

64
5. Place tube A in water bath (95oC), tube B in a water bath of 38oC to 42oC and tube C
and D at room temperature.
What purpose does having tube C placed in water at room temperature?
Why isnt tube D placed at high temperature?
6. After 5 minutes, add one stained potato cube to each of the four test tubes. Start the
stop watch immediately.
Explain the significance of the 5 minutes of incubation before adding the cubes
in.
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.
Explain the significance of the 2 minutes of incubation.
8. Immediately separate the tissue from solutions. For each tube, quickly pour away the
liquid into another the corresponding test tubes labeled A to D. Immediately record
your observations of each test tube.
Explain the reason for separating the tissue from solutions after adding the tissue
in.
9. Record your observations of each test tube.

What is the texture and colour of the tissue?
What is the colour of the liquid and how much light can pass through?
How will you record the difference in intensity of colouration of the liquid?

65
REPORT GUIDELINES
Table of Observations
Data to tabulate:
Tube A, B, C
Observations (please number observations according to meaning assigned to values
in table below):
Compile at least 2 sets of data. Unless otherwise instructed by tutor, you may calculate
average where necessary.
*
Texture of tissue
Colouration of tissue
Colouration of solution
(potato cubes)
1
Very soft/ very flaccid
Colourless
Colourless
2
Soft/ flaccid
Light blue
Light blue
3
Hard/ turgid
Blue
Blue
4
Very hard/ very turgid
Dark blue
Dark blue
* You may use just 3 numbers if the differences between tubes are not that clear.
Discussion
From the data you have collected in the practical, account fully for the observations you
have made and draw clear conclusions, using your knowledge and understanding. You
will need to use the following questions as guidelines.
1. Why are the potato cubes stained with methylene blue?
2. What happens to the stained potato cubes when they are placed in water at room
temperature?
3. What are the effects of temperature on potato cubes cell components in tubes A, B
and C?
4. What are the effects of the ethanol on the potato cubes cell components? [Hint: is
ethanol hydrophobic or hydrophilic? Which part of it?]
5. State the reason for using equal volumes of liquids in all tubes.
6. Explain why the potato cubes added after the tubes are left at the respective conditions
for 5 minutes.
7. What is the purpose of repeating the experiments by doing 2 sets?

66

Respiration of Germinating Beans
______________________________________________________________________
Objective:
To investigate the aspects of respiration in germinating mung bean seeds.
To understand the concept of respiratory quotient.
Each group is requested to germinate green beans 2 3 days before your practical
class.
Syringe
Gauze with soda lime
rubber tube
capillary tube
colour dye
germinating beans
Introduction:
Respiratory quotient
Metabolic energy comes primarily from oxidative reactions. As a result, the more highly
reduced a respiratory substrate, the higher potential it has for generating biological energy.
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).
RQ =
volume of CO2 produced

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.

67
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
You are required to investigate some aspects of respiration in germinating green bean
seeds, using the apparatus shown in Figure 1.
Figure 1
Warning: Do not remove the soda lime from the syringe as it will burn your skin.
Flowchart
Procedures:
1. You are required to germinate the green bean seeds 2 to 3 days before the practical
class.
2. Place four or five of the germinating green 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. Dip the end of the glass capillary tube into the coloured liquid so that a drop of liquid
enters the capillary tube. Remove any excess liquid with paper towelling.

68
5. Place the apparatus on a sheet of white paper alongside a milimeter ruler. Your
assembled apparatus should now look like that shown in Fig. 1.
6. Place the assembled apparatus on a piece of white paper. Wait until the drop of
coloured liquid starts to move.
7. Mark the position of the coloured liquid on the paper with a pen.
8. Measure the distance of the liquid movement in one minute. Repeat the measurement
every minute for the next nine minutes.
9. Record your results.
10. If you do not get any liquid movement after 3 minutes, adjust the apparatus (e.g., add
one more germinating seed, readjust the rubber connecting tube and syringe tip etc.).
Do NOT touch the point of connection between the tube and glass capillary.
REPORT GUIDELINES
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.
3. From your table, calculate the mean distance travelled in mm min-1. Show your
working.
4. Assume the diameter of the capillary tube hollow is 0.2 mm. The area of the end of the
capillary tube can be calculated by using the formula r2, 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.
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. You may provide
answers other than the ones below as long as you follow this tabulated format.

69
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.
Theory to apply: Refer to relevant information from lecture topics which may or may not
have been covered yet.

70
Practical 9 Exercise 1 (FHSB 1214 Biology I ONLY)

Microscopic Examination of Cells at Various Stages of Plant Mitosis and Meiosis
Experiment 1 Microscopic Examination of Cells at Various Stages of Plant Mitosis

Objective:
To examine the cell at various stages of the mitotic cell cycle microscopically.
Equipment:
Binocular microscope
Slides provided:
Onion mitosis Root tip, Allium l.s.
Onion mitosis Root tip, Allium c.s.
Note to instructor: this practical may be divided into two sessions, one for mitosis and
another for meiosis.

71
Introduction
The primary root system
i.
The apical meristem of the root.
The most obvious differences in appearance
between longitudinal section of stem and root
apices is the absence of bulges comparable to leaf
and bud primordial on the root apex. The root apex
is also covered by root cap (Fig 24.6). There is,
however, a marked similarly in appearance and
behaviour of the apical cells which constantly
divide by mitosis, in most roots it is possible to
distinguish a number of zones of cells at the apex.
The outermost zone is called the protoderm. It
produces cells which differentiate into the root
epidermis and root cap. Inside the protoderm is
the ground meristem, the derivatives of which
differentiate into the root cortex. Just behind the
root apex a single procambial strand can be seen
at the centre of the root. Some roots have an
additional meristematic layer, the calyptrogen,
which gives rise to the cells of the root cap. The
meristematic zones radiate from a clump of cells
called the quiescent centre situated immediately
behind the root cap. The significance of the quiescent centre is not as yet fully
understood. Its cells divide slowly and it is probably the site from which the other
meristematic layers arise.
ii.
Tissue differentiation in the root.
Differentiation of vascular tissue begins near the root apex. Several strands of
sieve-tube elements and companion cells appear near the outside of the
procambial strand. Shortly afterwards a similar number of strands of protoxylem
cells alternating with the primary phloem strands differentiate, Metaxylem cells
differentiate last of all at the centre of the procambial strand. The outermost
procambial cells undergo litter change and retain their ability to divide. They
become the pericycle which may later produce lateral roots.

Biology I & Fundamentals of Cell Biology
72

73
Acknowledgment:
Student name: Hon Lair Teng
Foundation in Science, CFS-PJ
Intake: Jan 2010
Title: Onion mitosis root tip, allium sp. (l.s.)
Magnification power: 10x.100x
Interphase

74
Acknowledgment:
Student name: Joanne Liew Hui Qi
Foundation in Science, CFS-PJ
Intake: Jan 2010
Title: Onion Mitosis Root Tip, Allium sp. (l.s.)

Magnification power: 100x.10x
Photo 1
Photo 2

75
Interphase
Photo 3
REPORT GUIDELINES
Results/ observations: Drawing
1. Make one detailed drawing showing at least one cell undergoing 1 mitotic phase.
Include also several surrounding cells (they can be of the same or different phase).
Please refer to Sem 2 lab manual, In making high-power detailed drawings,
repeated features need not all be drawn but only a small representative portion
showing a few large accurate cells (3 or 4 adjacent cells) of each type must be
indicated.
2. As part of an on-site assessment, identify for or show your lecturer 2 stages of
mitosis when called on.
Discussion: Answer the following questions
1. From the slides viewed, which is the most:
a. Frequently observed phase? Why?
b. The least? Why?
(2 marks)
(2 marks)
2. Figure 1 shows drawings of cell at various stages in the mitotic cell cycle.
Figure 1
a) List the letter shown in Figure 1 in the order in which these stages occur during
a mitotic cell cycle. The first stage has been entered for you.
(1 mark)
A _____ _____ _____ _____
Explain what is happening in stage D in Figure 1.

(2 marks)
76
b) Describe in outline what happens to the DNA in the nucleus during stage A in
Figure 1.
(2 marks)
c) State the importance of mitosis in the growth of a multicellular organism, such
as a flowering plant or a mammal.
(2 marks)
3. Figure 2 shows four animal cells in different stages of mitotic division.
Figure 2
b) Name the structures labelled A, B, C and D.
(4 marks)
c) Name the stages of division shown by cells 1 and 3.
(2 marks)
d) Using the number given to each cell above, arrange the stages as they occur
in the mitotic sequence.
(1 mark)

77
Experiment 2 Microscopic Examination of Cells at Various Stages of Plant

Meiosis
Slides provided:
Lily Anther early Prophase c.s.
Lily Anther late Prophase c.s.
Lily Anther First Meiotic division c.s.
Lily Anther second Meiotic division c.s.
Lily Anther Pollen Tetrad
Introduction:
This final histology schedule will deal with structures directly associated with
reproduction. Unlike asexual reproduction, where only single parent is involved and
where offspring are identical in hereditary characters to the parent, sexual reproduction
involves production of male and female gametes in specialized organs. This schedule
is concerned with these organs in mammals and angiosperms. Fusion of the nuclei of
these gametes results in the formation of the zygote that ultimately develops into the
offspring showing a combination of characteristics from both parents.
Brooker 1e

78
Brooker 1e
Lily Flower Bud TS:
This slide should be examined with the naked eye and then under low power. Prepare
a tissue map only. You may draw more than one ovule. However, you need only label
one ovule.
Note the following:
1. There are 6 stamens (anthers specifically), each a 4-lobed structure. Note the
pollen grains within each lobe.
2. The 3-loculate (i.e., chamber of three parts) ovary in the centre with the ovules.
Transverse Section of Flower Bud

79
Lilium Anther 2-cell Stage TS:

This is a dehisced stage of the anther. Comparison with the 4-celled stage seen in the
preceding slide should be made. Dehiscence is usually preceded by breakdown of the
partition between the locules of one half.
Note the following:
1. The bilayered wall of each locule made up of the outer epidermis and an inner
fibrous layer (endothecium).
2. The stomium or opening from the break is slit-like.
3. The single vascular bundle (connective) found in between the two lobules.
4. The thick exine of each pollen grain. In many of the pollen grains two nuclei will be
seen the vegetative and generative nuclei.
5. Use an appropriate objective lens with which you can make a detailed drawing with
labels such as the anther filament, wall-tissue, pollen sac and grains.
http://www.cartage.org.lb/en/themes/Sciences/BotanicalSciences/ClassificationPlants
/Spermatophyta/Angiosperms/FlowerStructure/anther.gif
Microspore tetrads in Lilium anther.

http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumAntherDiv1.jpg

80
Second division in microsporocytes of a Lilium microsporangium

http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumAntherDiv2a.jpg

81
Lilium Embryo Sac, 4-nucleate Stage TS:

This slide is a TS of the entire ovary which is 3 loculate and has 2 ovules per locule.
Compare this with the younger stage seen in the slide of Lily flower bud TS where the
ovules are only small groups of undifferentiated cells or ovular buds. Ovules in this
slide are already differentiated into the typical shape of an anatropous ovule.
1. Focus the embryo sac under high power and note, in some of them, the 4 nuclei
present. This will be the stage just before the formation of the typical 8-nucleate
embryo sac seen in most angiosperms. The 4-nuclei seen will probably be in
interphase.
2. Draw a tissue plan map of the embryo sac as viewed under high power (your
choice) to the best of your ability. Be sure to draw the nucleated portions of the
embryo sac distinctly. Include relevant labels for portions related to the ovule, to
the best of your ability.
Lilium embryo sac development - four nucleate stage.

http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumFourNucleate.jpg
Lilium embryo sac development - two nucleate stage

http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumTwoNucleate.jpg

82
Lilium Early Embryo TS:

After fertilization, in general, the triple fusion nucleus divides to form endosperm tissue
first and the zygote divides shortly afterwards to form the embryo.
1. Observe, under low power, the young embryo (proembryo) embedded in the
endosperm tissue. Cells of both embryo and endosperm are densely stained. Note
the very large nuclei relative to cell size in the young embryo. No distinction into
basal cell, suspensor, etc. can be made here.
2. Using an appropriate objective lens, draw a tissue map clearly indicating the
position of embryo and approximate the region of the endosperm as well. If visible,
include relevant labels for portions related to the fertilized ovule, to the best of your
ability.
http://www.botany.hawaii.edu/faculty/webb/Bot201/Angiosperm/MagnoliophytaLab99/
EmbLilyEndoOverview240Lab.jpg
Figure Transverse Section of Lily Ovary

For your assignment, one to two slides may be drawn [please consult your lecturer].
If two drawings are to be done, each student is allowed only 30 mins per slide for
drawings to be done in the 1st half. You are required to label according to the
instructions given.
83
Title: Meiotic phases of lily anther (c.s.)

Magnification: 10x.60x
(Picture & labels by Amy Tan En Ling, 2010)

84
Title: Meiotic phases of lily anther cross section.


85


86


87
Title: Photo of Lily Anther Second Meiotic Division (c.s.)

Magnification Power: 10x.40x
(Picture & labels by Gan Hui XIn, 2010)

88
Practical 10 (FHSB 1214 Biology I ONLY)

DNA, Mitosis and Meiosis Modelling
Experiment 1 Mitosis and Meiosis Modelling

Objective:
To understand and compare the different events occurring in Mitosis and Meiosis.
Students are divided into 3 groups to carry out the following activities.
Apparatus and Material:
6 pairs of chromosomes
16 triangular genes
16 circular genes
Introduction:
Comparison between Mitosis and Meiosis
Mitosis
Meiosis I
Prophase
Chromatin
Synapsis and
condense to form
Crossing over occur
chromosome
Metaphase
Individual
Homolog align at
chromosome align
metaphase plate
at metaphase plate
Anaphase
Separation of sister Separation of
chromatids
homolog
Telophase
Form 2 daughter
Form 2 daughter
cells
cells
Meiosis II
Chromatin condense
to form chromosome
Individual
chromosome align at
metaphase plate
Separation of sister
chromatids
Form 4 daughter
cells
Procedure:
Group 1: Mitosis
1. You are provided with a pair of duplicated homologous chromosome in Prophase
of a cell. The homologs contain different combination of genes.
2. Based on the given chromosomes, model the cell when it is in Metaphase,
Anaphase and Telophase. You are provided with the empty cells (without
chromosomes) in each phase to assist you in the modelling.
Group 2: Meiosis I
1. You are provided with a pair of duplicated homologous chromosome in Prophase
I of a cell. The homologs contain different combination of genes.
2. Based on the given chromosomes, model the cell when it is in Metaphase I,
Anaphase I and Telophase I. You are provided with the empty cells (without
Group 3: Meiosis II
1. You are provided with a duplicated chromosome in Prophase II in each daughter
cell arisen from Meiosis I.
2. Based on the given chromosomes, model the cell when it is in Metaphase II,
Anaphase II and Telophase II. You are provided with the empty cells (without

89
REPORT GUIDELINES
Discussion:
1. From the model cell that you have assembled, identify the following structures to
your instructor: sister chromatids, chromatins, chromosomes, centromere,
centrosome, kinetochore microtubules, non-kinetochore microtubule, aster and
metaphase plate.
2. Predict the possible chromosome for the daughter cells of Meiosis I and Meiosis
II if crossing over does not occur at Prophase I.

90
Experiment 2 DNA replication modelling

Objective:
To study and learn to construct a replication bubble using DNA Simulation Student
kit
Apparatus and Material:
44 Red beads phosphate
44 White beads Deoxyribose sugar
13 Orange beads Adenine
8 Pink beads Ribose sugar
13 Yellow beads Thymine/ Uracil

13 Green beads Guanine
13 Blue beads Cytosine
24 Clear connectors Hydrogen bonds
Introduction:
The structure of DNA
A nucleotide consists of the pentose sugar deoxyribose, a phosphate, and one of four
nitrogenous bases. The nucleotides are linked by covalent bonds to form an alternating
sugar-phosphate backbone. No matter how long the chain may be, the 5 end has a 5
carbon attached to a phosphate and the 3 end has a 3carbon attached to a hydroxyl
group.
DNA replication
Replication starts at origins of replication, where the two DNA strands are separated,
forming a replication bubble. DNA polymerases add nucleotides only to the free 3
end of a growing strand or RNA primer. Thus, a new DNA strand can elongate only in
the 5 to 3 direction.
Procedure:
1. Group yourselves into 2 to 3 students per group.
2. Construct a single stranded DNA with the base sequence as follows:
5 AGCACGTAACGTTCGA 3
3. Construct the complementary DNA strand based on the base sequence shown in
Step 2.
4. Join the two strands together by using clear connectors (hydrogen bonds).
5. Slightly twist the DNA molecules to observe the double helix structure of DNA.
6. Lay the DNA molecule flat on your bench.
7. Break open 12 base pairs from the two strands in the middle (origin of replication)
to form a replication bubble.
91
8. Attach a 2-nucleotide RNA primer (use pink beads to represent ribose sugar) to
each of the leading strand and lagging strand.
9. Add the respective DNA nucleotides one by one based on the template strand.
10. Before dismantling your completed DNA replication bubble, show and identify the
template strand, leading strand, lagging strand, Okazaki fragments and replication
fork to your instructor.
REPORT GUIDELINES
Discussion:
1. Based on the procedure, name the enzymes that participate in:
a) Step 7
b) Step 8
c) Step 9
2. Predict how long it will take to produce a 100-nucleotides-long DNA if the
elongation process is done by
a) you
b) the enzyme involve in Step 9.

92
Practical 10 (FHSC 1214 Cell Biology ONLY)

Respiration of Yeast
___________________________________________________________________
Objective:
To investigates 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)
Wash bottle
Materials:
20% sucrose solution
20% lactose solution
10% yeast suspension
20% glucose solution

10% starch suspension
Distilled water
Procedures:
1. Label five test tubes used for fermentation from A to E.
2. Using teat 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.

93
6. Place all the tubes in the incubator at 37 oC 40 oC for 30 minutes (in the absence
of an incubator, place your tubes outside the lab or in a non-air-conditioned room,
being careful not to bump into anything that will displace your Durham tubes).
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.
REPORT GUIDELINES
Tabulate a table on the difference in height between the initial and final readings for
each of the tubes.
Table 1 (title)
Tube
Nutrient
Distilled
water
B
C
D
E
Initial height of
fluid, x/mm
Final height of
fluid, y/mm
Difference
(x-y)/mm
Glucose
Sucrose
Lactose
Starch
solution

Present your graph (pasted from Excel). DONT use a best fit curve/ line. (think about
what type of graph to use)
Discussion:
In your report, be sure to address the following:
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 Q2.
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.

94

BioI Cell Bio LabManual SL Version 6 201505

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Table of Contents:

Writing of Lab Reports

Identification of Unknown Carbohydrate

Synthesis of Starch Using an Enzyme

Extraction of Cell Organelles by Cell

Effects of Different Treatments on

Respiration of Germinating Beans

Microscopic Examination of Cells at

Lab manual version 6_201505

Important rules on tests and lab assignments

Lab manual version 6_201505

How YOU can do well in BIOLOGY

Based on a true story

ttendance for lectures, tutorials and practicals

Lab manual version 6_201505

Lab manual version 6_201505

Format of a lab report

Lab manual version 6_201505

Your lab report should contain the following sections:

1 Apparatus, materials and procedure

2 Results and observations

Lab manual version 6_201505

2.1 Recording Qualitative Data

KI solution was added to the starch suspension

Amount of light penetrating solution

Colour of precipitate - green, yellow, brick red precipitate

Note: Particles cannot be regarded as precipitate. (e.g. groundnut particles in water.)

Lab manual version 6_201505

2.2 Recording Quantitative Data

Precipitate mass (g)

Lab manual version 6_201505

x-axis : labelled, including units (independent variables)

y-axis : labelled, including units (dependent variables)

appropriate scale used

points plotted according to table of data

best fit line/ curve

Average mass of precipitate of standards at various

Ave. precipitate mass (g)

Concentration of glucose solution (%)

Mass of eggs laid in a week (g)

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2.3 What if I do not obtain desired results?

3 Discussion with citations

Fox in 1988 investigated the hormones on the nest-building behavior of catbirds.

3.1 General Comments on Style

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17 sunfish, 13 bass, and 2 trout.

Every sentence must have a subject and a verb.

Avoid the use of slang and the overuse of contractions.

"The sample was incubated in mixture A minus B plus C."

Does the mixture lack both B and C or lack B and contain C?

"Protection against Carcinogenesis by Antioxidants"

As temperature increases from 25 oC to 50OC, rate of yeast respiration/ mean number of

Lab manual version 6_201505

Lab manual version 6_201505

(n.d.). Retrieved from

Practical 1 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)

Test tube rack

Lab manual version 6_201505

Test for reducing sugars

Benedicts test for reducing sugars:

[Note: report after

Reducing sugar test