Biology 1 Lab Manual 201405

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

Exercise 2: Notes on Biological Drawings
Identification of Biochemical in Their Pure
Form
6
21
Investigation of Action of Saliva and HCl

in Two Carbohydrate Solutions
30
Investigation of the Effects of Catalase

Concentration on Hydrogen Peroxide
34
Synthesis of Starch Using an Enzyme

Extracted from Potato Tuber
37
Investigation of the Enzymatic Effects of

Materials on Hydrogen Peroxide
39
43
49
50
52
Practical 6
Cell studies II
Practical 7
Cell studies III
Exercise 1: The Microscope and Its Uses

Exercise 2: On-site Assessment
Exercise 3: Preparation of Wet Mount
Exercise 4: Preparation of Microscopic
Slides
Exercise 5: Measurement with a
Microscope
Exercise 6: Observation of Starch Grains
(Additional practice tasks if time permits)
Exercise 7: Observation of Hair
Extraction of Cell Organelles by
Differential Centrifugation
Determination of Solute Potential of
Potato Cell Sap
Practical 8
Cell studies IV
Effects of Various Treatments on Pieces

of Stained Potato Cells
83
Practical 9
Energetics I
Respiration of Germinating Beans
87
Exercise 1: Microscopic Examination of

Cells at Various Stages of Plant Mitosis
Exercise 2: Reproductive Tissues in
Plants (Histology of Plant - Lily
Reproductive Structures) [Meiosis]
92
Optional:
Practical 3
Enzyme studies
I (Experiment 2)
Practical 4
Enzyme studies
II
Practical 5
Cell studies I
Lab manual version 4_201405

Biology I & Fundamentals of Cell Biology
58
64
65
66
73
99
FHSB 1214
Biology I
Practical 10
Cell Biology
Studies X
-
FHSC 1214
Cell Biology
-
Practical 10
Energetics II
Experiment Description
Page
Exercise 1: Mitosis and Meiosis Modelling

Exercise 2: DNA Replication Modelling
110
112
Respiration of Yeast
114
Appendix : Case study

E-lab manuals: towards saving the earth

More and more people all over the world and in Malaysia are getting involved in saving
the planet electric cars, wind and solar energy, and paper-less projects. What can
YOU do?
Beginning January 2011, CFS PJ Biology has piloted test e-lab manuals in an attempt
to join global conservation efforts to save the planet by saving trees. Less paper means
more trees will be left standing to absorb carbon dioxide and reduce the greenhouse
effect. Dont you & your future loved ones deserve to enjoy a cooler planet? Another
advantage is that full-colour biology pictures will be accessible to students for the first
time.
Important rules on tests and lab assignments
Summary:
No MCs or any valid reasons accepted for late/missed assignments and tests.
It is the students responsibility to submit another assignment in-lieu or sit for a
replacement test when announced (on different topic).
The lecturer will NOT remind students to submit late/missed assignments nor attend
replacement tests. (Treating you as adults.)
Details:
Unacceptable: MCs, valid reasons (chicken pox, met with an accident, menstrual
cramps, stomach ache, dog died etc, non-valid reasons (forgetfulness; lateness;
server/ IT problems). This is to be fair to everyone as fake MCs and liars are present
in Malaysia.
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 to choose one other assignment to be submitted
later in the semester when all students are so busy with tests and assignments.
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. If youre late, a link for
Late submissions is available if you dont mind getting a 50% discount, or you may
choose to submit another assignment which the lecturer will not bother to remind
you about.
I acknowledge reading the above & agree to be bound by terms therein.
Your signature:

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
Exercise 1 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 and methods (if your assignment is submitted online, this
step may be omitted)
Observations and/or results with discussion
Conclusion
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 methods
As the name implies, the materials and methods used in the experiments should be
reported in this section. The difficulty 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 Observations and/or results with discussion
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 text 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, emulsion etc. Often,
mixture is a safe descriptive term to employ. 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 transparent, translucent and opaque. 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 mixture 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 opaque.
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 need not 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
Average
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
10
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 but
both have gram in their units gram eggs vs. gram nutrients per gram plain feed)
Amount of nutrients
(g/ g plain feed)
Mean
0.30
78.0
Mass of eggs laid in a week (g)

0.25
0.20
0.15
0.10
74.0
69.3
62.7
0.00
59. 7 58.0
2.3 What if I dont 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.

11
3 Discussion
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.
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.
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.
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.

12
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.
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.
5 Literature Cited
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.
13
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.
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 Literature Cited, all names would be listed,
usually last name preceding initials.

14
Introduction
Exercise 2
Notes on Biological Drawings
______________________________________________________________________
Drawings are an aid to precise observations and for this reason they are an important
part of laboratory work. In the practical examination, the examiner will have only your
written recordings and drawings to assess you. Therefore full recordings and neatly
labelled drawings are of great importance.
Keep the following points in mind when making drawings:
1. Use a sharp, pointed HB/2B pencil.
2. Drawings should be as large as possible and made to fit into the space available.
3. Attention must be given to the general shape and proportion of the specimen.
First consider what you want to show. Then plan your drawing so that various parts
are in proportion and fit on the page. Small marks indicating the length and breadth
of the drawing are of great help in planning, and a faint outline can be rapidly drawn
to show the relative positions of the parts.
4. The final outline should be drawn with clean firm lines (not sketchy broken lines).
Details should be put in clearly with a sharp pencil. If important details are too small
to be shown in proportion, they can be shown in an enlarged drawing on the side.
5. Drawings should be accurate records of your observations. The biologist is not
expected to be an artist, but to become, in some degree, a draughtsman. Clear and
accurate line drawings are needed.
6. Shading and colouring should be avoided. It should be possible to make the
drawing perfectly clear by the judicious use of thick and thin pencil lines and careful
cross-hatching. Get into the habit of making your drawings large and clear.
7. As important as the drawing is the labelling. This should be done neatly in pencil
and the letters printed. Each label should be connected to the appropriate part of
the drawing by a clear guideline without arrowheads. Do not label too close to the
drawings, and never write on the drawing itself. Always make sure that each
drawing is fully labelled before you leave it. Guidelines to the labels must be drawn
with pencil and ruler and never crossing one another.
8. Each drawing must always have a title. The title should specify whether it is a
transverse section, s longitudinal section, whole mount, etc.
9. Magnification of drawing can be stated if necessary. Calculate using the formula,
Magnification =
size of drawing___
Actual size of specimen
10. Plan diagrams of microscopic sections should not include any cell structure.
They are outline drawings showing relative amounts and distribution of various
tissues.
11. 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.
12. It is sometimes appropriate, particularly when drawing live specimens, to make
succinct notes to the labels. These are called annotated drawings, which are
particularly valuable as they combine a record of structure with functional
observations. Annotations must be beneath the labels.

15
Example of an annotated drawing

Title: Detailed drawing of a Hydrilla leaf cell
Magnification power: 10x. 40x
Chloroplast
(moves in cytoplasm, site of
photosynthesis; stained brownish with
iodine solution)
Cytoplasm
(granulated; found at
periphery of cell;
stained light yellow
with iodine solution)
Cell vacuole
(contains salt and
sugar solution;
bound by
membrane
tonoplast;
colourless)
Cell wall
(made of
cellulose,
stained yellow)
Nucleus
(doubled membrane organelle embedded
in the cytoplasm; control center of the cell;
stained orange brown with iodine solution.)
Note:
The distinction between a plan diagram and a drawing:
A drawing is an exact and accurate representation of an object, unlike a diagram
which is a simplified outline.
Warning: Memorized textbook drawings or diagrams, bearing little likeness to the
specimens or observations will not be awarded marks.

16
A tissue map or plan diagram refers to a generalized outline of the tissue regions of a
specimen. If such is requested, no detailed drawings of individual cells are required.
The illustration below is a cross section of a leaf. The items in the square box are
detailed drawings of cells whereas those of the vascular bundle reflect a tissue map or
plan diagram.
Is the picture below a drawing or diagram?

17
General Marking Scheme

It is your responsibility to read the guidelines in the introduction to this lab manual.
In the past many clever students lost marks simply because they failed to look up
instructions already provided. Would you like to be as clever as them?
This is an example of what a marking scheme may look like for:
General instructions for students:
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.
The second half of the practical will be used to assess students microscope and / or
identification skills on one type of slide. Students are required to identify at least any
three structures which they choose.
For online submissions, students are required to upload pictures of their drawings
(more instructions on WBLE).
In order not to lose marks unnecessarily, please ensure that you comply with the
instructions on writing lab reports at the beginning of this manual, including your
particulars (e.g., name, group, etc.) as stated in the instructions.

18
Checklist for biological drawings

This section may comprise 10 marks out of 20 marks. Any mistake will result in
subtraction of 1 mark.
Items
1. Appropriate &
comprehensive (detailed and
complete) title
2. Written indication of
objective used (multiplied by
__x ocular lens)
3. Requirement for detailed
drawing or plan diagram
instruction adhered to
Please read full version for your personal success

Remarks from Lab Introduction
The title should specify whether it is a transverse
section, s longitudinal section, whole mount, etc.
A tissue map or plan diagram refers to a

generalized outline of the tissue regions of a
specimen. If such is requested, no detailed
drawings of individual cells are required.
4. Correctly labelled items

5. Annotations if requested
6. Drawing is as what is seen

under microscope (i.e., not
textbook-perfect picture)
7. Magnification if requested
annotated drawings, which are particularly

valuable as they combine a record of structure with
functional observations.
Memorized textbook drawings or diagrams,
bearing little likeness to the specimens or
observations will not be awarded marks.
Magnification =
8. Method of calculation if
requested
9. Early submission up to tutor
to delete marks according to
lateness
10. Overall impression of
drawings
(e.g., neatness)
a. Satisfactory
b. Quite good
c. Good
d. Very good
e. Excellent

___size of drawing
Actual size of specimen
19
General guidelines for biological photo(s)
Items
1. Title - Appropriate &
comprehensive (detailed
and complete)
2. Magnification - Written
indication of objective used
(multiplied by __x ocular
lens)
3. Label - Correctly labelled
items
please read full version for your personal success

Remarks from lab introduction
Specify whether it is a transverse section/cross
section, longitudinal section, whole mount, etc.
10x. 4x or 40x / 10x. 10x or 100x
Label box should not overlap the photo.

Label line should point to the specific area.
Label line should not overlap each other.
4. Annotations if requested
Include the structure and function

Annotation should be beneath the label.
5. Images and tidiness
Photos taken must be clear.
6. Late submission - up to
tutor to delete marks
according to lateness
General instructions for lecturers:

Two areas of assessment:
1) Biological drawings
1st hour allocated for drawing
Kindly instruct students to draw slides according to availability (as supplied).
The manual provides options to lecturers to request 1 detailed drawing or 2 plan
diagrams. Note: if students are to submit drawing on the spot, 1 drawing should
suffice.
Recommended time allocation: Briefing (5-10 mins) + Slide familiarisation &
reading (30 mins) + drawing (30 min if 1 detailed drawing; longer if 2 plan
diagrams required)
2) On-site assessment
2nd hour allocated for on-site assessment.
On-the-spot identification of 3 structures (10 marks); 2-3 students assessed
within 3 minutes.
Drawing (10 marks)
Recommended time allocation: On-site assessment (50 min).
General instructions for lab reports:
Prepare your answers on your own A4 sheets of paper.
You are not required to re-write questions.

20
Practical 1 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)

Identification of Biochemical in Their Pure Form
______________________________________________________________________
Objective:
To identify the components of the solution in its pure form with various food tests and
state the justifications.
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 taken place.
Apparatus & Equipments:
Test tubes
Water bath, 95oC
Test tube holder
Materials:
Iodine
1 M hydrochloric acid
Sudan III
Starch solution
Corn oil
Egg albumin
1% copper sulphate solution
DCPIP
(dichlorophenolindophenol)
solution
Ascorbic acid (or vitamin C
tablet, or lemon juice)
Test tube rack

Spatula
1% sucrose solution
(Analar sucrose must be used to
avoid contamination with a
reducing sugar Benedicts
reagent
1 M sodium hydroxide (or
potassium hydroxide or sodium
hydrogen carbonate)
Millons reagent
1% glucose solution
Absolute ethanol
Introduction
The nutrients in the food you eat supply your body with energy for growth and repair.
These principle substances include carbohydrates, proteins, fats, minerals and vitamins.
We can test for the presence of these important compounds in food by using chemical
reagents that react in predictable ways in the presence of these nutrients.
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.

21
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.
Procedures:
The following tests are to be done in pairs unless otherwise specified.
Part 1: Identification of Carbohydrates

Test for reducing sugars
The reducing sugars include all monosaccharide, such as glucose and fructose, and
some disaccharides, such as maltose and lactose, use 0.1 1% sugar solutions.
Common tests for reducing sugars include Benedicts test (described below) and
Fehlings test (not done 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. 9]
Reducing sugar test

Add 2 cm3 of any one
solution of the reducing
sugar provided to testtube. Add an equal
volume of Benedicts
solution.
Using a test-tube holder,
shake and heat at a high
temperature for one
minute (a water bath is
provided), 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.
Test for non-reducing sugars
The most common non-reducing sugar is sucrose, a disaccharide. If reducing sugars
have been shown to be absent (negative result in above test), a brick-red precipitate in
22
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 for testing for an unknown carbohydrate sample for non-reducing
sugars involves:
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 to a test
tube. Add 1 cm3 O.1 M
hydrochloric acid. Using a
test-tube holder, heat at a
high temperature for one
minute.
Carefully neutralize with
equal volume (1 cm3) of 1
M sodium hydroxide or
sodium hydrogen
carbonate or potassium
hydroxide.
(Care is required because
effervescence occurs.)
A polysaccharide or
disaccharide can be
hydrolyzed to smaller
component constituents
by boiling with O.1 M
hydrochloric acid.
Sucrose is hydrolyzed to
glucose and fructose,
both of which are
reducing sugars and give
the reducing sugar result
with the Benedicts test.
Finally, add an equal

volume (4 cm3 ) of
Benedicts solution to the
acid-hydrolysed sugar
solution.
shake continuously to
minimize spitting when
heating at a high
temperature for one
provided).

23
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?
provided.
Observe and report characteristics of tube contents before and after precipitate settles to
bottom of tube, taking note of liquid, colour and precipitate.
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 an object such
as your pen cover.
provided.

24
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 testtube. Add a few drops of
Sudan III and shake.
Fat globules are stained

red and are less dense
than water.
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 an
4 cm3 volume of cold (or
tap) water.
Lipids are immiscible with

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

shaking and after contents
settle down]
provided.

25
Part 3: Identification of Proteins

A suitable protein for these tests is egg albumen.
Procedure*
Basis of test
Observation
Millons Test
Add 2 cm3 protein
(albumin) solution or
suspension to a test-tube.
Add 1 cm3 Millons
reagent.
heat at a high
temperature for one
provided).
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 to a test
tube. Add an equal
volume of 5% sodium
hydroxide (or potassium
hydroxide) solution and
mix. Add 2 drops of 1%
copper sulphate solution
and mix. No heating is
required.
A test for peptide bonds.

In the 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.

26
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.

27
Assignments
Please check with your tutor which option is required for you.
Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other
options below)
Option 2: Skills Based Assessment: 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?
transparent, translucent, opaque?
o Colour
state initial and final colours?
o Precipitate (if any)
colour of precipitate?
amount of precipitate?
Better understanding of terms:
Mixture
Solution
Suspension
Option 3:
Skills Based Assessment: Critical thinking/ 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.
28
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?
For those who have done Sem 3
3. How would you make 100 cm3 of a 10% glucose solution?
4. Starting with stock solutions of 10% glucose and 2% sucrose how would you make
100 cm3 of a mixture of final concentration 1% sucrose and 1% glucose?

29

Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two Carbohydrate
Solutions
______________________________________________________________________
Objective:
Students are expected to state the objective of this experiment.
Apparatus & Equipments:
Boiling tubes
Pipette filler
Water bath, 37-40oC
Beaker
Metal test tube racks

Graduated glass pipette, 10ml
Water bath, ~90-95oC
Pasteur pipette
Materials:
Carbohydrate solution A
Carbohydrate solution B
Benedicts solution 3 M Hydrochloric acid
3 M Sodium hydroxide (or potassium hydroxide)
Flowchart
Procedures:
This experiment is to be done in pairs. To avoid congestion, each pair should collect the
following before beginning the experiment:
8 ml NaOH
16 ml Benedicts Solution
2ml Solution A
42ml Solution B
2ml HCl
1 pipette and 1 rubber teat (to be washed with distilled water each time before
reuse)
5 ml measuring cylinder (to be washed with distilled water each time before reuse)
Metal test tube racks (not wooden)
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 test tubes containing 2 ml solution A and 2 ml solution B respectively.

Add 2 ml Benedicts solution to each test tube. Heat both tubes together in the
hotter (~90-95oC) water bath for two minutes. Record the results in table 1.
2.
Pipette 10 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.

30
3.
Place tubes 1 and 2 in a water bath of ~37o, and tubes 3 and 4 in a water bath of
~95oC (It doesnt matter how long you put it in at this stage as no saliva or HCl
have been added yet).
4.
Salivate into a separate test-tube till it reaches a height of about 1 cm - 1.5 cm.
Dilute the saliva with an approximately equal volume of distilled water.
5.
Ensure that the following two steps (5 and 6) 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?)
Note: for the following, please ensure that the respective tube into which saliva is going
to be dropped does NOT leave the water bath (especially 95 oC) for more than 30
seconds! (Why is this necessary?)
6.
Use a 5 ml measuring cylinder to measure out 2 ml of the diluted saliva prepared in

(3) and pipette 1 ml each into tubes 1 and 4. Shake the contents of the tubes well
to ensure thorough mixing.
7.
Use a measuring cylinder to measure out 2 ml HCl and pipette 1 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.
8.
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 one-third 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.). Ensure that the
volume in each of the tubes 1-4 is approximately the same (why is this
important?). Straightaway, place back the original tubes (labelled 1-4) back into the
respective temperatures of incubation.
9.
Neutralize the acid in each of tube labelled 2 and 3 with 2ml of sodium hydroxide
(or potassium hydroxide) (each). Shake each tube (2 and 3) to ensure uniform
mixing.
10.
Remove 1ml of the solution from each tube (1 to 4) into new tubes and label
appropriately as you wish as long as you dont get confused. To carry out
Benedicts test, add an equal volume of Benedicts solution (1 ml) for each tube.
Using a test-tube holder, shake and heat at a high temperature for one minute (use
the hotter water bath provided), shaking continuously to minimize spitting. Record
your observations in table 2.
11.
Wash the test tubes 1 to 4. After 35 minutes of incubating tubes 1 to 4, pour out
about one-third of the total volume from test samples from all the tubes into the
respective tubes labelled 1- 4.
12.
Neutralize the acid in each test tube labelled 2 and 3 with 1ml of sodium
hydroxide (or potassium hydroxide). (Why is neutralization necessary?) Remove
1ml of solution from each tube 1 to 4 and carry out Benedicts test with an equal

31
volume of Benedicts solution (1 ml) for each tube. Remember to heat your sample
(please see previous. Record your observations in table 2.
13.
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 an object such as your pen cover. Record your observations in the
table 1.
Note: no penalization for unexpected results.

Please refer to Practical 1 Exercise 1 Writing lab report.
Table 1: (title)
Observations
Conclusions
Benedicts test:
Solution A
Iodine test:
Benedicts test:
Solution B
Iodine test:
Table 2: (title)
Benedicts TestColour Observation
Tube
Contents
Temp
(C)
10 ml solution B
1 ml saliva
37
10 ml solution B
1 ml 3 M HCl
37
10 ml solution B
1 ml 3 M HCl
95
10 ml solution B
1 ml saliva
95

After 5th min

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

(from tubes 1 4 into
1 4)
32
Guidelines
Observations
For Benedicts test and Iodine tests, please follow lab manual guidelines for students on
writing lab report on the following:
o Liquid
mixture, solution, suspension, emulsion?
transparent, translucent, opaque?
o Colour
state initial and final colours?
o Precipitate
colour of precipitate?
amount of precipitate?
Conclusions
Absence/presence of what type of carbohydrate?
Results and Discussions:

1. The results and discussion sections of your report should not exceed 2 pages.
2. Ensure that the guidelines for constructing tables and recording results for this
experiment are adhered to (see introduction).
3. If youre required to write a discussion straight-to-the-point, follow the numbering
below (please check with your lab tutor).
If your report is full-length, write your discussion in prose form (please check with
your lab tutor).
Theory to apply: Refer to relevant information from lecture topics on biological
molecules and enzymes.
Discussion should contain:
1) Name of enzyme involved
2) Specific action(s) of enzyme involved
3)
4) Effect of HCl on substances
(e.g., Solution B)
5) Effect of temperature on substances
(e.g., Solution B, saliva content)
6) Product:
a. Identification (make suggestion(s)/ educated guesses)
b. Structure (e.g., chemical classification etc.)
7) Basis of test() used
8) Which carbohydrate is more complex, A or B? Give a reason.
9) Conclusion: Summary of results

33

Investigation of the effects of catalase concentration on hydrogen peroxide
Objective:
To investigate the effects of different catalase concentration on the decomposition of
hydrogen peroxide.
Apparatus and Materials:
5 test or boiling tubes
Scalpel/ pen knife
1 beaker (500cm3)
White tile
1 beaker (250cm3)
Mortar and Pestle
1 Retort Stand (optional)
Weighing boat
1 rubber bung with delivery tube
4 filter funnel and filter paper (optional)
4 test tubes or plastic vials (if provided)
Potato
1% hydrogen peroxide solution
Hand-held pipette
**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.
Introductory instructions:
You may perform this experiment in groups of 3-5.
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. Optional: Set up an electric water bath at 37oC. (If this is not provided, its ok.)
2. 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.
3. From the potato sample given, cut (with a pen knife/ knife/ scalpel) and weigh 5g of
potato using a weighing boat so as not to dirty the weighing balance.

34
4. Cut the 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 it doesnt have to be KFC mashed potato quality! Add 6 cm3 of
distilled water to the potato samples after the mashing process.
5. You can do two things: (i) separate the solid mashed potato from the liquid in any
way you choose and 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 or
plastic vial (if provided) [using filter paper and funnel is more time-consuming].
6. Fill an empty test tube with tap water (see picture below).
7. Add 5cm3 of hydrogen peroxide into Tube A using the hand-held pipette provided.
8. Draw 1cm3 of the filtrate from the mashed potato samples and add to Tube A.
Immediately close the test tubes with the rubber bung. Seal the end of the delivery
tube furthest away from the bung with parafilm.
9. Set up the apparatus as shown below (if retort stand is provided; if not just use each
others hands). Note: You need neither the water bath nor retort stand.
10. Remove the parafilm and immediately immerse the tube in the water bath quickly
(use a beaker for this and pour into it water from an electric water bath) and start
your watch. 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.]
11. Optional, depends on time available: To get a 2nd measurement, dispose the
contents of tube A. Repeat step 7 to 10. After you finish, return the water you took
back to the electric water bath.
12. Repeat Step 2 to 11 but with 10g of potato, then 15g and finally 20g (optional,
depends on time available).
35
13. Record the data in table 1. Your class is to record their data on the whiteboard.
Calculate the averages in order to plot graphs.
Table 1: (title)
5g
2nd
(What heading should you write here?)

10g
15g
*20g
*3rd 1st 2nd *3rd 1st 2nd *3rd *1st *2nd *3rd
Number of
1st
Attempt
Number of gas
bubbles produced
*Optional, depends on time available.
After the experiment

Please dispose of potato pieces, masking tape, parafilm etc. into the dustbins provided.
Please clean the sink, removing any potato pieces, masking tape, parafilm etc.
Assignments
options below)
Option 2: Skills-Based Assessment: Tabulation of quantitative data (Table 1)
Option 3: Skills-Based Assessment: 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. To get full marks, please observe the
guidelines given on pp6-7 as well. No need to write procedure, draw table, write a
discussion or conclusion.
Option 4A: Skills-Based Assessment: Discussion
Data provided to students to discuss
Write your discussion in prose form and without numbering.
Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4
page of Word document (standard/ default size). Anything in excess will NOT be graded.
Font Arial, size 11.
Margins: 1 inch from top, bottom, left and right (no need to change if youre using the
standard/ default size when MS Word opens).
Theory to apply: Refer to relevant information from lecture topics which may or may not
have been covered yet.
*Option 4B: Skills-Based Assessment: Discussion
Students use own data to discuss
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.

36
Practical 3 Experiment 2 (FHSC 1214 Cell Biology ONLY)

Synthesis of Starch Using an Enzyme Extracted from a Potato Tuber
______________________________________________________________________
Objective:
To find out 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
Pipette
White tile
Glucose-1-phosphate (2%)
Glucose solution (2%)
Maltose solution (2%)
Iodine solution
Potato tuber
Procedures:
Introductory instructions:
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.
You may perform this experiment in pairs.
Take 5 ml iodine only when ready to begin the reaction.
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
starch-free 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.

37
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.
Assignments
options below)
Option 2: Skills-Based Assessment: Discussion
Discuss the following questions:
1. Draw the structural formula of the substrates. What features of the starchsynthesizing substrate molecule might have been recognized by the starchsynthesizing 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?

38

Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide Solution
Objective:
To investigates the enzymatic effect of various materials in the hydrogen peroxide
solution.
Apparatus & Equipment:
Beaker
Test tubes
Either: water bath (95oC) or Bunsen burner
Materials:
Fresh Liver
Manganese dioxide
Wood splints
Potato cubes
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:
Wear gloves when handling liver tissue, so as not to be contaminated by any pathogen
associated with the liver tissue used. Please stick to using one pair of gloves per person
to prevent wastage.
[Note: using boiling tubes may provide better results.]
1. Label six fresh empty test or boiling tubes 1, 2, 3, 4, 5, 6 and stand them in a rack.
2. Using a razor blade, cut the provided liver into several pieces of roughly 0.8 cm x 0.8
cm x 0.5 cm.
3. Place one piece of liver into tube 1.
4. Boil 100 cm3 of water in a beaker. (If youre using a water bath set at 95oC, this step
is not necessary).
5. Place the second piece of liver into the bottom of tube 2. Using a wooden splint,
gently spread the liver, without mashing it, over as wide an area as possible of the
bottom of the test or boiling tube. Place tube 2 in the boiling or water bath (95oC) for
about five minutes.
6. Using the weighing balance, measure out two 0.5 g portions of manganese dioxide
powder each onto a weighing boat. Pour each portion into tube 5 and tube 6.
39
7. Put tube 6 in the beaker of boiling water or water bath (95oC) for five minutes.
8. After five minutes let cool tube 2 and 6.
9. *Now put the third piece of liver into test or boiling tube 3. With the wooden stick
provided, mash it gently into a pulp.
10. Now put the third piece of liver onto a white tile. With a mortar and pestle, mash it
gently into a pulp. Scoop the pulp into tube 3.
11. Cut potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into a tube 4.
12. Prepare another six fresh empty test or boiling tubes and stand them in a rack. Put 5
cm3 of hydrogen peroxide into each of them.
13. Next, quickly add hydrogen peroxide into the test or boiling tubes 1, 2, 3, 4, 5, and 6.
If needed, you may push down some materials with one end of the wood splints
provided.
**Step 12 and 13 are to be done quickly.
14. Using the parafilm provided, stretch it quickly seal the mouth of the test or boiling
tubes by stretching the film over it. 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 masking tape.
15. Leave for 20 minutes or till when you see quite a lot of gas being produced in some
test or boiling tubes as evidenced by the bulging of parafilm from the test or boiling
tube mouths.
16. Once enough gas has accumulated in some test or boiling tubes, insert a glowing
splint (flame extinguished but glow remains) into each tube one at a time by just
penetrating the parafilm with it. You may use the same splint.
Why is it important to test each test or boiling tube at least without too much
difference in the duration of sealing among the tubes?
17. Record all your observations in the table. Record your observations on each tube
immediately after the reaction has started.
[Note: be sure to use the following terms correctly: glowing splint glowed brighter, flame
rekindled, effervescence (bubbles) observed, reference to sound, etc.]

40
Table 1: (title)
Test
Contents with 5 cm3
Tube
hydrogen peroxide
1
2
3
Fresh liver
Boiled liver (cooled)
Pulped liver
Potato cubes
Manganese dioxide
(untreated)
Observations before and after using wood splint
Boiled manganese
dioxide (cooled after
heating)
Washing up
Thoroughly wash and scrubbed all apparatus containing liver pieces with detergent or
Dettol solution provided to rid it of unpleasant odours.

41
Assignments
Option 1: (please refer to WBLE/Turnitin for instructions which may incorporate other
options below)
Option 2: Skills-Based Assessment: Tabulation of qualitative data
Tabulate the results you obtained (unexpected results accepted). The results table
should not exceed 1 page.
have been covered yet. Please be sure to address the following:
1. What is the equation of the reaction observed?
2. What plant or animal organelle is involved?
3. What effect does pulping the liver have upon the reaction? Account for this.
4. What effect does boiling the liver have upon the reaction? Account for this (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).

42
Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I)

Exercise 1
The Microscope and Its Uses
Microscope usage (revision)

Note to lecturer:
Before any microscope work (viewing of histological slides) commences, please ensure
students have gone through this introductory session.
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 (1 page)
Wash bottle
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.

43
Parts of the Microscope:
Component
Arm
Base
Body tube
Eyepiece or
ocular lenses
Revolving
nosepiece
Objective
lenses
Function
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.
Located at the lower end of the body tube, it carries 3 objectives of
different lengths. Rotating this part changes the magnification of the
objectives.
They are of different magnifications with the following visible
properties:
Objectives
Magnification Length
Lens opening
Scanning lens
4x
Shortest
Widest
Low power lens
10x
short
wide
High power lens
60x
longest
narrowest

44
Component
Focusing
adjustments
Stage
Mechanical
stage
Function
These comprise two knobs located on either side of the microscope
which are used to change the distance between the object being
viewed and the objective lens. Changing the distance determines the
focus.
For the object to be viewed in focus under high magnification, the
lens must be much closer to the object than when it is under low
magnification.
Coarse adjustment
Made by the large knob beside the body tube
for focusing under low power magnification.
Fine adjustment
Made by the small knob, which is for focusing
under high power magnification and accurate
focusing.
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 60x objective lens.
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.
Specimen
holder
Vertical feed Rotating this moves the glass slide in the vertical direction.
knob
Horizontal
This moves the glass slide in the horizontal direction.
feed knob
Condenser
Located just beneath the stage of the microscope, it incorporates a
lens which collects light on the stage to bear on the object.
Built-in light This is situated below the iris-diaphragm to provide light for
source
illuminating the object. It can be switched on or off.
Brightness
This provides adjustment to the illumination brightness.
adjustment
knob
Main switch
This ensures that power is turned on or off.

45
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. You should
never use any other type of paper or cloth. Discard the lens paper after use.
6. The microscope is now ready for use.
7. If youre using a binocular compound light microscope like the diagram above,
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.

46
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.
2. Most microscopes have parfocal objectives. This means that if one switches from
viewing a specimen in sharp focus under a lower power objective to a higher one,
the object should automatically come approximately into focus. Only some slight
further focussing with the fine adjustment knob is required to see the specimen
clearly. Therefore, if youre using the higher power objectives, do not use the
coarse knob to refine focus or youll risk breaking the slide and lenses.
If the objectives are not parfocal, adjust the stage such that it is about 1cm from
the low-power objective. Change to the high-power objective and then adjust the
stage with the coarse adjustment knob until it is about 1mm away from the
objective. This is determined by looking from the side of the microscope.
Using the fine adjustment knob and looking through the eyepiece now, slowly
bring the object into focus.
Repeat the procedure carefully if the first attempt at finding an object under highpower magnification is unsuccessful.
3. When changing from one objective to another, you will hear a click when the
objective is set in position.
4. You are now ready to switch from the scanning objective to a higher power objective
after obtaining a sharp focus of the object.
5. If required, adjust the fine adjustment knob to see the specimen clearly.
6. If youre going to switch to the next higher power objective, look from the side of the
microscope and move the revolving nosepiece slowly till that higher power objective
clicks into position. Be careful that it does not touch the slide (normally it shouldnt
unless the specimen is too thick and also covered by a thin cover slip).
7. Take care that the lower end of the high power objective does not touch the cover
slip. If this happens, you must repeat the whole procedure focusing again, starting
with the scanning objective.
Trouble-shooting
Below are some common problems associated with not being able to find and focus on
an object under high-power magnification.
Is the objective lens in position?

Is the cover slip on the slide facing upwards?
Is the object in the centre of the stage?
Are the lenses clean and free from dirt and moisture?
Is the condenser adjusted and focused?

47
Oil Immersion:
If your microscope comes with a 100 x objective, please do NOT use it. Used the
improper way, it will break.
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.
Microscope Care:
Like all laboratory instruments, the microscope needs proper care for best service.
Observe the following:
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.
Activity: Manipulation Skill practice task

Note to lecturer: this activity may be graded. Any mistake will result in subtraction of 1
mark.
Microscope manipulation checklist
Observed
Yes No
Skill: Manipulation
1. Position compound light microscope so that the stage faces you.
2. Ensure that the microscope stage is at its lowest position.
3. Position the specimen holder such that it is roughly in the middle
of the stage and not at either left or right extremes.
4. Secure the slide in position correctly with the specimen holder
5. Ensure that the scanning objective is first employed.
6. Ensure that the field of view is a complete circle and not totally
dark or an illuminated crescent.
7. Both eyes open and used to look through the eyepieces.
8. 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).
9. Focus on image accurately and sharply by using the coarse and
fine adjustment knobs.
10. When using the next higher power objective, look from the side of
the microscope to ensure that it does not touch the slide.
11. When using higher power objectives (e.g., 40 X onwards), only
the fine adjustment knob is used (i.e., not the coarse adjustment
knob).

48

Exercise 2
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
12. Position compound light microscope so that the stage faces you and
ensure that the microscope stage is at its lowest position.
13. Position the specimen holder such that it is roughly in the middle of
the stage and not at either left or right extremes.
14. Ensure that the scanning objective is first employed.
15. Ensure that the field of view is a complete circle and not totally dark
or an illuminated crescent.
16. Both eyes open and used to look through the eyepieces.
17. 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).
18. When using the next higher power objective, look from the side of
the microscope to ensure that it does not touch the slide.
19. When using higher power objectives (e.g., 40 X onwards), only the
fine adjustment knob is used (i.e., not the coarse adjustment knob).
20. Focus on image accurately and sharply by using the coarse and fine
adjustment knobs.
Skill: Identification
21. Able to name the specimen from the slide or identify two - three
structures from the slide.
Total marks

49

Exercise 3 Preparation of Wet Mount
Materials for microscopic examination are usually placed on the glass slide of
standard size, the microscope slide. In most cases, 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. Dirty
handkerchiefs will not do.
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.
REMEMBER
Always handle glass slides and cover slips by
their edges, never by their flat surfaces.
REMEMBER
Always handle glass slides and cover
slips
by the
their
edges,
never
by oftheir
flat cut a
Prepare a microscope slide
to view
letter
e. Using
a pair
scissors,
piece of newspaper about
3
mm
square
that
includes
a
tiny
sharp-lined
letter
surfaces.
Focusing the Microscope - e slide

1.
(an R or "e" is best).
2. Place this square piece of newspaper in the centre of the slide with the printed
side up.
3. Add one or two drops of water onto the newspaper using a dropper. The
water should be sufficient for the newspaper to absorb and still leave
some remaining around it.
4. Place t he cover slip carefully over the newspaper. If this is done properly, the
remaining water should spread out evenly with minimum formation of air bubble
between cover slip and slide.
This may take bit of practice. One effective method is to 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. Use a mounted needle if
necessary.
50
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.
Anyway, a few bubbles should not hinder most observations to be made.
5. Make a drawing of the image under 4x magnification.
How to Mount an Object for Microscopic Examination

Carry out the observations as follows:
1. Compare the position of image as seen through the eyepiece with that of the printed
letter as seen with the unaided eye. Does the image appear to be reversed, i.e. as it
would appear if seen in a mirror?
2. Slowly move the slide from left to right, observe and describe the way the image
moves. Repeat right to left.
3. Move the slide away from yourself and describe observe the movement of the image
again.

51

Exercise 4
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. (The final image is
theoretical focused at infinity as any book on optical instruments will tell us). This will cut
out eye-strain caused by continual forcing of one eye to remain closed.
Apparatus and Equipments:
Binocular Microscope
Microscope slide
Plastic millimeter ruler
Forcep
Cover slips
Soft tissue papers (lens cleaner)
Wash bottle
Materials:
Potato
Hair
Safranin
Onion
Iodine
Observation of Onion Cells:

The onion scale leaf (see figure below) 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.
(Mackean, D. G., 1973. Introduction to biology, pg 25.)

52
Part 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. You may use forceps, scissors or a scalpel (used against a white tile) to pull out 2
small-sized portions of the inner epidermis (roughly 5 mm X 5 mm). Make sure that it
is not too big so as to reach the width of the cover slip or of such a size that it rolls up
easily.
6. Using a dropper, place 1-2 drops of water on the slide and place the epidermis on
the water. Make sure that there is some water covering the specimen and
surrounding it.
7. If there are any bubbles, try to get rid of them by pricking them with a mounting
needle provided. Why are bubbles undesirable?

53
8. Referring to the diagram below, hold a cover slip at about 45 to the slide and lower
it so that one edge touches the water droplet.
9. Slowly lower the cover slip onto the slide using the points of a pair of fine forceps,
pencil or a mounted needle. 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.
Anyway, a few bubbles should not hinder most observations to be made.
10. Remove excess water from on top or around the cover slip with a piece of tissue
paper provided be careful not to absorb all the water from under the cover slip.
Removal of excess water ensures that when the slide is viewed under the
microscope, water will not spill onto the stage.
11. The mounting of a specimen on a slide with solution is called a wet mount. Avoid
tilting the microscope when using a wet mount.
Part 2 - Viewing the slides

1. Place the slide carefully on the stage and position such that the specimen is in the
centre of the hole in the stage and also in the middle of the circle of light emanating
from the lamp through the stage hole.
2. Ensure that the scanning objective is in place by moving the revolving nosepiece.
The revolving nosepiece is in correct position if the objective lens is felt to click to fit
an unseen internal groove that aligns the lenss field of view with the eyepieces. If
not, the field of view is totally dark or an illuminated crescent instead of a complete
circle.
3. If the bifocal compound light microscope youre using has eyepiece lenses which can
be slid horizontally, slide the eyepieces to the maximum length away from each other
first.
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.

54
4. Adjust the brightness adjustment knob to give the right amount of light for viewing the
object clearly. Some materials are best viewed in dim light, others in bright light.
A common cause of poor definition of (i.e., not being able to see details clearly) the
image is that the object is over-illuminated. Best definition is often obtained by
cutting down the amount of light and not increasing it. If the condenser is not
adjusted and focused, the specimen may appear too dark or too bright, obscuring the
objects finer details.
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. If the details of the specimen are not clear, adjust the brightness adjustment knob
and/or condenser.
8. Once the object is in sharp focus, its time to view it at higher magnification (i.e., the
10X objective).
9. Never to lower the body tube while looking into the eyepiece and using the coarse
adjustment. If for some reason you miss the image, look up and repeat the whole
procedure of focusing.
10. For viewing under every objective lens, get a sharp focus first. Use the fine
adjustment to sharpen the focus of the specimen.
11. Count the number of cells you see at 10X magnification.
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.
12. Turn again to the scanning objective and remove the slide from the stage.
Staining with iodine

13. Stain the grains by the technique of irrigation. This is done by placing 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.

55
Tissue paper
The Technique of Irrigation

14. Examine first under low power (begin with the scanning objective first) and then
under high power.
Answer these questions:
1. What are the effects of the iodine stain on the cells?
2. Can you observe any changes in the cells? If so, describe them.
3. Are there starch grains in the cells?
4. How can you identify the starch grains if they are present?
Staining with safranin
15. Prepare another slide of the onion epidermis. This time add a drop of safranin onto
the epidermis instead of distilled 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.
1. What are the effects of the safranin stain on the cells?
2. 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.

56
Assignments
Option 1: On-site assessment
This option may be used for stained (iodine or safranin) cells.

Students will be graded based on microscope skills stated in Activity:
Manipulation Skill practice task under Exercise 1 The Microscope and Its Uses and
Exercise 2 On-site Assessment.
For Exercise 2 On-site Assessment, students have to identify at least TWO
structures - cell wall, nucleus or starch grains (if visible) in under 1.5 minutes.
There should be minimal folding of epidermal tissue.
Mark allocation: 5 marks.
(Minus marks for any violation of microscope usage or unsatisfactory
specimen preparation)
Option 2: Drawing
For specimens stained with either iodine or safranin, make a drawing of 4 6 cells,
each 2 3 cm long. Include only the details you can observe in your preparation.
Label at least TWO structures: cell wall, nucleus or starch grains (if visible).
Students will be graded based on Introduction Exercise 2: Notes on biological
drawings above.

57

Exercise 5
Measurement with a Microscope
Quantitative observations under the microscope making microscopic

measurements
A more exacting type of observation introduces the idea of amount and is called
quantitative. How big? How fast? Measuring instruments such as rulers, balances and
thermometers must be used to extend the range and accuracy of the senses. The
microscope can be used for the dual purpose of measuring very small objects (too small
to be measured with an ordinary ruler) and observing them accurately.
Magnification
When looking through the microscope, it is important to know how much the object being
observed is magnified. To find the degree of magnification, multiply the magnification on
the objective being used by that on the eyepiece. For example, if the magnification of
the eyepiece is 10x, and that of the objective is 40x, then the total magnification is
10x40, or 400x.
Objects viewed under the microscope will be seen in three-dimensions. For a given
object, the distance that allows it to be in focus should thus be investigated using the fine
adjustment knob. This distance is the focal depth of the object being observed. The
focal depth decreases with increasing magnification.
Determining the diameter of the field of view by means of a stage graticule/
micrometer or an eye-piece graticule/ micrometer
A graticule / micrometer is a micro-ruler. It is a piece of plastic or glass slide which is
finely calibrated to 0.1mm or even smaller (0.01mm). It is used for measuring
microscopic structures. There are two types of graticule / micrometer:
1.
Stage graticule/ micrometer

This is placed on the stage over the specimen slide. When viewed under the
microscope, the finely calibrated division marks can be seen clearly and are
magnified according to the magnification of the two lenses used. Since it is more
finely calibrated than a ruler, it more accurately measures the size of a specimen
under the microscope.
2.
Ocular/ Eyepiece graticule/ micrometer

This is placed in the body tube together with the eyepiece lens. It is also used to
measure microscopic structures under the microscope. However, it is magnified
differently from that of an object on the stage. The object on the stage is
magnified by both the objective lens and the eyepiece lens, while the eyepiece
graticule is only magnified by the eyepiece lens. Thus, there is a need to further
calibrate the scale when different objective lens is used.

58
Calibrating the stage graticule/ micrometer

Each graticule/ micrometer, whether it is the stage type or the eyepiece type; must be
calibrated before it can be used.
(i)
Place a stage micrometer on the microscope stage, and using the lowest
magnification (4X), focus on the grid of the stage micrometer.
(ii)
Rotate the ocular micrometer by turning the appropriate eyepiece. Move the
stage until you superimpose the lines of the ocular micrometer upon those of the
stage micrometer. With the lines of the two micrometers coinciding at one end of
the field (align the start (0 mm) of divisions on the stage graticule with the start
(0mm) of divisions on the eyepiece graticule).(Figure 1)
(iii)
Count the spaces of each micrometer to a point at which the lines of the
micrometers coincide again (Figure 2) (i.e. find the division /scale of eyepiece
graticule which is parallel with a particular division/scale of the stage
micrometer).
Since each division of the stage micrometer measures 10 micrometers
(m)/0.01mm, and since you know how many ocular divisions are equivalent to
one stage division, you can now calculate the number of micrometers in each
space of the ocular scale.
*Alternatively: Suppose the full length of the ocular / eyepiece graticule covered
250 divisions of the stage micrometer.
Then the full length of the ocular/eyepiece graticule is equivalent to (250 x
0.01mm) = 2.5mm long.
For an ocular/eyepiece graticule with 100 divisions, each division will measure
25m at the stage for this magnification.
(iv)
Record your calculations as shown below.

(Using the lowest magnification (4X objective / total magnification 40x)
_____number of divisions on eyepiece graticule corresponds to_____divisions of
stage micrometer.
Value for each division of the stage micrometer
= __________mm
= __________m
Thus value/size for each division of eyepiece micrometer = ____________m
(give your answer in m)
(v)
Repeat your measurements for 10x, 40x/60x, and 100 x objectives.
(vi)
Fill in your calibrated values for 1 ocular / eyepiece graticule scale at various
magnifications in Table 1.

59
Figures 1 & 2 illustrate the calibration of the eye-piece/ ocular graticule/

micrometer.
Figure 1
Note: the display of scales of stage and ocular micrometers shown here may be
different from the actual ones provided in your laboratory
60
Figure 2
Table 1 Value of Eyepiece Graticule / Micrometer Division for Various Objective

Magnifications
Objectives magnification
4X
*Calibrated values of eye piece graticule

1 eyepiece graticule unit = ___________ mm
= __________ m
10X
1 eyepiece graticule unit = ___________ mm

= __________ m
40X/60x
1 eyepiece graticule unit = __________ mm

= __________ m
100x
1 eyepiece graticule unit = __________ mm

= __________ m
*Note: Please keep these calculations for your on-site assessments (see below).
Note: all values to have precision up to 4 decimal places for mm and 2 decimal places
for m)

61
Application exercise
Option 1: Mock on-site-assessment
Use any given specimen slide provided which may not be used for the on-site
assessment or assignment this semester (e.g., skin, trachea etc.).
In 2 minutes, identify any three structures of your choice.
You will be assessed according to the Microscope manipulation checklist under
Activity: Manipulation Skill practice task above.
For the final structure, determine the number of eyepiece divisions spanning the size
(e.g., length, breadth, diameter etc.) of the structure.
Multiply by the correct factor according to the corresponding lens (see Table 1: value
of eyepiece graticule above) to obtain the actual size of the structure in micrometers.
Option 2: Biological drawing with scale bar

Name of specimen:
Magnification power:
Draw your specimen and show the measurement/size of the specimen in m using the
scale bar as shown in the figure below (refer to the eyepiece micrometer and convert
the length into m according to the objective lens used):

62
http://www.mun.ca/biology/Help_centre/1001_2_tutorialpages/Measuring_scope.html
http://www.nature.com/nature/journal/v474/n7349/full/nature09974.html

63

Exercise 6
Observation of Starch Grains
1. Place a clean glass slide on the white tile. Place a small piece of potato in the centre
of the slide and rub the piece of the potato in a circular pattern 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. Examine them under low or high power. Make a drawing of 4 6 starch
grains to illustrate their typical shape with any other observable details.
5. After completing your drawings, turn again to the scanning objective. Remove the
slide from the stage and place it on a white tile. Stain the grains with iodine using the
technique of irrigation earlier mentioned.
6. Examine various aspects of the iodine-stained mount first under the scanning
objective and then under low and high power. Note the effects of different iodine
concentration on the starch grains. Draw 4 6 typical starch grains to illustrate their
shape and structure.
1. What observable changes may be seen in the starch grains exposed to relatively
high iodine concentration?
2. What observable differences are there between these starch grains when
compared to those exposed to lower iodine concentration?
3. Can the internal grain structure better observe in strained grains or unstained
ones?
7. 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.
Skills-Based Assessment: Discussion

Biological materials are often stained before examination under a microscope. Based on
your experience in this exercise suggest reasons for such use of stains.

64

Exercise 7
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.
1. While shifting the focus with the fine adjustment, what changes in the image can
be observed? Explain why these changes take place.
2. Does higher-power magnification allows greater detail to be seen?
3. Is the depth of focus as great with higher power as with low power?
4. Is the resolving power increased or decreased when magnification is increased?
Using the procedure outlined in Exercise 2, estimate the width of the hair. State his
measurement in millimeters as well as in micrometers.
4. As an interesting corollary of this exercise you could examine hair from different
members of the class and try to determine differences between fine and coarse hair,
curly and straight hair, and between hair of different shades or colours. The
differences and similarities could be represented by diagrams based on your
observations
5. The exercise in step 4 above can be further extended by using hair from different
animals, e.g. dog, cat, mouse, rabbit, guinea pig, etc.

65
Practical 6 (FHSC 1214 Cell Biology ONLY)

Extraction of Cell Organelles by Differential Centrifugation
___________________________________________________________________
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.

66
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)

67

68
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.

69
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.
70
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.

71
20. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one
15 ml graduated plastic tube.
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).

72

Determination of the Solute potential of the Potato Cell Sap
______________________________________________________________________
Objectives:
To find out the solute potential of potato cell sap as a function of changes in the
concentration of sucrose solution and in the length of potato strips.
To prepare sucrose solutions of various concentrations from a stock solution of 1.0 M by
dilution technique for the determination of the solute potential.
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.).

73
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

74
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.).

75
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. 4
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
4
Pic. and text from Campbell (2002). Chp 36. (6th Ed.). pressure (tension) decreases water potential
76
Essential knowledge for practical: Osmosis in living plant cells

77
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%.

78
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.]
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 (5 per pair)
Ruler
Knife
Forceps
Glass rod
Boiling tube rack

Petri dish
White tile
Graduated glass pipette, 10cm3
Materials:
Sucrose solution, 1.0 M
Distilled water
Potato
Tissue paper
Graph paper
Flowchart
Procedures:
1. Each group should have 35cm3 of sucrose. In labelled boiling tubes, prepare a series
of 20 cm3 of sucrose solutions at the concentrations of 0.1 M, 0.2 M, 0.3 M, 0.4 M,
and 0.5 M from the stock solution using dilution technique. Record the volumes of
solution and of distilled water used in the table below.
You may find this formula helpful:
M = molarity
V = volume
M1V1=M2V2
Existing solution: 1 M (M1) of sucrose
Desired solution: 0.1M (M2) of 20cm3 sucrose
What is the volume of 1 M sucrose in cm3?
(1M) (V1) = (0.1M) (20 cm3)
V1 = ?

79
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)
2. Prepare 15 strips of potato tissue of about equal length (choose any single length
between 4 cm to 6 cm) with a cross-section of about 0.5 cm x 0.5 cm. Using a table,
record the average length of the potato strips.
(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?)
3. Using a ruler and simple eye estimation, measure the initial level of 0.1 M sucrose
solution in the boiling tube before adding the potato strips. Record this in your
manual. Take 3 potato strips, record the length of each strip, and place the strips into
the boiling tube. Repeat these steps using 0.2 M, 0.3M, 0.4 M and 0.5 M sucrose
solutions.
4. After 30 minutes, remove the strips with the forceps provided. Wipe them gently with
tissue paper and record the final length and change in length of each potato strip in
your table.
5. In your manual, record the final level of the sucrose solution in each boiling tube and
record any changes to the physical condition of the potato strips.
6. Calculate the average change in the length of the potato strips and record it in your
table.
7. On a piece of graph paper, 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
8. 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 curve/ line.

80
Assignments
Please check with your tutor which option is required of you.
options below)
Option 2: Skills-Based Assessment: Tabulation of quantitative data
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
4. Adherence to the specifications mentioned in the introduction of this manual.
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 pp6-7 as well. No need to write
procedure, draw table, write a discussion or conclusion.

81
General
Note: Students must relate results with theory (no separate

paragraphs on theory without reference to expected results)
Definition of relevant terms:
a. Osmosis
b. Water potential
c. Solute potential
d. Turgor pressure
Positive change in
length
Zero change in
length
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
Negative change in
length (shortening of
strip)
Comment on:
a. Change in length
Conclusion
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

82

Effects of various treatments on pieces of stained potato tissues
______________________________________________________________________
Objective:
To investigate the effects of various treatments on pieces of stained potato tissues
1 pen-knife or scalpel (if provided)
1 white tile
A pair of forceps
A thermometer (if no incubator used)
A plastic ruler
10 test tubes
3 syringe (5ml)
2 beakers
1 stop watch
1 Potato rod (if provided)

Methylene blue solution
50% ethanol
Flowchart
Procedures:
Unless otherwise instructed by your tutor, you may conduct this experiment in pairs.
1. Use a sharp scalpel, cut four cubes from the potato rod provided, each
approximately 1 cm (breadth) x 1 cm (length) x 2 to 5 mm thick (select one size of
thickness. 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 them in a 50mL size small beaker (or petri dish or specimen tube), 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, pour off the methylene blue solution and wash the cubes with tap
water until the water contains little or no stain. Then cover the cube with tap water.
Why is a coloured stain chosen?

83
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.
5. Place tube A in boiling water or 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 2 minutes, add one stained potato cube to each of the four test tubes. Start the
stop watch immediately.
Explain the significance of the 2 minutes of incubation before adding the tissue 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. [Some guidelines (non-exhaustive): Whats the
texture (i.e., soft/ flaccid, hard/ turgid?) and colour of the tissue? Whats 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?]
Explain the reason for separating the tissue from solutions after adding the tissue
in.
9. Repeat the experiment using fresh potato cubes.

Why repeat the experiment again?

84

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.

85
Assignments
options below)
Option 2: Skills-Based Assessment: Tabulation of qualitative data
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 2 minutes.
7. What is the purpose of repeating the experiments by doing 2 sets?

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Respiration of Germinating Beans
______________________________________________________________________
Objective:
To investigate the aspects of respiration in germinating mung bean seeds.
To understand the concept of respiratory quotient.
Syringe
Gauze
rubber tube
capillary tube
colour dye
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

87
In general, the lower the respiratory quotient, the more oxygen is required for complete
oxidation of a respiratory substrate, and hence the greater the potential for generating
ATP from that respiratory substrate.
The table given illustrates some common values and their significance.
When RQ is
>1.0
Carbohydrates are used as a respiratory substrate with some anaerobic

respiration occurring simultaneously.
1.0
Carbohydrates are used as the respiratory substrate.
0.7
Mainly fats are being used as the respiratory substrate.
0.8 0.9
A mixture of carbohydrates, lipids and proteins are used as respiratory

substrate
0.85
A mixture of carbohydrates and lipids are used as respiratory substrates.
0.9
Proteins are the respiratory substrates. Note that the composition of

proteins is too varied for them to give the same RQ. However, most of
them have a value around 0.9
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 provided with some germinating green bean seeds and a coloured liquid.
2. Place four or five of these 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.

88
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.
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. Wait until the drop of coloured liquid starts to move.
7. Mark the position of the coloured liquid on the capillary tube with a marker pen.
8. Measure how far the liquid moves in one minute. Repeat the measurement every
minute for the next nine minutes.
9. If you do not get any liquid movement after 3 minutes, adjust the apparatus (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.
10. If youve obtained reasonable readings, you may dismantle the apparatus and
dispose the used soda lime (without touching it) in the syringes into the beakers
provided.
11. Record your results also on the whiteboard.

If you are required to prepare an individual report, complete with a write up on
procedures, results and discussion. The results and discussion section will be your
answers to questions a to h. Questions c to h not exceed 2 pgs of your report. A
conclusion is not required.
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
89
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.
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.

90
Assignments
options below)
Tabulate a table on how far the liquid moves in one minute over ten minutes
Present your graph (pasted from Excel). Use a best fit curve. To get full marks, please
observe the guidelines given on pp6-7 as well. No need to write procedure, draw table
and write a discussion or conclusion.

91
Practical 9 Exercise 1 (FHSB 1214 Biology I ONLY)

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.

92
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.

93

94
Acknowledgment:
Student name: Hon Lair Teng
Foundation in Science, CFS-PJ
Intake: Jan 2010
Title: Photo of onion mitosis root tip, allium longitudinal section
Magnification power: 10x.100x

95
Acknowledgment:
Student name: Joanne Liew Hui Qi
Foundation in Science, CFS-PJ
Intake: Jan 2010
Title: Detailed Photos of Onion Mitosis Root Tip, Allium (Longitudinal Section)
Magnification power: 100x.10x
Photo 1
Photo 2

96
Photo 3
Assignment:
Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate
other options below)
Consult your lecturer on which assignment is required.
Students are allowed to make either drawings or photographs.
For either presentation, please ensure the general conventions are followed (please
refer to Notes on Biological Drawings) with at least the following appearing:
Title
Magnification power
Labels, where visible, plus annotations (descriptive): chromosomes, nucleolus,
nuclear envelope, cell wall etc.
Option 2: Photography
1. Take one or more photographs showing any one of the following:
At least three cells each showing any 3 mitotic phases
At least two cells each showing any 2 mitotic phases, for each l.s. and c.s. slide
viewed
2. Label in a Word or Powerpoint file.
3. Upload to the given WBLE link.
4. As part of an on-site assessment, identify for or show your lecturer 2 stages of
mitosis when called on.
Option 3: 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.

97
Option 4: 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)
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
a) Name the structures labelled A, B, C and D.
(4 marks)
b) Name the stages of division shown by cells 1 and 3.
(2 marks)
c) Using the number given to each cell above, arrange the stages as they occur
in the mitotic sequence.
(1 mark)

98

Reproductive Tissues in Plants (Histology of Plant - Lily Reproductive
Structures) [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

99
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

100
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

101
Second division in microsporocytes of a Lilium microsporangium

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

102
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

103
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.
104
Title: Meiotic phases of lily anther cross section

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

105
Title: Meiotic phases of lily anther cross section.


106
Title: Meiotic phases of lily anther cross section.


107
Title: Meiotic phases of lily anther cross section

Magnification: 10x60x

108
Title: Photo of Lily Anther Second Meiotic Division C.S.

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

109

Mitosis and Meiosis Modelling
Objective:
To understand and compare the different events occurring in Mitosis and Meiosis.
Apparatus and Material:
6 pairs of chromosomes
16 triangular genes
16 circular genes
Introduction:
Comparison between Mitosis and Meiosis
Mitosis
Meiosis I
Prophase
Chromatin condense Synapsis and
to form chromosome Crossing over occur
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:
Students are divided into 3 groups to carry out the following activities.
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

110
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.

111

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.

112
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.
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.

113
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.

114
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-airconditioned 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.
Results: (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
Washing up
Please rinse the small fermentation glass tubes within the beaker provided so that
none will slip down the sink hole.

115
Assignments
Please check with your tutor which option is required of you.
Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate
other options below)
Tabulate a table on the difference in height between the initial and final readings for
each of the tubes.
Present your graph (pasted from Excel). DONT use a best fit curve/ line (think about
what type of graph to use). To get full marks, please observe the guidelines given on
pp6-7 as well.
Excluding your cover page, your discussion and conclusion should NOT exceed ONE
A4 page of Word document (standard/ default size). Anything in excess will NOT be
graded.
Margins: 1 inch from top, bottom, left and right (no need to change if youre using
the standard/ default size when MS Word opens).
Theory to apply: Refer to relevant information from lecture topics which may or may
not have been covered yet.
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.

116

Biology 1 Lab Manual 201405

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Table of Contents:

Exercise 1: Writing of Lab Reports

Investigation of Action of Saliva and HCl

Investigation of the Effects of Catalase

Synthesis of Starch Using an Enzyme

Investigation of the Enzymatic Effects of

Exercise 1: The Microscope and Its Uses

Effects of Various Treatments on Pieces

Respiration of Germinating Beans

Exercise 1: Microscopic Examination of

Lab manual version 4_201405

Exercise 1: Mitosis and Meiosis Modelling

Appendix : Case study

Lab manual version 4_201405

E-lab manuals: towards saving the earth

Lab manual version 4_201405

How YOU can do well in BIOLOGY

Based on a true story

ttendance for lectures, tutorials and practicals

Lab manual version 4_201405

Lab manual version 4_201405

Format of a lab report

Lab manual version 4_201405

Your lab report should contain the following sections:

experimental apparatus. Generally, this section attempts to answer the following

KI solution was added to the starch suspension

Amount of light penetrating solution

Lab manual version 4_201405

Colour of precipitate - green, yellow, brick red precipitate

Moderate amount of brick red precipitate suspended in solution, which bore a

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

Lab manual version 4_201405

Precipitate mass (g)

Ave. precipitate mass (g)

Concentration of glucose solution (%)

Mass of eggs laid in a week (g)

2.3 What if I dont obtain desired results?

Lab manual version 4_201405

3.1 General Comments on Style

Every sentence must have a subject and a verb.

Avoid the use of slang and the overuse of contractions.

Lab manual version 4_201405

Be consistent in the use of tense throughout a paragraph--do not switch between

Be sure that pronouns refer to antecedents. For example, in the statement,

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

Lab manual version 4_201405

Lab manual version 4_201405

Example of an annotated drawing

Lab manual version 4_201405

Is the picture below a drawing or diagram?

Lab manual version 4_201405

General Marking Scheme

Lab manual version 4_201405

Checklist for biological drawings

Please read full version for your personal success

A tissue map or plan diagram refers to a

4. Correctly labelled items

6. Drawing is as what is seen

annotated drawings, which are particularly

Lab manual version 4_201405

Actual size of specimen

General guidelines for biological photo(s)

please read full version for your personal success