BIOLOGY LABORATORY MANUAL

MINISTRY OF EDUCATION
MATRICULATION DIVISION

BIOLOGY

LABORATORY MANUAL

SB015 &
SB025

13th EDITION

MATRICULATION DIVISION
MINISTRY OF EDUCATION MALAYSIA

BIOLOGY

LABORATORY MANUAL
SEMESTER I & II
SB015 & SB025

MINISTRY OF EDUCATION MALAYSIA
MATRICULATION PROGRAMME
THIRTEENTH EDITION

First Printing, 2003
Second Printing, 2004
Third Printing, 2005 (Sixth Edition)
Fourth Printing, 2006 (Seventh Edition)
Fifth Printing, 2007 (Eighth Edition)
Sixth Printing, 2011 (Ninth Edition)
Seventh Printing, 2013 (Tenth Edition)
Eighth Printing, 2018 (Eleventh Edition)
Ninth Printing, 2020 (Twelfth Edition)
Tenth Printing, 2022 (Thirteenth Edition)
Copyright © 2022 Matriculation Division
Ministry of Education Malaysia

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Matriculation Division, Ministry of Education Malaysia.

Published in Malaysia by

Matriculation Division
Ministry of Education Malaysia,
Level 6 – 7, Block E15,
Government Complex Parcel E,
Federal Government Administrative Centre,
62604 Putrajaya,
MALAYSIA.
Tel : 603-8884 4083
Fax : 603-8884 4028
Website : www.moe.gov.my

Printed in Malaysia by

Malaysia National Library
Biology Laboratory Manual
Semester I & II
SB015 & SB025
Thirteenth Edition

e ISBN 978-983-2604-73-0

NATIONAL EDUCATION PHILOSOPHY

Education in Malaysia is an on-going effort towards
further developing the potential of individuals in a
holistic and integrated manner, so as to produce
individuals who are intellectually, spiritually and
physically balanced and harmonious based on a firm
belief in and devotion to God. Such an effort is
designed to produce Malaysian citizens who are
knowledgeable and competent, who possess high
moral standards and who are responsible and capable
of achieving a high level of personal well-being as
well as being able to contribute to the betterment of
the family, society and the nation at large.

NATIONAL SCIENCE EDUCATION
PHILOSOPHY

In consonance with the National Education Philosophy,
science education in Malaysia nurtures a science and
technology culture by focusing on the development of
individuals who are competitive, dynamic, robust and
resilient and able to master scientific knowledge and
technological competency.

ii

FOREWORD

I am delighted to write the foreword for the Laboratory Manual,
which aimed to equip students with knowledge, skills, and the
ability to be competitive undergraduates.

This Laboratory Manual is written in such a way to emphasise
students’ practical skills and their ability to read and understand
instructions, making assumptions, apply learnt skills and react
effectively in a safe environment. Science process skills such as
making accurate observations, taking measurement in correct
manner, using appropriate measuring apparatus, inferring,
hypothesizing, predicting, interpreting data, and controlling
variables are further developed during practical session. The
processes are incorporated to help students to enhance their
Higher Order Thinking Skills such as analytical, critical and
creative thinking skills. These 21st century skills are crucial to
prepare students to succeed in Industrial Revolution (I.R.) 4.0.

The manipulative skills such as handling the instruments, setting
up the apparatus correctly and drawing the diagrams can be
advanced through practical session. The laboratory experiments
are designed to encourage students to have enquiry mind. It
requires students to participate actively in the science process
skills before, during and after the experiment by preparing the pre-
report, making observations, analysing the results and in the
science process skills before, during, after the experiment by
preparing the pre-report, making observations, analysing the
results and drawing conclusions.

It is my hope and expectation that this manual will provide an
effective learning experience and referenced resource for all
students to equip themselves with the skills needed to fulfil the
prerequisite requirements in the first-year undergraduate studies.

DR HAJAH ROSNARIZAH BINTI ABDUL HALIM
Director
Matriculation Division

iii

CONTENTS

1.0 Student Learning Time (SLT) Page
2.0 Learning Outcomes v
3.0 Guidance for Students v
4.0 Introduction to Microscopy viii
x
Semester I
Page
Experiment Title 1
8
1 Basic Techniques of Microscopy 15
2 Plant Tissue 18
3 Transport Across Membrane 23
4 Cell Division - Mitosis 31
5 Inheritance
6 Isolating DNA Page
36
Semester II 42

Experiment Title 51
57
7 Diversity of Bacteria 60
8 Plant Diversity - Bryophytes 66
and Pteridophytes 86
9 Biocatalysis 87
10
11 Cellular Respiration
12
References Chromatography

Mammal Organ System

Acknowledgements

iv

1.0 Student Learning Time (SLT)

Students will be performing the experiment within the time allocated for
each practical work.

Face-to-face Non face-to-face
2 hours 0

2.0 Learning Outcomes

2.1 Matriculation Science Programme Educational Objectives

Upon a year of graduation from the programme, graduates are:

i. Knowledgeable and technically competent in science disciplines
study in-line with higher educational institution requirement.

ii. Able to apply information and use data to solve problems in
science disciplines.

iii. Able to communicate competently and collaborate effectively in
group work to compete in higher education environment.

iv. Able to use basic information technologies and engage in life-long
learning to continue the acquisition of new knowledge and skills.

v. Able to demonstrate leadership skills and practice good values and
ethics in managing organisations.

2.2 Matriculation Science Programme Learning Outcomes

At the end of the programme, students should be able to:

i. Acquire knowledge of science and mathematics as a fundamental
of higher level education.
(MQF LOC i – Knowledge and understanding)

ii. Apply logical, analytical and critical thinking in scientific studies
and problem solving.
(MQF LOC ii – Cognitive skills)

iii. Demonstrate manipulative skills in laboratory works.
(MQF LOC iii a – Practical skills)

v

iv. Collaborate in group work with skills required for higher
education.
(MQF LOC iii b – Interpersonal skills)

v. Deliver ideas, information, problems and solution in verbal and
written communication.
(MQF LOC iii c – Communication skills)

vi. Use basic digital technology to seek and analyse data for
management of information.
(MQF LOC iii d – Digital skills)

vii. Interpret familiar and uncomplicated numerical data to solve
problems.
(MQF LOC iii e – Numeracy skills)

viii.Demonstrate leadership, autonomy and responsibility in managing
organization.
(MQF LOC iii f – Leadership, autonomy and responsibility)

ix. Initiate self-improvement through independent learning.
(MQF LOC iv – Personal and entrepreneurial skills)

x. Practice good values attitude, ethics and accountability in STEM
and professionalism.
(MQF LOC v – Ethics and professionalism)

2.3 Biology 1 Course Learning Outcome

At the end of the course, student should be able to:

1. Explain the basic concepts and theories in cells, biomolecules,
inheritance, genetics and biological development.
(C2, PLO 1, MQF LOC i)

2. Solve problems related to cells, biomolecules, inheritance,
genetics and biological development.
(C4, PLO 2, MQF LOC ii)

3. Conduct biology laboratory work on microscopy, biological
molecules, histology and genetics information by applying
manipulative skills.
(P3, PLO 3, MQF LOC iii a)

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4. Use basic digital and technology applications to prepare a
laboratory report.
(A2, PLO 6, MQF LOC iii d)

2.4 Biology 2 Course Learning Outcome

At the end of the course, student should be able to:
1. Explain the basic concepts and theories in transport system

processes, mechanisms for adaptations in living things,
ecological and environmental issues in biology.
(C2, PLO 1, MQF LOC i)

2. Solve problems related to transport system processes,
mechanisms for adaptations in living things, ecological and
environmental issues in biology.
(C4, PLO 2, MQF LOC ii)

3. Conduct biology laboratory work on diversity of bacteria and
plant, biocatalysis, cellular respiration, chromatography and
dissecting technics by applying manipulative skills.
(P3, PLO 3, MQF LOC iii a)

2.5 Biology Practical Learning Outcomes

Biology experiment is to give the students a better understanding of
the concepts of Biology through experiments. The aims of the
experiments in this course are to be able to:

1. know and practice the necessary safety precautions to be taken.
2. use the correct techniques of handling apparatus.
3. plan, understand and carry out the experiment as instructed.
4. observe, measure and record data consistency, accuracy and units

of the physical quantities.
5. define, analyse data and information in order to evaluate and

deduce conclusions from the experiments.
6. discuss data and information logically and critically.
7. analyse and draw conclusions from biological data.
8. develop solution to biological problems.
9. acquire scientific skills in measuring, recording and analysing

data as well as to determine the uncertainties (error) in various
physical quantities obtained in the experiments.
10. understand the limitations to the accuracy of observations and
measurements.

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3.0 Guidance for Students

3.1 Laboratory Regulation

1. Always wear laboratory coats and covered shoes in the lab.
2. Do not eat or drink in the laboratory.
3. Use the apparatus and materials wisely.
4. Do not throw rubbish and residues into the sink. Wrapped and

throw them into the dustbin provided.
5. At the end of the experiment, students must

a) clean the apparatus using the detergent provided.
b) soak the apparatus in acidic solution containing mild

hydrochloric acid.
c) wash the sink and work station.
d) make sure that all the tables are clean and neat.
e) place the materials and apparatus in their respective places.

3.2 Sectioning and Staining Plant Tissues

1. Sectioning of plant tissues or parts must be made and stained
before they are examined under the microscope.

2. Use sharp blade or microtome to make a thin slice of the
specimen.

3. Clean the blades with water and dry them using tissue paper after
being used.

3.3 Preparation for experiment

1. You are advised to read the manual before carrying out the
experiment. You are also advised to make additional references
about the topic.

2. Prepare a rough layout of the experiment that consists of tables,
graphs and space for drawing.

3. Identify the equipments and materials that are going to be used
in the experiment. This will maximise the time used for
experiment.

4. Follow strictly the instructions in the manual.
5. Record only what you observe in the experiment.

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3.4 Laboratory Report and Evaluation

1. Report should contain the following:-

Title
Objective(s)
Introduction (hypothesis/variable/problem statement)
Procedures (in passive voice, past tense, in reporting style)
Observation (tables, graphs, data, drawing)
Analysis / Discussion regarding tables, graphs, data or
drawings
Conclusion
Questions
References

2. Reports must be handwritten or typed.
3. Diagrams should be drawn on the blank sheet using a 2B

pencil. All diagrams must be labelled.
4. Metric system must be used in writing numerical data.
5. Data can be presented in the form of graphs, tables, flow

charts or diagrams. Give suitable titles to the graph, table,
flow charts and diagrams.
6. Record the following on the front page of the report.

College’s name:
Student’s name:
Matriculation number:
Practicum group:
Title:
Date:
Tutor’s / lecturer’s name:

7. Submit your report to your lecturer at the end of the practical
session.

The report for each lab will be evaluated and included in the
assessment.

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3.5 Scientific Drawing

1. Diagrams drawn must be based on the observation of the
specimen and not copied from books.

2. All parts of the specimens observed must be drawn using the
right scale.

3. An overall drawing or plan drawing must be made to show the
parts where the drawings are made.

4. Show clear orientation of the specimen so that the position and
the relationship with other organs can be determined.

5. Use a sharp 2B pencil to draw thin, clear and continuous lines.
6. Drawing must not be coloured or shaded to differentiate the

systems from tissues. For this purpose, students are allowed to
use various patterns to differentiate systems.
7. Label all your drawings. All labels must be written on the right
and left side of the diagram. Do not write the labels on or in
the diagram. Labels must be written horizontally. Straight line
must be used to connect the structure.
8. Magnification used in the drawing from observation under the
microscope must be mentioned; e.g.: 40x or 100x actual
magnification.

Reminder: NO PLAGIARISM IS ALLOWED.

3.6 Caring for Plants and Animals

1. Water the plants every day. Make sure the soil is damp and
wet.

2. Clean the animal cages every day. Make sure the cages are in
good condition.

3. Feed the animal daily.

4.0 Introduction to Microscopy

The discovery of microscope started a new era in biology since for the first
time man was able to observe cells, the basic units of life.

The optical properties of lenses have been known for the last 300 years B.C. ,
but these knowledge were not used to the fullest until the seventeenth century
when Antonio Van Leeuwenhoek (1632-1732), a Dutch, and his colleagues
discovered a simple workable microscope. With the discovery of the simple
microscope, many people were able to observe minute living organisms in great
details. One of them was Robert Hooke who in 1665 gave the first extensive
description of his experience in observing cork tissue using the simple

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microscope. This marked to the beginning of the study of cells. Below is the
excerpt from the journal Micrographia by Hooke of what he observed from the
cork tissue under the microscope:

“I could exceedingly plainly perceive it to be all perforated and porous…..
these pores, or cells, ….. were indeed the first microscopical pores I ever saw,
and perhaps, that were ever seen, for I had not met with any writer or person,
who has made any mention of them before this”.

Although the description by Hooke about the cork tissue might sound hilarious,
you may have described them in the same way had you lived in the seventeen
century when the concept of cell as the fundamental unit of life was something
unknown.

4.1 What is a Microscope?

Microscopes are precision instruments, and therefore need to be
handled carefully. Many people think that microscopes can only be
used to observe objects in higher magnification. If a microscope can
only be used to observe a magnified image, then its usage is limited.
In fact, microscope can be used to magnify an object, determining the
size of an object and observing fine details of an object, all of which
are not discernible to our naked eyes. Therefore, before one can
properly use a microscope, first he has to be familiar with the
microscope and be able to identify the components of the microscope
and their functions.

With the advancement of technology in microscopy, many high-
quality microscopes have been designed for many specific uses.
Nowadays, many microscopes are of the compound types which use
two sets of lenses. The first set of lens constitutes the objective lens
which supplies the initial real magnified image. The second set of lens
constitutes the ocular lens which magnifies further the image formed
by the first set of lens and converts the real image into virtual image
which is in turn viewed by the user’s eyes. In compound microscopes,
the actual magnification is calculated as the magnification of objective
lens multiplied by the magnification power of the ocular lens.

Today there are many types of light microscope, for example the
phasecontrast microscope that allows user to view living cells or
specimens without the use of stains to increase the contrast. Contrast
is based on the differential absorption of light by parts of the specimen.
There are compound microscopes, which employ ultraviolet light as
the source of light, making it possible to view specimens that emit
fluorescence. Such microscopes are now commonly used in
diagnostics laboratories and research. There are also other compound

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microscopes which use either dark field or light field. Another type of
microscope is compound microscope with inverted objective, called
inverted microscope, which is used to observe living cell cultures.

A microscope is not only capable of producing the image of an object
but also capable of distinguishing between two adjacent points on the
object. This capacity is termed as the resolving power of the lenses or
the resolving power of the microscope. The higher the resolution of
the microscope, the higher is the ability to distinguish details of the
object. Microscope quality depends upon the capacity to resolve, not
magnify, objects. Magnification without resolving power, however, is
not worthless in the field of microscopy.

The resolving power of a light microscope depends upon the
wavelength of light (colour) being used, and not on a value called the
numerical aperture (N. A) of the lens system used. The numerical
aperture is derived from a mathematical expression that relates the light
transferred to the specimen by the condenser to the light received by
the objective lens. This relationship is given by the following
expression:

Resolving power structure = Shortest diameter of the observed

= Wavelength (λ)
Numerical Aperture (N.A)

Thus, the resolving power is increased by reducing the wavelength of
the light used. The shorter the wavelength used, the shorter will the
diameter of the structure being observed, or in other words, the
resolving power is increased. The resolving power cannot be increased
substantially because the light spectrum is narrow (500 nm). However,
we can increase the resolving power by increasing the numerical
aperture in the lens system of the microscope. When the specimen is
illuminated with light from direct or oblique direction, the relationship
is given as follows:

Resolving power = Wavelength (λ)
where 2 x N.A

λ - wavelength of light
N.A - Numerical Aperture

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The condenser located below the mechanical stage or slide holder can
transfer oblique and direct light sources to the specimen and this can
approximately double the numerical aperture (N.A). Thus, the
resolving power can be increased. Therefore, the condenser has to be
properly focused to achieve high resolving power.
Light enters the specimen, and some of it will be refracted as it goes
through the air. This light will not enter the objective lens. By placing
oil of immersion in the space between specimen and objective lens, we
can reduce the light refraction and increase the amount of light entering
the objective lens resulting in a brighter and clearer image. The oil of
immersion used should have the same refractive index (R.I) as the glass
to reduce refraction.
After understanding some principles of microscopy, we need to
identify the components of the microscope and know their functions.
The microscope to be used in the laboratory is the bright field light
compound. The diagram of the microscope is shown in Figure 1.
Familiarise yourself with a microscope and its functions before using
it (refer Table 1).

Figure 1: Compound light microscope

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Each objective lens will generate an image in a specific field of view.
The size (diameter) of the field of view depends on the type of
objective lens used. As the magnifying power of the objective lens
increases, the size of the field of view decreases, and the working
distance, the distance between the slide and the objective lens, also
decreases. When the specimen field of view is wide, more light will
enter the objective lens, so it is important to regulate the amount of
light. Figure 2 shows the relationship between objective lens, fields of
view and working distance for each of the objective lens.

Figure 2: Comparison of working distance at three different
objective magnification
Table 1: Components of microscope and their functions

Component Function

1a. Ocular lens or eyepiece lens: 1a. Magnifies the real image and
This lens is found at the top of the converts it to a virtual image to be
microscope. It normally has a viewed by user’s eyes.
magnification power of 10x or 15x.
1b. To adjust and compensate the
1b. Ocular control knob differences in binocular image of
the eyes.

2. Body tube Body tube is the hollow housing which
supports the ocular lenses at the top and
connects them with the objective lenses
below it.

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3. Rotating nosepiece: The structure to which the objective
You will hear clicking sounds when the lenses are mounted. By gently rotating
objective lens is in its correct position the nosepiece, you may choose the
above the specimen. You may practise objective lens you want and correctly
this by rotating and changing the place it over the specimen.
objective lenses.

4. Objective lenses: a. Used to scan the specimen before
Normally there are 3-4 objective lenses identifying the specific part to be
mounted on the nosepiece, and these viewed further.
can be rotated and changed as you
require.

a. Scanning objective lens (4x):
Coarse specimen field = 5 mm

b. Low-power objective lens (10x): b. Used to view major part of the
Small specimen field = 2 mm specimen.

c. High-power objective lens (40x): c. Used to view specific part of the
Very small specimen field = 0.5 mm specimen.

d. Oil immersion objective lens (100x): Used to view microorganisms such as
Immersion oil is placed in the space bacteria and microstructures in the cells.
between the specimen and objective lens
to reduce the light refraction from the
specimen.

Very small specimen field = 0.2 mm

5a. Stage: a. The horizontal surface on which a
The horizontal surface which has a hole specimen is placed.
in the centre to allow light from below
to focus on the specimen.

5b. Slide clips b. The stage is usually equipped with
slide clips to hold the slide in place.

5c. Slide adjustment knob c. Two knobs are used to move the
slide to the left, right, forward or
backward. Move these knobs to

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learn how the slide is moved into
position.

6a. Condenser: a. Used to focus and deliver light to the
Condenser is located immediately specimen.
under the stage
b. Used to adjust the condenser.
6b. Condenser control knob c. Used to focus the light.

6c. Condenser lens knob

7. Iris diaphragm: Controls the amount of light entering
An adjustable light barrier of iris type and leaving the condenser.
built into condenser. The size of the
diaphragm is controlled by rotating the
knob either to the left or right. Rotate
the knob to the left and to the right and
observe what happens.

8a. Off/on switch. The source of light is a tungsten bulb
located at the base of a microscope.
8b. Light control knob.
Please ensure that either of the switches
is OFF or MINIMUM, respectively
before you use the microscope.

9. Body arm The metal part used to carry a
microscope.

10. Base The heavy cast metal part used as the
base and for support.

11. Coarse adjustment knob: Used to bring specimen into focus by
Use this knob only when using moving the stage to the specimen.
lowpower objective lens. Rotate this
knob carefully, and observe what
happens. Does the stage or the body
tube move?

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12. Fine focus adjustment knob Used to bring specimen into focus while
using high-power or oil immersion
objective lenses.

Now that you have become familiar with the component parts of the
microscope, you can proceed to use the microscope. Check the
microscope to ensure that it is in good working conditions.

4.2 Setting Up of a Light Microscope

a) Plug the microscope to a power source. Before switching on
the plug, check that the light switch is OFF or the light control
knob is set at MINIMUM

b) Switch on the power. Turn on the light control knob or adjust
the light diaphragm to deliver the light to the specimen field
(but not too much light). To focus the condenser, do the
following:
i) Take a prepared slide and place it on the stage.
ii) Rotate the nosepiece and put the coarse objective lens
into position above the specimen.
iii) Move the stage upwards by rotating the coarse
adjustment knob until it stops completely.
iv) While looking through the oculars, move the stage
downwards using the fine adjustment knob until the
specimen is in focus.
v) To focus the condenser, you need to bring the
specimen and the condenser into focus in the same
plane. Close down the iris diaphragm and reduce the
amount of light.

4.3 Focusing a Specimen

a) Place a prepared slide on the stage (for this exercise you may
use any of the prepared slides available in the lab). Move the
slide so that the specimen is placed in the centre and under the
objective lens.

b) First you need to ensure that either the scanning objective lens
(4x) or low-power objective lens (10x) is placed above the
specimen.

c) While looking at the slide from the side, move the stage
upwards until it stops completely. Use the coarse adjustment
knob to do this.

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d) Now observe the specimen through the ocular lens. The
specimen will appear blur because it is still not focused. To
focus the specimen, gently move the stage downwards until
the specimen comes into sharp focus and clear. Use the fine
adjustment knob to do this.

Look through the ocular lens with both eyes. You may see the
image differently between your right and left eyes. Do the
following to adjust the ocular lenses for the differences
between your eyes. Determine which ocular lens is adjustable.
Close the eye over that lens and bring the specimen into sharp
focus for the open eye (right eye). Open the other eye (left
eye) and close the first eye (right eye). If the specimen is still
not in sharp focus, turn the adjustable ocular control knob (1b)
until the specimen is in focus. You may now look with your
eyes through both ocular lenses.

e) After the specimen has been focused by the low-power
objective lens, rotate the nosepiece to change to the high-
power objective lens (40x). You will hear a clicking sound
when the objective lens comes into its correct position right
above the specimen. The microscope used should be of
PARFOCAL type, that is once a specimen has been focused
using a particular objective lens, it will stay focused for the
other objective lenses. Using this microscope, you do not need
to refocus the specimen when you change the objective lens.
You just need to adjust the fine focus adjustment knob.

f) If the field of view is dark or too bright, adjust the amount of
light by using the light control or diaphragm knob.

g) When you have finished using the microscope, rotate the
nosepiece to place the coarse objective lens (4x) back in position over
the centre of the stage. Remove the last slide and clean the stage if
necessary.

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4.4 Using Oil Immersion Objective Lenses

The oil immersion objective lens is used when you want to observe a
specimen at the highest resolution with the light microscope or when
the resolution of other objective lens is not sharp and clear enough. The
objective lens is usually used to observe microorganisms such as
bacteria and protozoa or to observe microorganelles in the cell. Before
using the objective lens, the specimen has to be fixed and stained to
increase its contrast.

a) Follow steps (a) to (d) in procedure (3).
b) Rotate the nosepiece to bring the high-power objective lens

(40x) half way as shown in Figure 3.
c) While holding the nosepiece in this position, apply a single

small droplet of immersion oil to the illuminated spot on the
slide.

Figure 3: Using oil immersion objective lenses

d) Rotate the nosepiece again to move the high-power objective
lens into position until you hear a clicking sound. The
objective lens is now right above the specimen and will be
immersed in the oil.

e) Open up the iris diaphragm to increase the amount of light.
f) While looking at the specimen through the ocular lens, use the

fine adjustment knob until the specimen comes into sharp
focus and become clear. If you have any problems, consult the
instructor / tutor.
g) When you have finished using the oil immersion objective
lens, do the following steps:
i) Carefully move the stage downwards.
ii) Clean the oil immersion objective lens by gently

wiping it with clean lens tissue. If the objective lens

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is still dirty, clean it with a little amount of xylene and
rub it gently with clean, dry lens tissue.
iii) Remove the slide off the stage.
iv) Gently rotate the nosepiece again to place the low-
power objective lens back in position over the centre
of the stage.
v) If oil is found on the stage, wipe the oil off with lens
tissue and with some alcohol.

4.5 Storage of Microscopes

When you have finished using the microscope, do the following to
store the microscope.
a) Check that you have not left a slide on the stage.
b) Check that the stage is clean without any trace of water or dust

on it. If there is any water on the stage, wipe it off with dry
tissue. If it is oil, wipe it off with dry tissue with some alcohol.
c) If you use oil immersion objective lens, gently wipe it with
clean lens tissue.
d) Check that the scanning objective lens (4x) is placed back in
position over the centre of the stage.
e) Turn off the light switch or close down the iris diaphragm to
reduce the amount of light to a minimum and then switch off
the power.
f) Tie up the power chord below the body arm.
g) Ensure the slide clips are placed on the stage and they are not
protruding.
h) HOLD THE MICROSCOPE WITH BOTH HANDS, that is
hold the body arm of the microscope with one hand and the
base of the microscope with the other hand.

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4.6. The Dissecting Microscope (Stereoscopic Microscope)

The dissecting microscope (Figure 4) is used for observations at low
magnification in binocular view (involving 2 ocular lenses) or in three
dimensions. Specimens are often viewed in a fresh state and need not
be placed on a slide. The microscope is ideal for dissection of a small
specimen. The procedures of using a dissecting microscope are
basically similar to the procedures for a light microscope; however, it
is simpler to use than a light microscope.

1. Place a specimen on the specimen plate at the base (5).
2. Illuminate the specimen, by switching on the light source.

a) The eyepiece lenses need to be adjusted to suit your
eyes and to ensure that the image remains clear at
different magnifications. Turn the adjustment knob
(2) to position ‘O’

b) Adjust ocular 1 so that the two oculars fit well with
both eyes.

c) Turn the magnification control knob (4) to select the
magnification to 4x.

d) Look through both ocular lens and focus the specimen
by turning the focus knob (8).

e) Change the magnification to 0.8x by turning the control knob
(4).

f) Observe the image with your right eye and focus using the
adjustment knob located on the right ocular until the image
becomes clear.

The microscope has now been adjusted to suit your eyes so that you
can take advantage of the stereoscopic effect.

3. Look through the oculars with both eyes. Focus the image by
turning the focus knob (8). Specimen as high as 20 mm may
be focused using the adjustment knob.

4. For specimen higher than 20 mm, the microscope may be
focused by moving its body (3) upwards. This is done by
turning the body screw (7) loose and moving the body upwards
or downwards along the stand (6) as far as the stop screw (9).
Tighten the body screw (7) when the body is at the right
position.

5. The stop screw (9) prevents the body of the microscope from
crashing on to the specimen plate at the base.

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Figure 4: A dissecting microscope

4.7. Electron Microscope

This microscope makes use of the electron beams instead of light
source. Electron beams have very short wavelength of approximately
0.005 mm, and therefore theoretically, the microscope can resolve
objects as small as 0.0025 nm in diameter. The resolution of an electron
microscope is usually 1 to 1.2 nm. With electron microscope,
magnifications up to 250,000 are commonly obtained with biological
materials. The shorter wavelengths of electrons are said to have greater
resolving power than those of light microscope. There are two types
of electron microscope, namely the transmission electron microscope
and the scanning electron microscope.

In transmission electron microscope, the electron beams are used
instead of light source. An image will be formed on a photographic
film screen. The microscope uses an electromagnetic lens as a
condenser and the electron source is focused by the condenser lens
through the specimen. The image is then magnified by the objective
lens and the projector lens. An image taken from the electron
microscope is called a transmission electron micrograph. In
transmission electron microscope (TEM), only very thin sections of
specimen of < 30 nm are used for microscopic observation. They are
placed on a copper grid used for support. Electrons cannot be seen
with the human eye, so the image is made visible by shinning the
electrons on to a fluorescent screen. This will only produce black-and-
white pictures. The electron microscope can be used only for dead
tissue materials because they are viewed in vacuum.

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In scanning electron microscope, specimens are coated with a heavy
metal such as gold. Electron beams will not be focused through the
specimen, and when the electron beams collide with the specimen,
some electrons will be absorbed while some are deflected or scattered.
Those parts of the specimen which are denser will absorb more
electrons and will appear darker in the final pictures. Density
differences are due to differences in the contour of the coated surfaces
of the specimen. The image produced will be in three dimensions, and
the pictures are called scanning electron micrograph (SEM).

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BIOLOGY 1
SB015

SB015 Lab Manual

EXPERIMENT 1: BASIC TECHNIQUES OF MICROSCOPY

Objectives:

At the end of this lesson, students should be able to:
i. obtain accurate images;
ii. determine the depth of field;
iii. determine the field of view;
iv. calculate the actual magnification; and
v. apply the use of oil immersion with high magnification

(oil immersion lens)

Before doing the following exercises, you must read and understand the basic
techniques of using a microscope.

Introduction:

Compound light microscope is an instrument that can be use to focus
and magnify small specimen.

Depth of field refers to the thickness of the plane of focus. With a large
depth of field, all of the threads can be in focused at the same time. With a
smaller or narrower depth of field, only one thread or a part of one thread can
be focused, everything else will be out of focus. In order to view the other
threads, you must focus downward to view the ones underneath and upward
view the ones that are above.

Field of view is the observable visible circular scope when looking
through the microscope. The simplest method of estimating linear dimension
is to compare the size of the image to the diameter of the field of view. You
can make a rough estimation of the field of view diameter by focusing on the
millimetre scale of a transparent ruler using the scanning power and low power
objective lens.

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Experiment 1.1: Images, Depth of Fields and Field of View of the
Microscope

Apparatus:

Compound light microscope

Materials:
‘e’ prepared slide
Cross threads prepared slide (3 colours e.g.: yellow, red and blue)
Transparent ruler (10 mm size) or graph paper prepared slide

Procedures and Observation:

1.1.1 Images under the microscope
1. Observe the ‘e’ prepared slide using the 4x objective lens.

2. What do you observe using the 4x objective lens? Draw what you have
observed.

3. Determine the position of ‘e’ (inverted/original position) (Figure 1.1).

Figure 1.1: Letter ‘e’ to be observed under microscope

1.1.2 The depth of field

1. Observe the position of the thread on the slide with your naked eyes.
Identify the colour of thread
a) at the top
b) in the middle
c) at the bottom

2. Observe the cross threads under the microscope using 4x and 10x objective
lens.

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3. Determine what happens to the depth of field when the power of objective
lens increases.

Bottom: Yellow

Middle: Red

Top: Blue

Figure 1.2: Cross thread prepared slide
1.1.3 The field of view
1. Place a transparent ruler on the stage. Make sure the ruler scale is placed

at the centre of the field of view.
2. Observe the transparent ruler using the 4x, 10x and 40x objective lenses.

(Increase the amount of light by adjusting the control knob to the
maximum).
3. Draw your observation at 4x objective lens.

Figure 1.3: Diameter field of view

4. Determine the diameter of field of view at 4x, 10x and 40x objective lens.

5. Use this formula below to calculate the diameter of field of view.

Diameter of field of view under = high magnification power
low magnification power low magnification power

Diameter of field of view under
high magnification power

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6. Record your result in Table 1.1.

Table 1.1 Diameter field of view

Objective lens Diameter in mm Diameter in μm
4x
10x
40x

Experiment 1.2: Magnification

Procedures and Observation:

1. Determine the actual magnification of a specimen by using the formula
below.

Magnification Magnification
power
Actual = power x
magnification of objective lens of ocular lens

2. Calculate the actual magnification in Table 1.2.

Table 1.2 Actual magnification of a specimen

Magnification Actual magnification
power of ocular Magnification power of objective lens

lens 4x 10x 40x 100x

10x

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Experiment 1.3: Oil Immersion Objective Lens

Apparatus:

Compound light microscope

Materials:

Immersion oil
Lens tissue papers
Prepared slide of bacteria
Methylated spirit (only for specific use)

Procedures and Observation:

1. Observe the prepared slide under the microscope using 100x objective
lens.
(Refer to the method in Introduction: to Microscopy: Using Oil
Immersion Objective Lenses)
(Caution: Use immersion oil only for 100x objective lens).

2. Draw your observation.
(Caution: Draw only the bacteria and not artifacts such as air bubbles,
dust, fibre, etc.)

Questions:

Part A

For Questions: 1 to 7, choose the correct answer from the following list:

A Scanning objective lens (4x)
B Low-power objective lens (10x)
C High-power objective lens (40x)
D Oil immersion objective lens (100x)

1. Which is the shortest objective lens?

2. Which objective lens should you use when you begin to focus a
specimen?

3. Which objective lens should be in position before you store a
microscope?

4. Which objective lens will deliver the highest amount of light?

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5. Which objective lens requires immersion oil to fill up the space between
the specimen and the lens?

6. Which objective lens will still remain in focus when placed at the longest
working distance from the specimen?

7. When using an ocular lens with 10x magnification power, which
objective lens should be used to obtain the following actual
magnification?
a. 100 times of its diameter
b. 1000 times of its diameter

Part B

1. Using 40x objective lens, determine the length and width of a cell from
a piece of cork tissue with approximately 20 cells in horizontal position
and 10 cells in vertical position.

Figure 1.4 Cork tissue
(Adapted from http://www.saomarcosdaserra.com/cork.php)

2. Based on laboratory practices, what do you use to clean the microscope
lenses?

3. While observing a moving microorganism under a microscope, you
found that the organism has moved out of the field of view to the right.
In order to keep observing the microorganism, which direction do you
move your slide (right/left)?

4. How do you adjust the slide when the specimen is out of the field of
view to the top?

Part C

1. Complete the following sentences.

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a. A microscope is called a compound microscope when it consists of
more than one set of ……………………………

b. Condenser and iris diaphragm are useful to coordinate
…………………………………………...

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SB015 Lab Manual

EXPERIMENT 2: PLANT TISSUE

Objectives:

At the end of this lesson, students should be able to:
i. identify different types of plant tissues; and
ii. compare the structure and distribution of plant tissue in monocots

and dicots

Introduction:

Plant tissues are divided into two types: meristematic tissues and permanent
tissues.

Meristematic tissues are located in the apical meristem, or zone of active cell
division. The division of cells on apical meristems, located at the tips of the
roots and shoots, cause elongation of the root or the shoot, known as primary
growth. The cells are smaller in size, with large nucleus, thin walls, large
amount of cytoplasm and without intercellular space.

Permanent tissues consist of mature cells that have specialized structure and
functions. Permanent tissues can be divided into three: dermal tissues
(epidermal and peridermal), ground tissues (parenchyma, sclerenchyma and
collenchyma) and vascular tissues (xylem and phloem).

Apparatus:

Compound light microscope

Materials:

Prepared slides of cross sections of dicot stem and root
Prepared slides of cross sections of monocot stem and root

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Procedures and Observation:

1. Examine the prepared slides of cross sections of dicot and monocot
stems and roots focus using the 4x objective lens.

2. Identify the distribution of the tissues (epidermis, parenchyma,
collenchyma, sclerenchyma, phloem, xylem and cambium).

3. Draw arrangement of the tissues observed.

4. Select a section and observe it under 10x or 40x objective lens. Draw
and label a part of the tissue showing different type of tissues (epidermis,
parenchyma, collenchyma, sclerenchyma, phloem, xylem and
cambium). (Note: the differences in the following characteristics: cell
size, shape, wall thickness and stained parts). Use the figures provided
to assist you in your tissue investigation.

primary
cell wall

intercellular
space

Figure 2.1: Parenchyma tissue (c.s.)
Adapted from https://image.slidesharecdn.com/groundtissue-
170907142850/95/ground-tissue-5-638.jpg?cb=1504795152

primary
cell wall

cytoplasm

Figure 2.2: Collenchyma tissue (c.s.)
Adapted from gopher://wiscinfo.wisc.edu:2070/I9/.image/
.bot/.130/Cells_and_Tissues/Medicago_Stem/Collenchyma.

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primary
cell wall
secondary
cell wall

lumen

Figure 2.3: Sclerenchyma tissue (c.s.)
http://en.wikipedia.org/wiki/File:Plant_cell_type_sclerenchyma_fibers.png

tracheid

vessel
element

Figure 2.4: Structure of xylem vessel element and tracheid (l.s.)

sieve
tube

companion
cell

sieve
plate
Figure 2.5: Structure of phloem sieve tube and companion cell (l.s.)

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Figure 2.6: Cross section of monocotyledon stem
(Source:http://www.phschool.com/science/biology_place/biocoach/plants/im

ages/monstmlb.gif)

Figure 2.7: Structure and distribution of vascular bundles in cross section of
monocotyledon stem

(Source: https://i.pinimg.com/564x/dd/84/d3/dd84d3e61465aa559f0c
42c62fd497e3.jpg)

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Figure 2.8: Structure and distribution of vascular bundles in cross section of
dicotyledon stem

(Source:http://3.bp.blogspot.com/Pr_pAO5SlOo/UVKowiOM9II/AAAAAAAA
FVs/9OctyttLZy8/w1200-h630-p-k-no-nu/Dicot+stem.jpg)

Figure 2.9: Illustrated diagram of cross section of dicotyledon stem
(Source: http://botanystudies.com/wp-content/uploads/2017/03/Cell-Types-

Tissues.jpg)

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Figure 2.10: Structure and distribution of vascular bundles in cross section
of monocotyledon root

(Source: https://i.pinimg.com/564x/3f/e3/50/3fe3508d08430
dfccfd8656d4ba81c96.jpg)

epidermis
cortex

phloem
xylem

Figure 2.11: Structure and distribution of vascular bundles in cross section
of dicotyledon root

(Source: https://biology4isc.weebly.com/uploads/9/0/8/0/
9080078/439610706.jpg)

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Questions:

1. You are given two slides of cross sections of Angiosperms stems and
roots. How would you differentiate between the following?
a. the stems of monocot and dicot plants
b. the roots of monocot and dicot plants

2. Explain the structures and functions of parenchyma, collenchyma and
sclerenchyma.

3. Explain the structures and functions of xylem and phloem.

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SB015 Lab Manual

EXPERIMENT 3: TRANSPORT ACROSS MEMBRANE

Objectives:

At the end of this lesson, students should be able to:
i. determine the sucrose concentration which is isotonic to potato cells;

and
ii. determine the osmotic pressure of potato cells in atmospheric unit.

Introduction:

The cell membrane is a selective permeable structure because only selected
Materials: can pass through it. Water molecules can easily pass through the
membrane and the movement of water is called osmosis. The direction of
movement of water molecules is determined by the concentration of the solutes
of both sides of the membrane. The water potential inside and outside of the
cell is said to be isotonic, that is the movement of water molecules in both
direction is at the same rate. The vacuolar membrane is also a selective
structure and the condition in the vacuole is isotonic to the cell environment.

In a hypertonic environment, water molecules will move out of the cell and
the cell shrinks. The shrinking of cell is due to the hypertonic environment
outside the plant and animal cells. The shrinking of plant cell is called
plasmolysis while the shrinking of animal cell is called crenation.

When a plant cell is in a hypotonic environment, it will expand but the increase
in size is restricted by the cell wall (turgid). On the other hand, animal cells
which are in the hypotonic environment will expand and burst and this is called
lysis or haemolysis in red blood cell.

Apparatus:

Boiling tube
Beaker
Cork borer
Electronic balance
Forceps
Measuring cylinder (25 ml)
Petri dish and Pipette (10 ml)

Materials:

Distilled water 15
Filter paper
Fresh potato tuber
Graph paper

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SB015 Lab Manual

Labelling paper
Razor blade
Ruler
Sucrose solutions 1.0 M (40 ml per student)
Tile

Procedures and Observation:

1. Prepare 20 ml of sucrose solution with different molarities in boiling
tubes using the dilution method. The molarities required are 0.1 M, 0.2
M, 0.3 M, 0.4 M and 0.5 M. Record in Table 3.1 the volumes of sucrose
solution (1.0 M) and the distilled water used in preparing the sucrose
solutions.

Table 3.1 Determination of molarities of sucrose solutions using dilution
method

Final molarity of the sucrose solutions
0.1 M 0.2 M 0.3 M 0.4 M 0.5 M

Volume of 1.0 M
sucrose (ml)

Volume of distilled
water (ml)

2. Prepare 15 pieces of potato strips using cork borer to have 3 replicates
for each concentration. The length of each strip is 4 cm.

3. For every concentration, take 3 potato strips, record their average weight
in a table.

4. Put all potato strips into the boiling tubes containing different sucrose
concentrations.

5. After 30 minutes, remove the three strips from the boiling tube, wipe
with filter paper for a second and immediately record their average
weight.

6. Based on your results, draw a graph to show the changes in weight of
the potato strips against the molarities of the sucrose solutions.

7. From the graph obtained in step 6, determine the sucrose concentration
which is isotonic to potato cells.

8. Next, draw a standard graph of osmotic pressure against the molarity of
sucrose solution based on the values given in Table 3.2.

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Table 3.2 Values for constructing a standard graph

Molarity 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
(M)

Osmotic

pressure 1.3 2.6 4.0 5.3 6.7 8.1 9.6 11.1 12.6 14.3 16.0

(atm)

9. From the graph obtained in step 8, determine the osmotic pressure of
potato cells in atmospheric unit (atm).

Questions:

1. Identify which molarity of sucrose gives the highest weight of
potato strips? Explain your answer.

2. From the experiment, what is the molarity of sucrose that is equal to
the internal concentration of potato cell? Explain your answer in terms
of water potential.

Extension (Do-It-Yourself)

Have you ever wondered if we could revive wilted vegetable such as salad or
celery stalk? By using the concept of osmosis try to design an experiment how
you can revive wilted veggies. Good luck!

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SB015 Lab Manual

EXPERIMENT 4: CELL DIVISION – MITOSIS

Objectives:

At the end of this lesson, students should be able to:
i. prepare onion root tip slides; and
ii. identify stages in mitosis.

Introduction:

In most tissues, new cells are formed as a result of mitosis. If the chromosomes
of such cells are selectively stained with a dye such as aceto-orcein, stages in
mitosis can be observed. An example of a tissue that undergoes mitosis is the
meristematic tissue. This tissue is located in the cell division zone of the apical
meristem at the root tip and shoot apex.

Experiment 4.1: Prepared slides of mitosis

Apparatus:

Compound light microscope

Materials:

Prepared slide of stage of mitosis

Procedures and Observation:

1. Examine the prepared slides of various stages of mitosis.
2. Draw and label the stages of mitosis observed.

Experiment 4.2: Preparation of onion root tips slide

Apparatus:

Beakers
Blades
Compound light microscope
Hot plate/ Bunsen burner/ spirit lamp
Needle
Prepared slides of various stages of mitosis
Pipette/ Dropper
Slides and cover slips
Watch glass

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Materials:

Aceto–orcein (Freshly prepared. Heat 45 ml 70% acetic acid and when the acid
is hot, add 2g of orcein. Allow the solution to cool and dilute with 55 ml of
distilled water. Filter the solution prior to use.)
1 M hydrochloric acid (HCl)
Filter paper
Onion root tips (3-4 days old)
Tissue paper

Safety Precaution:

1. Be careful while using blade or scalpel.

2. Handle acidic and stain solution with care.

3. Use forceps or wooden thong to hold the heated slide/ watch glass.

Procedures and Observation:

1. Using a blade or scalpel, carefully cut 5 mm from the apical tip of five
lateral onion roots.

2. Place the root tips in a watch glass containing of one drop of
1 M hydrochloric acid and 10 drops of aceto-orcein stain.
(Warning! Aceto-orcein is an acidic stain that turns protein purple.
Any stain that gets on skin or natural clothing will stain the material
purple).

3. Warm the prepared watch glass in step 2 for 5 minutes on a hot plate
at medium heat (50oC) to macerate the tissues but DO NOT allow the
solution to boil. Alternatively, gently heat the preparation by moving
the watch glass over the flame above 30 seconds and let it cool down
for 5 minutes.

4. Place the root tip onto a clean slide.

5. Using a sharp knife, cut transversely 1-2 mm of the apex of the onion
root tip. Be careful not to touch the root tip.

6. Using a pipette, carefully add two or three drops of aceto-orcein stain
to the root tip on the slide.

7. Carefully lower a cover slip over the root tip.

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8. Use a mounted needle or similar gently break the tissue of the root tip
apart.

9. By using a filter paper or paper towel, gently press the cover slip down
onto the slide so that the root tip is squashed but do not twist the cover
slip.

10. Gently blot any excess stain from the slide.

11. Warm the slide on a hot plate at medium heat (50oC) for about 10
seconds to intensify the staining. Alternatively, gently heat the slide
for 10 seconds over the heat.
(Caution: The slide should be very warm but not too hot to touch).

12. Examine the slide using a microscope using 40x or 100x objective lens
and observe for the different stages in mitosis.

13. Draw the stages of mitosis that you observed.

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Onion SB015 Lab Manual
root tip Step 2

Step 1

Step 3 Step 4

Step 5 Step 6

Figure 4.1 Steps in preparing onion root tip slide
(Source: https://www.youtube.com/watch?v=K2_FZRdjQH4)

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Figure 4.2 Stages of mitosis seen under light compound microscope
(Source:

https://fthmb.tqn.com/uWS8dstnohIso29NAme1H0iugYg=/768x0/filters:no_u
pscale()/139812087-56a2b3cd3df78cf77278f2cb.jpg)

Questions:

1. What is the purpose of using HCl in this experiment?

2. What is the stage in the mitosis that is frequently observed? Why?

3. Explain the chromosome behaviour at each stage in mitosis.

4. Where does mitosis actively take place in plants?

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SB015 Lab Manual

EXPERIMENT 5: INHERITANCE

Objectives:

At the end of this lesson, students should be able to:
i. determine the inheritance of genetic traits controlled by single genes in

human; and
ii. determine the inheritance of ABO blood groups.

Introduction:

A number of human characteristics are determined by single genes. These
characteristics include the earlobe, the ability of tongue rolling, the presence
of dimple, hairline shape, chin shape and thumb bending (Figure 5.1). A single
gene also determines pigmentation of iris and the ability to taste
phenylthiocarbamide (PTC).

Experiment 5.1: Inheritance of genetic traits in human
Procedures and Observation:

1. The exercise will require information to be gathered from every
student in the class.

2. Below are six inherited characteristics in human:
(i) Earlobe:
Free earlobe (P_) is dominant to attached earlobe (pp).

(ii) Tongue Rolling:
Ability of tongue rolling into “U” shape (C_) is dominant to
inability of tongue rolling into “U” shape (cc).

(iii) Dimple:
Individual with dimple is genotypically (D_) dominant
compared to those without dimple (dd).

(iv) Hairline shape:
The widow’s peak hairline (H_) is dominant to straight hairline

(hh).

(v) Chin shape:
The cleft chin is (S_) is dominant to smooth chin (ss).

(vi) Thumb bending:
The normal thumb-bending (T_) is dominant to ability to bend
thumb at 60° angle or more (tt).

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3. Based on the above characteristics, fill in the information below in
Table 5.1.
(i) Determine your genotype for each of the six characteristics.
(ii) Calculate the observed phenotype frequencies for each of the six
characteristics.

Earlobe

Free earlobe(detached) Attached earlobe
https://upload.wikimedia.org https://www.popsci.com

Tongue rolling

Ability to roll tongue Inability to roll tongue
https://www.gannett-cdn.com https://ctsciencecenter.org

Dimple

With dimple Without dimple
https://www.news.makemeheal.com https://www.sharewhy.com

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