e-BOOK MANUAL AMALI BIOLOGY

MATRICULATION
DIVISION

BIOLOGY

LABORATORY MANUAL
SEMESTER I & II
SB015 & SB025

TWELFTH EDITION

MATRICULATION DIVISION
MINISTRY OF EDUCATION MALAYSIA

BIOLOGY

LABORATORY MANUAL
SEMESTER I & II
SB015 & SB025

MINISTRY OF EDUCATION MALAYSIA
MATRICULATION PROGRAMME
TWELFTH 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)
Copyright © 2020 Matriculation Division
Ministry of Education Malaysia

ALL RIGHTS RESERVED. No part of this publication may be reproduced
or transmitted in any form or by any means, electronic or mechanical,
including photocopying, recording or any information storage and retrieval
system, without the prior written permission from the Director of
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 : 603-88844083
Fax : 603-88844028
Website : http://www.moe.gov.my/v/BM

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

eISBN 978-983-2604-48-8

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 Malaysia citizens who
are knowledgeable and competent, who posses 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 on 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.

iii

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 skills are crucial to prepare students
to face upcoming challenges in the 21st century era.

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

iv

CONTENTS Page

Foreword iv
Content v
Learning Outcomes vii
Introduction x

SEMESTER I 1
7
Experiment Title 14
1 Basic Techniques In Microscopy 17
2 Plant Tissues 21
3 Transport Across Membrane 29
4 Cell Division – Mitosis
5 Inheritance
6 Basic Techniques in Isolating DNA

v

SEMESTER II

Experiment Title

7 Diversity of Bacteria 32
8 Plant Diversity – Bryophytes and Pteridophytes 38
9 Biocatalysis 48
10 Cellular Respiration
11 Photosynthesis 53
12 Dissection 56
61
References 76
Acknowledgements 77

vi

1.0 Learning Outcomes

1.1 Matriculation Science Programme Educational Objectives

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

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

2. Communicate competently and collaborate effectively in group
work to compete in higher education environment.

3. Solve scientific and mathematical problems innovatively and
creatively.

4. Engage in life-long learning with strong commitment to continue
the acquisition of new knowledge and skills.

1.2 Matriculation Science Programme Learning Outcomes

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

1. Acquire knowledge of science and mathematics fundamental in
higher level education.
(PEO 1, MQF LOD 1)

2. Demonstrate manipulative skills in laboratory work.
(PEO 1, MQF LOD 2)

3. Communicate competently and collaborate effectively in group
work with skills needed for admission in higher education
institutions.
(PEO 2, MQF LOD 5)

4. Apply logical, analytical and critical thinking in scientific studies
and problem solving.
(PEO 3, MQF LOD 6)

5. Independently seek and share information related to science and
mathematics.
(PEO 4, MQF LOD 7)

vii

1.3 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 LOD 1)

2. Conduct biology laboratory work on microscopy, biological
molecules, histology and genetics information by applying
manipulative skills. (P3, PLO 2, MQF LOD 2)

3. Solve problems related to cells, biomolecules, inheritance,
genetics and biological development.
(C4, PLO 4, CTPS3, MQF LOD 6)

1.4 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 LOD 1)

2. Conduct biology laboratory work on diversity of bacteria
and plant, biocatalysis, cellular respiration,
chromatography and dissecting technics by applying
manipulative skills.
(P3, PLO 2, MQF LOD 2)

3. Solve problems related to transport system processes,
mechanisms for adaptations in living things, ecological and
environmental issues in biology.
(C4, PLO 4, CTPS 3, MQF LOD 6)

viii

1.5 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:
• know and practice the necessary safety precautions to be

taken.
• use the correct techniques of handling apparatus.
• plan, understand and carry out the experiment as instructed.
• observe, measure and record data consistency, accuracy and

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

deduce conclusions from the experiments.
• discuss data and information logically and critically.
• analyse and draw conclusions from biological data.
• develop solution to biological problems.
• 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.
• understand the limitations to the accuracy of observations
and measurements.

ix

INTRODUCTION

A. General Guidelines

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.

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.

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.

x

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

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.

xi

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.

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.

B. 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 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.

xii

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 phase-
contrast 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 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.

xiii

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 = Shortest diameter of the observed
structure
= Wavelength ()
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 ()
2 x N.A

where

λ - wavelength of light
N.A - Numerical Aperture

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.

xiv

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
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.

xv

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
microscope. It normally has a converts it to a virtual image to be
magnification power of 10x or 15x. viewed by user’s eyes.

1b. Ocular control knob 1b. To adjust and compensate the
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.

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:
Normally there are 3-4 objective lenses
mounted on the nosepiece, and these can
be rotated and changed as you require.

a. Scanning objective lens (4x): a. Used to scan the specimen before
Coarse specimen field = 5 mm identifying the specific part to be
viewed further.

xvi

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): d. 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 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.

xvii

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
10. Base microscope.

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 low- moving the stage to the specimen.
power objective lens. Rotate this knob
carefully, and observe what happens.
Does the stage or the body tube move?

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.

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.

xviii

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.

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.

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.

xix

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 is still dirty, clean it with a
little amount of xylene and rub it gently with clean, dry lens
tissue.

xx

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.

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.

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.

xxi

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.

Figure 4 A dissecting microscope

xxii

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.

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).

xxiii

BIOLOGY 1
SB015

SB015 Lab Manual

EXPERIMENT 1: BASIC TECHNIQUES IN MICROSCOPY

Course Learning Objective: Conduct biology laboratory work on
microscopy, biological molecules, histology and genetics information by
applying manipulative skills.
(P3, CLO 2, PLO 2, MQF LOD 2)

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

(oil immersion lens)

Student Learning Time (SLT):

Face-to-face Non face-to-face

2 hour 0

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

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

Apparatus

Compound light microscope

Materials

‘e’ prepared slide
Crossthreads prepared slide (3 colours eg: yellow, red and blue)
Transparent ruler (10 mm size) or graph paper prepared slide

Updated: 13/09/2017 1

SB015 Lab Manual

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

The 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.

Do the following exercises to determine the depth of field of
microscope.
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 crossthreads under the microscope using 4x and 10x
objective lens.
3. Determine what happens to the depth of field when the power of
objective lens increases.

Updated: 13/09/2017 2

SB015 Lab Manual

Bottom: Yellow
Middle: Red

Top: Blue

Figure 1.2 :Cross thread prepared slide

1.1.3 The field of view

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 estimate of the field diameter by focusing on the millimetre
scale of a transparent ruler using the lowest power objective. To
calculate the field of view, use this formula:

Diameter of field of view under low High magnification power
magnification power Low magnification power

=
Diameter of field of view under high
magnification power

Do the following exercises to determine the diameter of the field of
view for each of the objective lens on your microscope.
1. Place a transparent ruler on the stage.
2. Observe the transparent rulerusing the 4x, 10x and 40x objective

lenses. (Increase the amount of light by adjusting the control knob
to the maximum).
3. What do you observe using the 4x objective lens? Draw what you
have observed.

Updated: 13/09/2017 3

SB015 Lab Manual

Figure 1.3 : Diameter field of view

The diameter of field of view for the 4x objective lens is mm or m.
Determine the diameter of field of view for the 10x and 40x objective
lens in mm or m.
The diameter of field of view for the 10x = ____ mm = ____ m.
The diameter of field of view for the 40x = ____ mm = ____ m.
4. Using 40x objective lens, determine the size of a cell from a piece

of cork tissue with approximately 20 cells in horizontal position
and 10 cells in vertical position.

Exercise 1.2: Magnification

Procedures and Observation

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

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

2. Calculate the actual magnification in Table 1.1.

Table 1.1 Actual magnification of a specimen

Magnification Actual magnification
power of ocular Magnification power of objective lens

lens 4x 10x 40x 100x

10x

Updated: 13/09/2017 4

SB015 Lab Manual

Exercise 1.3: Oil Immersion Objective Lens

Apparatus

Compound microscope

Materials

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

Procedures and Observation

1. Observe the prepared slide under the microscope.
(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.)
(Refer to the method in Introduction to Microscopy)

Questions

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

between the specimen and the lens?

Updated: 13/09/2017 5

SB015 Lab Manual

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

B) Answer the following questions.

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

2. 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)?

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

C) Complete the following sentences.

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

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

Updated: 13/09/2017 6

SB015 Lab Manual

EXPERIMENT 2: PLANT TISSUES

Course Learning Objective: Conduct biology laboratory work on
microscopy, biological molecules, histology and genetics information by
applying manipulative skills.
(P3, CLO 2, PLO 2, MQF LOD 2)

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

monocots and dicots

Student Learning Time (SLT):

Face-to-face Non face-to-face

2 hour 0

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 inter-
cellular 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, collenchyma and endodermis) and
vascular tissues (xylem and phloem).

Updated: 13/09/2017 7

SB015 Lab Manual

Apparatus

Compound light microscope

Materials

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

Procedures and Observation

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

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

3. Draw the arrangement of the tissues. Note the differences in the
following characteristics: cell size, shape, wall thickness and
stained parts.

4. Select a section and observe it under 40x objective lens. Draw
and label the section showing the different tissues (epidermis,
parenchyma, collenchyma, sclerenchyma, phloem, xylem and
cambium). Use the figures provided to assist you in your tissue
investigation.

Updated: 13/09/2017 8

SB015 Lab Manual

starch
granule
primary
cell wall

intercellular
space

Figure 2.1 Parenchyma cells (c.s)

primary
cell wall

Figure 2.2 Collenchyma cells (c.s)

primary
cell wall
secondary
cell wall

lumen

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

Updated: 13/09/2017 9

SB015 Lab Manual

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)
epidermis

phloem
xylem

parenchyma

Figure 2.6 Cross section of monocot stem
(Source:http://www.phschool.com/science/biology_place/biocoach/pla
nts/images/monstmlb.gif)

Updated: 13/09/2017 10

SB015 Lab Manual

Figure 2.7 Illustrated diagram of cross section of monocot stem
(Source :
https://farm9.staticflickr.com/8504/8426623226_56e9e1f488_o.jpg)

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

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

Updated: 13/09/2017 11

SB015 Lab Manual

Figure 2.9 Cross section of dicotyledon stem
(Source : http://botanystudies.com/wp-content/uploads/2017/03/Cell-
Types-Tissues.jpg)

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

(Source:http:// http://cssmith.co/monocot-root-cross-section-diagram/)

Updated: 13/09/2017 12

SB015 Lab Manual

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

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 cells.

Updated: 13/09/2017 13

SB015 Lab Manual

EXPERIMENT 3: TRANSPORT ACROSS MEMBRANE

Course Learning Objective: Conduct biology laboratory work on
microscopy, biological molecules, histology and genetics information by
applying manipulative skills.
(P3, CLO 2, PLO 2, MQF LOD 2)

Learning Outcomes:
At the end of this lesson, students should be able to:
i. To determine the sucrose concentration which is isotonic to

potato cells
ii. To determine the osmotic pressure of potato cells in

atmospheric unit

Student Learning Time (SLT):

Face-to-face Non face-to-face

2 hour 0

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.

Updated: 13/09/2017 14

SB015 Lab Manual

Apparatus

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

Materials

Distilled water
Filter paper
Fresh potato tuber
Graph paper
Labelling paper
Razor blade
Ruler
Sucrose solutions 1.0 M (40 ml per student)
Tile

Procedures and Observation

Exercise: Osmotic pressure of potato cells

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

Updated: 13/09/2017 15

SB015 Lab Manual

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

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

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

Updated: 13/09/2017 16

SB015 Lab Manual

EXPERIMENT 4: CELL DIVISION - MITOSIS

Course Learning Objective: Conduct biology laboratory work on
microscopy, biological molecules, histology and genetics information by
applying manipulative skills.
(P3, CLO 2, PLO 2, MQF LOD 2)

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

Student Learning Time (SLT):

Face-to-face Non face-to-face

2 hour 0

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.

Apparatus

Compound light microscope
Beakers
Blades
Needle
Prepared slides of various stages of mitosis
Slides and cover slips
Watch glass
Water bath

Updated: 13/09/2017 17

SB015 Lab Manual

Materials

Filter paper
Tissue paper
1M hydrochloric acid (HCl)
Onion root tips (3-4 days old)
Acetic alcohol (3 parts of absolute alcohol:1 part of acetic acid, freshly
prepared)
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.)

Procedures and Observation

Exercise 4.1: Prepared slides of mitosis

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

Exercise 4.2: Preparation of onion root tips slide

1. Rest onion bulb on the rim of a container of water. Leave until
the roots develop for 3 to 4 days.

2. Cut off the root tips 1 - 2 cm long. Put them in a small volume of
acetic alcohol for 10 minutes.

3. Wash root tips in ice cold water for 4 - 5 minutes, then dry them
on filter paper.

4. Transfer root tips to pre-heated 1M HCl at 60°C for 5 minutes.
Repeat step 3.
Caution - they will be very fragile.

5. Transfer two root tips onto a clean microscope slide. Cut each
root tip about 1 mm from the growing tip. Keep the tips, discard
the rest.

6. Tease the root tips with a mounted needle. Add one small drop of
aceto–orcein stain for 2 minutes.

7. Cover with a cover slip, and blot firmly with several layers of
tissue or filter paper and press gently to spread root tips.

8. View under the microscope using 40x objective lens and observe
the chromosome behaviour in the mitotic stages.

Updated: 13/09/2017 18

SB015 Lab Manual

9. Identify, draw and label cells showing the stages of mitosis:
prophase, metaphase, anaphase and telophase.

Step 1 Step 2

Step 3 Step 4

Step 5 Step 6
Updated: 13/09/2017
19

SB015 Lab Manual

Figure 4.2 : Stages of mitosis seen under light compound microscope

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

Questions

1. Why is the root tip placed in acetic alcohol?
2. What is the purpose of using HCl in this experiment?
3. What is the stage in the mitosis that is frequently observed?

Why?
4. Explain the chromosome behavior at each stage in mitosis.
5. Where does mitosis actively take place in plants?

Updated: 13/09/2017 20

SB015 Lab Manual

EXPERIMENT 5: INHERITANCE

Course Learning Objective:
Conduct biology laboratory work on microscopy, biological molecules,
histology and genetics information by applying manipulative skills
(P3, PLO 2, MQF LOD 2)

Learning Outcomes:
At the end of this lesson, students should be able to:
i. To determine the inheritance of genetic traits controlled by

single genes in human
ii. To determine the inheritance of ABO blood groups

Student Learning Time (SLT):

Face-to-face Non face-to-face

2 hour 0

Introduction

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

Procedures and Observation

Exercise 5.1: Inheritance of genetic traits in human

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

2. Below are six inherited characteristics in human:
(i) Shape of nose :
Straight nose (E_) is dominant to curved nose (ee).
(ii) Earlobe :
Free earlobe (P_) is dominant to attached earlobe (pp).
(iii) Tongue Rolling :
Ability of tongue rolling into “U” shape (C_) is dominant
to inability of tongue rolling into “U” shape (cc).

Updated: 13/09/2017 21

SB015 Lab Manual

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

(v) Left-handed :
The right-handed characteristic (H_) is dominant to left-
handed (hh).

(vi) Hitch hiker thumb:
The ability to bend thumb at 60 angle or more are
genotypically(tt) recessive compared to normal thumb-
bending (T_).

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 and expected frequencies for each
of the six characteristics.

Updated: 13/09/2017 22

SB015 Lab Manual

Shape of nose

Curved nose Straight nose

Earlobe

Free earlobe(detached) Attached earlobe
https://www.users.rowan.edu https://www.users.rowan.edu

Tongue rolling

Ability to roll tongue Inability to roll tongue
https://askabiologist.asu.edu https://askabiologist.asu.edu

Updated: 13/09/2017 23

SB015 Lab Manual

Dimple

With dimple Without dimple

https://www.news.makemeheal.com https://www.sharewhy.com

Left-handed

Left handed Right handed
http://www.edquest.ca http://www.edquest.ca

Hitch hiker thumb

Normal thumb Hitch hiker thumb
https://askabiologist.asu.edu https://askabiologist.asu.edu

Figure 5.1 The six inherited characteristics in human

Updated: 13/09/2017 24

SB015 Lab Manual

Results :

Table 5.1 Observed and expected frequencies of each genotype for
six characteristics in the class

Characteristic Phenotype Genotype Tick (√) Observed Expected
your frequency frequency
Shape of nose Straight nose E_ own
Earlobe Curved nose ee of each of each
Free earlobe P_ genotype genotype in genotype in
Tongue Attached pp
Rolling earlobe the class the class
Ability of C_
Dimple tongue rolling
Left-handed into “U” shape cc
Inability of D_
Hitch hiker tongue rolling dd
thumb into “U” shape H_
Have dimple hh
Without T_
dimple
Right-handed tt

Left-handed
Normal
thumb-
bending
Ability to bend
thumb at 60
angle or more

Updated: 13/09/2017 25