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MATRICULATION DIVISION

MINISTRY OF EDUCATION MALAYSIA

BIOLOGY LABORATORY MANUAL

SEMESTER I & II

SB015 & SB025

MINISTRY OF EDUCATION MALAYSIA

MATRICULATION PROGRAMME

ELEVENTH EDITION

2

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)

Copyright © 2017 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/bmkpm

Printed in Malaysia by

Malaysia National Library Cataloguing-in-Publication Data

Biology Laboratory Manual

Semester I & II

SB015 & SB025

Eleventh Edition

ISBN 978-983-2772-81-1

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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.

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FOREWORD

I am delighted to write the foreword for the third edition of the Ministry

of Education, Two-Year Matriculation Programme Laboratory Manual.

The first edition was published in 2011 which aimed to equip students

with knowledge, skills, and the ability to be competitive undergraduates.

Now, seven years later, the Laboratory Manual has been carefully updated

to be in line with the outcome-based education (OBE) which is an

educational process focused on achieving certain specified outcomes in

terms of individual learning. This means starting with a clear picture of

what is important for all students to be able to do, then organizing

curriculum (outcome), instruction (activity) and assessment to make sure

learning ultimately happens (Spady, 1994:1).

The third edition reflects the guidelines, standards and codes of practice

developed by Malaysian Qualifications Agency (MQA) which inculcate a

strong culture of OBE good practices that translated into the eight

domains of learning outcomes (Malaysian Qualifications Framework,

MQF) in which the Matriculation Programme focuses on five domains.

This Laboratory Manual is written in such a way to emphasize students’

practical skills (MQF LOD 2) 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. Such 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.

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

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students to have enquiry mind. It requires the learners 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 drawing conclusions. Students are encouraged to apply the

findings to new situation. The experiments in the manual are carefully

scheduled in accordance to the syllabus specification. The learning

objectives are clearly stated at the beginning of each experiment as

guidance. Hence, involving students in the science process skills through

investigation is amongst the best way of teaching.

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. BAIDURIAH BINTI YAAKUB Director


Ministry of Education

April 2018

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CONTENTS

Page

Foreword i

Content iii

Learning Outcomes v

Introduction

viii

SEMESTER I

Experiment Title

1 Basic Techniques In Microscopy

1

2 Plant Tissues

7

3 Transport Across Membrane

14

4 Cell Division – Mitosis

17

5 Inheritance

21

6 Basic Techniques in Isolating DNA

29

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SEMESTER II

Experiment Title

7 Diversity of Bacteria

32

8 Plant Diversity – Bryophytes and Pteridophytes 38

9 Biocatalysis

48

10 Cellular Respiration

54

11 Photosynthesis

57

12 Dissection

62

References 76

Acknowledgements 77

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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)

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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)

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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.

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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.

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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.

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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.

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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.

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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.

xiii

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.

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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:

This lens is found at the top of the

microscope. It normally has a

magnification power of 10x or 15x.

1b. Ocular control knob

1a. Magnifies the real image and

converts it to a virtual image to be

viewed by user’s eyes.

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:

You will hear clicking sounds when the

objective lens is in its correct position

above the specimen. You may practise

this by rotating and changing the

objective lenses.

The structure to which the objective

lenses are mounted. By gently rotating

the nosepiece, you may choose the

objective lens you want and correctly

place it over the specimen.

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):

Coarse specimen field = 5 mm

a. Used to scan the specimen before

identifying the specific part to be

viewed further.

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b. Low-power objective lens (10x):

Small specimen field = 2 mm

c. High-power objective lens (40x):

Very small specimen field = 0.5 mm

d. Oil immersion objective lens (100x):

Immersion oil is placed in the space

between the specimen and objective lens

to reduce the light refraction from the

specimen.

Very small specimen field = 0.2 mm

b. Used to view major part of the

specimen.

c. Used to view specific part of the

specimen.

d. Used to view microorganisms such as

bacteria and microstructures in the cells.

5a. Stage:

The horizontal surface which has a hole

in the centre to allow light from below

to focus on the specimen.

5b. Slide clips

5c. Slide adjustment knob

a. The horizontal surface on which a

specimen is placed.

b. The stage is usually equipped with

slide clips to hold the slide in place.

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:

Condenser is located immediately

under the stage

6b. Condenser control knob

6c. Condenser lens knob

a. Used to focus and deliver light to the

specimen.

b. Used to adjust the condenser.

c. Used to focus the light.

7. Iris diaphragm:

An adjustable light barrier of iris type

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.

Controls the amount of light entering

and leaving the condenser.

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8a. Off/on switch. 8b. Light control knob.

Please ensure that either of the switches

is OFF or MINIMUM, respectively

before you use the microscope.

The source of light is a tungsten bulb

located at the base of a 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: Use this knob only when using low-

power objective lens. Rotate this knob

carefully, and observe what happens.

Does the stage or the body tube move?

Used to bring specimen into focus by

moving the stage to the specimen.

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.

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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.

xviii

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.

xix

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.

xx

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

xxi

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).

BIOLOGY 1

SB015

1

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

2

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.

3

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:

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.

Diameter of field of view under low

magnification power

Diameter of field of view under high

magnification power

High magnification power

Low magnification power =

Bottom: Yellow

Middle: Red

Top: Blue

4

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


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

formula below.

Actual magnification =

2. Calculate the actual magnification in Table 1.1.

Table 1.1 Actual magnification of a specimen

Actual magnification

Magnification

power of ocular

lens

Magnification power of objective lens

4x 10x 40x 100x

10x

Magnification power

of objective lens

Magnification

power

of ocular lens

x

a

g

n

i

f

i

c

a

t

i

o

n

p

o

w

e

r

5

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)


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?

6

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…………………………………………...

7

EXPERIMENT 2: PLANT TISSUES






i. To identify different types of plant tissues

ii. To compare the structure and distribution of tissues in

monocots and dicots



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).

8

Apparatus


Materials

Prepared slides of cross sections of monocot stem and root

Prepared slides of cross sections of dicot stem and root


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.

9

Figure 2.1 Parenchyma cells (c.s)

Figure 2.2 Collenchyma cells (c.s)

Figure 2.3 Sclerenchyma cells (c.s)

(Source:

http://en.wikipedia.org/wiki/File:Plant_cell_type_sclerenchyma_fibers.png)

starch

granule

primary

cell wall

intercellular

space

primary

cell wall

primary

cell wall

secondary

cell wall

lumen

http://en.wikipedia.org/wiki/File:Plant_cell_type_sclerenchyma_fibers.png

10

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

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

Figure 2.6 Cross section of monocot stem

(Source:http://www.phschool.com/science/biology_place/biocoach/plants/images/monstmlb.gif)

tracheid

vessel

element

sieve

tube

companion

cell

sieve

plate

epidermis

phloem

parenchyma

xylem

11

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/AAAAAAAAFVs/9OctyttLZy8/w1200-

h630-p-k-no-nu/Dicot+stem.jpg

12

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/)

13

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.

14

EXPERIMENT 3: TRANSPORT ACROSS MEMBRANE






i. To determine the sucrose concentration which is isotonic to

potato cells

ii. To determine the osmotic pressure of potato cells in

atmospheric unit



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.

15

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


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)

16

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

(M)

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55

Osmotic

pressure

(atm)

1.3 2.6 4.0 5.3 6.7 8.1 9.6 11.1 12.6 14.3 16.0

9. From the graph obtained in step 8, determine the osmotic

pressure of potato cells in atmospheric unit (atm).

17

EXPERIMENT 4: CELL DIVISION - MITOSIS






i. To prepare onion root tip slides

ii. To identify stages in mitosis



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


Beakers

Blades

Needle

Prepared slides of various stages of mitosis

Slides and cover slips

Watch glass

Water bath

18

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.)


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.

http://www-saps.plantsci.cam.ac.uk/worksheets/ssheets/ssheet17.htm#acetic

19

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

20

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?

21

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)


i. To determine the inheritance of genetic traits controlled by

single genes in human

ii. To determine the inheritance of ABO blood groups



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).


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).

22

(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.

23

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 https://askabiologist.asu.edu

Inability to roll tongue

https://askabiologist.asu.edu

24

Dimple

With dimple

https://www.news.makemeheal.com

Without dimple

https://www.sharewhy.com

Left-handed

Left handed Right handed

http://www.edquest.ca http://www.edquest.ca

Hitch hiker thumb

Normal thumb https://askabiologist.asu.edu

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

Figure 5.1 The six inherited characteristics in human

25

Results :

Table 5.1 Observed and expected frequencies of each genotype for six

characteristics in the class

Characteristic Phenotype Genotype

Tick (√)

your

own

genotype

Observed

frequency

of each

genotype in

the class

Expected

frequency

of each

genotype in

the class

Shape of nose Straight nose E_

Curved nose ee

Earlobe

Free earlobe P_

Attached

earlobe pp

Tongue

Rolling

Ability of

tongue rolling

into “U” shape

C_

Inability of

tongue rolling

into “U” shape

cc

Dimple

Have dimple D_

Without

dimple dd

Left-handed Right-handed H_

Left-handed hh

Hitch hiker

thumb

Normal

thumb-

bending

T_

Ability to bend

thumb at 60

angle or more

tt

26

Questions

1. Individuals with certain heterozygous characteristics are usually

called a carrier. What does a carrier mean?

2. A student inherited left handedness from parents who are both

right handed. Explain the pattern of inheritance.

3. What is the expected frequency for a person having tongue

rolling ability and attached earlobe?

4. What is the expected frequency for a person to have all six

recessive characteristics?

Exercise 5.2: ABO blood group inheritance

ABO blood groups in human are examples of multiple alleles of a

single gene and also codominant alleles. Each individual inherited any

one of four blood types, i.e. A, B, AB or O. Type A groups are

determined by the presence of antigen A found on the surface of red

blood cells (erythrocytes), while the blood plasma contains B antibody

which agglutinates type B blood. Individuals with type B blood have

antigen B and antibody A which agglutinates type A blood. Individuals

with type AB blood have both antigen A and antigen B but without

antibodies A or B. Finally, individuals with type O blood have

antibody A and antibody B but without any antigen. Table 5.2 shows

individual characteristics for all ABO blood groups.

Table 5.2Individual characteristics for all ABO blood groups

B

lood

group

(phenotypes)

Antigen

present

on

erythrocytes

Antibodies present

in blood plasma

(serum)

Agglutinated

blood group

A A Anti-B B

B B Anti-A A

AB A and B none none

O none Anti-A and

Anti-B A and B

27

Apparatus

Depression slide/Pallet

Lancing device

Materials

Anti-A and Anti-B serum/ Blood test kit

Alcohol swab

Sterilized lancet

Toothpicks


1. Label two clean and dry slides/pallet (no. 1 and 2).

2. Wash your hands with soap and let them dry.

3. Swing your hand for 10 – 15 seconds.

(Caution: Do not use the same lancet twice or exposed lancet)

4. Apply alcohol to your middle finger. Prick the tip of the middle

finger using sterilized lancet.

5. Wipe off the first blood drop.

6. Place the next drop at the center of slide 1 and 2.

7. Drop an Anti-A serum near the blood on slide 1 and Anti-B

serum on slide 2.

8. Mix the blood and serum on slide 1 with a toothpick. Use another

toothpick for slide 2.

9. You belong to A blood group if agglutination occurs on slide 1

only; B blood group if agglutination is observed on slide 2 only;

AB blood group if agglutination occurs on both slides 1 and 2; O

blood group if no agglutination is seen on both slides.

10. Calculate the frequency of each blood group in the class. Record

your observation in Table 5.3.

Your blood group:

28

Table 5.3Frequency of blood group in the class

Blood group Possible genotypes Frequency of each

blood group

A

B

AB

O

Questions

1. Why do you swing your hand for 10 to15 seconds before

pricking the tip of your middle finger?

2. Why can’t you use the same lancet twice?

3. Why do you need to wipe off the first blood drop?

4. Why do you need different toothpicks to mix the blood and

serum on slides 1 and 2?

5. Can an individual with O blood group donates his blood to an A

blood group person? Give reason to your answer.

6. A mother with O blood group gave birth to a baby girl having the

same blood group. However, she is not convinced that the baby

belongs to her because her husband has AB blood group. She

claimed there might be swapping of babies in the nursery.

Explain.

29

EXPERIMENT 6: BASIC TECHNIQUES IN ISOLATING DNA






i. To isolate DNA from plant tissue.



2 hour 0

Introduction

Each chromosome is a single thread-like structure made up of long

molecules of DNA combined with histone protein. The DNA molecule

is made up of many small sections called genes. Shortly before cell

division occurs, each DNA molecule replicates itself. So one thread of

the chromosome becomes two identical chromatids. As the two

chromatids are identical, they will have identical genes. These identical

genes are known as allele. In this experiment, you will rupture fruit

cells, thus releasing their contents such as protein, DNA, RNA, lipids,

ribosomes and various small molecules. DNA is then suspended by

alcohol as supernatant layer.

The purity of DNA will require further steps. After the isolation of

nucleic acids, the solution is still contaminated with proteins which can

be removed. To check the success of the removal, a purity

determination is performed, which is based on the different absorption

characteristics of the proteins and the nucleic acids using UV

spectrophotometer.

30

Apparatus

Mortar and pestle

500 ml beaker

Muslin cloth

Boiling tube

Boiling tube rack

Water bath (60 °C)

Materials

Kiwi /banana/onion/tomato/watermelon

Ice-chilled 95% alcohol

Ice cubes

50.000g sodium dodecyl sulfate or sodium lauryl sulfate (SDS or SLS)

8.770g sodium chloride

4.410g sodium citrate

0.292g ethylenediaminetetraacetic acid (EDTA)

1 liter water


Exercise: Isolation of crude DNA.

1. Prepare the salt-detergent solution. Stir gently to completely

dissolve the salt without producing foam.

2. Pour 10 ml of ice-chilled alcohol into a boiling tube and place it

into a beaker containing ice cubes. (Remarks: place the ethanol

in the freezer overnight)

3. Peel, slice and mash kiwi/onion/tomato/banana/watermelon with

mortar.

4. Transfer mashed fruit into a beaker and add 100 ml of the salt-

detergent solution. Incubate the mixture in the water bath for 15

minutes.

5. After 15 minutes, sieve the mixture with muslin cloth and collect

the liquid in a beaker.

6. Fill in half of the boiling tube with sieved liquid.

salt-

detergent

solution

31

7. Very carefully pour 10ml of ice-chilled alcohol into the side of

the boiling tube (at flat angle). (Remark: make sure both liquid

do not mix and alcohol form a separate layer on top of the sieved

liquid)

8. Put the boiling tube into a rack and observe it. Observe the

extracted DNA between alcohol and the sieved liquid. Crude

DNA should be found in between the alcohol and sieved liquid.

Questions

1. What is the purpose of using the following?

(a) salt-detergent solution

(b) ice chilled alcohol

(c) water bath

2. Why do we need to mash the fruits?

BIOLOGY 2

SB025

32

EXPERIMENT 7: DIVERSITY OF BACTERIA


diversity of bacteria and plant, biocatalysis, cellular respiration,

chromatography and dissecting technics by applying manipulative skills.



i. To demonstrate Gram staining technique in classifying bacteria

ii. To identify Gram-positive and Gram-negative bacteria

iii. To identify different shapes of bacteria



2 hour 0

Introduction

Gram stain is a widely used method of staining bacteria as an aid to

their identification. It was originally devised by Hans Christian

Joachim Gram, a Danish doctor. Gram stain differentiates two major

cell wall types. Bacterial species with walls containing small amount

of peptidoglycan and characteristically, lipopolysaccharide, are Gram-

negative whereas bacteria with walls containing relatively large

amount of peptidoglycan and no lipopolysaccharide are Gram-positive.

Apart from Gram staining technique, the identification of bacteria can

also be based on shapes. The three most common shapes are spheres,

rods and spirals.

33

Apparatus

Compound microscopes

Slides

Wash bottle

Bunsen burner

Bacterial loops

Petri dish

Forceps

Staining racks

Materials

Prepared slides of different types of bacteria

Cultures of Escherichia coli

Cultures of Staphylococcus aureus

Yoghurt (diluted in water 1:10)

Immersion oil

Safranin

Crystal violet

95% ethanol

Iodine

Filter paper

Labelling stickers

Figure 7.1 Comparative staining and cell wall structures of Gram-positive

and Gram-negative bacteria. (Adapted from www.quia.com)

34

Figure 7.2 Gram staining of bacteria

(Adapted from http://enfo.agt.bme.hu/drupal/node/9460)

35

Figure 7.3 Different shapes of bacteria

(Adapted from commons.wikimedia.org/wiki/File:OSC_Microbio_03_03_ProkTable.jpg)

36


1. Put a slide into the petri dish. Pour 95% alcohol and soak for

about 30 seconds. Then use forceps to take out the slide. Let the

slide dry and heat it by placing above the flame.

2. Place a loop of sterile distilled water on the slide and put a little

bit of bacterial colony.

3. Gently heat the slide to fix the bacteria onto the slide.

4. Place the slide on the staining rack. Cover the smear with single

drop of crystal violet and wait for 30 seconds to one minute.

5. Gently, rinse the slide with slow running water.

6. Cover smear with 2 drops of iodine. Rotate and tilt the slides to

allow the iodine to drain. Then, cover again with iodine for 30

seconds to one minute. Since the iodine does not mix well with

water, this procedure ensures that the iodine will be in contact

with the cell walls of the bacteria on the slide.

7. Rinse the slide with water as in step 6.

8. Place several drops of 95% alcohol (decolouriser) evenly over

the smears, rotate and tilt the slide. Continue to add alcohol until

most of the excess stain is removed and the alcohol running from

the slide appears clear.

This is the most critical step of the procedures! If the smears

are too thick, or if the alcohol is kept on the slide for too long or

too short a time, the results will not be accurate. Although there

is no recommended time for this step, it usually takes 10-20

seconds to decolourise if exposed to a sufficient amount of

decolouriser.

9. Add few drops of safranin on the bacterial smear and leave it for

approximately 30-45 seconds.

Colourless Gram-negative cell will readily accept the light

red safranin stain, while the already dark coloured Gram-

positive cell will undergo no change at all.

10. Rinse off with water and blot dry with filter paper.

11. Observe the slide under oil immersion magnification and

describe your observation in terms of types of bacteria, shape,

colour and determine whether it is Gram-positive or Gram-

negative.

12. Repeat steps 2-11 for microorganisms found in yoghurt.

37

Table 7.1 Observation results on the type of bacteria, shape, colour

and Gram-positive/ Gram-negative

Bacteria Shape Colour Gram +ve/-ve

E. coli

S. aureus

Bacteria from

yoghurt

Questions

1. Why Gram-positive bacteria purple in colour while Gram-negative

are red?

2. List some examples of beneficial and harmful Gram-positive

bacteria and Gram-negative bacteria.

3. If the iodine step were omitted in the Gram-staining procedure,

what colour of stain would you expect from Gram-positive and

Gram-negative bacteria?

38

EXPERIMENT 8: PLANT DIVERSITY - BRYOPHYTES AND

PTERIDOPHYTES






i. To observe the diversity of species in bryophytes and

pteridophytes.

ii. To construct scientific drawing of bryophytes and

pteridophytes.



2 hour 0

Introduction

Bryophytes and pteridophytes are two large groups of spore producing

terrestrial plants. Compared to the flowering plants, they have a longer

history of evolution.

Bryophytes

There are three main divisions of bryophytes, namely Bryophyta

(mosses), Hepatophyta (liverworts), and Anthocerophyta (hornworts).

Bryophytes are the most primitive among the terrestrial plants. They

are non-vascular and are confined to moist areas because they lack

well developed tissues for transporting water and nutrients. Bryophytes

have a root-like structure, which is called rhizoid and have no true

stem and leaves. Bryophytes are characterized by clear alternation of

generation in its life cycle where the gametophyte generation is

dominant. The male reproductive organ is called antheridium and

produces flagellated sperms (antherozoids). The sperm fertilizes the

egg (oosphere), which is produced by the archegonium that is the

female reproductive organ.

39

After fertilization, the zygote develops in the archegonium to produce

sporophyte, which grows out from the gametophyte. The sporophyte

produces haploid spores, which will eventually give rise to mature

gametophytes.

Pteridophytes

Pteridophytes are the only non-flowering seedless plants possessing

vascular tissues – xylem and phloem. This enables pteridophytes to

achieve larger sizes than the bryophytes. In the tropics, ferns may grow

up to 18 m (60 ft). A major difference between pteridophytes and

bryophytes is that the diploid sporophyte generation is dominant in

pteridophytes. The gametophyte generation retains two traits that are

reminiscent of the bryophyte. Firstly, the small gametophytes lack

conducting vessels. Secondly, as in bryophytes, the flagellated sperms

(antherozoids) require water medium to reach the egg (oosphere), so

pteridophytes still depend on the presence of water for sexual

reproduction. Pteridophytes have true stems with vascular tissues, and

also true roots and leaves.

Exercise 8.1 Bryophytes

Apparatus

Compound microscope

Materials

Prepared slides

Marchantia sp. - capsule l.s

Marchantia sp. - male gametophyte (antheridium) l.s

Marchantia sp. - female gametophyte (archegonium) l.s

Polytrichum sp. - capsule l.s

40


1. Examine the prepared slides which show the longitudinal

sections of Marchantia sp. capsule, antheridium and

archegonium. Draw and label the seta, foot, sporangium, spores

and calyptra.

2. Examine the prepared slides which show the longitudinal

sections of Polytrichum sp. capsule. Draw and label the

operculum, spore, peristome, annulus, calyptra, seta and capsule.

Figure 8.1 Capsule of Marchantia sp. (l.s)

https://www.morton-pub.com/customize/images/immature-and-mature-

sporophytes-callouts

41

Figure 8.2 Archegonia of Marchantia sp. (l.s)

(Adapted from http://www.bio.miami.edu/dana/dox/altgen.html)

Figure 8.3 Archegonia of Marchantia sp. (l.s) 400x

(Adapted from majorsbiology202)

42

Figure 8.4 Antheridia of Marchantia sp. (l.s)

(Adapted from www.vcbio.science.ru.nl)

Figure 8.5 Antheridia of Marchantia sp. (l.s)

(Adapted from www.vcbio.science.

43

Figure 8.6 Capsule of Polytrichum sp. (l.s)

(Adapted from www.k-state.edu)

Questions

Bryophytes

1. State the unique characteristics of bryophytes.

2. How is the transport of substances carried out in bryophytes

tissue? How is this feature related to the general size of these

plants?

3. What is the process involved in spore formation of bryophytes?

4. Explain the adaptations of bryophytes to the terrestrial

environment.

44

Exercise 8.2 Pteridophytes

Apparatus

Compound microscope

Dissecting microscope

Magnifying glass

Razor blade

Tiles

Materials

Fresh specimens:

Selaginella sp. (Division Lycopodiophyta)

Dryopteris sp. (Division Pteridophyta)

Prepared slides:

Lycopodium sp. – strobilus l.s

Selaginella sp. – strobilus l.s


1. Examine the specimens of Selaginella sp. Observe the

dichotomous branching, types and arrangement of sporophyll and

strobilus.

2. Examine the specimens of Dryopteris sp. Draw and label the

rhizome, rhizoid, rachis, frond, pinna and sorus.

3. Examine the prepared slides showing longitudinal sections of the

strobilus of Lycopodium sp. and Selaginella sp. Draw and label

sporophyll, sporangium and spore (homosporous or

heterosporous).

45

Figure 8.7 Selaginella sp.

Figure 8.8 Dryopteris sp.

46

Figure 8.9 Strobilus of Lycopodium sp. (l.s)

(Adapted from www.stolaf.edu)

Figure 8.10 Strobilus of Selaginella sp. (l.s)

(Adapted from www.sfsu.edu)

47

Questions

Pteridophytes

1. State the unique characteristics of pteridophytes.

2. Fern sporophytes have an underground stem called rhizomes. How

do you distinguish that rhizomes are stems and not roots?

3. Compare the spores of Lycopodium sp. and Selaginella sp.

4. Division Pteridophyta is considered to be more advanced than

Division Lycopodiophyta. Explain the advanced characteristic of

Division Pteridophyta.

48

EXPERIMENT 9: BIOCATALYSIS






i. To extract catalase from liver tissue

ii. To observe the qualitative activity of catalase.

iii. To measure the quantitative activity of catalase.

iv. To determine the factors affecting the catalase activity.



2 hour 0

Introduction

Enzymes are biological catalysts, normally proteins, synthesized by

living organisms. Enzymes speed up reactions by lowering the

activation energy. Enzymes are normally very specific. An enzyme

catalyses a single reaction that involves one or two specific molecules

called substrates. Each enzyme has evolved to function optimally at a

particular pH, temperature and salt concentration. Some require the

presence of other molecules called coenzymes, derived from water-

soluble vitamins, for its function. The rate of reaction also depends on

the amount of enzymes present.

In this experiment, the enzyme to be extracted and tested is catalase,

which present in almost all cells especially in the liver and red blood

cells. The substrate for this enzyme is hydrogen peroxide (H2O2). The

accumulation of hydrogen peroxide in the body is toxic. Catalase

renders the hydrogen peroxide harmless by breaking it down to water

and oxygen.

catalase

2H2O2 2H2O + O2

49

The chemical properties of catalase resembles most those of the

enzymes.

(Note: The success of this experiment depends on the amount of

catalase present in the prepared extract. The results of the catalase

reaction can be observed clearly if the amount of enzyme in the extract

is large. Use a boiling test tube to avoid spillage during the reaction).

Apparatus

Beakers (250 ml & 1000 ml)

Glass rod

Boiling tube

Boiling tube rack

Blade

Dropper

Filter funnel

Measuring cylinder (10 ml)

Mortar and pestle

Muslin cloth

Labelling stickers

Retort stand

Rubber stopper

Syringe (1 ml)

Thermometer

Tile

Tissue paper

Waterbath

Materials

Fresh liver of a cow/chicken

Distilled water

3 % H2O2 solution

1 M H2SO4

1 % KMnO4 solution

Ice cubes

Phosphate buffer solutions (pH 5, pH 7, pH 9 and pH 11)

50

Exercise 9.1: Estimation of catalase activity


A) Preparation of catalase extract

1. Cut 10-15 g of fresh liver tissue into small pieces and macerate

the tissue in a mortar and pestle.

2. Gradually add 20 ml of water.

3. Filter the mixture into a beaker using the muslin cloth.

4. The filtrate will be the enzyme stock solution to be used in the

experiment.

B) Qualitative test for catalase activity

1. Label two boiling tube as A and B

2. Pour 1 ml H2O2 solution into a boiling tube A.

(Caution: H2O2 is a toxic substance).

3. Pour 1 ml enzyme stock solution in boiling tube B.

4. Using a dropper, add the stock solution in boiling tube B into the

boiling tube A. Label the tube as boiling tube C.

5. Observe and explain the activity of the enzyme. Use this boiling

tube C for the following.

C) Estimation of catalase activity

1. Pour 1 ml H2SO4 into boiling tube D.

2. Transfer 1 ml of enzyme-H2O2 mixture from C into D. Shake

well the boiling tube.

3. The left over H2O2 solution that does not react can be measured

using KMnO4 solution.

4. KMnO4 solution reacts with H2O2 in acid medium.

5H2O2 + 2KMnO4 + 4H2SO4 2KHSO4 + 2MnSO4 +

8H2O + 5O2

5. Fill the syringe with KMnO4.

6. Using the syringe, add drops of KMnO4 into test tube D until the

red colour remains unchanged for 10 seconds.

7. Determine the amount of KMnO4 used. The value shows the

activity of catalase.

51

8. The more KMnO4 is used indicates that more H2O2 is present in

the mixture. It means that the H2O2 is not fully broken down by

catalyst to oxygen molecules.

Exercise 9.2: Factors affecting the activity of catalase

Procedures and observation

A) Temperature

1. Put the following boiling tubes in a beaker containing chilled

water (20°C).

a) boiling tube A containing 2ml of enzyme stock solution

b) boiling tube B containing 3 ml of H2O2

c) empty boiling tube labelled C

2. Prepare 1 ml H2SO4 in boiling tube 5.

3. When the temperature in the boiling tube B containing H2O2

drop to 20°C, pour the chilled H2O2 into boiling tube C.

4. Pour the cooled enzyme stock from boiling tube A into boiling

tube C. Make sure that the reaction takes place in the iced-chilled

beaker. Record the time.

5. After 4 minutes, transfer 1 ml of solution from boiling tube C

into boiling tube 5 and then plug with rubber stopper. Shake

well and estimate the activity of catalase as conducted in

Exercise 15.1C.

6. Repeat the steps 1 to 5. (Set at different temperatures: 30oC, 40

oC

and 50oC). Use different boiling tubes.

7. Record the values obtained in Table 9.1 and plot the graph of the

approximate catalase activity against temperature.

52

Table 9.1 The effects of temperature on catalase activity

Temperature

20oC

30oC

40oC

50oC

Amount (ml)

of KMnO4

used

Approximate

catalase

activity

(1/amount of

KMnO4 used)

B) pH

1. Label four boiling tubes as 6, 7, 8 and 9.

2. Pour 1 ml of H2SO4 into each boiling tube.

3. Label four boiling tubes as F, G, H and I.

4. Pour 1 ml of H2O2 into each boiling tube.

1. Add 2 ml phosphate buffer solution with pH 5, pH7, pH 9 and

pH 11 to boiling tube F, G, H and I, respectively. Shake them

well.

2. Pour 1 ml enzyme stock solution into boiling tube F. Record the

time for 4 minutes.

3. After 4 minutes, transfer 1 ml of solution from boiling tube F

into boiling tube 6. Shake well and estimate the activity of

catalase as conducted in Exercise 15.1C.

4. Repeat the above steps for boiling tubes G, H and I using boiling

tubes 7, 8 and 9 respectively.

5. Record the values obtained in Table 9.2 and plot the graph of the

approximate catalase activity against pH.

53

Table 9.2 The effects of pH on catalase activity

pH

5

7

9

11

Amount (ml) of

KMnO4 used

Approximate

catalase activity

(1/amount of

KMnO4 used)

Notes:

1. Ensure all apparatus are clean, in order to obtain accurate results.

2. Measure precisely the volume of the solutions used.

Questions

1. What is the role of H2SO4 in the reaction?

2. Explain the effects of the following factors on the enzymatic

reaction:

(a) temperature

(b) pH

54

EXPERIMENT 10: CELLULAR RESPIRATION






i. To organize the experiment setting for redox reaction procedures

ii. To conduct an experiment on redox reaction in cellular

respiration

iii. To explain the biochemical processes in yeast suspension



2 hour 0

Introduction

Aerobic cellular respiration produces ATP from glucose. As an

organism breaks down the glucose, most of the energy comes as the

hydrogens of glucose are removed by enzymes in glycolysis and the

citric acid cycle. The electrons of the hydrogens are carried to the

electron transport chain (ETC) in the forms of NADH and FADH2. We

can demonstrate these redox reactions by substituting NAD+

with

methylene blue. In the oxidized state, this dye has a blue colour. When

it is reduced, it becomes white or light blue as indicated below, hence

the reduction has taken place.

Methylene blue decolourised methylene

(blue/greenish blue) (white/light blue)

reduction

oxidation

55

Apparatus

Beaker (250 mL)

Boiling tubes

Bunsen burner

Cork or rubber stopper

Dropper

Labelling paper

Measuring cylinder (10 mL)

Pasteur pipette

Stopwatch

Thermometer

Tripod stand

Water bath (380C – 42

0C)

Materials

Methylene blue 0.1%

Yeast suspension (5%) added to 1% glucose (freshly prepared)


1. Label 3 boiling tubes as A, B and C.

2. Fill in tube with 10 mL of yeast suspension.

3. Heat tube C in boiling water for 5 minutes.

4. Add 5 drops of methylene blue into each of the tubes using

Pasteur pipette. Shake gently to ensure the colour is evenly

distributed.

5. Incubate all tubes in the water bath (40oC) for 15 minutes.

6. Observe the colour changes in all tubes.

7. Heat tube B in boiling water for 5 minutes.

8. Plug tube A, B and C with cork or rubber stopper. Press it with

your thumb and shake the tube vigorously for 30 seconds.

Observe the colour changes. Remove the stopper and incubate all

tubes in water bath (40oC) for 15 minutes.

9. Observe the colour of the yeast suspension precipitate in each

tube.

Note: Observations are based on the colour changes.

10. Record your observations in Table 10.1.

56

Table 10.1 Colour changes observed for demonstrating redox

reactions in yeast using methylene blue

Treatments

Colour

Tube A Tube B Tube C

Boiling 5 minutes √

5 drops of

methylene blue √ √ √

First incubation

(40oC)

√ √ √

After first

incubation

Boiling 5 minutes √

Vigorous shaking

Second incubation

(40oC)

√ √ √

After second

incubation

Questions

1. Explain the redox reaction.

2. What is the substance in a living cell that has the same function

as methylene blue?

3. Name the important process which involves substances in

question no.2 above.

4. Explain the biochemical processes based on the observations in

boiling tubes A, B and C.

5. Are enzymes responsible for the colour changes? State your

reason.

57

EXPERIMENT 11: PHOTOSYNTHESIS






i. To demonstrate chromatography technique to separate the

photosynthetic pigments

ii. To calculate Rf value



2 hour 0

Introduction

The chloroplasts in green plants contain many pigments such as

chlorophyll a, chlorophyll b, carotene, phaeophytin and xanthophylls.

These pigments have different solubility in certain solvent and they can

be separated by chromatography.

Paper chromatography is a useful technique for separating and

identifying pigments and other molecules from cell extracts that

contain a complex mixture of molecules. Typically, a drop of the

sample is applied as a spot to a sheet of chromatography paper. The

solvent moves up the paper by capillary action, which occurs as a

result of the attraction of solvent molecules to the paper and the

attraction of solvent molecules to one another. As the solvent moves up

the paper, it carries along any substances dissolved in it. The pigments

are carried along at different rates because they are attracted to

different degrees, to the fibres in the paper through the formation of

intermolecular bonds, such as hydrogen bonds. Another factor that is

taken into account is molecular size.

58

Apparatus

Beaker (100 mL)

Blade

Boiling tube rack

Boiling tube with cork stopper

Chromatography paper strip (Whatman No. 3) with pointed end

Dissecting pin

Filter funnel

Forceps

Hair dryer

Labelling paper

Measuring cylinder

Mortar and pestle

Muslin cloth

Spatula

Materials

Fresh leaves:

i. Sauropus sp. (Cekur manis)

ii. Pandanus sp. (Pandan)

iii. Erythrina sp. (Dedap)

iv. Coleus sp. (Ati-ati)

Solvent (mixture of ether petroleum-acetone at 9:1, freshly prepared)

Acetone 80% (Should be handled in fume cupboard, do not inhale the

fume)

59


Exercise 11.1 Chlorophyll extract preparation

1. Cut approximately 20g of fresh leaves using a blade.

2. Grind the leaves and add 5 mL acetone gradually.

3. Leave them for 10 minutes.

4. Grind again and add another 5 mL acetone.

5. Filter the extraction using muslin cloth.

Remarks : Extraction of the pigments also can be done by carefully

pressing and moving a coin back and forth more than 10 times on top

of the leaf onto the chromatography paper until enough pigments are

placed on the chromatography paper.

Exercise 11.2 Paper Chromatography

1. Using the tip of dissecting pin, place a drop of the chlorophyll

extract on the chromatography strip. Let the drop dry completely.

Repeat the process more than 15 times to build up a small area of

concentrated pigment.

(Caution: Use forceps to handle the chromatography strip

throughout the experiment).

2. Attach the paper strip to the stopper with a pin. Suspend the strip

straight into the boiling tube that contains 3-5 mL solvent. The

bottom of the paper should be dipped into the solvent, but make

sure that the pigment spot (point of origin) is not immersed in

the solvent. Place the chromatography paper strip vertically in

the tube rack.

3. Let the solvent rise until its front reaches 1cm from the top of the

strip.

4. Remove the chromatography paper strip and mark the solvent

front with pencil. Mark the pigmented area.

60

5. Calculate the Rf value for each pigment using the following

formula:

Figure 11.1 Paper chromatography set up using a boiling tube

6. Record your results in the Table 11.1.

Table 11.1 Photosynthetic pigments and the observed Rf values

Pigment Colour Standard Rf

value

Observed Rf

value

Chlorophyll b Yellow-green 0.45

Chlorophyll a Blue-green 0.65

Xanthophyll Yellow 0.71

Phaeophytin Grey 0.83

Carotene Orange 0.95

(Remarks – it is recommended that different groups of plant be used)

stop cork

pin

Whatman No. 3

filter paper

pigment extract

solvent

Rf = Distance moved by the pigment from the origin

Distance moved by the solvent from the origin

61

Figure 11.2 Paper chromatography shows the value for

each pigment.

Questions

1. Do the leaf extracts from different plants contain the same

pigments? Explain why.

2. Name the most common pigment which is usually found in many

plants. Explain your answer.

3. Why do plants have different types of pigment?

Solvent front

12 cm

Pigment origin

Solvent origin

4.1 cm

5.2 cm

6.4 cm

11.2 cm

62

EXPERIMENT 12: MAMMAL ORGAN SYSTEM






i. To demonstrate dissecting skill.

ii. To examine the organ systems in mammal: Digestive,

Circulatory, Respiratory, Urogenital and Nervous System.



2 hour 0

Introduction

An organ system is a group of anatomical structures that work

together to perform a specific function or task. Although we learn

about each organ system as a distinct entity, the functions of the body's

organ systems overlap considerably, and your body could not function

without the cooperation of all of its organ systems. In fact, the failure

of even one organ system could lead to severe disability or even death.

A mammallian body is composed of different organ systems which

include the following:

Integumentary

Muscular

Skeletal

Nervous

Circulatory

Lymphatic

Respiratory

Endocrine

Urinary/excretory

Reproductive

Digestive

63

Apparatus

Dissecting set

Dissecting pins

Dissecting tray

Petri dish

Note: Demonstration by the lecturer on how to use the dissecting kit.

Material

Chloroform

Cotton wool

Disposable gloves

Mice

Surgical mask


1. Put the mice to sleep.

2. Lay down the mice on a dissecting tray, with its ventral surface

facing upward. Spread the legs and pin at 45° angle as shown in

Figure 12.1.

3. Use forceps to lift the skin on the mid-ventral line (Figure 12.2).

Dissecting tray

Figure 12.1 Pin the legs of the mice at 45° angle

64

4. Slit the skin along the mid-ventral line.

Figure 12.2 Lifting the skin on the mid ventral line

Figure 12.3(a) Male mice

Penis

Scrotal sac

65

Note: Keep the scissors as low as possible to avoid from cutting

the body wall underneath the skin.

Male:

Cut straight up until you reach the lower jaw. Cut straight down,

till around the penis and end at the scrotal sacs (Figure 12.3a).

Female:

Cut the skin as described for the male, but continue to cut

straight down, passing on either side of the urinary and genital

apertures to the anus (Figure 12.3b).

4. Cut through the skin towards the end of each limb. Pull the skin

aside to expose the abdominal wall (Figure 12.4).

Note: Be careful not to tear off the nerves and muscles at the

axillary region.

Figure 12.3(b)

Female mice

66

Figure 12.4 Exposing the abdominal wall

67

5. Stretch the skin and pin it back as shown in Figure 12.5. Lift the

abdominal wall with forceps and make an incision as shown.

Using a pair of scissors, cut through the body wall to expose the

components of the abdomen.

Figure 12.5 Making an incision on the abdominal wall

68

6. Pin aside the abdominal wall (Figure 12.6).

7. Observe the digestive and reproductive systems of the mice.

8. Remove the fat bodies as shown in Figure 12.7 when necessary.

Figure 12.6 Exposing the internal anatomy of the abdomen

69

Note: Do not use sharp instruments while observing internal

organs.

Male:

i. Cut the ureters. Pin the bladder, seminal vesicle and rectum.

ii. Remove the fat body on the right of the mice.

iii. The blood vessels can be traced through the right groin by

easing away the muscle and connective tissue with forceps.

Trim with a pair of scissors if necessary.

iv. Remove the remains of the mesentery and fat to display the

aorta and posterior vena cava.

Figure 12.7 Exposing the lower abdominal region

70

Female:

i. Cut the ureters.

ii. Pin the rectum.

iii. Lay aside the vagina and bladder as shown and pin it if

necessary.

iv. The blood vessels can be traced through the right groin by

easing away the muscle and connective tissue with forceps.

Trim with a pair of scissors if necessary.

v. Remove the remains of the mesentery and fat to display the

aorta and posterior vena cava.

vi. Cut through the side wall of the thorax along the line

indicated as shown in Figure 12.8.

9. Continue the cut to the apex by turning the ventral part of the

thoracic wall aside and pull it slightly to avoid cutting the heart.

Repeat on the other side to remove the ventral part of the

thoracic wall entirely. Remove the loose parts of the pleura (refer

to Figure 12.8).

Figure: 12.8 Exposing the thoracic cavity

71

10. Observe and draw the components of the thorax as they appear at

this stage. Refer to Figure 12.9.

11. Remove the thymus gland as shown in Figure 12.10. Clear away

the fat tissues around the great vessels.

Figure 12.9 Components of the thorax

Figure 12.10 Removing the thymus gland Figure 12.10 Removing the thymus gland

72

12. Pin the heart to the right of the mice. Observe the structures in as

shown in Figure 12.11.Fig

13. .11.

13. Based on your observations, draw a labelled diagram of the

organ systems in mammal; Digestive System, Circulatory

System, Respiratory System, Urogenital System and Nervous

System.

Figure 12.11 Circulatory system of the mice

73

Figure 12.12 Digestive System

Figure 12.13 Circulatory System.

74

Figure 12.14 Respiratory System

Figure 12.15 Urogenital System (Male)

75

Figure 12.16 Urogenital System (Female)

Figure 12.17 Nervous System

76

REFERENCES

Campbell, N. A., Reece, J. B., Urry, L. A., Cain, M. L., Wassermen, S. A.,

Minorsky, P. V. & Jackson, R. B. (2016). Biology. (11th

Ed.). Pearson

Benjamin Cummings. USA.

Lawrence, E. (2016). Henderson’s Dictionary of Biological Terms (16th Ed.),

Prentice Hall.

Solomon, E. P., Berg, L. R. & Martin, D. W. (2018). Biology. (11th Ed.).

Nelson Education, Ltd, Canada.

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77

ACKNOWLEDGEMENT

The Matriculation Division wishes to extend heartfelt thanks and utmost

gratitude to the following individuals who have actively participated in

marking the review a success. We are grateful for the support and guidance

provided by those involved, namely:

Dr. Baiduriah binti Yaakub Director of Matriculation Division,

Ministry of Education

Mr. Azman bin Abd. Karim Deputy Director,


Dr. Shah Jahan bin Assanarkutty Principal Assistant Director,


Dr. Saharawati binti Shahar Senior Assistant Director,


Prof. Dr. Shahrul Hisham bin Zainal Ariffin Universiti Kebangsaan Malaysia

Prof. Dr. Aziz bin Arshad Universiti Putra Malaysia,

Prof. Dr. Mahmud bin Tengku Muda Universiti Putra Malaysia,

Mohamed

Mdm. Salbiah binti Mohd. Som Selangor Matriculation College,

Mr. Roslan bin Abu Bakar Selangor Matriculation College,

Mdm. Hajjah Hanita binti Ghazali Selangor Matriculation College,

Mdm. Mushitah binti Abu Penang Matriculation College,

Miss Norhayati binti Othman Penang Matriculation College,

Miss Hjh. Zuraidah binti Mohamed Kelantan Matriculation College,

Mr. Abdul Aziz bin Abdul Kadir Malacca Matriculation College,

Mdm. Rohana binti Hassan Malacca Matriculation College,

Mdm. Michelle Mariam binti Abu Bakar Malacca Matriculation College,

Mdm. Nizaha binti Zulkifli Negeri Sembilan Matriculation College,

Mdm. Fariza binti Zakaria Perak Matriculation College,

Mdm. Lena Maizura binti Basaruddin Perak Matriculation College,

Mdm. Rudziah binti Umar Selangor Matriculation College

Miss Siti Khadijah binti Ahmad Khairi Selangor Matriculation College

Mdm. Rohaiza binti Rozali Assistant Director,


Mr. Ruslan bin Achok Assistant Director,


biology - s5ee8b77f7f9dee0d.jimcontent.com...1.2 matriculation science programme learning outcomes...

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