Faculty of Science

Laboratory Manual
Basic Immunology

Bachelor of Biotechnology (Hons.)

Copyright © 2018
Lincoln University College, Malaysia
All rights reserved
No part of this book can be reproduced or transmitted by any means, electronic or
mechanical, including photocopying recording or by any information storage and retrieval
system without prior written permission from the publisher.

Edited By: Dr. Idris Adewale Ahmed

LINCOLN UNIVERSITY COLLEGE

Wisma Lincoln, No. 12,14,16 & 18,
Jalan SS 6/12, Off Jalan Perbandaran
47301 Petaling, Jaya,
Selangor Darul Ehsan, Malaysia
Tel.: +603-7806 3478
Fax: +603-7806 3479
Toll Free: 1-300-880-111
E-mail: [email protected]
[email protected]
Web: www.lucp.net
www.lincoln.edu.my

ISBN: 978-967-2257-04-2
 Basic Immunology

Table of Content

Experiments: Page

1 Laboratory safety rules 1

2 Agglutination reaction 6

3 Complement fixation test 9

4 Radial immuno-diffusion techniques 12

5 Dot enzyme linked immunosorbent assay (Dot ELISA) 15
 LINCOLN UNIVERSITY COLLEGE
FACULTY OF SCIENCE (DEPARTMENT OF BIOTECHNOLOGY)
LABORATORY SAFETY RULES

The following rules must be obeyed by all students in the science laboratory of the faculty.
Wilful or repeated in advertent non-compliance may result in dismissal or suspension from the
laboratories

• No entry without permission:

- Outsiders are not allowed to enter the laboratory without permission.
- No student is allowed to enter the laboratory unless permission has been given by a laboratory
assistant or a lecturer.

• At work in the laboratory:

- No experiment may be attempted without the knowledge and permission of a lecturer.
- Students must wear shoes in the laboratory. Students wearing slippers or sandals are not allowed to
work in the laboratory.
- Lab coat must be worn at all times during practical work in the laboratory.
- Do not mouth pipette chemicals.
- Do not eat or smoke in the laboratory.
- Do not taste any chemicals, including dilute solutions. If any acid or alkali accidentally enters your
eyes or mouth, wash immediately with plenty of tap water. Inform your lecturer, and seek medical
attention if necessary.
- Paper should be used to light up the Bunsen burners.
- Used match sticks, filter papers, and other solid waste must never be thrown into the sinks. They
must be thrown into the dustbins provided. Lighted match sticks and smoldering materials must be
extinguished with tap water before thrown in to the dustbins.
- Any equipment broken or damaged must be reported to the laboratory assistant.

• Before leaving the laboratory:

- All the equipment and benches must be cleaned at the end of each practical session.
- Wash hands and arms with soap and water before leaving the laboratory.
- No student is allowed to take away any chemicals, equipment or other property of the laboratory.

1
 INTRODUCTION

1. The Scientific Method

- Making observations
- Generating hypotheses
- Making predictions
- Designing and carrying out experiments
- Constructing scientific models

2. Practical Exercises
To get the most out of the practical exercises, you need to follow carefully the instructions
given. These instructions have been designed to provide you with the experience in the following skills:
- Following instructors
- Handling apparatus
- Having due regard for safely
- Making accurate observations
- Recording results in an appropriate form
- Presenting quantitative results
- Drawing conclusions

3. Following Instructions
Instructions are provided in the order in which you need to carry them out. We would advise
that before carrying out the instructions, you read through the entire exercise. This will help you to
remember what you have learned.
Each practical exercise in the book begins with a few lines describing its purpose in most cases
the following headings are also used:
- Procedure-numbered steps that need to be carried out.
- For consideration -some questions to help you think carefully about the results you have obtained.
- Materials-a list of the apparatus, chemicals and biological materials you need.

4. Handling apparatus
Biologists need to able to use many different types of apparatus, for example, photometers (to
measure water uptake by plants), respirometers (to measure oxygen uptake or carbon dioxide
production), Petri dishes (for plating out bacteria and other microorganisms) and the light microscope
(to magnify specimens). Many of the practical exercises are designed to help you derive the maximum
benefit from a piece of apparatus.

5. Having Due Regard for Safety

Surveys have been shown that science laboratories are among the safest places to be.
Nevertheless, this is no cause for complacency.
- Always move slowly and carefully in a laboratory.
- Never put your fingers in your mouth or eyes after using chemicals or touching biological specimens
until you have washed your hands thoroughly with soap and warm water, and dried them.
- Make sure glass objects (e.g, thermometers, beakers) cannot roll off tables or be knocked onto the
floor.
- Wear safely goggles whenever there is a risk of damage to the eyes.

Situations of risk include:

- Heating anything with a Bunsen burner (even heating water has its dangers’)
- Handling many liquids, particularly those identified as corrosive, irritant, toxic or harmful

2
 - Handling corrosive or irritant solids
- Some dissection work
- Allow Bunsen burners, tripods, gauzez and beakers to cool down before handling them.
- Never allow your own body fluids (especially blood and saliva) to come into contact with someone
else, or theirs into contact with you.
- Keep long hair tied back and do not wear dangly earrings.
- Do not allow electrical equipment to come into contact with water.
- If you are unsure how to carry out a scientific procedure, ask.
- Make sure you understand why you are going to do something before you do it.
- Wear a lab coat when using chemicals or handling any biological specimens.
- Follow exactly agreed procedures with regard to cuts, burns, electric shocks and other accidents
(e.g. with chemicals).
- Follow exactly all specific safely instructions given in this book or provided by your teacher for
particular practical exercises (e.g. use of gloves, disinfection)
With practice, these procedures should become second nature to you. They will enable you to carry out
practical work in safety.
6. Making Accurate Observations
In most cases the practical exercise will make it clear what you need to observe, e.g. the time
taken for a certain volume of gas to be evolved or the width of a sample cells. Ensure that you know
how to use any necessary equipment before starting practical. Think carefully about the precision with
which you will make your observations.

7. Recording Results in an Appropriate Form

Results can be recorded in various ways. Often it is helpful to record raw data in a table. Most
data will be in the form of numbers, i.e. they will be quantitative data (also known as numerical data).
However, some data, e.g. flower colour, will be qualitative data.
One form in which some biological findings can be recorded is a drawing. You don’t need to be
professional artist to make worthwhile biological drawings. If you follow the following guidelines, a
drawing can be of considerable biological value:
- Ensure that your completed drawing will cover at least a third of A4 page.
- Plan your drawing so that the various parts are is proportion and will not be drawn too small.
Small marks to indicate the length and breadth of the drawing are a great help in planning and a
faint outline can be rapidly drawn to show the relative positions of the parts.
- The final drawing should be made with clean, firm lines using a sharp HB pencil and, if needed, a
good quality eraser (not a fluid).If important details are too small to be shown in proportion, they
can be put in an enlarged drawing at the side of the main drawing.
- Avoid shading and the use of colour unless you are an excellent artist and they really help, for
example when drawing soil profiles.
- When drawing structures seen with the naked eye or hand lens, use two lines to delineate such
things as blood vessels and petioles. This will help you to indicate the relative widths of such
structures.
- When drawing low power plan drawings from the light microscope, do not attempt to draw
individual cells-just different tissues.
- When drawing plant cells at high power under the light microscope, use two lines to indicate the
width of cell walls, but a single line to indicate a membrane.
- Always put a scale on each drawing.

3
8. Presenting Quantitative Results
Presentation of data is all about using graphs or other visual means to make it easier to see
what your results tell you. The following four ways of presenting data are the most frequently used in
biology: line graphs, bar charts, histograms and scatter graphs (Figure 1).

Figure 1: Line graphs, bar charts, histograms and scatter graphs

9. Drawing Conclusions
Finally, you will need to draw conclusions. If your practical exercise has involved the testing of
a hypothesis, for example that the enzyme pepsin works better at low pH than in neutral or alkaline
conditions, your conclusion should indicate whether the hypothesis has been refuted (i.e. shown not to
be the case) or supported. Of course, even if your hypothesis has been supported, it doesn’t mean that
it has been confirmed with 100% certainty- in other words it isn’t proved. Science proceeds more by
showing that certain ideas are wrong than by showing that others are right (think about that!). Your
conclusion might therefore include further ways of testing the original hypothesis, or might raise new
possibilities to be investigated.
Often you will only be able to arrive at your conclusions after statistically analysing your data.

10. Writing a Scientific Lab Report

Title
- Communicate the subject investigated in the paper.

Introduction
- State the hypothesis.
- Give well-defined reasons for making the hypothesis.
- Explain the biological basis of the experiment.
- Cite sources to substantiate background information.

4
 - Explain how the method used will produce information relevant to your hypothesis.
- State a prediction based on your hypothesis. (If the hypotheis is supported, then the results will be.)

Materials and Methods

- Use the appropriate style.
- Give enough detail so the reader could duplicate your experiment
- State the control treatment, replication and standardized variables that were used.

Results
- Summarize the data (do not include raw data).
- Present the data in an appropriate format (table or graph).
- Present tables and figures neatly so they are easily read.
- Label the axes of each graph completely.
- Give units of measurement where appropriate.
- Write a descriptive caption for each table and figure.
- Include a short paragraph pointing out important results but do not interpret the data.

Discussion
- State whether the hypothesis was supported or proven false by the results, or else state that the
results were inconclusive.
- Cite specific results that support your conclusions.
- Give the reasoning for your conclusions.
- Demonstrate that you understand the biological meaning of your results.
- Compare the results, with your predictions and explain any unexpected results.
- Compare the results to other research or information available to you.
- Discuss any weaknesses in your experimental design or problems with the execution of the
experiment.
- Discuss how you might extend or improve your experiment.

Conclusion
- Restate your conclusion.
- Restate important results.

Literature Cited
- Use the proper citation form in the text.
- Use proper citation form in the Literature Cited section.
- Refer in the text to any source listed in this section.

Acknowledgement
- State any appropriate acknowledgement that you think is necessary.

5
Practical 1
Title: Agglutination reaction

Objective:
After completing the practical, you will be able:
1. To demonstrate the agglutination reaction through blood grouping test (blood group and Rh factor)

Introduction:
Agglutination is the interactions between insoluble particles (e.g. intact bacteria and cells) or
soluble antigens that attached to particles and their corresponding specific antibodies that result in
some visible agglutinates if given enough time and the proper concentration of electrolyte. The
agglutination of RBCs (hemagglutination) is a result of an immune reaction between the RBCs and
antibodies against the corresponding blood type. Hemagglutination caused by antibodies is detected
either by human eyes or by imaging techniques in conventional blood typing methods (Ashiba et al.,
2015).

Blood grouping is the classification of blood based on the presence or absence of two inherited
antigenic substances on the surface of red blood cells (RBCs). The ABO and Rh are the major clinically
significant and the most important of all the blood group systems. ABO and Rh(D) blood typing is also
one of the most important tests that are performed prior to blood transfusion. The ABO blood group
system was first discovered by Karl Landsteiner in 1900. The associated Anti A and Anti B antibodies
usually belong to IgM class of immunoglobulins. The Rhesus system (Rh) is the second most important
blood group system in humans. The most significant and immunogenic Rhesus antigen is the RhD
antigen. The individuals carrying the Rh antigen are considered to have positive blood group whereas
those individuals that lack this antigen are considered to have negative blood group.

Slide agglutination reaction is the direct agglutination carried out on the slides, by directly
mixing the antibody with a particle antigen under the certain concentration of electrolyte. The result is
positive when there is visible agglutinate, otherwise negative.

The ABO blood group antigens are O-linked glycoproteins in which the terminal sugar residues
exposed at the cell surface of the red blood cells determine whether the antigen is A or B. Blood group
A individuals have A antigens on RBCs and anti-B antibodies in serum. Similarly, blood group B
individuals have B antigens on RBCs and anti-A antibodies in serum. Blood group AB individuals have
both A and B antigens on RBCs and neither anti-A nor anti-B antibodies in serum. Whereas, blood
group O individuals have neither A antigens nor B antigens, but possess both anti-A and anti-B
antibodies in serum. The Rh antigens are transmembrane proteins in which the loops exposed on the
surface of red blood cells interact with the corresponding antibodies.

ABO Blood
Antigens Antibodies Genotype
Group
A Anti B A A/A or A/O
B Anti A B B/B or B/O
Neither Anti A
A and B AB A/B
nor Anti B
Anti A, Anti B,
Neither A nor B O O/O
Anti AB

6
Materials
• Human peripheral blood
• Standard sera: Anti-A, anti-B sera and Anti RhD
• Saline
• Slides, needles, cotton ball, etc.
• Reagents: 70% Alcohol/ Spirit

Procedure
1. Dangle the hand down to increase the flow of blood in the fingers.
2. Clean the fingertip to be pierced with spirit or 70% alcohol (usually ring or middle finger).
3. With the help of the sterile lancet, pierce the fingertip and place one drop of blood in each of the
cavities.
4. Add one drop of antiserum into each cavity as shown below:
Blood drop Blood drop Blood drop
+ + +
Anti A Anti B Anti RhD

5. Mix each blood drop and the antiserum using a fresh mixing stick.
6. Observe agglutination in the form of fine red granules within 30 seconds. Anti RhD takes slightly
longer time to agglutinate compared to Anti A and Anti B.
7.

Results and Observation:

1. Record your group results in the table below:

ID No. Blood
Student Name Anti A Anti B Anti RhD Group

7
1. Paste a photograph of your blood grouping result in the space provided below:

Questions:

1. Is ABO blood compatibility enough for the safety of blood transfusion? Justify your response.
2. Describe the relevance of Rh factor.

8
Practical 2
Title: Complement fixation test

Objective:
After completing the practical, you will be able:
1. To determine whether the complement has been fixed using sheep RBCs and antibodies against
sheep RBCs

Introduction:
The complement fixation test (CFT) is a common serological test which is used to detect the
presence of specific antibodies or antigens to diagnose infections, particularly diseases caused by
microbes that are not easily detected by standard culture methods (Li et al., 2016)

The complement fixation test was extensively used in syphilis serology after being introduced
by Wasserman in 1909. Erythrocytes are used as the target cell, because complement-induced
leakiness of the membrane can be visualized or measured calorimetrically as an increase in free
hemoglobin.

In the presence of specific antibodies to an infectious agent, any complement in the system is
bound, leaving no residual complement for reaction with antibodies to the erythrocytes. Thus, the
presence of specific antibody is indicated by the absence of hemolysis.

It is the nature of the complement to be activated when there is formation of antigen-antibody

complex. The first step is to heat the serum at 56°C to destroy patient’s complement. A measured
amount of complement and antigen are then added to the serum. If there is presence of antibody in the
serum, the complement is fixed due to the formation of Ag-Ab complex. If no antibody is present then
the complement remains free.

In the positive test: The available complement is fixed by Ag-Ab complex and no hemolysis of
sheep RBCs occurs. So the test is positive for presence of antibodies. (Figure 1)

In the negative test: No Ag-Ab reaction occurs and the complement is free. This free
complement binds to the complex of sheep RBC and it’s antibody to cause hemolysis, causing the
development of pink color. (Figure 1)

9
 Figure 1: Positive and negative test

Materials:
• Sheep erythrocytes suspension (5% suspension of washed sheep RBCs)
• Hemolysin (rabbit anti-sheep red-cell antibody)
• Guinea pig complement, free of antibodies to the agent of interest (Note: Guinea pig is the
commonest source of fresh complement)
• Barbital-buffered diluents
• Plastic microtitre plate
• Centrifuge adapter for microtitre plates
• Water bath for incubation of plates
• Color standards for judging hemolysis (prepared by lysing various concentrations of red
cells)

Procedure:

Complement Fixation Test (CFT) consists of two stage:

Complement fixation stage:
1. Heat the serum at 56°C for 30 minutes to destroy patient’s complement.
2. Incubate a known antigen and inactivated patient’s serum with a standardized, limited amount
of complement. If the serum contains specific, complement activating antibody the complement

10
 will be activated or fixed by the antigen- antibody complex. However, if there is no antibody in
the patient’s serum, there will be no formation of antigen-antibody complex, and therefore
complement will not be fixed but will remain free.

Indicator Stage:
3. The second step detects whether complement has been utilized in the first step or not. This is
done by adding the indicator system including sheep erythrocytes suspension (5% suspension
of washed sheep RBCs) and haemolysin
4. If the complement is fixed in the first step owing to the presence of antibody there will be no
complement left to fix to the indicator system. There won’t be any lysis of RBCs.
However, if there is antibody in the patient’s serum, there will be no antigen-antibody complex,
and therefore, complement will be present free or unfixed in the mixture. This unfixed
complement will now react with the antibody- coated sheep red blood cells to bring about their
lysis.

Results: Place the photographs of relevant result from your work:

Question:

1. Discuss the advantages and disadvantages of complement fixation test.

11
Practical 3
Title: Radial immuno-diffusion techniques
Objective:
After completing the practical, you will be able:
1. To determine the concentration of immunoglobulin (IgG) in a dry lab experiment

Introduction
Gel precipitation is an immunologic assay in which soluble antigen and antibody are allowed to
diffuse through a gel medium. As the antigen and / or antibody diffuse from the point of application
(usually a well cut in the agar) through the gel, their concentration decreases until they eventually arrive at
their own zone of equivalence (optimal concentration of antigen and antibody) and forming visual
precipitation within the gel.

Explanation of terms often applied to gel diffusion tests:

Diffusion - refers to the number reactants are moving through the gel. For instance, single-diffusion tests
have only one reactant (usually the antigen) moving, while the other reactant is fixed in the gel. Double-
diffusion testing has both of the reactants moving though the medium.
Dimension - refers to the direction of movement. Reactions in tubes essentially have only one effective
dimension, up and down and are called single dimension. When a reactant is able to move both up and
down as well as radially through a gel the term double dimension can be applied. Therefore using this
criterion, there are four combinations or classification of reactions in gels:
1. single diffusion - single dimension
2. single diffusion - double dimension
3. double diffusion - single dimension
4. double diffusion - double dimension

Principle:
When the antigens and corresponding antibodies are allowed to react in gels or other mediums, they
will diffuse toward one another, and at the point in which they meet in optimal proportions, they will form a
visible precipitate (Burtis, Ashwood, & Bruns, 2012).
Ouchterlony gel diffusion (double diffusion, double dimension)

When an antigen solution is placed in a well / hole cut into an agar plate and the corresponding
antibody is placed in an adjoining well, they will diffuse radially from their respective wells toward one
another. When an optimal ratio of the two materials is reached, a visible line of precipitation is formed in
the gel. The test provided qualitative identification of individual proteins.

Radial immunodiffusion (RID) [single diffusion, double dimension]

RID testing is used to obtain a quantitative level of an antigen when agar containing an
appropriate antiserum (antibody is “fixed” in the agar) is poured in plates and carefully cut circular wells
are removed. A series of three (3) standards containing known concentration of antigen are placed in
three of the wells, while control and “unknown” samples are placed in other wells. As the antigen diffuses
radially, a ring of precipitate will form in the area of optimal antigen - antibody concentration. A standard
curve is prepared using the ring diameters of the standards versus their concentrations. This curve is then
used to determine the concentration of the control and unknown samples.

12
 The relationship of immunoprecipitation ring size and antigen concentration was described in the
mid- 1960s by Mancini et al. Mancini observed that the precipitin ring diameter stopped increasing at the
point where diffusible antigen had been reduced and antigen-antibody complexing had attained
equivalence. At equivalence, or endpoint, a linear relationship exists between the antigen concentrations
and their corresponding ring diameters squared. A reference curve on linear graph paper is constructed
by plotting the square of the precipitin ring diameters of reference sera against their corresponding
concentrations.

Materials:
• Lab paper representing the RID rings of standards, controls, and patients
• Ruler capable of providing accurate measurement in millimeters
• Calculator
• Linear graph paper

Procedure:
1. For each diagram of a precipitin ring below, measure the diameter in millimeters and record
the results in the space provided.

2. Square the diameter of the precipitin ring and record in the space provided.
3. Use the graphing paper provided, create a standard curve of the results of the standards provided.
4. Read the results of the controls and patients from the standard curve.
5. Record the results in the spaces provided using correct units.
6. Using product insert or other reference information, determine acceptability of the controls. Indicate
your evaluation of each control by circling YES or NO.

13
7. Using textbook or other references, evaluate each patient result as to being low, normal, or high.

Results:

Record your results in the format provided below:

Sample & standards (mg/dl) Diameter (mm) Diameter2 (mm2)

200
400
800
1600
Sample A
Sample B

Place the photographs of the standard curve:

Questions:

1. What do the circular precipitin rings represent?

2. Why do the ring sizes change until equilibrium is reached?
3. Predict the results if a very low concentration of antigen were loaded into a well. What would
happen if not enough antibody was incorporated into the agarose?

14
Practical 4
Title: Dot enzyme linked immunosorbent assay (Dot ELISA)

Objective:
After completing the practical, you will be able:
1. To learn the technique of Dot ELISA for the detection of an antigen

Introduction
Enzyme linked immunosorbent assay (ELISA) is a sensitive immunological technique
commonly used to detect the presence of a specific antigen (Ag) or antibody (Ab) in a biological
sample. ELISA is extensively used for diagnostic purposes. It requires an immobilized antigen/antibody
bound to a solid support (e.g. microtitre plate or membrane).

There are different types of ELISAs for the detection of a protein of interest in a given
sample. One of the most common ELISA is dot ELISA which can visually detect the presence of an
antigen very quickly. The nitrocellulose dot technique was first developed for screening large number
of hybridoma antibodies in 1983.

Principle
Dot ELISA, a qualitative ELISA test, can be performed very quickly with the end
detection done visually (Li, Huang, Zhang, Ye, & Li, 2017). Because of its relative speed and simplicity,
the dot ELISA is an attractive alternative to standard ELISA. In Dot-ELISA, small volumes of
antibodies are immobilized on a protein binding membrane (Nitrocellulose) and the other antibody is
linked to an enzyme Horse radish peroxidase (HRP). The test antigen at first reacts with the
immobilized antibody and later with the enzyme-linked antibody. The amount of enzyme linked
antibody bound is determined by incubating the strip with an appropriate substrate (Hydrogen
peroxide, H2O2) and a chromogen [Tetramethylbenzidine (TMB)]. HRP acts on H2O2 to release
nascent oxygen, which oxidizes TMB to TMB oxide, which gives, a blue colored product. The latter
precipitates onto the strip in the area of enzyme activity and appears as a colored dot, hence the
name Dot-ELISA. The results can be visualized in naked eye. The enzyme activity is indicated by
intensity of the dot, which is directly proportional to the antigen concentration. Figure 2 describes the
principle of Dot ELISA.

15
 Figure 2: Principle of Dot ELISA

Materials
• HiPer® Dot ELISA Teaching Kit
• Glassware: Test tubes
• Distilled water,
• Micropipette and tips

Procedure:
1. Pour agarose solution containing the antiserum on to a grease free glass plate.
2. Take 2 ml of 1× Assay Buffer in a test tube and add 2 µL of the test serum sample. Mix thoroughly
by pipetting. Insert a Dot-ELISA strip into the tube.
3. Incubate the tube at room temperature for 20 minutes. Discard the solution.
4. Wash the strip two times by dipping it in 2 mL of 1× Assay Buffer for about 5 minutes each. Replace
the buffer each time.
5. Take 2 ml of 1×Assay Buffer in a fresh test tube, add 2 µL of HRP conjugated antibody to it.
Mix thoroughly by pipetting. Dip the ELISA strip into it and allow the reaction to take
place for 20 minutes. Wash the strip as in step # 3 for two times.
6. In a collection tube (provided in the kit) take 1.3 ml of TMB/H2O2 and dip the ELISA strip into
this substrate solution.
7. Observe the strip after 5 - 10 minutes for the appearance of a blue spot.
8. Rinse the strip with distilled water.

Results and Observation:

Look for the appearance of blue dot as shown below:

Zone Spot
Positive
Negative
Test

16
Question:

1. Illustrate the various types of ELISA.

References

Ashiba, H., Fujimaki, M., Awazu, K., Fu, M., Ohki, Y., Tanaka, T., & Makishima, M. (2015).
Hemagglutination detection for blood typing based on waveguide-mode sensors. Sensing and Bio-
Sensing Research, 3, 59-64.

Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2012). Tietz textbook of clinical chemistry and molecular
diagnostics-e-book: Elsevier Health Sciences.

Li, M., Shi, Z., Fang, C., Gao, A., Li, C. M., & Yu, L. (2016). Versatile microfluidic complement fixation
test for disease biomarker detection. Analytica Chimica Acta, 916, 67-76.

Li, R., Huang, H., Zhang, X., Ye, S., & Li, Q. (2017). Monoclonal antibody based Dot-ELISA and indirect
fluorescence antibody technique for detecting Edwardsiella ictaluri infection in yellow catfish
(Pelteobagrus fulvidraco). Aquaculture and Fisheries, 2(5), 207-212.

17
Notes
LINCOLN UNIVERSITY COLLEGE
Wisma Lincoln, No. 12, 14, 16 & 18,
Jalan SS 6/12,47301 Petaling Jaya,
Selangor Darul Ehsan, Malaysia.
Tel.: +603-7806 3478 Fax: +603-7806 3479
Toll Free: 1-300-880-111
E-mail: [email protected]
Web.: www.lucp.net

www.lincoln.edu.my www.lucp.net