ENVIRONMENTAL HEALTH

EH 215 ENVIRONMENTAL MICROBIOLOGY

LABORATORY PRACTICAL MANUAL

FOR

STUDENTS

Written By:

Francie Mahap

Senior Tutor

Environmental Health

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 Table of Content

Content Pages

Introduction 3

Laboratory Safety Procedures 5

- House Keeping 6
- Personal Safety Rules 7
- Safety Procedures 8
- Safety Contract 9

Format of Writing up Laboratory Practical Report 10

Some Useful Laboratory Chemicals and their Preparations 11-12

Microscopy 13 - 23

PART A: MICROSCOPY TECHNIQUES 24 - 25

PART B: MICROBIOLOGICAL TECHNIQUES 26 - 32

PART C: MICROBES IN ACTION (APPLICATION) 33 - 37

PART D: FUNGI 38 - 47

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Introduction

One cannot expect to get a ‘feel’ for science by just reading about it. Hence most science
courses involve you in doing things. Laboratory is a place where facts studied in the lectures are
proven experimentally. Experiments not only give the first-hand information about facts but
also provide a better understanding and develop scientific attitude in you.

Besides offering the hands-on experience, science laboratory equipment teaches students how

to make a scientific argument. Conducting experiments, reviewing them closely, developing

logical reasoning, and responding to analytical comments, are some of the valuable skills that

help in preparing the next generation of scientists and/or medical/health professionals.

Work done with hands makes you more inquisitive about various facts of nature and allows you

to interact with your surrounding in a scientific way. In addition, your experiment work will be

important in deciding your continuous assessment marks.

The scientific method often involves a kind of trained or learned way of thinking about

evidences and facts. Gathering information involves a keen observation and accurate

measurement. Through the practical exercises you will learn to investigate and measure the

data and interpret the results. You will also learn how to critically assess the accuracy of your

results.

Laboratory experiences should also help students learn to work independently and

collaboratively, incorporate and critique the published work of others in their communications,

use scientific reasoning and appropriate laboratory techniques to define and solve

problems/challenges of life.

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Laboratory practicals should correlate closely with lectures and not be separate activities and all
students should have the opportunity to participate in laboratory
investigations/experimentations in a safe environment.

USE THIS MANUAL FOR EVERY PRACTICAL CLASS AND USE IT IN CONJUCTION WITH
LECTURE NOTES AND OTHER READING MATERIAL.

Also bring with you, a notebook, pencil, pen, eraser and a calculator.

ATTENDANCE AT ALL PRACTICAL CLASSES ARE STRONGLY RECOMMENDED. TESTS

AND OTHER ASSESSABLE WORK WILL BE SET FROM THE PRACTICAL WORK FROM
TIME TO TIME.

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Laboratory Safety and Handling

Inherent in many laboratory settings including medical laboratories is the potential for injury

and possible litigation. These issues can be avoided or reduced by knowing and applying of

safety procedures.

Accidents in the Laboratory can be prevented when we:

1. Stop to think!

2. Use common sense.

3. What will be the consequence of what I’d do next?

4. If in doubt, ask the instructor.

The purpose of this section is to highlight the basic safety requirements that must be observed

in laboratory settings when individuals are handling and/or conducting laboratory-based

practicals involving chemicals/reagents.

Working in a laboratory environment requires that the student observe certain housekeeping

procedures and safety precautions. These procedure and precautions are outlined below. The

student needs to review and follow the procedure so as to ensure the safety of himself/herself,

the instructor, fellow students, the equipment and the facilities. Safety in the lab demands that

lab directions be followed carefully. Please read these 'General Laboratory Practices' carefully

and be sure you understand each one. If you aren't sure, ASK!!!

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 a) Laboratory House Keeping Practices

1. Do not work in the laboratory in the absence of your instructor or his authorized
representative.
2. Each student is responsible for keeping the bench he/she uses clean. Benches must be
left clean and dry at the end of a laboratory practical.
3. All apparatus should be handled with care. If any accidental breakages occur they must
be reported immediately to the Lecturer.
4. No apparatus are to be taken out of the laboratory.
5. Do not touch chemicals or equipment that other classes may have left behind.
6. NO chemicals are to be flushed down a drain unless specifically instructed to do so by
the lab procedure. Wastes are to be poured into the appropriately labeled waste
container (e.g., solvent waste, halogenated solvent waste, etc.).
7. Do not mix any chemicals except as instructed. Do not do unauthorized experiments.
8. DO NOT mix wastes from different categories.
9. Clean up solid and liquid spills immediately, but only after checking with your laboratory
instructor about possible safety hazards.
10. Read the label on chemical bottles carefully. Ensure that you have the correct chemical.
11. Do not insert a pipette or medicine dropper into a stock bottle. Avoid contamination by
pouring a small quantity into a flask or beaker before taking a sample.
12. Take containers to the stock of chemicals. Do not bring stock chemicals to your
laboratory table.
13. Take no more of a chemical than an experiment requires.
14. Use special care when handling stoppers or tops of bottles so as not to pick up
contamination.
15. Never return an unused chemical to a stock bottle. Dispose it as waste.
16. Set up your glassware and apparatus away from the front edge of your laboratory bench.
17. Follow any other housekeeping, safety, or disposal rules given by your instructor.

Applying strict guidelines for handling, storage, and disposal of hazardous chemicals wastes is
imperative. Because of the wide variety of chemicals used in a teaching laboratory, it is a must
that all students follow proper disposal procedures so as to not pollute our environment and
groundwater supply. Willful violations of these rules can result in a zero being given for that lab.
Repeated willful violations can result in an "F" being given for the practical component of the
Unit/Course. Every effort has been made to minimize the hazardous chemicals used in the Lab.
All chemicals provided can be used safely by following correct procedures. However, any

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chemical can be dangerous if used improperly [Material Safety Data Sheets (MSDS) will be
provided for any chemical on request].

b) Laboratory Safety Rules

1. No food or drinks in the Lab.

2. No smoking or chewing in the lab.
3. Wear all safety equipment as required by the lab procedure or your instructor. Learn the
location of all lab safety equipment. Wear safety glasses or goggles at all times. DO NOT
WEAR CONTACT LENS AT ANY TIME. Students without footwear will not be allowed into
the laboratory.
4. Use fume hoods when required.
5. Tie long hair back to keep it out of flames or harmful liquids.
6. Wear shoes that cover all of your feet. No open toed shoes or sandals.
7. Do not taste any chemical.
8. Do not smell chemicals directly. Use your hand to waft the odor to your nose.
9. Do not pipette solutions by mouth. Use a rubber suction bulb or special pipette filler.
10. Handle glass tubing and thermometers with care.
11. Wash your hands before leaving the laboratory.

NB: In case of injury seek help from staff member.

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 c) Laboratory Safety Procedure

Safety is one of the concerns in any lab. Safety for humans is always a primary concern. The

location of safety equipment should be common knowledge to anyone working in the lab. In

order to become familiar with the safety equipment in the lab, make a map on the reverse side

of this sheet showing the location of the following safety items. Indicate the fire escape route

with an arrow.

1. Emergency shower and eye wash station

2. Fire extinguisher(s)

3. Exits from the room

4. Fire escape route

5. Fire alarm boxes

6. Container for broken glass

7. Electrical power cut off switch(es)

8. First aid box

9. Biohazardous Waste Container

Laboratory Safety Contract

After you have read the 'General Laboratory Safety Rules and turn it into your lab
instructor.

_________________________________________________________________________________________
I have read and understand the general lab practices and procedures and am familiar
with the location and operation of safety items in the lab.

________________________________ _________________________________
Printed Name Lab Instructor

________________________________ _________________________________
Signature Class Section

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Format of Writing up the Practical Experiments

It is expected that after completion of each laboratory practical sessions each student is to write
a report of what he/she did with interpretation of what they observed in each of the practical
sessions. This activity report is part of your academic Unit Assessment.

The format of writing and presenting the activity report is outlined below:

Abstract
This is a summary of what you did and importantly, what you found. It is usually one or two
paragraphs long. It should be a summary of the whole study, but only of the study.
Keep background information to the introduction and speculation for the discussion.

Introduction
Explain why you did this practical
 Provide subject background and
 Provide rational for conducting the practical

o Internal Referencing
The use of information from the lab manual or your text book must be
acknowledged in the text of your paper. Whenever you make a statement of
fact that is not a finding this study you should quote a reference for that fact:
eg: Proteins are broken down into short chain polypeptides by the enzyme
trypsin (Knox, Ladiges and Evans, 1994). The author’s surname (s) and the date
of publication only should appear in the text, next to the information you
obtained from that reference.
If you have made a direct quote from a book or paper, that quote should be in
inverted commas and the page number added to the reference eg: Knox et al
(1994, p. 398) state that “pepsinogen is activated to pepsin by either acid or
existing pepsin.”.

Procedures
Describe how you carried out the practical
 Step-by-step procedure (in past tense)

Results
Record your observations (draw/or describe what you observed)
 Describe what you observe as a result of your practical

Discussion
What do the results you obtained mean:
 Explain the results your obtained from your practical session
 Explain what the results mean or their implications

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  Make connection to life application

Quality Control/Recommendation:

1. Ensure correct procedures are followed as described in the practical handbook

 List activities that would ensure quality of the practical Safety Precautions

2. Ensure safety of yourself and others during the practical session

 List potential risks of physical, chemical and/or biological injuries during the practical
 Discuss ways to avoid the potential risks

Referencing
Do not plagiarize, or copy paste any one’s work.
This section only contains references to books or articles that were cited in report. So if
you referred to the text books as given in the example above, this section should contain a
complete reference to this source of information giving the authors surname, and initials,
the year of publication, the name of the book, the name of publishers and the place of
publication. See examples as follows:

Knox, B., Ladiges, P., and Evans, B. 1994. Biology. MacGraw Hill Book Company, Sydney

Similarly if you refer to this lab manual, the complete details about the lab manual must
also be in the reference list,

Mahap, F. 2017. Environmental Microbiology. Divine Word University Printery, Madang

Questions
You are required to answer the questions given. You will probably find that all of these
questions will answered as you write up the various sections of the report. If the
questions are not answered there then have a section under Questions are state your
answers there.

Cover Page
Ensure to record name, the title of the practical, practical Unit number, and the date of
practical conducted.

Make sure the name is clearly written on the cover sheet of your practical activity report
and submit to the relevant academic staff member.

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Some Useful Laboratory Chemicals and their Preparation

Many chemicals used in the Laboratory are solutions made as homogenous mixtures of two
or substances. The concentration of a solution is expressed in the units of weight, volume
or moles.
A Molar Solution is 1 gram molecular weight of a substance dissolved in 1 Litre of solution.

Example:
1M NaCl solution contains 58.5 g of NaCl in 1L of solution.
(Mol. Wt. of NaCl = 23 + 35.5 = 58.5 g )

Chemicals commonly used in Biology Lab Are:

1. Acid water
Dissolve 2 drops of conc. HCl in 100.0 mL of solution
2. Starch solution
Make a paste of 1.0 g starch in 5 mL of distilled water. Boil 95.0 mL distilled water
and add starch paste to it. Stir it and store.
3. Iodine solution
Put 2.0 g potassium iodide and 1.0 g iodine in 100.0 mL distilled water.
4. Benedict’s solution
Take 17.3 g of sodium citrate and 10.0 g of anhydrous sodium carbonate in 90 mL of
water till they dissolve. Filter the solution. Dissolve 1.7g of copper sulphate in
10mL of water and add it to the above solution and mix them well.
5. Fehling’s solution A
Dissolve 34.65g of copper sulphate in 500mL of distilled water.
6. Fehling’s solution B
Dissolve 175g of potassium hydroxide and 173g of potassium sodium tartarate in
500mL of distilled water.
7. Biuret Reagent
Dissolve 95mL of 3% copper sulphate solution in 1L of 10% potassium hydroxide
solution.
8. Millon’s Reagent
Dissolve 100g of mercury and 200mL of Nitric acid in 600mL of distilled water.
9. Ringer’s Solution
Take NaCl 6g, KCl 0.42g, CaCl2 0.24g, NaHCO3 0.1g in a beaker and dissolve in 1L of
distilled water.
10. Glycerine
Add 250 mL of glycerine to 250 mL of distilled water.

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 11. Buffer Solution
It is made by mixing M/15 Na2HPO4.12H2O and M/15 KH2PO4 in proper proportion.

a) M/15 Na2H.PO4.12 H2O (Disodium hydrogen phosphate)

Dissolve 23.88 g of Na2HPO4.12H2O in distilled water to make 1 L solution.
b) M/15 KH2PO4 (Potassium dihydrogen phosphate)
Dissolve 9.08 g of KH2PO4 in distilled water to make 1 L solution.

Required pH can be obtained by mixing; the above two solutions in the amount given
below:

pH M/15 Na2HPO4 (ml) M/15 KH2PO4 (ml)

5.2 1.0 99.0
5.4 3.0 97.0
5.6 5.0 95.0
5.8 7.8 92.2
6.0 12.0 88.0
6.2 18.5 81.5
6.4 26.5 73.5
6.6 37.5 62.5
6.8 50.0 50.0
7.0 61.1 38.9
7.2 71.5 28.5
7.4 80.4 19.6
7.6 86.8 13.2
7.8 91.4 8.6
8.0 94.5 5.5

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MICROSCOPY

The availability of Microscope has been the single most important tool used in the study of
microbiological material and organisms. It is essential that you become thoroughly familiar
with the instrument and how to set it up so that material can be examined to the best
advantage.

Although a variety of microscopes are used in the Microbiology laboratories they have a
similar structure and mode of operation; they will however, differ in detail.

PARTS OF THE MICROSCOPE

The microscope consists of:

1. A stand and base which incorporates a quadruple revolving nosepiece, a plain stage,
and an in-base illuminator.

2. A mechanical stage with coaxial right hand controls.

3. The optical system includes a Binocular observation tube inclined 45°, with x10
eyepiece fitted.

Ordinarily there are four (4) objectives fitted into the rotating nosepiece. These
are D Achromat x4 and x10 low power objectives, and D Achromat, spring loaded,
x40 and x100 (oil immersion) high power objectives.

4. The illumination apparatus consists of a halogen light source, a condenser and an iris
diaphragm.

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Figure A1 demonstrates the correct position of the observation tube to the operator. Do
not reverse it, thereby turning the microscope around. With constant use, you will become
accustomed to using the microscope correctly and with either hand. Reversing the
observation tube creates an additional problem of hindering the hand grip making handling
of microscope unsafe.

Figure A2: Anatomy of Microscope

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Taking Care of Microscope

1. The microscopes provided for your use are sturdily built, but are delicate adjusted.
Always handle the microscope with the care it deserves, and avoid abrupt motions
or any impact.

2. The stage, mirror, eyepieces and objectives must be kept clean, dry and free from
dust at all times. Lenses may be cleaned by wiping gently with lens tissue (not
Kleenex tissue or anything else). If strong acids or bases are being used, ensure all
traces of fluid are removed. Check with your Demonstrator for correct methods.
Once a lens is scratched or corroded it is useless. TAKE CARE.

3. Do not take the objective, eyepieces apart. Avoid removing the eyepieces from the
holders if possible. Dust in the eyepiece holders can be a problem.

4. Avoid applying undue force on any microscope mechanism:

 Rack and pinion should operate smoothly and easily.
 The nosepiece revolves and clicks into position with a light touch. Turn
it by holding the rim of the nosepiece, not the objectives.
 The iris diaphragm is a delicate structure, stop it down gradually.

5. Switch off the power and remove the glass slides or other material from the stage.

6. When lifting the microscope always hold it upright so that the eyepieces and mirror
do not fall off. Hold the microscope by the hand grip while supporting the base
with your free hand.

7. Report all problems to your demonstrator or to the technical staff. Most problems
are easily rectified on the spot or alternatively a replacement microscope can be
found for you. Do not put away microscope that are causing problems or are in an
unclean condition, and always remove slides or other material from the stage.
Remember: Other classes use these microscopes too.

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SETTING UP THE MICROSCOPE

The following steps should become routine every time the microscope is set up for use.

1. Place the microscope squarely in front of you on the bench with the front of the
microscope toward you.
2. Illumination is one of the most important factors for effective microscopy. The
resolving power of an objective is largely influenced by the correct adjustment of
the light source and the condenser. Either natural or artificial light may be used.
3. Place a specimen slide on the stage, securing it in place using the slide holder or
slide clips.
4. Adjust the condenser to its highest point.
5. Open the iris diaphragm as far as it will go (but see notes below).
6. Turn the revolving nosepiece until the low power, x10 objective is in the light path.
7. Course focus: Look down through the eyepiece(s) and turn the course focusing
control until the specimen is in focus.
8. The condenser is generally used in the top position for use with the x4 and x10
objectives, however it is recommended that the condenser be lowered slightly to
eliminate uneven field illumination.

When the condenser is correctly focused, light is concentrated in the plane of the
specimen being viewed. Lowering the condenser enables the viewer to observe near
transparent or unstained specimens in greater contrast. However, this is not the
best position for general microscopy, and you should re – position the condenser
when transparent conditions no longer prevail.

9. Eyepieces
These should be positioned correctly as this will enable the operator to use the
microscope for long periods of time without experiencing fatigue and eyestrain.
Operators who normally wear spectacles to read and draw should get used to using
the microscope with their glasses on.

Look through the eyepiece with both eyes. Hold the dovetail slides at each end of
the observation tube with both hands and slide literally in either direction until
perfect binocular vision is obtained. Note the value on the inter pupillary distance
scale.

Rotate the tube length adjustment ring on the right eyepiece to match your inter
pupillary distance setting which you obtained from the scale.

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 Cover the left eyepiece with a piece of paper and look at the image through the
left eyepiece. Rotate the tube length adjustment ring of the left eyepiece to focus
the specimen. Do not use the course and fine focus controls.

All ocular adjustments should now be complete and no further adjustments to the
eyepiece are required. Check by observing the specimen using both eyes and
eyepieces. You should now be able to change specimens easily and adjust the focus
with the coarse and fine focus knobs only.

10. Specimens
When placing a specimen slide on the stage it is important to observe the following:

a. A coverslip must always be on the specimen slide.

b. Swing the x10 objective only into the light path.
c. Place the specimen on the stage and focus with coarse and fine controls.
d. Change to high power if required (see below).
e. Before removing the specimen from the holder, swing the nosepiece to bring the
x10 objective back into the light path.
Do not remove a slide from the holder while the high power objectives are
in position.

11. High Power - Magnification

Focus the specimen with the low power (x10) objective making sure to center the
specimen. Turn the revolving nosepiece to bring x40 objective into the light path.
Adjust using the fine focus ONLY. The condenser should be in the highest position.
Increase the light intensity if required.

To use the x100 oil immersion objectives, focus on the specimen using
objectives:

a. Low power objective (x10) with coarse and fine focus adjustments.
b. High power objective (x40), using fine focus adjustments only. Make the
necessary light, condenser and diaphragm adjustments, and position the portion
of the specimen to be examined into the center of the field view.
c. Swing the x40 objective out of position and place a small drop of immersion oil
onto the coverslip.
d. Swing the x100 objective into the light path. Increase the light intensity if
required. Focus the specimen with fine focus only.

When examination is complete, wipe off the immersion oil from the objective using lens
cleaning tissue only. Clean the oil from the slide.

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TROUBLE SHOOTING

1. Image is blurred and cannot be focused.

 Lens dirty or wet – clean with lens cleaning tissue
 Dirty coverslip or slide – clean with lens cleaning tissue
 Dirty condenser or mirror – clean with lens cleaning tissue

2. No image
 Objective is out of alignment with body and draw tubes – align correctly
 The nosepiece is marked with a notch which should click into place –
make sure the nosepiece clicks into place
 Iris diaphragm closed right down – allow the diaphragm to be opened up
 Lamp switch off – lamp should be turned on

3. Object lost when switching from low to high power

 The object is alive and moves – do not attempt to kill it by applying
force!
 The object was not centered in the field of view before switching lenses
– center carefully and fix the position of the slide with clips as even the
vibration involved in changing lenses may move the slide. Do not search
under high power for lost objects as you may lift the slide and scratch
the lens. Go back to low power.

NOTES

Aperture Iris Diaphragm

An aperture iris diaphragm is provided on the condenser and is usually opened to match the
numerical aperture of the objective in use in order to achieve optimum objective
performance as depth of focus, image contrast and resolution.

However, since microscope specimens generally are low in contrast, their image lacks
contrast if the objective is used with its full numerical aperture. Therefore it is often
preferable to stop down the aperture diaphragm slightly more than indicated by the
objective numerical aperture.

An aperture setting at 70 – 80% of the maximum possible is recommended.

To observe the numerical aperture, remove one of the nosepieces and observe the
aperture as you adjust the diaphragm with the aperture diaphragm lever. This should be
done each time you change objective lenses.

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Observation Tube
The observation tube can be rotated to allow other people to observe the specimen being
examined. However, the tube should never be rotated too far in any one direction as the
eyepieces become out of focus as the tube is rotated. Ensure that the clamping screw is
secure before putting away the microscope at the end of each practical session.

Tension adjustment of coarse focus adjustment knobs

There is a tension adjustment ring next to the right hand course focus adjustment knob.
With this device, the tension of the coarse focus is freely adjustable for either heavy or
light movement, depending on the preference of the operator. However, do not loosen the
ring too much, as this may cause the stage to drop or the fine focus knob to slip. Be
careful not to rotate the right and left coarse focus adjustment knobs in the opposite
directions simultaneously.

Pre – focusing lever

This lever is provided to prevent possible contact between specimens and objectives as
well as to simplify coarse focusing. The lever is locked after coarse focus has been
accomplished. This prevents further upwards travel of the stage by means of the course
adjustment knobs and provides a limiting stop if the stage is lowered and then raised again.
The pre – focusing lever does not restrict fine focusing.

Once the pre – focusing lever is set there is no need to reset the lever for further
examination. In fact, this leer is usually set by laboratory staff when the microscopes are
serviced and it is preferred that students do not make further adjustments unless it is
essential for their work.

MAGNIFICATION
The magnification of most objectives and eyepiece is engraved on them.

On the eyepiece, the marking can be found on the top edge or on the smooth cylinder
which fits inside the body tube. On the objectives, the magnification is engraved on the
side of the cylinder. The marking ‘x10’ means that the element so marked yields an image
ten times larger in each dimension than the object being viewed.

The total magnification for any combination of objective and eyepiece can be computed
simply as the product of the magnification of each element.

19
Preparation of Biological Material for Examination with the Optical Microscope

Biological materials to be examined with the optical microscope require special

preparation. Generally, the observations are made on dead tissues. However, some
materials like cells in tissue culture, some free cells as blood cells, and some small
organisms can be studied while they are alive.

Following steps are to be followed for the preparation of material for examination under
the microscope.

1. Embedding
2. Sectioning
3. Fixation
4. Staining
5. Mounting
6. Labeling

Embedding and Sectioning

Biological materials that are either too soft or small enough to be sectioned without
support must be embedded before cutting in slightly harder materials such as ice or wax
to make them rigid.
Sections are usually cut in a particular plane. Following types of sections are commonly
made:
1. Transverse Section (T.S)
o At right angles to the longitudinal axis (similar to vertical section or V.S)
2. Longitudinal Section (L.S)
o Section parallel to the longitudinal axis.
3. Radial Longitudinal Section (R.S)
o Parallel to the longitudinal axis and in radial plane (common in wooden logs)
4. Tangential Longitudinal Section (T.L.S)
o Parallel to the longitudinal axis and along tangent (common to wooden logs)
5. Sagittal Section
o Median longitudinal section

20
 Fixation
There are certain chemicals that kill and fix cells and tissues in such a manner that
morphological organization and chemical composition of the cell remains unaltered up the
greatest extent. These chemicals are called Fixatives. Fixation can be brought about
either by use of chemicals or just cooling.

A fixative performs the following functions:

i) Prevents bacterial decay and autolysis of the cell.
ii) Makes the components of cell insoluble.
iii) Reduces the cell distortion and shrinkage.
iv) Increases the visibility of different cellular components.
v) Prepares the cell for staining.

List of some common fixatives, their composition and cytological studies associated with is
given below:

Fixative Composition Cytological Study

Acetic acid This is made from 0.3% to 50% solution. Is used for fixing nucleus and
For 50% sol. add 50ml of acetic acid. chromosomes.

Bouin’s solution It has got picric acid, 40% formaldehyde Fixes chromosomes, precipitates
and glacial acetic acid in 5:5:1 ratio. the proteins and causes little
shrinkages of cell.

Cornoy’s solution It is made up of ethanol and acetic acid in Fixes nucleus and chromosomes.
3:1 ratio.

F.A.A It contains ethyl alcohol, formalin and Fixes polysaccharides

glacial acetic acid in 9:1:5 ratio. nucleoproteins. It is a good
preservative for cells and tissues.

Formalin 40% 40ml of formaldehyde mixed in with 60ml It is good fixative and
water makes 40% formalin preservative for plant and animal
specimens.

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 Staining

The process of colouring of cells, tissues or animal and plant bodies by certain organic and
inorganic dyes is known as staining. Stains could be acidic, basic or neutral. The action of
dyes or stain on a particular material usually depends upon its chemical nature, the pH
value of the fixative used and chemical selectivity of the stain to the material. For
example, chromosomes are stained by basic stains like safranine, acetocarmine, feulgen,
etc. Cytoplasm, mitochondria and protein are stained by acidic dyes like Eosin, Janus
green B, Methylene Blue, etc.

List of commonly used stain is give below:

Stain Composition Result

Safranine 2.5% solution of safranine is dissolved Lignified, cutinized tissues,

in 100mL of 90% alcohol.

Acetocarmine 45mL glacial acetic acid is mixed with 55mL Chromosomes stain in dark red.
of distilled water and boiled 2g carmine Cytoplasm remains unstained.
powder is added to it and filtered.

Methylene blue It is mixture of 0.3g methylene blue stain In plant and animal temporary
and 30mL of 95% ethanol and 100mL of D.H 2O. preparation cytoplasm stains blue
and nucleus dark blue.

Eosin Aqueous solution contains 1g eosin in 100mL of Animal’s tissues stain red.
distilled water.

Leishman stain 15g of Leishman’s powder is dissolve in Specially for blood – R.B.C stain
100mL of methyl alcohol. Pink nuclei of W.B.C stain blue and
Platelets stain purple.

Crystal violet Dissolve 14g of crystal violet dye in 100mL Bacteria and protozoan stain
Of 95% ethanol. Filter and store it. violet.

22
 Mounting a Specimen

To Mount a Specimen for Examination under the Compound Microscope

The specimens must be very thin, so that in most cases a section out with a razor blade is
used. The specimen is placed on a microscope slide in a drop of water, 20% glycerol or
other mountant and covered with a coverslip. The coverslip is necessary for the optical
system of the microscope and also to prevent the mountant from damaging the objective
lens.

Take care not to use too much mounting fluid; the correct amount should form a thin film
under the coverslip but not ooze out at the edges. (It if does, gently soak up the excess
with a small piece of blotting paper.) Also take care not to trap air bubbles under the
coverslip; they can obscure the specimen. Gentle application of the coverslip is important;
the recommended method is shown below:

needle

specimen

glass slide coverslip

Figure A1: Preparing a Slide for microscope viewing

23
PART A. MICROSCOPY TECHNIQUES

Experiment 1: Viewing of Prepared Slides - Malarial Slide

Instructions:
In this exercise, choose a microscope, make sure you have read on how to use the
microscope, which was set out for use. Using the instructions given in this manual, you are
required to view the slide accordingly.

Experiment 2: Preparation of Wet Mount

Practical 1.Preparation of Wet Mount of Onion cell from the Epidermal tissue of an Onion Bulb

Instructions:
Peel off a small piece of epidermal tissue from the onion as shown in the diagram below:

4.
5. Place the peeled epidermis on a watch glass containing 2 drops of distilled water.
6. Using a toothpick/brush transfer the epidermis onto a drop of safranine on a
separate watch glass.
7. Transfer the epidermis to the watch glass containing distilled water to remove
excess stain.
8. Using a dropper place 3 drops of glycerine on a dry glass slide.
9. Using a toothpick transfer the epidermis to the glass slide containing the glycerine.
10. Place the cover slip at 45° and cover the epidermis to avoid bubbles.
11. Using paper tower soak excess liquid.

24
Practical 2: Making a Wet Mount of Animal Cell

Instructions:
You will make a slide with your cheek epithelial cells.

1. Use a clean tooth pick to gently scrape the inside of your cheek, making sure that
you do not scratch the skin tissue otherwise it would hurt.
2. Put the scrapings on a slide, add a tiny droplet of saline solution to separate the
cells, so they are not in a lump.
3. Add a drop of methylene blue and hold a cover slip by its edge on the water film,
gently lower it to cover the epithelial scrapings.

(Note: if you use too much water or stain you may wash off all the cells from the slide).
Examine the slide as before and draw the cells.
Observe the some of the differences between plant and animal cells.

25
PART B. MICROBIOLOGICAL TECHNIQUES

Introduction
Micro – organisms include the prokaryotic Bacteria, Viruses and single celled eukaryotes
and their relatively simple multicellular relatives. Being microscopic in size, they are
invisible to the unaided eye. However, their colonies when grown on solid media, after a
day or two, are large enough to be seen easily. The size, shape, texture and color of these
colonies provide clues to identify bacteria. Further identification can be done by
microscopic examination of bacteria after fixing and staining them. Bergey’s Manual
contains a key for identifying bacteria.

Bacteria can be grown in the Lab by culturing them in Petri dishes or test tubes containing
liquid or solid media of known composition. The media or the apparatus is sterilized before
use to avoid growth of unwanted microbes. This is done by autoclaving (pressure cooking
at 120 °C). This kills the endorespores of bacteria which can survive the boiling
temperatures.

Though the composition of culture medium varies with the organism being cultivated, all
media should meet certain requirements. All must contain water, carbon source, a nitrogen
source, minerals and a source of energy. Some organisms need vitamins for growth.
Buffers are often added to media to maintain a suitable pH (optimum pH 6 – 8). The
common media used in the lab are – Malt Extract Agar (MEA) or the Nutrient Agar (NA)
or Eosin Methylene Blue Agar (EMB).

Precautions to be taken while working with microbes:

As some of the microbes are pathogenic causing diseases in the host organisms, it is
important that the apparatus and the place is sterilized thoroughly before use. This is
done by dipping the equipment or wiping the surface with a cotton swab dipped in 70% or
95% alcohol. (Make sure that you sterilize the equipment and your hands after use.)

26
Experiment 1. Growing cultures of Bacteria using Different Agents

New cultures are grown by transferring a small portion of an old colony (Inoculum), to a
fresh, sterile medium. A sterile inoculating loop or needle is used to transfer microbes
from a liquid culture. The culture plates or tubes are then covered by a sterile cap to
prevent contamination.
Two methods commonly in use are:
i) Streak – Plate Method
The loop with inoculum is drawn over the agar plate making streaks.
ii) Spread – Plate Method
Plate is inoculated by spreading the inoculum over the surface of the medium.
Note: In any case the inoculum should not be pierced through the Agar medium. It should
be very gently touched over the surface of agar medium.

Materials Required:

- prepared agar plates in multiples of six,

- inoculating loop or glass rod,
- 70% alcohol,
- Drain water
- DWU Playing Field soil sample

Procedure:
1. Each group should pick six (6) agar plates of one type of media.
Group 1 – Nutrient Agar
Group 2 – Malt Extract Agar
Group 3 – Blood Based Agar
Group 4 – Eosin Methylene Blue Agar
Later the results could be compared for all the agars used.

2. Label the plates as A, B, C, D, E, F and G.

3. Give the following treatment to each plate.

Plate A. CONTROL
Leave the plate as it is, without opening it. DO NOT THIS PLATE.

Plate B. MICROBES IN AIR

To catch the micro – organisms in air, open the agar plate, remove the lid and
expose it to the air outside the laboratory for 2 – 3 minutes. Close the lid and
bring it inside.

27
 Plate C. MICROBES IN SEWERAGE DRAINS
Place the sterilized loop into the sample to get a drop of drain water on the already
sterilized loop and make a long zigzag streaks on the agar with it (streak plate
method). If you are using a bent glass rod then you could spread the drop on agar
(spread plate method). Close the lid after inoculation.

Plate D MICROBES IN SOIL (FIELD)

Get a pinch of fine soil from Divine Word University Playing Field and sprinkle a
small amount on the agar plate. Close the lid.

Plate E. MICROBES ON YOUR HAND

Place your fingers gently on the surface of agar on plate C. Close the lid and put
the plate aside. Make sure that the finger does touch the surface of the agar but
not pierce through it.

Plate F. MICROBES ON YOUR HAND AFTER USE OF BACTERIOCIDAL

SOLUTION
Wash your hand thoroughly with bacteriocidal solution provided and repeat the
treatment as for C.

Once you have inoculated all the plates, seal the plates tightly with cellotape and put them
in the oven set at optimum temperature for the growth of microbes (between 30 – 40 C).
After two (2) to three (3) days, you observe the plates for the growth of colonies.

OBSERVATIONS:

Observe the colonies for following characteristics:

1. Size, color and elevation (whether flat or raised) of isolated colonies.

2. Shape of the margin or edge of colonies whether smooth, lobed, with hair – like or
root – like projections, regular or irregular.

28
COLONY DESCRIPTION CHART

Note
Escherichia coli is a universal intestinal inhabitant of human and other animals; its
presence in water indicates recent contamination with raw sewage. Colonies of E. coli are
usually dark with metallic sheen.

Colonies of Aerobacter aerogenes another normally non – pathogenic inhabitant of

intestine look pink on EMB agar.

29
Fungal colonies look like cottony structures on agar plates.

Tabulate your results as shown:

Plate Label Characteristic of Colonies

Nutrient Agar Malt Extract Agar or
EMB Agar

(You may observe the colonies of more than one microbe, you should write about all of
them. If need be, increase the size of boxes.)

Questions
1. There should not be any growth on the control plate. Give reasons for your answer.
2. If there is any growth on the control plate, suggests reasons for it.
3. Is there any difference in the type of microbes that grow on different media? If
so, give reasons for the same.
4. Comment on the importance of type of media used for the growth of different
microbes.
5. How do the bacteriocidal solutions work?
6. Did you isolate any E. coli colonies from the plate? If so which plate was it? What
kind of pollution does this indicate?

30
Experiment 2. Preparing Agar Plates with POUR PLATE TECHNIQUE

Agar is an ideal solid medium for many bacteriological purposes. It cannot be digested
except by a few marine species. It does not melt until its temperature rises to 100 °C and
does not solidify until cooled to 40 °C. Therefore, it can be used both in solid and liquid
form. Nutrients are added to it for the growth of microbes. The agar medium provided to
you is the Nutrient Agar which contains Nutrient Broth (mixture of peptone and meat
extract) with agar.

Materials Required:
Molten Nutrient Agar medium, Petri dishes.

Procedure:
1. Sterilize your bench by wiping surface (where you need to work) with a cotton swab
dipped in 70% Alcohol.
2. Collect about 15mL of molten agar in a measuring cylinder or a test tube.
3. Pick up a sterile petri dish provided. Open the lid and pour molten Nutrient Agar
into it. Without over – exposing the agar to air half close the lid. Make sure that
you allow some space for the water vapor to escape from the hot agar, otherwise
the water vapors will condense on the lid spoiling your plate.
4. After the agar has cooled for some time, further close the lid still allowing a very
small space for water vapors to escape.
5. Once the agar has cooled and set, close the lid firmly using sticky tape. Leave the
plate in a corner and check it after 3 – 4 days.

In practice plates are autoclaved once the agar has set to sterilize them.

NOTE: Do not rush for closing the lid until all water vapors have escaped.

31
QUESTION

Are you successful in making a good plate? If no, suggest what could have gone wrong with
your experimentation.
(Indication of a preparation of a good plate is where there is no growth of microbes.)

Experiment 3 Making a Bacterial Smear Slide

Bacterial smear slides are prepared for observing bacteria under the microscope. The
smear is then stained for easy identification of bacteria. Since bacteria are minute
organisms they should be fixed on the slide before staining (fixing of bacteria preserves
them with a minimum distortion of cellular components and adheres bacteria to the slide).
Bacteria cells are usually fixed by heating by passing it quickly over Bunsen burner flame
three to four times. This fixes the smear to the slide.
The fixed smear is now flooded with a stain solution and then rinsed with distilled water.
The common stains used are methylene blue and crystal violet. This is simple staining.
Sometimes more than one stain is used after the other. This is called differential
staining. For e.g. Gram Stain which is used to differentiate between Gram Positive and
Gram Negative bacteria uses three different dyes – Violet dyes, Iodine and a Red dye.

Materials Required:
Grease free slides which have been washed in pyroneg, stored in jar or alcohol, Bacterial
cultures, inoculating loops, methylene blue stain.

Procedure:
1. Remove a slide from the jar with a forcep, pass it over the flame once and place on
a sheet of paper towel.
2. Sterilize a loop and pick up a bit of bacterial culture on it. DO NOT scratch
through the agar medium while picking culture.
3. Smear the culture on the slide so that it forms a thin, even layer on the slide. The
smear looks like an opaque film on the slide.
4. Fix the slide by passing gently over the flame 3 – 4 times.
5. Place the fixed slide on the rack over the sink and flood it with Leoffler’s
Methylene Blue stain. Leave the slide for a few minutes to let it get stained
6. Wash the slide under a very gentle running tap, blot dry it over the paper towel and
observe under the microscope, first with low power and then with high power lens.

Observations:
Draw the type of bacteria you seen under the microscope.

32
PART C. MICROBES IN ACTION (APPLICATION)

Microbes cause changes in foods and other materials when they act on them. Some of
these changes are harmful while others are useful. The diversified metabolic capabilities
of microbes have been exploited to use them for practical purposes. This has led to a
branch of science called Industrial Microbiology.
Now we use microbes to make desirable changes to foods or produce certain chemicals
some of which can be used against their pathogenic activities.
Microbes also play a very important role in Geochemical cycles enabling an exchange of
elements between organisms and their environment. Microbes in the soil and water are
largely responsible for digestion of complex organic molecules trapped in the bodies of
living organisms to convert them to inorganic compounds. In this practical, an attempt is
made to study some activities of microbes.

Experiment 1: Microbes in the Root Nodules of Leguminous Plants

Microbes live in a symbiotic relationship (living together) with the roots of leguminous
plants. The relationship is mutualistic one, where both the organisms benefit from the
presence of each other. Lumps on the roots of legumes called nodules are home to
bacteria which fix nitrogen from the air which is used by the host. The plant reciprocates
with a steady supply of sugar and other organic nutrients to bacteria. The most common
Symbiotic Nitrogen fixing microbes are Rhizobium bacteria and blue green algae.
Alternation of leguminous crops with non – leguminous crops enriches the soil with
nitrogen. Other bacteria like Clostridium, Azotobacter, Rhodospirillum, Desulfovibrio can
also fix nitrogen but they are non – symbiotic.

Materials Required:
Roots of leguminous plants, razor blades, slides, coverslips, watch glass, glycerine and
methylene blue stain.

Procedure:
1. Select a root with the nodules.
2. Cut thin sections of the nodules and place one on the slide. Stain it with methylene
blue.
3. Wait for two (2) minutes and then wash away the extra stain.
4. Pour a drop of glycerine on the section and cover with a cover slip.
5. Observe under the microscope.

Observations:
You may be able to see the cells of root studded with cylindrical bacteria.

33
Questions:
1. What does the plant gain from this association?
2. How does the bacteria gain from the relationship?
3. What does the mutualistic relationship mean?
4. Draw the Nitrogen cycle.
5. Name the three steps in nitrogen cycle where bacteria are specifically involved.
Also name the bacteria responsible for each step.

Experiment 2: Testing for Milk Quality against Spoilage by Microbes.

Milk contains Lactose, Casein (milk protein), fats and vitamins and has a pH of 6.8. It is an
excellent food both for us and microbes.
Non – pathogenic species of Streptococcus, Lactobacilllus are among the normal flora of
milk. However, the pathogenic flora of milk spread diseases like Tuberculosis, Q fever,
typhoid fever, Diphtheria, and scarlet fever.
Growth of micro – organisms in milk which is not refrigerated is so rapid that it can reach
million per mL within 24 hours.
To prevent quick spoilage of milk, the milk is heated to 70 °C or 161 °F for 15 seconds –
PASTEURISATION.
This treatment kills all the pathogenic bacteria in the milk but does not sterilize the milk
completely. Therefore, the milk should be refrigerated before or after pasteurization to
slow down the growth of remaining bacteria. On the other hand, UHT treatment (Ultra
High Temperature) makes the milk virtually sterile and the milk does not require
refrigeration until the packet is opened.
In order to test milk for quality, methylene blue is added to the milk. Blue color indicates
good quality. Slowly due to microbial activity in the milk the dye is reduced which makes it
colorless. White colour with coagulation indicates poor quality milk. Decolorization time
for good quality milk is more than eight (8) hours at 35 °C.

Materials Required:
Pasteurised and UHT milk both refrigerated and non – refrigerated, test tubes bath at 35
C, methylene blue.

Procedure:
1. Use a separate sterile pipette for each milk sample.
2. Put 10mL of each type of milk in four separate test tubes and label them
accordingly. Make sure that you do not mix the pipettes used or the samples of the
milk which could affect your results.
3. Add 1mL of methylene blue to each test tube and mix it well.
4. Place the test tubes in a water bath at 35 °C.

34
Observations

Observe the test tubes for a change in colour and note down the time for the colour
change. Tabulate your results as shown.

Results:

Milk Sample
Observation
Refrigerated Non – Refrigerated
Pasteurised UHT Pasteurised UHT
Colour of the
dye in the
beginning.
Time taken to
decolourize.

Q6. Interpret the results in terms of quality of milk of the two samples.

Experiment 3 Sensitivity of Bacteria to different Antibiotics

Antibiotics are the chemical compounds that are produced by organisms and can be used to
inhibit the growth of other organisms. Most useful antibiotics are produced by micro –
organisms, particularly by three genera; Penicillium, Steptomyces, and Bacillus. Some
antibiotics are more economically produced synthetically nowadays.

There are other chemicals which are not antibiotics, but can be used effectively against
pathogens for the treatment of diseases, such sulfonamides, nitrofurans, and isoniazid.
These do not occur naturally.

Most antibiotics interfere with bacterial cell wall synthesis thus inhabiting growth,
particularly in Gram positive bacteria. Cell walls of many Gram negative bacteria are much
thicker and difficult to invade which makes these bacteria more pathogenic.
Antibiotics produced by Streptomyces interfere with protein synthesis of bacteria thus
stopping their growth.
The potency of antibiotics is tested by Assays Method.
In microbiology assays, the microbes are exposed to various dilutions of the antibiotic to
be tested; the amount of growth in each of the dilutions gives a measure of the potency of
the antibiotic.

35
In this exercise, you would compare the sensitivity of a particular bacteria towards
antibiotics. You are provided with sterile paper discs, each impregnated with a coded
antibiotic. These discs are placed on a culture of Bacillus subtilis. If the growth of
bacteria is inhibited, the bacteria is sensitive to the antibiotic.
Although the size of the zone of inhibition is used to indicate the relative sensitivity of
bacteria to different antibiotics, it is not an accurate measurement. There are many
factors which together determine the activity of antibiotic on a particular bacterium.

Tabulate your results as shown:

Antibiotic Sensitive Moderately Resistant

Resistant
Amphicillin (PN)
Chloramphenicol (C )
Cloxacillin (OB)
Erythromycin (E)
Novobiocin (NV)
Penicillin G (P)
Streptomycin (D)
Tetracycline (TE)
Methicillin (CB)
Suphafurazole (G)

Questions:

Say why it is necessary to use different antibiotics to combat and control different
diseases.

Experiment 4 Fermentation of Sugars by Bacteria

Many microbes can ferment sugars in anaerobic conditions. The sugar is first oxidized to
pyruvic acid by the removal of hydrogen. The hydrogen is later used to reduce pyruvic
acid to various organic compounds. The products of fermentation are often alcohols or the
organic acids. The carbon dioxide may or may not be produced. The release of acids and
gases can sometimes be used like glucose, lactose and sucrose. The sugars are then
inoculated with bacteria from the family Enteriobacteriaceae (anaerobic bacteria which
inhabit human intestine). If an acid is produced there is a colour change of the sugar
solution. If gas is also produced it displaces the medium in the inverted Durham tubes in
the test tubes containing sugars.

36
Materials Required:

Colour coded sugar samples with inverted Durham tubes, inoculum, inoculating loops.

Procedure:

1. Inoculate the colour coded sugars with the inoculum using the inoculating loops.
2. Leave one uninoculated set for the control.
3. Observe the change in colour of the sugar solution or the accumulation of gas in the
Durham tubes, after 24 hours of inoculating.
4. Tabulate your results.

37
PART D. FUNGI

Fungi belong to kingdom Fungi and can be clearly distinguished from other four kingdoms of
life: Animalia (animals), Plantae (plants, including algae), Monera (including bacteria) and
Protista (including amebae) by a combination of the following characteristics:

1. Fungi contain membrane-bound nucleus (like plants and animals but unlike bacteria),
so they are eukaryotes (Greek Eu = true;karyon = nucleus).
2. They contain cell wall made of chitin (unlike plant cells with cellulose walls and animal
cells, which have no cell wall).
3. They have no chlorophyll (like plants and algae have), so they do not produce food by
photosynthesis, but they have to obtain it from organic matter from their
environment, so they are heterotrophes (Greek heteros = another; trophe = nutrition).
4. They do not ingest food, but secrete enzymes, which digest food outside of their bodies,
and then absorb obtained nutrients.
5. Multi-cellular fungi grow by extension of thin, tubular threads called hyphae through
which they absorb nutrients. Hyphae may branch and form mycelium.
6. Fungi reproduce by sexual or asexual spores (which are usually spread by air), or by
budding.

Kingdom fungi includes five phyla (classes) or divisions (1):

 Chytridiomycota (Greek chytra = pipkin) or chytrids, which mainly live in fresh and
seawater
 Zygomycota (Greek zygon = yoke, bond); example: Rhizopus stolonifer or black bread
mold
 Glomeromycota (Greek glomus = ball shapped mass) or soil fungi are connected to roots
of some plants
 Ascomycota (Greek askos = sac) or sac fungi; examples: Saccharomyces cerevisiae or
baker’s yeast, colored molds on foods, Penicillium, morels, truffles, yeasts. Lichens are
symbiotic associations between Ascomycetes and certain green algae.
 Basidiomycota or club fungi; examples: mushrooms, puffballs; some species break down
wook and some cause plant diseases like rusts and smuts

Fungi included all organisms that look like plants but lack chlorophyll, therefore feed
heterotrophically like animals. Most fungi are saprophytes and help recycle the nutrients
in the environment and produce some economically important products. However, parasitic
fungi cause considerable damage to agricultural crops and forest trees. On the other
hand, the mutually symbiotic relationship of the fungi as lichens and mycorrhiza is of great
importance to the ecosystems.
Fungi are classified into:

38
1. Myxomycetes

2. Oomycetes

3. Phycomycetes

39
 4. Ascomycetes

5. Basidiomycetes

True fungi have cell wall made of polymer Chitin (such as exoskeleton of insects).
However, the cell wall of Myxomycetes and Oomycetes cells is made up of cellulose and
they behave more like amoeba. These fungi engulf food like other parasites but produce
fruiting bodies like fungi.
Therefore, some scientists group them under Kingdom Protista. They are not true fungi.

40
NOT TRUE FUNGI
Myxomycetes – slime moulds
Oomycetes – water moulds

Observe the water mould Seprolegnia growing on a dead insect.

Does the Mycelium look like any other fungus? ________________

Most of the life cycle, these fungi behave like Amoebas but produce brightly pigmented
fruiting bodies like fungi.

Do you observe fruiting bodies on the fungal mycelium? __________________

Oomycetes are recognized by the oogonium and the oospores they produce.
Prepare a slide of Seprolegnia to look for Oogonium and sperm nuclei attaching to it for
fertilization.
Otherwise observe a prepared slide.

True Fungi

A. Phycomycetes

Most members of this group are saprophytes. Common examples are Mucor (black mould)
and Rhizopus (bread mould).

The distinguishing characteristics of Phycomycetes are:

1. Non – septae hyphae

2. Produce asexual spores in fruiting bodies called sporangia that look like black pin –
heads.
Use water to mount a few sporangium with stalks on a slide and observe under a
microscope. Try to differentiate between the sporangium of two.
Sporangium of Rhizopus and Mucor will look like this:

41
 3. Phycomycetes mostly reproduce by asexual reproduction. During unfavorable
conditions only the sexual reproduction takes place forming zygosporangium that
later produce zygospores.
The life cycle of Phycomycetes is dominated by Haploid stages.
Observe the Rhizopus culture provided to locate the zygosporangium.

Procedure:
1. Instructor will inoculate (+) and (-) strains of bread mold onto the agar.
2. Where the two strains will meet, gametes fuse, producing a line of pigmented
3. Examine the petri dish with a dissecting microscope.

42
 B. Ascomycetes

Observe different specimens of Ascomycetes provided and familiarize yourself with the
different types of fruiting bodies they produce. The members of this group are both
Saprophytes and Parasites causing serious plant diseases

1. The hyphae are much finer that Phycomycetes and have cross – walled in them
(septa).

Make a wet mount of fungal mycelium stained with methylene blue and observe
under the microscope for septate hyphae. Draw a few hyphae.

2. Ascomycetes produce open asexual spores (conidia) on stalks called conidiophores.

You are provided with cultures of Penicillin and Aspergillus.
Prepare a slide and observe for the conida and conidiophores as shown below.
(Do not confuse the conidiospores of Aspergillus with a sporangium of Rhizopus –
look under high magnification.)

Aspergillus

43
3. Sexual reproduction
Ascomycetes produce cup – like structures called Ascocarps on fertilization which
bear Asci (sac – like structures) with eight spores in it.
Observe the prepared slide of Peziza under low and high magnifications to see
ascocarp and asci and draw a diagram of each one of them.

44
C. Basidiomycetes

Basidiomycetes have septae mycelium and lack asexual reproduction generally. They produce a
range of different shaped fruiting bodies that bear basidium (club like structure) with four
basidiospores. These includes:

i) Bracket fungi

ii) Crust fungi

iii) Mushrooms

45
 iv) Puffballs

v) Club fungi

Observe the different specimens provided to familiarise yourself with fruiting bodies of
basidiomycetes.

D. Lichens

Lichens represent a mutualistic relationship between fungi and algae, where alga provides
photosynthetic products and fungi provides protection of the alga. They are important as
pioneers of life on barren rocks. Lichens mostly consists of blue – green algae in association
with ascomycetes. Only a few species of basidiomycetes form lichens. Algae and fungi in lichen
either reproduce asexually independently (appearance of ascocarp on surface) or reproduce
asexually together by producing spores called Soredia (one fungal hypha enclosing one algal
cell).

There are three types of lichens:

a) Crustose – paint smear like

46
 b) Foliose – leaf like

c) Fruiticose – shrub like

Observe the specimens of three different lichens provided. Try to recognise the ascocarp
produced by the fungi partner. Draw the specimens.

Also observe the prepared slides of cross – section of lichen to see the internal structure of
lichen.

47