My Cology Manual

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Mycology Manual
Book · January 1998

DOI: 10.13140/RG.2.1.3550.7683
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Mycology Manual
Mohammed S. Ali Shtayeh

Professor of Biological Sciences, Department of Biological Sciences, An-Najah University, Nablus.
Rana Majid Jamous

MSc., Biologic. Sc.; Educator, Ministry of Education, Nablus.
Reem M-R. Yaghmour

MSc., Biologic. Sc.; Lecturer, Department of Biology, Al-Quds University, Jerusalem.
Authors, Nablus, Department of Biological Sciences, An-Najah University
IV
Published and distributed by: Ali-Shtayeh M. S.,
Jamous R. M., &Yaghmour R. M-R.
Department of Biological Sciences,
An-Najah National University,
Nablus POB 696.
Tel: Office 00-972-9-2381115
Fax: 00-972-9-2383589
E-mail: shtayeh@najah.edu
Copyright  Ali-Shtayeh M. S., Jamous R. M., &Yaghmour R. M-R. 1998.

All rights are reserved. No part of this publication may be reproduced, stored in a
retrieval system, or transmitted, in any form or by any means, electronic,
mechanical, photocopy, recording or otherwise without the prior permission of the
copyright owner.
IV
TABLE OF CONTENTS
PREFACE…………….……………………………………………………………………... V
PART ONE: GENERAL MYCOLOGICAL LABORATORY PROCEDURES…………1
1.1 CULTIVATION OF FUNGI…………………………………………………………… 1

1.1.1 Nutritional needs of fungi ……………………………………………………… ……… 1
1.1.2 Culturing fungi - general principles ………………………………………………… …1
1.1.2.1 General equipment ……………………………………………………… …………….2
1.1.2.2 Sterilization …………………………………………………………………………...3
1.1.2.3 Cleaning glassware……………………………………………………………………..5
1.1.2.4 Culture media ………………………………………………………………………….5
1.1.2.5 Culture techniques……………………………………………………………………...6
1.1.2.6 Preservation of fungi …………………………………………………………………...7
1.2 ISOLATION OF FUNGI…………………………………………………………………7
1.2.1 Introduction………………………………………………………………………………7
1.2.2 Isolation of plant pathogenic fungi……………………………………………………….7
1.2.3 Isolation of soil fungi……………………………………………………………………..8
1.2.4 Isolation of aquatic fungi ………………………………………………………………...8
1.2.5 Isolation of air-borne fungi ………………………………………………………………8
1.2.6 Isolation of medically important fungi …………….……………………………………. 9
1.2.7 Purification of fungal cultures…………………………………………………………….9
1.3 EXAMINATION OF FUNGI …………………………………………………………...10
1.3.1 Microscopic preparation ………………………………………………………………...10
1.3.2 Mounting fungi from agar media……………………………………………………..….11
1.3.3 Mounting fungi from the surfaces of plant parts ………………………………………...11
1.3.4 Identification of species………………………………………………………………….11
1.3.5 Fungal systematics…………………………………………………………………….... 12
PART TWO: EXERCISES
EXERCISE 1. Preparation of Media, Pouring of Plates, and Preparation of Agar Slants…….13

EXERCISE 2. Cultivation and Microscopic Examination of Fungi………………………….15
EXERCISE 3. Morphology of Fungi ………………………………………………………...21
EXERCISE 4. Kingdom fungi: Phylum Chytridiomycota ………………………………….29
EXERCISE 5. Kingdom Fungi: Phylum Zygomycota……………………………………....31
EXERCISE 6. Kingdom Fungi: Phylum Ascomycota……………….……………………...33
EXERCISE 7. Kingdom Fungi: Phylum Basidiomycota …………………………………...43
EXERCISE 8. Kingdom Fungi: Deuteromycetes: Asexual Ascomycetes and
Other Asexual Fungi ……………………………………………………... 59
EXERCISE 9. Kingdom Protista: Phylum Plasmodiophoromycota…………………………63
IV
EXERCISE 10. Kingdom Stramenopila: Phylum Oomycota ………………………………...65
EXERCISE 11. Soil-Borne Fungi …………………………………………………………. ..81
EXERCISE 12. Aquatic Fungi ……………………………………………………………….87
EXERCISE 13. Air-Borne Fungi ……………………………………………………………..91
EXERCISE 14. Plant Pathogenic Fungi…………………………………………………….. .93
EXERCISE 15. Inter Fungal Parasitic Relationships: Mycoparasitism……………………. 99
EXERCISE 16. Fungi as Human and Animal Pathogens ………………………………….103
EXERCISE 17. Keratinolytic Activity of Fungi……………………………………………...111
EXERCISE 18. Antibiotic Properties of Fungi (Penicillin)……………………………… .115
EXERCISE 19. Observation of Mycorrhizae……………………………………………….117
EXERCISE 20. Antifungal Susceptibility Testing…………………………………………...119
EXERCISE 21. Sporulation and Spore Germination ……………………………………….125
EXERCISE 22. Entomopathogenic Fungi in Soils …………………………………………..127
EXERCISE 23. Detection of Aflatoxin Contamination Produced by Aspergillus
Species in Food Products …………………………………………………...131
REFERENCES ……………………………………………………………………………...133
APPENDICES……………………………………………………………………………….137
IV
PREFACE
The manual includes two parts: General Mycological Laboratory

Procedures and Exercises. Part 1“General Mycological Laboratory
Procedures” includes sections on procedures for the cultivation,
examination, and isolation of fungi. Part 2 “Exercises” includes exercises
on fungi: handling, cultivation, microscopic examination, morphology,
and taxonomy. The most commonly found fungi are generally employed
for study in the laboratory. It also includes exercises on different
ecological groups of fungi: soil-borne, aquatic, air-borne, keratinolytic
fungi. Exercises on plant, animal, and human pathogenic fungi are
provided. Other exercises are centered on fungi as biocontrol agents
against insects and other fungi, relationships between fungi and other
organisms, sporulation and spore germination, control of fungal growth,
and production of antibiotics and aflatoxins by fungi.
The design and content of the manual provides an introductory text in
laboratory mycology, as well as a prerequisite course for a better
understanding for more advanced textbooks in mycology. The manual is
especially useful for biologists, plant pathologists, laboratory technologists,
environmentalists, and particularly to those who are welling to pursue a
career in mycology or engage in research and advanced studies.
The authors are indebted to Mrs. Hana Hilmi Yaish for making the
illustrations.
Department of Biological Sciences, Mohammed S. Ali
Shtayeh
An-Najah University, Nablus. Rana Majid Jamous
Department of Biology, Reem M-R.
Yaghmour
Al-Quds University, Jerusalem,
Palestine
August 1998
IV
PART ONE
GENERAL MYCOLOGICAL LABORATORY

PROCEDURES
MYCOLOGY MANUAL / PART 1:GENERAL MYCOLOGICAL LABORATORY PROCEDURES 1
PART ONE
PROCEDURES
1.1 CULTIVATION OF FUNGI Fungal growth may be prevented or
1.1.1 Nutritional Needs of Fungi controlled by several approaches including
Unlike autotrophic organisms (e.g., green (Deacon, 1984):
plants), fungi are unable to synthesize 1. Manipulation of the fungus
organic materials from carbon dioxide, itself or its physical environment.
mineral ions and water. Their protoplasm is 2. The use of fungicides.
encased in a cell wall and therefore only 3. The use of antibiotics.
substances in solution can be taken in by 4. Plant breeding for disease
absorption (Ingold, 1984; Hudson, 1988). control.
All fungi require a set of 5. The use of other organisms in
environmental conditions (aeration, light, biological control.
moisture, temperature, etc.) under which However, the use of antifungal agents
they grow and sporulate best. Water and is still the main approach for the prevention
oxygen are both essential for growth of and control of fungal growth. An antifungal
fungi. Other elements are also required. agent is a chemical that kills fungi or
Some are known as macroelements (carbon, inhibits their growth. The term covers a
nitrogen, phosphorus, potassium and wide range of compounds, not all of which
magnesium), since they are required in large are suitable for use in a particular set of
amounts. And some are known as circumstances because of such factors as
microelements (iron, zinc, copper, phytotoxicity, mammalian toxicity, color,
manganese, and molybdenum), being unpleasant odor etc. (Deacon, 1984).
required in concentrations of parts per
million. Some fungi may also require 1.1.2 Culturing Fungi - General
additional microelements such as calcium Principles
and gallium (Ingold, 1984). Most fungi are In pure culture work, dust is the most
self-sufficient in vitamins. They can frequent cause of contamination. It is,
synthesize the vitamins they need for their therefore, necessary before proceeding with
growth. Others require an external source of culture work, to wipe free of dust the surface
one or more vitamins (e.g., thiamin, B). of the working area of the bench. A towel
Other factors that affect the development of soaked in a suitable antiseptic or water-
a fungus include temperature, light, and pH. detergent solution may be used for this
purpose. Dusty objects should be either
Many fungi can therefore, be grown cleaned or removed.
on culture media in the laboratory. In Materials containing the fungus
addition to water, a complete medium must should be processed so they can be
provide the fungus with a suitable organic introduced into or onto liquid or solid sterile
substance as a source of carbon (a soluble culture media. To facilitate later purification
utilizable carbohydrate); a source of and identification of fungi, it is
nitrogen, certain inorganic ions, and certain recommended that small samples of the
organic growth factors in very low material be plated out on a large number of
concentrations. The medium must be free of plates (Onions et. al., 1981).
inhibitory chemicals, and has a pH at which When choosing media for culturing of
the fungus can reproduce. fungi, original material, and the target
2 ALI-SHTAYEH, JAMOUS, & YAGHMOUR
fungus and its suspected contaminants wool, so that any air, which enters, will be
should be taken into consideration. filtered from all contaminating organisms. A
Temperature of incubation, except in plug should project about 2 cm into the tube
thermophilic fungi or fungi that require very and should have a tuft outside the tube by
low temperatures, is not critical. However, it which it can be taken out. The plug should
may be useful to set up plates at both lower also fit accurately and tightly.
and higher temperatures.
Sub-cultures should be made as soon c. Screw top bottles (universals)
as fungi are seen to be growing. Tiny pieces Their usual size is about 75 X 20 mm and
of the colony are picked off and plated out the screw caps are fitted with rubber pads to
(inoculated) in new sterile media. Resulting effect an airtight seal. The caps must be
pure cultures can be left to grow to maturity loosen during sterilization and they may be
and be identified. Pure cultures can then be screwed down as soon as the bottles come
transferred to an agar slope in a tube or out of the sterilizer. Culture medium can be
bottle. When the fungus has grown, the stored for a long time in these bottles
culture may then be stored for some time. without drying out. Bottles containing
cultures are easy to store, and are thus more
1.1.2.1 General equipment suitable for sending cultures by post.
It is essential to obtain pure cultures of the Disposable plastic screw-top bottles are also
fungi before they could be properly available and may be purchased in a sterile
identified. Each species is grown on a sterile condition.
substratum (culture medium) free from other
organisms and with suitable precautions to d. Petri dishes
prevent subsequent contamination. Cultures These are flat, shallow, glass or plastic
may be grown in Petri dishes, plugged test dishes with perpendicular sides, provided
tubes, and screw-top bottles. with covers of the same shape, but of
slightly larger diameter. Petri dishes may be
a. Tubes obtained in various sizes but the most
These are either ordinary test tubes, or the convenient size is 10 cm by 1.5 cm.
much preferred bacteriological test tubes. Disposable gamma-radiation sterilized
They are usually made of glass (disposable plastic dishes are now extensively used.
sterile tubes are also available) which has a These are nearly unbreakable and easier to
high degree of resistance to chemical and handle.
mechanical action, and will withstand
repeated sterilization and comparatively
e. General apparatus
rough handling. It is recommended to have The general equipment necessary for
two sizes (e.g., 150 X 19 mm; 150 X 24 handling fungi includes beakers and flasks
mm) of these tubes in the laboratory. The of various sizes, measuring cylinders,
smallest size is used for slopes, and the pipettes, funnels, Bunsen burners and
larger size is used to hold medium for tripods, and balances. Other equipment
plating out. Tubes are usually packed into includes needles and loops (required for
rectangular baskets of stainless steel or iron inoculation) fine-pointed scissors, scalpels
wire, galvanized or tinned, during and forceps. For labeling petri dishes and
sterilization. culture tubes suitable markers are also
necessary.
b. Plugs
Tubes containing pure cultures of fungi are
f. Incubators
usually plugged with non-absorbent cotton
These are containers that provide fungi with
the required temperatures necessary to from Bunsen burner or alcohol lamp.
induce maximum rate of growth, and in Tempered metal can be heated to red-hot.
some cases, to promote the formation of Cutting tools are best treated by dipping in
certain types of spores. alcohol and then carefully flaming-off the
alcohol. Glass objects are passed through
1.1.2.2 Sterilization the flame several times and should not be
Pure culture technique, which provides placed immediately on a cool surface or
sterile conditions, is essential for the they will crack.
isolation, identification and cultivation of
fungi, and for studying their biology. 2. Moist heat (steam
Sterilization involves the inactivation or sterilization). Moist heat is provided by
elimination of all living cells and infective saturated steam under pressure in an
agents from the targeted environment. autoclave or pressure cooker. It is the most
In order to avoid contamination it is reliable method of sterilization for most
necessary to use aseptic (pure culture) materials. However, the method is not
techniques throughout all operations suitable for materials damaged by moisture
concerning the cultivation of fungi. This or high temperature, or culture media
means that all tools, apparatus, utensils, containing compounds hydrolyzed or
containers, culture media, must be sterilized. reactive with other ingredients at higher
Also, inoculation and subsequent operations temperature. Steam sterilizers by
should be carried out in a sterile coagulation of protein.
environment (sterile room or hood) to Steam under pressure is a better
prevent the contamination of the sterile sterilizer than steam at 100 oC, because
apparatus by un-sterile air. latent heat of the trapped steam is given up
The choice of method of sterilization when steam condenses resulting in a higher
depends on the desired efficiency and the temperature. Culture media are altered by
physical nature of the material to be heat temperature. For example, the medium
sterilized and the reason for doing it, pH usually is changed by 0.2-0.4 units;
whether materials are being prepared for use carbohydrates are partially hydrolyzed and
or for disposal and cleaning. The methods the nature of proteins may be changed.
used for sterilization are heat, gas, and in the
case of liquids, ultra filtration. a. Steam at 100 oC
This is known as steaming or free steaming.
A. Heat sterilization The method is used to sterilize culture media
This is the most reliable method for that might be spoiled by exposure to higher
sterilization when the material to be temperatures. Exposure to heat is carried out
sterilized is not modified by high for 1 hour, or for 30-60 minutes on each of
temperature. Using either dry or moist heat three successive days (intermittent
can attain high temperature. The chief steaming).
mechanism of cell destruction by heat is In the latter method, most of the
oxidation or coagulation of proteins. vegetative growth, but not the spores, is
destroyed by the first heating. Spores then
1. Flame sterilization. This is break dormancy and germinate rapidly in the
used for metal tools such as inoculating medium. The second heating destroys the
wires, loops, and tips of forceps, and glass new growth and the third heating kills any
objects such as the lips of culture tubes and spores whose germination has been delayed.
microscope slides. This is done by holding b. Autoclaving
the object in the upper portion of a flame The autoclave is a device for boiling water
under pressure. Autoclaving at 15 lb per sq. B. Chemical sterilization
inch (1.05 Kg per sq. cm) for 10-30 minutes Chemicals used for sterilization are either
is the usual method of sterilizing culture termed disinfectants or antiseptics.
media. Disinfectants are toxic and can only be
Excessive autoclaving partially applied to inanimate objects. Antiseptics are
hydrolyzes agar-agar, which can inhibit sufficiently non-toxic to be applied to the
microbial growth. Acidification of agar superficial areas of living tissue. Chemical
media should be done after autoclaving sterilizing agents operate in one of the
since acidified agar does not gel properly. following ways: coagulation of protein,
Antibiotics, hormones, vitamins, and other inactivation of enzymes, hydrolysis,
similar compounds may be destroyed by breaking cell membranes, altering the
heating and therefore should be sterilizes by permeability on the bacterial surface, and
filtration or other means after autoclaving oxidation, usually by removal of
the media. sulphohydryl groups from enzymes on the
bacterial surface.
3. Dry heat (hot air sterilization). Chemical sterilizing agents most
This is used for the sterilization of dry commonly used in the laboratory are:
glassware, such as tubes, flasks, pipettes, 1- Antiseptics of the phenol group
and petri dishes, metal instruments, certain (including lyso1). These are used for
plastics and heat stable compounds. The hot sterilizing instruments, discarded cultures,
air oven sterilizes by dry heat, by oxidizing and washing down the bench after cultures
protein. The oven can be heated by gas or have been spilt.
electricity. Heat conduction by dry heat is 2- Ethyl alcohol 50-70 %. Sterilized
slower than that of moist heat and thus the instruments can be stored in alcohol.
former requires higher temperatures for 3- Soap and detergents, the action is due to
longer duration than the latter. their concentration at the interface between
A hot air oven can be loaded when hot water and the object, leading to a lowering
or cold. When loading the oven, space must of surface tension and therefore the
be left between loaded articles, as loading to disruption of the cells.
capacity prevents air circulation and results 4- Potassium dichromate; sulfuric acid
in hot or cold spots. Heating of objects is by solution; 1:1000 mercuric chloride.
radiation from the oven walls, unless Glassware can be sterilized by dipping in
equipped with a fan to circulate the air one of these solutions for 1 minute or more.
within a chamber; heating of object is Sterilization techniques commonly
uneven. used in laboratories include:
Three hours heating at 160 oC or 1 hr 1- Inoculation needles and loops and points
at 180 oC is sufficient for successful of forceps are sterilized by holding them in
sterilization. Glassware should be the flame of Bunsen burner until they are
completely dry before placing in a hot air red-hot.
oven since wet glassware may break. 2- Mouths of culture tubes flasks and
Objects such as glass culture plates, and bottles, cover slips, scalpels, and needles are
glass pipettes should be placed in sealable sterilized by passing through the Bunsen
metal. After sterilization, the oven and its flame without allowing the object to become
contents should be allowed to reach ambient red-hot.
temperature before opening to prevent 3- Dry glass petri dishes, test tubes, flasks,
breakage and recontamination by cool air pipettes, instruments and glass syringes are
rushing into the chamber. sterilized in a hot air oven at 160 oC for 3
hours.
4- Culture media, cotton wool and other dichromate 100 g, sulfuric acid 500ml, and
porous materials are sterilized in an water 1000 ml. Potassium dichromate is
autoclave. dissolved in hot water. The solution is then
5- Benches and old cultures may be cooled and the sulfuric acid is slowly added
sterilized by a suitable antiseptic (e.g., with constant stirring. This solution should
Lysol). be handled with great care since it could
injure the skin. Glassware is soaked in
C. Filtration cleaning solution for several hours, cleaned
Filtration, which physically separates thoroughly with a brush, and rinsed several
microorganisms, cells, and debris from times (the final rinse in distilled water).
liquids, but not viruses or metabolic by-
products, is superior to other methods since 1.1.2.4 Culture media
there is no change in the properties of the Fungi may be grown in the laboratory on a
filtrate. Aqueous solutions, organic variety of media or substrata. Culture
solutions, and oils can be sterilized by medium is the major factor influencing
filtration. Filters generally used are cellulose fungal cultivation. Its constituents
ester membrane, sintered glass, asbestos concentration determines the quality and
pads, unglazed porcelain, and diatomaceous quantity of growth and whether vegetative
earth disks or candles. Microorganisms and growth or sporulation will dominate. A
other large particles are retained on the filter weak medium with a low C and /or N source
by the small size of the pores and dry suppresses vegetative growth and stimulates
adsorption onto pore walls. sporulation. Natural media are generally
Membrane filters. These are made more favorable to growth and sporulation
from cellulose ester, very thin and delicate, than synthetic ones. Media may be either
need careful handling and are used once. liquid or made solid by the addition of agar.
They are available in various diameters and Most fungi usually grow best on media high
pore sizes (a pore size of 0.22 m is suitable in carbohydrates, with a pH range between 5
for most sterilization). Since the gravity flow to 6. There is no one medium ideally suited
rate of liquids through these membranes is for the growing of all fungi, because the
slow, it is necessary to have negative nutritional requirements vary with the
pressure i. e., suction. All liquids should be species.
clarified by passing it through a coarse filter
before filtering for sterilization. a. Types of media
On the basis of their composition culture
1.1.2.3 Cleaning glassware media may be classified into two main
Routine cleaning of glassware is usually groups natural media and synthetic media.
carried out using a detergent solution. Natural media usually contain
Where mineral content of water is high, infusions of natural substances (animal or
washed glassware should be rinsed with plant extracts). Such media are prepared as
distilled water. To remove wax pencil decoctions, extracts or juices from plant
marks, paraffin, balsam or similar materials, parts or powdered plant or animal materials
from glassware, xylol may be used added to the medium. The chemical
effectively before the glassware is washed. composition of these media varies from time
A cleaning solution can be used to to time. Natural media, such as corn meal
clean very dirty glassware, or when it is agar (CMA) potato dextrose agar (PDA),
necessary to have the glassware potato carrot agar (PCA), beef extract agar
exceptionally clean. Cleaning solution may (BEA), malt extract agar (MEA), oatmeal
be prepared as follows: potassium agar (OA), and glucose yeast extract (GYE)
contain all of the nutrients necessary to the Agar plates
growth of most fungi. However, these media Fungi are usually grown in Petri dishes as
may need to be supplemented for good well as on slopes for morphological studies.
growth or sporulation of some fungi. Natural Plates inoculated with single colonies are
media are easy to make. useful for determining rate of growth and
Synthetic media such as Czapek-Dox colony characteristics. Plates inoculated with
Agar (CzA), are media of known several colonies are more suitable for
composition, made of pure chemicals, and microscopical examinations (Onions et al.,
each component and concentration can be 1981). The amount of medium per the usual
controlled as desired. 9-cm Petri dish necessary to give adequate
The pH of all media is usually thickness is approximately 12 ml. this
measured and adjusted to approximately 6- should be fairly standardized.
6.5 before autoclaving. The composition of
selected useful media is given in Appendix 1.1.2.5 Culture techniques
A. a. Slide cultures
These are used to study fungal species that
b. Preparation of media produce very small and delicate
The general routine procedure for the sporophores. Using these methods it is
preparation of media is similar for most possible to make microscopic preparation
media. The procedure for the preparation of for the fungus under consideration. Two of
media, which are purchased as dry powders, such methods are: Riddell's method (1950)
is outlined on the label of the container. which is outlined in Exercise 2 and the
method outlined below.
c. Sterilization of media A drop of melted agar is poured on the
Media are prepared mainly to grow fungi in center of a sterile slide placed in a moist
pure culture. Therefore, all microorganisms, chamber (See Exercise 2). To study spore
other than the one to be grown should be germination a little spore material may be
excluded (killed) by sterilizing the media mixed with the agar before pouring onto the
and the glassware containing them. slide. If spore production is to be observed,
Sterilization can be achieved by placing the it is better to inoculate the medium at two or
media in an autoclave for 15-20 minutes at three points. When the desired stage of
120 oC and 15-psi pressure. Agar plates or growth has been reached the slide is
slants are usually used for the cultivation of transferred to a similar dish containing little
fungi. formalin, or osmic acid solution, in order to
kill the fungus and partially fix its structures,
d. Types of culture the slide may then be examined either dry or
Agar slopes (slant) with a coverslip laid on gently. Sometimes it
An agar slant can be prepared by permitting is possible to make permanent mounts in
the agar in a tube (or a suitable bottle) to lactophenol, afterwards by removing the
solidify while the tube is in a slanted agar around the coverslip and sealing with
position. Care should be taken not to permit cement.
the molten agar to come in contact with the
cotton plug. Agar slopes (in tubes or bottles) b. Hanging drop cultures
are usually used for keeping culture for These are especially useful for studying
some time or which are to be stored in a spore germination and for checking spores
culture collection. for appressoria formation (See Exercise 2).
c. Transferring fungi to slants and and incubated. Once growth has been
Petri dishes established, the sterile liquid paraffin
Fungal cultures can be perpetuated in the (mineral oil) is added so that the entire slope
laboratory by aseptically transferring spores is covered and the culture is then stored at
or fragments of the mycelium from one low temperature.
culture to sterile media. Suitable inoculating
needles or scalpels may be used efficiently. 1.2 ISOLATION OF FUNGI
1.2.1 Introduction
1.1.2.6 Preservation of fungi Measuring the quantity of pathogen
The preservation and maintenance of fungal inoculum (inoculum density or inoculum
cultures is essential in order to keep original potential) can help determine disease
cultures available. Techniques for preserving intensity and severity, and control measures
fungi include continuous growth methods to be used. Inoculum density is the number
(frequent transfer, storage under mineral oil, of propagules per unit volume of soil, air or
soil storage, water storage) and methods of other medium. Inoculum potential includes
drying, and induced dormancy (silica gel the energy available to the inoculum for
storage, freeze-drying or lyophilization, colonizing the host that is determined by its
liquid nitrogen storage). Preserving fungi by age, nutritional status, propagules
periodic transfer requires that they be grown organization, and any antagonism to the
alternately on rich and poor media with a pathogen. These factors contribute to the
low carbohydrate content, at neutral pH capacity of the inoculum to colonize a
and/or media prepared from natural product susceptible host under conditions optimum
extracts; they be grown on a medium which for disease development.
allows for minimum mycelium growth and The methods used for the isolation of
maximum sporulation; transfers be made fungi and /or measuring the quantities of
using spore mass only from the youngest their inocula depend largely upon their
portion of the colony; if screw cap culture natural environments or habitats such as
tubes are used, the cap is not closed tight; soil, water, plant, human, and animal tissues.
and fungi that are not cold sensitive should It is generally easier to isolate saprophytic
be refrigerated (for 10 oC) after the colony fungi than specific animal or plant
has covered the agar surface. pathogens. If the fungus is producing either
Cultures producing resistant spores sexual or asexual fructifications isolation
are the easiest to keep healthy. The cultures could be made by single spore isolations.
is grown on a slope of suitable medium and
incubated until most of the surface area is 1.2.2 Isolation of Plant Pathogenic Fungi
covered by actively growing mycelium. The Superficial saprophytes found naturally on
cultures can then be stored in a refrigerator the surface of infected plant tissues could
at 4 oC. Cultures prepared in this way may interfere with the isolation of the plant
be stored for many months before further pathogenic fungi. Surface contaminants can
sub-culturing is necessary. be eliminated or reduced by treating the
For all other cultures it is essential to tissue with surface sterilants such as 5.75 %
reduce the metabolic activity in order to sodium hypochlorite solution (1 Clorox: 9
prevent rapid exhaustion of the medium. By water) for different durations (30-120
storing the cultures at low temperatures (2 to seconds). Other surface sterilants include 95
4 oC) the growth rate is reduced. The rate of % ethyl alcohol (used for dips for 3 seconds
respiration can be reduced by the addition of or more), mercuric chloride 1:1000 in 50 %
sterile medicinal paraffin. The culture is ethyl alcohol (Rada's solution). The treated
inoculated on to a slope of suitable medium
tissues should be blotted dry with sterile growing fungus may then be sub-cultured on
paper towel when treated with first two a suitable nutrient medium.
solutions, but they must be rinsed in three Lupine radicles and citrus leaf discs
changes of sterile water then blotted dry have been used as baits for the isolation of
with sterile paper towel when treated with Phytophthora cinnamomi and other Ph.
the last sterilant (Agrios, 1988). species from soil of citrus orchards (Weste,
When the treated tissue has been 1983; Grimm & Alexander, 1973).
sterilized with Clorox or Rada's solution
small parts of the tissue are cut out and 1.2.4 Isolation of Aquatic Fungi
placed in one or more plates containing Zoosporic fungi may be isolated by a variety
nutrient medium. of methods. These include baiting
techniques, centrifugation and filtration
1.2.3 Isolation of Soil Fungi techniques (Gareth-Jones 1971).
a. Soil dilution plates (SDP) a. Baiting: Water samples, are
This is probably the most widely used collected in sterile containers from rivers,
method for the isolation of soil fungi. The streams, and ponds. The baiting procedure is
method consists of shaking a known amount outlined in Exercise 11.
of soil in sterile water or sterile 0.08 % b. Centrifugation: A number of
water agar (Ali Shtayeh et. al., 1986), then zoosporic fungi have been isolated by
preparing a progressive series of dilutions. continuous flow centrifugation. By this
Known volumes (usually 1 ml aliquots) method large water samples can be
from one or more of the dilutions are concentrated and then plated onto suitable
incorporated in melted cooled agar (Warcup, agar plates.
1967). c. Filtration techniques: Zoospores
b. Surface-soil-dilution can also be isolated from smaller quantities
plates (SSDP) of water by filtration through Millipore filter
This can be used as an alternative to the discs (0.45 - 3.0 m) and the concentrate on
SDP for the isolation of soil fungi. The the filter discs resuspended in a known
SSDP consists of transferring known volume of sterile water or 0.08 water agar.
aliquots (e.g. 1 ml) of the soil dilution on to Portions of the suspension are then plated
the surface of a suitable solidified medium onto suitable media.
in each of a number of replicate plates (Ali-
Shtayeh et. al., 1986). The SSDP has the 1.2.5 Isolation of Air-Borne Fungi
advantage that the developing colonies are Air represents an important medium
easy to pick off from the plates for dispersal of fungal propagules (Jamous,
subsequent purification. 1998). These fungi may be pathogenic or
opportunistic. The most important factors
c. Baiting that may influence both concentration and
Baits used for the isolation of fungi include composition of air spora, in the
surface sterilized roots, stems, leaves, fruits, atmosphere, are local ecology and human
seeds, pollen grains, insect wings, hair, activity. The most common genera in air-
snake skin casts etc. The baiting procedure dust samples, are Alternaria, Aspergillus,
consists of placing a few baits (3-5 per plate) Drechslera, Fusarium, Phoma, and
on soil in sterile, petri dishes moistened with Stachybtrys. Air fungal spores may cause
sterile distilled water (in case of chitinous allergic reactions and several other
and keratinous materials) (MI, 1983) or clinically diagnosed illnesses in human
covered with a thin layer of water (in case of being. Some fungal species, which are
zoosporic fungi) (Ali-Shtayeh, 1986). The considered as non-pathogenic, may cause
severe and even fatal infections in weak or from ulcers, pus, cerebrospinal or other body
immuno-compromised patients. fluids, urine, sputum, blood, bone marrow,
Methods and apparatus most and stools.
commonly used to trap, detect and estimate
concentration of spore/unit volume of air are 1.2.7 Purification of Fungal Cultures
similar to those used for trapping airborne Dilution cultures: This method
dust and pollens. These methods employ involves making a series of dilutions in
impacting, impingement or sedimentation. sterile distilled water or 0.08 % water agar,
The term impinger is used to describe a and then plating out known aliquots of the
device in which the sampled airstream is different dilutions onto agar plates.
blown into liquid culture fluid or sterile Developed isolated colonies can then be
water, while in an impactor the sampled air transferred to new plates.
stream is directed onto a solid surface such
as a sticky faced microscope slide or agar Hyphal tip cultures: This method can
medium in a Petri dish. be used when the initial growth rate of the
target fungus is greater than that of the
1.2.6 Isolation of Medically Important contaminant. A single colony is plated in the
Fungi center of an agar plate. When the colony is
Laboratory diagnosis of mycoses (fungal about 1 cm in diameter a small block of agar
infections of man and other vertebrates) is containing the tip of one of the radiating
carried out by demonstration of the fungus hyphae at the edge of the colony is cut out
in the skin, exudates or deeper tissues. and transferred to a fresh agar plate.
Isolation and identification of the causative
agent in culture is usually necessary to Bacterial contamination: Various
confirm this. antibacterial substances may be incorporated
Proper collection and rapid transport into the media in order to eliminate bacteria
of clinical specimens are very important for from fungus cultures. These include
successful diagnosis of mycotic infections penicillin, rifampicin, ampicillin, . . . etc.
and the recovery of etiological agents.
Prompt delivery of specimens to the Single-spore cultures: A number of
laboratory is important to avoid overgrowth methods may be used to obtain a single-
of bacteria or rapidly growing saprophytic spore culture (see Exercise 2). Such cultures
fungi. Specimens should be transported in a are usually used for the purification of
sterile container that provides a moist mixed cultures, in genetical investigations
environment. Sterile saline can be added if and for establishing relationships between
necessary. If storage of specimens is perfect and imperfect states.
necessary, they should be stored at 4 oC for
no longer than 24 hours. However, some 1.3 EXAMINATION OF FUNGI
loss of specimen viability may occur. 1.3.1 Microscopic Preparation
Specimen container should be properly For the study of fungi, it is usually necessary
labeled. It should be accompanied by a slip to make microscopic preparations. The
that provides the name of the patient, method to be employed in making the
address, the name of referring physician, the preparation is determined by the nature of
source of the specimen, and information on the fungus itself and its host if any. The
the clinical condition. Sufficient material of method also depends on whether the
specimens should be collected for both preparations required for a temporary
direct examination and culture. Specimens examination or whether it is intended for a
may include: skin scraping, nails, scraping
permanent preparation. Many fungi may be mounting fungi (see Appendix A for recipe).
mounted directly without sectioning.
However, it is sometimes necessary to slice b. Staining of fungal nuclei
the fungus in / on the material, which the Acetic-orcein. Uredospore nuclei
fungus is found. of Puccinia graminis f. sp. tritici may be
stained by spreading the spores on a slide
a. Mounting fluids. Water may be coated with fresh egg white. The spores are
used as a mounting fluid. Its application for then fixed by placing them in a mixture of 4
the majority of fungi is however limited formalin and 25 mM potassium phosphate
because it evaporates rapidly, causes buffer (pH 7.0) for 30 min at room
swelling of hyphae by osmosis and causes temperature and then for 90 min at 45 oC.
parts of the fungal material to adhere The slide is washed in water and placed in
together. Slides made with water as the alcoholic KOH (1:7 KOH: 95% ethanol) for
mounting fluid may be protected from 1 to 2 hrs at 45 oC. Spores are washed and
evaporation by sealing the edges of the mounted in a drop of acetic acid-orcein (see
cover slip with nail polish. Appendix A for recipe). The coverslip is
Lactophenol (Aman's medium) is placed and sealed. Nuclei are stained in
used for mounting most fungi so that about 2-3 hrs.
mycelium and other fungal structures may Fluorescent staining using
be more easily seen (see Appendix A for acridine orange. This method may be used
recipe). for nuclear staining in mycelium and young
A stain may be added to this medium, gametangia of Phytophthora and Pythium
e.g. 0.05-g cotton blue, or trypan blue per species and in some structures of certain
100 ml (Borelli & Salas, 1975 In: Beneke & other fungi. The fungus is grown on a glass
Rogers, 1980). Other dyes include picric coverslip placed on an agar medium. The
acid, and acid fuchsin. Cotton blue is coverslip with the fungal growth is mounted
perhaps the most widely used of all stains in the stain (see Appendix A for recipe) on a
for fungi (Onions et al., 1981). microscope slide and then examined
Lactophenol is an excellent medium, immediately with a fluorescent microscope
since it rarely causes shrinkage or swelling with appropriate barrier filter. The nuclei
of the cells and it has no tendency to appear light to yellow-green in contrast to a
evaporate. It is also viscous enough to faint green or orange background depending
permit the use of oil-immersion lenses upon the barrier filter used.
without improper movement of parts of the
specimen c. Microscopic measurement.
Lactophenol mounts can be sealed by Measuring the size of objects under the
nail varnish as mentioned for water mounts. microscope can be made by means of an
Slides sealed carefully can keep for a long eyepiece micrometer and a stage
time. Also, permanent slides should be micrometer. The unit of length used is 1
protected by sealing the edge of the cover micron () which is equal to 0.001 mm. The
glass with suitable cement. This process can ocular micrometer is a small glass disc
be done using a turntable when circular inserted into the eyepiece of the microscope.
cover-glass are used, or free hand if square Uniformly spaced lines are etched into the
cover-glass are used. The slides should be glass; thus it functions as a ruler that is
labeled properly, by giving the name of the superimposed on the object being measured.
species, date of preparation, the stain used, Ocular micrometers must be calibrated for
and the mounting fluid. the specific microscope in which they are to
Hoyer’s medium is also used for be used, since microscopes may vary in their
actual magnification. This calibration is 2. Color and color changes of the colony.
achieved with use of a stage micrometer in This may be described as uniform, in zones,
conjunction with ocular micrometer. The or patchy.
stage micrometer acts as a ruler. 3. Color and color changes of the reverse
of the colony.
1.3.2 Mounting Fungi From Agar Media 4. Color changes in the medium (whether
Slide culture techniques provides a useful confined to the colony or diffusing).
method for the examination of fungi 5. Texture of the colony surface. This can
particularly for fungus such as Penicillium be described as loose or compact, plane,
and Aspergillus, whose conidiophores and wrinkled or buckled, velvety, floccose,
chains of conidia are fragile. In this gelatinous, leathery, etc.
technique reproductive structures and 6. Odder, if any.
mycelium adhering to the glass left on the 7. Character of drops of fluid frequently
slide often discarding the agar from the found on aerial growth.
microscopic slide. Mounting fungi from agar 8. Characteristics of hyphae, color
culture may be made by taking a block of septation, diameter, and presence of special
agar from the culture, melting it on the slide structures.
and the molten agar replaced by a mounting 9. The stage at which reproductive
fluid. structures form.
10. The character and arrangement of
1.3.3 Mounting Fungi From the Surfaces reproductive structures.
of Plant Parts 11. Color, sizes, and shape of mature
Pieces of fungi growing on the surfaces of reproductive structures.
plant parts may be scraped off with a needle 12. Details of structure of the reproductive
and mounted in a suitable mounting fluid. organs or components, including
An alternative method is to press a measurement of essential parts and
short piece of adhesive, transparent tape arrangement of spores.
such as sellotape, or scotch tape, over the 13. Characteristics of spores: color, shape,
surface of the plant on which the fungus is separation, surface markings, and size
growing. The tape is removed, placed on a (including both average and range).
drop of lactophenol on a slide, another drop Details numbered 1-7 are obtained by
of lactophenol is added on top of the tape the examination of cultures with the unaided
and a cover glass is added. eye or with a hand lens, 8-11 by the
examination of living cultures with the aid
1.3.4 Identification of Species of a microscope, and 12-13 by the
Once a pure culture of the fungus has been examination of microscopic preparations
obtained, the fungus may now be identified. under the highest powers of the microscope.
The fungus is grown on one or two suitable The information recorded is usually
media in Petri dishes. Several cultures sufficient to identify the fungus especially
should be made; some are used for when a good monograph of the genus is
measurement of growth rate and for available.
recording the general appearance of
colonies, the remainder may be used for 1:3:5 Fungal Systematics
microscopic examination and observations A number of changes have taken place in
recorded. The following details may be fungal systematics in the last few decades
recorded (Onions et al. 1981): including the recognition of the artificial
1. Growth rate (mm/day) at a specified nature of three or even five kingdom
temperature. classification systems, and the polyphyly of
organisms traditionally known as fungi and
the development and application of
molecular techniques in mycology. A KINGDOM FUNGI
hierarchical classification for fungi was Phylum Chytridiomycota
made by Whittaker (1969) by placing them Phylum Zygomycota
in kingdoms (Fungi and Protista) that more Phylum Ascomycota
nearly reflected their presumed evolutionary Phylum Basidiomycota.
relationships.
However, organisms once classified as KINGDOM STRAMENOPILA
fungi are now considered in three different Phylum Oomycota
groups, the monophyletic kingdoms Fungi Phylum Hyphochytridiomycota
and Stramenopila and four protest phyla Phylum Labyrinthulomycota
(Barr, 1992; Hawksworth et al., 1994)
(Figure 1.1). KINGDOM PROTISTA
Although these organisms are not Phylum Plasmodiophoromycota
closely related and do not all share a Phylum Dictyosteliomycota
common evolutionary history, they do share Phylum Acrasiomycota
several characteristics related to their Phylum Myxomycota
morphology, nutritional modes, and ecology
(Alexopoulos et al., 1996).
The organisms studied by mycologists Figure 1.1 Classification system adopted in
usually satisfy the following criteria: (1) this manual. The term “phylum” replaces
absorptive mode of nutrition, (2) growth by “division” of equivalent rank.
polarized hyphal extension, and (3)
reproduction involving the formation of
spores (Money, 1998). For these reasons it
has been suggested that the organisms
classified under Kingdom Stramenopila
(Phylums Oomycota,
Hyphochytridiomycota,
Labyrinthulomycota) and Kingdom Protista
(Phylums Plasmodiophoromycota,
Dictyosteliomycota, Acrasiomycota,
Myxomycota) should continue to be studied
by mycologists (Alexopoulos et al., 1996;
Money, 1998).
In this manual the true fungi were
placed in the Kingdom Fungi which
includes the phyla Chytridiomycota,
Zygomycota, Ascomycota and
Basidiomycota. Additional phyla considered
include Plasmodiophoromycota of the
polyphyletic Protista, and phylum
Oomycota of the Kingdom Stramenopila
(Alexopoulos et al., 1996).
PART ONE

PROCEDURES
1
PART TWO
EXERCISES
IV
MYCOLOGY MANUAL / PART TWO: EXERSICES 13
EXERCISE 1: PREPARATION OF MEDIA, POURING OF PLATES, AND

PREPARATION OF AGAR SLANTS
Materials needed:
Culture media and chemicals: Corn Meal Agar (CMA); Potato Dextrose Agar (PDA); Malt
Extract Agar (MEA); Sabouraud Dextrose Agar (SDA); 95% Ethyl alcohol. See Appendix A for
recipes.
Antibiotics: Penicillin; Rifampicin; Ampicillin.
Procedure:
1. Prepare 500-ml of each of the following media: CMA; PDA; SDA; MEA.
1. Weigh the designated quantities of ingredients.
2. Dissolve in the required amount of distilled water in a flask.
3. Plug the flask with cotton and sterilize by placing the media in an autoclave for 15-20
minutes at 120 oC and 15-psi pressure.
What a complete medium should provide a fungus with?

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When preparing a medium its necessary to adjust its pH. Why?

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2. Prepare agar slants (slopes) in screw-capped bottles or in tubes as follows:

1. Sterilize the medium in the bottles or tubes (15 ml per bottle).
2. While the agar is still hot and molten lay the tubes on a bench with the plugged ends
supported (in a slanted position) so that the medium forms a layer of decreasing thickness
from the bottom of the bottle to within about 2 cm of the plug. Sloping the agar obviously
provides the growing fungus a relatively large surface.
Agar slants are some times superior over agar plates for growing fungi. Explain.
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3. Pour the media into 9-cm plates as follows:

1. Clean the working area of the bench with a detergent and disinfectant as mentioned
before.
2. Melt the agar medium in the bottles or flasks and then allow the medium to cool to about
60 oC. Antibacterial agents (e.g., penicillin 50 mg/L, rifampicin 10 mg/L, ampicillin 250
mg/L) may be incorporated in the medium before pouring in plates.
3. Place the Petri dishes on the bench.
4. Light a Bunsen burner and proceed as follows.
5. Remove the cotton or plastic plug and flame the mouth of the bottle or flask gently.
6. Pour the medium into the sterile plastic or glass Petri dishes and rotate each dish so as to
spread the agar evenly over its bottom. The lids of the dish is raised just enough to insert
the mouth of the bottle or flask, thereby reducing the danger of airborne contamination.
The dishes are allowed to cool sufficiently for the medium to harden before using.
`Store labeled agar plates and agar slants in the refrigerator at 10 oC until use.
You should cool the medium to about 60 oC before pouring in plates. Why?
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Agar plates are usually stored up side down. Why?

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EXERCISE 2: CULTIVATION AND MICROSCOPIC EXAMINATION OF

FUNGI
Materials needed:
Water cultures of: Pythium aphanidermatum, P. ultimum, Phytophthora parasitica, and
Saprolegnia sp. Agar cultures of: Fusarium, Penicillium, Microsporum canis, Trichophyton,
and Rhizopus stolonifer.
Culture media and chemicals: CMA; PDA; SDA; MEA; Sterile 20 % solution of glycerol;
Lactophenol; Suitable mounting fluid; Nutrient broth; 7% Aqueous safranin; 0.5% Basic fuchsin;
25, 50, 75, 95, and 100% Ethanol; 100% Xylene; Fingernail varnish or commercial water-based
PVA (polyvinyl acetate).
Other materials: Ocular micrometer; Stage micrometer; Cavity slide.
Procedure:
A. Transferring of fungi to Petri dishes and slants
Fungal cultures can be perpetuated in the laboratory as follows:
1. Using a sterile straight inoculating needle or a scalpel, transfer spores or fragments of the
mycelium from one or more of the provided fungal cultures to sterile media (in plates or
culture bottles) which you prepared in the last lab period.
2. Place the inoculated plates or slants in the incubator at 25 ºC until the next lab period.
After 7 days of incubation examine the inoculated plates or culture tubes and record your
observations by describing the growing colonies.
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B. Slide culture technique

1. Prepare a suitable agar medium (e.g. MEA, SDA, CMA, and PDA), and pour into a plate
to a depth of about 2-mm.
2. When it has set completely, cut out a small block, about 1-cm square using a sterile
scalpel and transfer to the center of a sterile slide.
3. Inoculate the block with the fungus on all four edges.
4. Place a sterile coverslip on top of the block of agar.
5. Incubate the slide in a moist chamber (Figure 2.1). This may be made from a sterile Petri
dish with filter paper in the bottom. Two sterile glass rods (or a bent glass rod) are placed on the
filter paper to function as supports for the slide. About 10 ml of a sterile 20 % solution of
glycerol is poured into the dish. This will keep the agar moist. The dish with the inoculated slide
is incubated until the growth reaches a desired stage (until the mycelium formed from each edge
of the block has reached the edge of the coverglass). The fungus usually spreads out from the
agar block and tends to attach itself to the two glass surfaces.
6. When the desired stage of fungal growth has been reached, lift off the coverslip and
carefully lower fungus side down onto a drop of mounting fluid such as lactophenol (with or
without stain) on a clean slide. Carefully remove the agar block, add a drop of mountant (e.g.,
lactophenol) to the growth adhering to the slide, and apply a clean coverslip. Then seal the slides
using nail varnish.
Examine your preparations under the microscope (10 X, 40 X).
B G D
A
F E
Figure 2.1 Slide culture technique: A, culture plate; B, blotter paper; C, bent glass rod; D, glass slide;
E, block of agar; F, inoculum; G, cover slip.
Record and illustrate your observations.

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C. Single spore cultures

Several methods for preparing single spore isolations are available. The following is a quick
method that does not involve the usage of micromanipulators.
1. Using a sterile looped wire needle place a few drops of sterile water on a sterilized slide.
2. Transfer a small amount of the fungus spores to the water drop using a sterile
inoculating needle and mix to form a suspension.
3. Using a sterilized wire needle, pick up a loopfull of the suspension, and streak it onto a
nutrient agar plates using 4 rapid strokes.
4. After incubation for e.g. 17 hr, study the plate with a dissecting microscope when colonies
just starting to develop from single spores. Pick off an isolated colony with an inoculating needle
and transfer to fresh petri dish. Look for isolated colonies at the end of the fourth streak where
the suspension is the finest.

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D. Hanging drop
Hanging drop cultures can be prepared as follows:
1. Place a few spores in water or nutrient broth on a coverslip.
2. Invert the coverslip over the cavity in a cavity slide or a suitable glass or plastic ring.
3. Place the slide in a moist chamber overnight.

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E. Microscopic preparations
a. Temporary mounts:
1. Place a small drop of mounting fluid in the center of a clean glass slide.
2. Pick off a very small portion of the fungal material from the culture, with a sterile needle,
place it in the drop of fluid, and tease it out with a pair of needles.
3. Place a cover glass over the preparation in a way as to avoid air bubbles. Placing the
object directly in the mounting fluid, particularly if this be lactophenol, may result in the
inclusion of air bubbles in the preparation. To avoid such a problem the fungal material is
placed on a slide and moistened with a drop of alcohol. Most of the alcohol is allowed to
evaporate before being replaced by the mounting fluid.
4. Seal the coverslip on the slide using fingernail varnish or commercial water-based PVA
(polyvinyl acetate). Apply two coats of nail varnish and when the nail varnish seal has
dried, apply a thick layer of PVA diluted with water (1:1) over the nail varnish.
Examine the preparation under the microscope. Record and illustrate your observations.
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b. Permanent mounts:
1. Using a sterile needle pick off a very small portion of the fungal material from an agar
culture of Penicillium or Fusarium species and place it in a drop of water, and tease it out with a
pair of needles.
2. By holding the slide with the fungus side upwards, pass the slide rapidly 10-cm above an
open flame a few times.
3. Add a few drops of 7% aqueous safranin and leave for 1 min or for 5 to 20 sec in 0.5%
basic fuchsin.
4. Wash the stained mount in distilled water.
5. Dehydrate the fungus material by dipping the slide for 30-60 sec through a 25, 50, 75, 95,
and 100% ethanol series, then through a 100% xylene and finally in 3:1 and 1:1 mixtures of
xylene and mounting fluid before mounting in the mounting fluid.
Examine the preparation under the microscope. Record and illustrate your observation.
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c. Measurement of microscopic fungal structures:

Determine the value for each of the lines in the ocular micrometer by matching them with lines
on the stage micrometer. This value is fixed for each objective lens.
Record your results in a tabulated form. The ocular micrometer can now function as a ruler in the
microscope.
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Using the method described in Part 1 (1.3.1 c) measure the dimensions of conidia and hyphae in
Penicillium and Fusarium. Record your results.
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Cultures prepared in this lab period may be used for the study of morphology of fungi in
the next lab.
Questions:
1. What conditions of nutrition and environment favor the growth of most fungi in the
laboratory?
2. What is meant by absorptive nutrition?
3. How are fungi commonly propagated in the laboratory?
4. Briefly discuss the role of light in the growth and reproduction of fungi.
EXERCISE 3. MORPHOLOGY OF FUNGI
Materials needed:
Fungal cultures: Four-day-old water cultures of Pythium aphanidermatum, P. ultimum,
Phytophthora parasitica, and Saprolegnia sp.; 1-2 week old agar cultures of Fusarium,
Penicillium, Microsporum canis, Trichophyton, and Rhizopus stolonifer; Cultures prepared in the
previous lab period.
Prepared slides: Claviceps purpurea (l.s., stroma; c.s., sclerotium), Puccinia graminis
(teliospores), Rhizopus stolonifer (sexual stage).
Procedure:
1- Mycelium
a. Coenocytic mycelium: Examine a culture of one or more of the following fungi:
Saprolegnia, Pythium, and Phytophthora, growing on corn kernels in water, by directly putting
the Petri dish on the stage of the microscope and examining it with 10 X objective. Prepare a wet
mount using a small excised portion of the mycelium, and examine under 10 X and 40 X
objectives and note the distribution of protoplast in the hyphae. Look for any septa. Are there
any? If yes, at what locations?
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Can you see any cytoplasmic movement?

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Illustrate.
Coenocytic mycelium
b. Septate mycelium: Examine microscopic preparations of excised portions of

mycelium from an agar culture of one or more of the following fungi: Fusarium, Penicillium,
Microsporum, and Trichophyton. Note the distribution of protoplast in the hyphae. Look for
septa (partitions) that divide hyphae into compartments. Note the pattern of branching in the
mycelium. Can you distinguish any differences in the branching of hyphae between different
fungi? Note the differences in the morphology of the colonies of different fungi that you
examined.
Illustrate.
Septate mycelium
2- Fungal tissues (plectenchyma)

a. Prosenchyma: Examine the stroma (longitudinal section, l.s.) of Claviceps purpurea and
observe its structure with reference to the organization of hyphae within it.
Illustrate.
L.s. in the stroma of Claviceps purpurea showing prosenchyma.
b. Pseudoparenchyma: Examine a cross section (c.s.) of a sclerotium of Claviceps

purpurea. Note its organization with reference to the relationships between hyphae in this
structure. What is the role of such a structure relative to the fungus survival?
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Illustrate.
C.S. of a sclerotium of Claviceps purpurea showing Pseudoparenchyma.
3- Main reproductive structures

1- Asexual structures
a- Chlamydospores: Examine under the microscope (45 X) an excised part of
mycelium of Trichophyton. Observe the thick-walled portions, usually intercalary, of the
mycelium. Illustrate.
Chlamydospores
b- Buds: These can be seen in actively growing yeast. Illustrate.
Yeast buds in actively growing yeast

c- Sporangiospores (formed inside sporangia)

(i) Aplanospores (non-motile): Examine a culture of Rhizopus and observe the
sporangia with the nonmotile spores (sporangiospores) within them. Illustrate.
Sporangia and sporangiospores (aplanospores, non-motile spores) of Rhizopus
How do you think these spores disseminate?

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(ii) Planospores (motile): Examine under the low power of a microscope chilled
undisturbed water culture of Pythium aphanidermatum, Saprolegnia, or Phytophthora parasitica
and observe the biflagellate moving zoospores. Pick a sporangium that is about to sporulate and
watch it for a few minutes until zoospore discharge takes place. Describe and illustrate the
process.
Sporangia, zoospores (planospores, motile spores), and zoospores discharge of … …. …………

d- Conidia (formed at tips of hyphae): Examine under the high power of a microscope a
small portion of mycelium of each of the following fungi: Microsporum canis, Epidermophyton
floccosum, and Fusarium sp. Observe the conidiophores and the various types of conidia.
Illustrate.
Conidia
2- Sexual structures:
a. Oospores (result of gametangial contact): Examine under the low powers of a
microscope a water culture of Pythium ultimum (or a similar fungus). Prepare a wet mount of the
fungus and examine under the high power of the microscope. Observe the sexual structures
present (especially oogonia, antheridia, and oospores). Illustrate.
Oospores of Pythium ultimum

b. Zygospores (result of gametangial copulation): Examine under the high powers of a

microscope the permanent slide of the zygospores of Rhizopus stolonifer. Observe the various
steps of the sexual process that ends up with the formation of zygospores. Illustrate.
Stages of Zygosporangium and zygospores formation in Rhizopus stolonifer zygospores.
c. Ascospores (spermatization): Examine the ascocarps of Claviceps purpurea.

Observe the asci and the ascospores. The ascospores (perithecia) are embedded in the
stroma. How many ascospores can you see in the ascus? Illustrate.
Ascospores, asci and ascocarps (embedded in the stroma) in Claviceps purpurea
d. Basidiospores: Examine a permanent slide of Puccinia graminis. Observe the

teliospores which give rise to basidia and basidiospores. Illustrate.
Teliospores in Puccinia graminis.
Questions:
1. Define the fungi by listing their characteristic features.
2. Differentiate between prosenchyma and psedoprosenchyma.
3. How can parasitic fungi obtain their nutrients?
4. Define heterokaryosis and explain how this phenomenon occurs in fungal thallus.
5. Define or explain: planogametes, hypha, mycelium, septum, mitospore, meiospore.
6. What is the distinguishing chemical component of the cell wall of most fungi?
7. How does the fungal cell wall differ in chemical composition from that of plants?
8. How does mitosis differ from that in plants?
9. Some fungal chromosomes are very minute and difficult to count accurately, what other
criterion is used to determine the occurrence of meiosis in fungi?
10. Where does growth take place in a hypha?
EXERCISE 4. KINGDOM FUNGI: PHYLUM CHYTRIDIOMYCOTA

Class Chytridiomycetes
Order Chytridiales
Family Synchytriaceae
Genus Synchytrium
S. endobioticum (causative agent of potato black wart disease)
Synchytrium endobioticum is a biotrophic parasite. It has not so far been cultured outside
living host cells.
Materials needed:
Infected plant parts: Potato tubers infected with Synchytrium endobioticum.
Prepared slides of: Potato tubers infected with Synchytrium endobioticum.
Procedure:
1. Examine potato tubers infected with Synchytrium endobioticum. Note the effect of
infection on the host.
2. Study the life cycle of the parasite from prepared slides of infected tubers. Search in the
slide for the following stages in the life cycle:
a. Young prosorus.
b. Mature summer spore (prosorus).
c. Germinating summer spore.
d. Sorus of sporangia.
e. Resting sporangium.
3. Observe the size of the nuclei in a, b, and c. Note the relative thickness of the walls of
the prosorus (summer spore) and the resting sporangium. Illustrate.
Stages in the life cycle of Synchytrium endobioticum in a prepared slide of potato tubers infected
with the parasite.
Label the following diagram of the life cycle of Synchytrium endobioticum
Questions:
1. What determines the production of sporangia and gametangia in S. endobioticum?
2. Explain what is meant by a biotrophic parasite.
3. Outline the disease cycle of the potato wart fungus.
EXERCISE 5. KINGDOM FUNGI: PHYLUM ZYGOMYCOTA

Class Zygomycetes
Order Mucorales
Family Mucoraceae
Genus Rhizopus
Rhizopus nigricans
Genus Mucor
Mucor sp.
Materials needed:
Agar cultures of: Rhizopus nigricans and Mucor on agar plates.
Culture media: CMA, Potato-carrot agar (PCA).
Prepared slides: Various stages in zygospore formation: Rhizopus nigricans, Mucor.
Procedure:
Rhizopus nigricans
1- Examine a culture of Rhizopus nigricans on an agar plate. The cottony growth consists of
hyphae, rhizoids, sporangiophores, and sporangia. Mount some of the mycelium on a slide and
observe these different structures. Find the rhizoids, the stolons, the sporangiophores, the
sporangia, collumellae, and sporangiospores.
2- Rhizopus nigricans is a heterothallic species; it requires the union of two mating types of the
fungus (- & +) for sexual reproduction. To demonstrate sexual reproduction in this fungus
crossings can be made on cornmeal agar (CMA) or potato-carrot agar (PCA). The compatible
mating types (- & +) are inoculated at opposite sides of an agar plate. Zygospores are formed
within a few days in a dense zone of contact of the mycelia.
3- Mount some of the thallus from the contact zone formed in 2, on a slide and study the various
stages in zygospore formation.
4- Study prepared slides showing various stages in zygospore formation and compare with what
you see in the slide and fresh culture.
5- Examine a culture or a prepared slide of Mucor and compare with Rhizopus nigricans.
Illustrate the different structures you observe in the provided cultures and prepared slides.
Label the following diagram showing the probable life cycle of Rhizopus nigricans.
Questions:
1. Construct a diagram showing zygosporangium formation.
2. Explain homothallism and heterothallism.
EXERCISE 6. KINGDOM FUNGI: PHYLUM ASCOMYCOTA
Materials needed:
Cultures: Schizosaccharomyces octosporus on ascospore agar.
Culture media: Ascospore agar.
Infected plant parts: Peach leaves infected with Taphrina deformans. Plant parts (e.g., leaves of
roses) infected with a powdery mildew.
Prepared slides: Different types of ascocarps: cleistothecia (c.s. in ascocarp of Erysiphe
graminis; cleistothesium, whole mount, Uncinula sallics), perithecia (median sec. in apple leaf
infected with Venturia inequalis), apothecia (l.s., apothecium, Morchella sp.), ascostroma (c.s.
stroma, Claviceps purpurea, causal agent of ergot of Gramineae). Schizosaccharomyces
octosporus; l.s. through the head of a sclerotium of Claviceps purpurea.
Ascospores, Asci, and Ascocarps:
Procedure
Examine under the low and high power objectives prepared slides showing different types of
ascocarps: cleistothecia (c.s. in ascocarp of Erysiphe graminis; cleistothesium, whole mount,
Uncinula sallics), perithecia (median sec. in apple leaf infected with Venturia inequalis; Xylaria
sp.), apothecia (l.s., apothecium, Morchella sp.), ascostroma (c.s. stroma, Claviceps purpurea,
causal agent of ergot of Gramineae). Illustrate.
Types of ascocarps
Label the different types of ascocarps shown in the following diagram
Questions:
1. Construct a diagram showing ascus formation.
2. Name the types of ascocarps.
3. Define or explain: ascus, conidium, ascocarp, pycnidium, antheridium, heterothallic,
spermatum, cleistothesium, ascogonium, trichogyne.
Class Archiasciomycetes
Order Schizosaccharomycetales (Ascospore-forming Yeasts)
Family 1 Endomycetaceae
Genus Schizosaccharomyces (Fission Yeasts)
S. octosporus
Procedure
From prepared slides of S. octosporus, study somatic cells, ascus and ascospores formation.
Search your slide for various stages of development that begin with the copulation of two cells
and end with the mature ascus. Note the variation in the number of ascospores in the asci.
Illustrate somatic cells, ascus and ascospores of S. octosporus. Illustrate various stages of
ascospores development.
Questions:
1. How does sexual reproduction take place in the yeast?
2. What does fission yeast mean?
3. What is the importance of yeast to man?
Order Saccharomycetacetales
Family Saccharomycetaceae (Budding Yeast)
Genus Saccharomyces
S. cervisiae
Procedure
1- Study a portion of a colony of the fungus and observe the somatic cells.
2- Ascospore formation can be induced by growing the yeast on a nutrient-rich medium
containing an assimilable sugar, a suitable nitrogen source for good growth, and vitamins of the
B group. Sodium or potassium acetate (0.1-1.0 % w/v) was found to stimulate sporulation (Haber
& Halvorson, 1975). Study a microscopic preparation made from such a culture and search your
slide for various stages of development beginning with the copulation of two cells and ending
with the mature ascus. Compare and contrast with Schizosaccharomyces octosporus.
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Illustrate various stages of ascospores development in Saccharomyces cervisiae.

Label the following diagrams showing the probable life cycles of Schizosaccharomyces
octosporus and Saccharomyces cervisiae.
Questions:
1. What do budding yeasts mean?
2. What ascosporogenous and sporogenous yeasts mean?
Order Taphrinales
Family Taphrinaceae
Genus Taphrina
T. deformans (Causative agent of leaf curl disease in peaches and almonds)
Procedure
1- Section infected peach leaves and mount the sections in water. Study the asci of Taphrina
deformans.
2- Study prepared slides showing hymenial layer with asci at different stages of development.
Note the stalk cell, the asci, and the ascospores.
Illustrate and record your observations.
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Label the following diagram showing the probable life cycle of Taphrina deformans
Questions:
1. How does the binucleate condition arise in Taphrina deformans?
2. How does Taphrina deformans reproduce asexually?
3. How does Taphrina deformans overwinter?
Filamentous Ascomycetes
Pyrenomycetes (Ascomycetes with perethesia)
Order Clavicipitales
Family Clavicipitaceae
Genus Claviceps
C. purpurea
Procedure:
1- Examine permanent preparations of l.s. through the head of a sclerotium under low
magnification and draw a general view, and then under higher magnification draw a section
showing a part of a perithecium. The surface of the stroma head is covered with sunken
perithecia (ascostroma). The inoperculate asci have a typical cap at their apex that is penetrated
by a pore.
2- Study the preserved specimen of the sclerotia available. Illustrate and record your
observations.
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Label the following diagram showing the probable life cycle of Claviceps purpurea
Questions
1. What is a sclerotium, what is its function?
2. What is ergot? Ergotism? Ergotamin?
3. How are the sclerotia of Claviceps purpurea used medicinally?
Other Filamentous Ascomycetes

Order Erysiphales
Family Erysiphaceae (Powdery Mildews)
Genera Erysiphe, Sphaerotheca, Microsphaera, Levillula,
Uncinula, Phyllactinia, Podosphaera.
Sphaerotheca pannosa (powdery mildew of roses)
Procedure:
1- Using a dissecting microscope examine plant parts (e.g., leaves of roses) infected with powdery
mildew. Note the hyphae that form a white mat over the infected portions. Strip a portion of the
epidermis and mount in water. Study under the microscope the hyphae of the fungus and note
that they are richly branched and septate, and that they penetrate the epidermal cells by means of
numerous haustoria. Illustrate and record your observations.
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2- Observe the erect conidiophores and the conidial chains under the dissecting microscope. Study
these structures under the compound microscope in a water mount. Illustrate and record your
observations.
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3- Mount some cleistothecia (formed in the late summer over the surface of the host plant;
easily seen with a lens) in water or lactophenol under a coverglass. Note the appendage
characteristics of the genus, paying more attention to the tips and basis of the appendages,
and to the manner in which the tips may be branched. While looking through the tube of the
microscope press on the coverglass with a mounting needle and watch the asci with
ascospores break out of the cleistothecia. Illustrate and record your observations.
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Label the following diagram showing the life cycle of Sphaerotheca pannosa.
EXERCISE 7. KINGDOM FUNGI: PHYLUM BASIDIOMYCOTA

Class Uredinomycetes
Order Uredinales
Family Pucciniaceae
Genus Puccinia
P. graminis var. tritici (causative agent of the black stem rust of wheat)
Materials needed:
Infected plant parts: Aboveground parts of corn plant infected with smut.
Infected wheat stems with the black stem rust.
Fresh basidiocarp of Agaricus sp., Pleurotus ostreatus.
Media and chemicals: MEA; Calcium sulfate (gypsum); Pasteurized medium clay loam or
peat / limestone mixture (calcium carbonate).
Plant and animal materials: Fresh Horse or chicken manure; Wheat or barley straw; Wheat
bran; Grain (e.g., rye, wheat, or sorghum).
Prepared slides: Barberry leaves infected with the black stem rust; C.s. of wheat stem bearing
uredinia; C.s. of a wheat stem bearing telia; C.s. of a part of a corn plant infected with Ustilago
maydis.
Other materials: Glass bell jar; 2-L flasks; Trays.
Procedure:
1- Under the microscope examine prepared slides of barberry leaves infected with the rust. The
spermogonia (Stage 0) can be seen on the upper epidermis. Observe the aecia on the lower
epidermis. Note the lip of the aecium. Illustrate and record your observations.
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2- Study the spermogonia under the high power (or the oil immersion) objective. Note the
spermatiophores, spermatia, and periphyses. Some sections may also show the drops of nectar
exuding from the spermogonia. Illustrate and record your observations.
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3- Study a mature aecium and note the basal aeciospore mother cells from which the chains of
aeciospores and the peridium form. Illustrate and record your observations.
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4- Examine infected wheat stems and find the uredinia (Stage II) which are rusty in color.
Scrape a uredinium with a scalpel and mount some urediniospores onto a slide in lactophenol
cotton blue. Study the spores under the oil immersion objective. Study a prepared slide showing
c.s. of a wheat stem bearing uredinia. Observe the origin of the uredinium and its effect on the
host epidermis. Note the long stalks of the urediniospores. Illustrate and record your
observations.
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5- If wheat straw bearing the teletial stage (Telia, Stage III) of the fungus is available you could
observe the black pustules from which the disease "black stem rust of wheat" takes its name.
Teliospores can then be studied in a lactophenol mount under the oil immersion objective; their
color, number of cells in each, and thickness of the wall could also be noted. Illustrate and record
your observations.
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6- Study prepared slides showing cross sections of a wheat stem bearing telia. Note how the
teliospores are borne in the telia and note their position in relation to the host cells and to each
other. Illustrate and record your observations.
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Label the following diagram showing life cycle of Puccinia graminis.

Questions:
1. Explain how the rusts obtain nourishment from the host?
2. Define basidium; clamp connection; dolipore septum; teliobasidium; dikaryotic;
spermatiophore; spermogonium; autoecious rusts; heteroecious rusts.
3. How do long-cycle rusts differ from short-cycled rusts?
4. How do the rusts reproduce sexually?
5. The uredospores are considered to be the conidia of the rusts. Why?
6. What structures represent the basidia of the rusts? Explain.
Class Ustilaginomycetes
Order Ustilaginales
Family Ustilaginaceae
Genus Ustilago
U. maydis (Corn Smut)
Procedure:
1- Examine various parts of an infected corn plant and note the smut balls on the above ground
plant parts. Illustrate.
2- Mount some smut spores (teliospores) in lactophenol and study under the oil immersion of
objective. The teliospores of the Ustilaginaceae produce a 3-septate (four-celled) promycelium
from which the basidiospores are formed laterally. The teliospores have minute spines on their
surfaces. They are thick-walled, and dark in color. Note the size of the spores. llustrate.
3- Transfer some teliospores into 3-5 ml water or aqueous Treacle medium (Esser, 1982) in a
sterilized test tube, incubate at 30 oC and begin to study the germination of the spores in a
hanging drop after 24 hours. Illustrate and record your observations.
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4- Study prepared slides showing a c.s. of a part of a corn plant infected with Ustilago maydis.
Note how the teliospores are borne in the telia and note their position in relation to the host cells
and to each other. Illustrate and record your observations.
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Label the following diagram showing the detailed life cycle of Ustilago maydis.
Questions:
1. Explain in some detail the method of teliospore formation in the Ustilaginales.
Class Hymenomycetes
Order Agaricales (Mushrooms and boletes)
Family Agaricaceae (Mushrooms)
Genus Agaricus
Agaricus bisporus
Family Tricholomataceae
Genus Pleurotus
Pleurotus ostreatus (oyster mushroom)
These mushrooms bear their basidia on gills (lamellae) which form on the underside of the
cap of an umbrella-like basidiocarp. Most of the edible mushrooms are members of the genus
Agaricus. Agaricus bisporus, the common edible mushroom, is the main species used for
growing on an industrial scale.
1. Main morphological features of Agaricus bisporus
Procedure:
1. Study the basidiocarp of Agaricus sp. observe the stipe, pileus, the viel, the annulus, and the
lamellae. Illustrate.
2. Remove a piece of lamella from the mushroom basidiocarp. Use forceps to peel off the skin
from the surface of the lamella (or alternatively make a cross section). Mount in a drop of water
or lactophenol cotton blue on a clean glass slide. Cover with a coverslip and examine for basidia
and basidiospores. Illustrate.
3. Choose a well-formed sporophore of any species of mushroom and prepare a spore print as
follows: with a sharp razor blade cut of the stem squarely with the edge of the gills. Place
mushroom cap down so that half of the gills rest on a piece of white paper and the other half on a
piece of dark paper. Cover with a bell jar or other cover and allow to stand until the next lab
period. Lift the bell jar and carefully remove the mushroom cap note the spore print and
determine the color of the spores. Illustrate.
Construct a life cycle diagram of Agaricus bisporus.
2. Cultivation of the edible mushroom Agaricus bisporus:

Procedure:
1. Spawn production. Pure cultures of A. bisporus may be derived from spores or tissues of
this mushroom and germinated on agar medium as follows.
a. For culture from spores (basidiospore collection and germination): Place the cap from fresh
mushroom gills down on a piece of clean paper or a microscope slide. Add a drop of water
to the cap surface to aid spore release. Place a container over the mushroom to decrease
evaporation and disturbance from air currents. After a few hours store the deposited spores
in a labeled airtight container until use. For spore germination, use one of the following
techniques. 1. Allow spores to fall onto a nutrient agar such MEA. 2. Collect fresh spores as
described above, mix them with sterile water and plate out a series of dilutions onto nutrient
agar. Incubate in darkness at 20-25 ºC.
b. For culture from basidiocarp tissue: Use a recently picked young basidiocarp and brush off
any surface detritus. Break the basidiocarp in half. Using a sterile needle, remove about 10-
mm3 pieces of tissue from different areas including the stipe. Partially, immerse the tissue
sample in the agar. Incubate in darkness at 20-25 ºC.
c. Subculture the new mycelium (after 3-7 days). Store the mycelium growing over the
surface of an agar slope in a small bottle. If necessary, submerge the surface in sterile mineral
oil and keep the bottle in a refrigerator at 10 ºC.
d. Production of grain-spawn: The grain recipe comprises grain (e.g., rye, wheat, or
sorghum), water, and calcium carbonate (1-3 % by weight).
1. Soak the grain in water overnight
2. Decant water.
3. Mix with calcium carbonate.
4. Autoclave for 30 minutes and repeat the process after 24 hours.
5. Inoculate the grain by placing mycelium in agar on the grain in a suitable container (e. g.,
2-L flasks). Alternatively, suspend the mycelium in sterile water and add the mixture to
the grain.
6. Close the container and shake to distribute fragments of mycelium.
7. Store at 20-25 ºC in darkness.
8. After a few days, when the mycelium becomes visible, shake to ensure an even
distribution.
9. Store the spawn in a refrigerator (but not frozen) until use.
2. Compost production. Composting is done outdoors and usually requires about 2 weeks to
complete. The compost (a substrate that is high in insoluble nitrogen-containing compounds
as well as cellulose and lignin) provides the nutritional requirements of the fungus as well as
the physical and chemical conditions that give the fungus a competitive edge over other
microorganisms. The raw materials for the compost are straw, manure, calcium sulfate
(gypsum), water, and nutritional supplements. Wheat straw is preferred, because it is resilient
and maintains an open well-ventilated compost structure. The following recipes are based on
using fresh materials but are given as dry weight ratios. Horse manure has proportion of
manure and straw 100 to gypsum 5 while synthetic compost is made up in these proportions:
wheat straw 52, chicken manure 45, gypsum 3.5. Prepare mushroom compost as follows.
1. First phase
a. Mix fresh manure and straw.
b. Soak the mixture and then stack. To prevent the development of anaerobic conditions
within the stack the vertical cross-section is generally not more than 2X2 meters. This
allows the microorganisms present in the raw materials to begin to degrade the stack.
Their activity heats up the stack, which in turn makes conditions ideal for further
decomposition by other microorganisms. The stack core is depleted of oxygen in 48-96
hours and it is then rebuilt.
c. Repeat this procedure 2-3 times during the first phase to allow decomposition to
continue, typically during the third, fifth and seventh day after initial mixing. The center
of the stack should reach 65-80 ºC. At the end of this phase the compost should be deep
brown, be flecked with whitish colonies of actinomycetes, smell of ammonia, have a pH
of 8.0-8.5, and release liquid when firmly squeezed.
2. Second phase:
a. Fill the compost into available trays, beds or bags and pasteurize (58-60 ºC). This step
kills nematodes, fly eggs and larvae, mites and competitor fungal mycelium and spores.
b. Allow the compost to ferment for a further 5-8 days. During this time maintain the
internal temperature of the compost at about 50 ºC by controlling ventilation.
c. When the ammonia concentration has dropped to 10-20 g per gram allow the
temperature to drop to 25 ºC.
3. Inoculation and spawn running

a. Add spawn to compost (usually as 1-2% by wet weight). About 50g of spawn is
sufficient for 9.1 kg of fresh compost.
b. Incubate at about 24 ºC for up to 14 days. Make sure that temperature in the compost
does not exceed 28 ºC.
4. Casing
a. Spread a layer of soil [pasteurized medium clay loam or peat/ limestone mixture
(calcium carbonate)] over the surface of the compost at a depth of 2-4 cm.
b. Incubate at 25 ºC for 10 days.
c. Ventilate to lower compost temperature to 14-18 ºC and carbon dioxide content of the
atmosphere, and incubate for 11 days. This induces the formation of initials and
primordia which, develop into buttons and mature mushrooms (basidiocarps). Primordia
form 14-18 days after casing.
5. Harvesting. Harvest over a three-day period commencing 21 days after the initial casing.
6. Terminal disinfection. After cropping 5-6 weeks dispose of the culture and disinfect the
trays.
Record your observations.
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2. Cultivation of the oyster mushroom Pleurotus ostreatus:

1. Spawn production. Pure cultures of P. ostreatus may be derived from spores or tissues of this
mushroom and germinated on agar medium as follows.
a. For culture from spores (basidiospore collection and germination): Place the cap from fresh
mushroom down on a piece of clean paper or a microscope slide. Add a drop of water to
the cap surface to aid spore release. Place a container over the mushroom to decrease
evaporation and disturbance from air currents. After a few hours store the deposited spores
in a labeled airtight container until use. For spore germination, use one of the following
techniques. 1. Allow spores to fall onto a nutrient agar such MEA. 2. Collect fresh spores
as described above, mix them with sterile water and plate out a series of dilutions onto
nutrient agar. Incubate in darkness at 20-25 ºC.
b. Subculture the new mycelium as described for Agaricus species.
c. Production of grain-spawn: The grain recipe comprises 240 ml grain (e.g., rye, wheat, or
sorghum), 170-200 ml water (adjust to 50 % moisture), and calcium carbonate 1-3 % by
weight). Mix the above-mentioned ingredients. Autoclave for 30 minutes and repeat the
process after 24 hours. This step can alternatively be carried out as mentioned for
Agaricus.
d. Inoculate the grain by placing mycelium in agar on the grain in a suitable container (e. g.,
2-L flasks or wide-mouth bottles). Alternatively, suspend the mycelium in sterile water
and add the mixture to the grain.
e. Close the container and shake to distribute fragments of mycelium.
f.Incubate at 22 ºC in darkness.
g. After a few days, when the mycelium becomes visible, shake to ensure an even
distribution.
h. After two weeks of incubation, store the spawn in a refrigerator (but not frozen) until
use.
2. Cultivation room. Use a well-ventilated clean dark room with windows provided with
wire gauze to prevent the entry of insects. Relative humidity should be kept at 80-85%. The
floors and walls of the room are disinfected with a suitable disinfectant (e. g., septol or detol).
3. Cultivation mixture or medium. The raw materials for the cultivation mixture are wheat
or barley straw, wheat bran, limestone (calcium carbonate) (w/w 20:1:1). Wheat straw is
preferred, because it is resilient and maintains an open well-ventilated medium structure.
Prepare cultivation mixture or medium as follows.
1. Cut straw into 8-15 cm long parts.
2. On a sheet of plastic mix the straw with bran and limestone.
3. Place the mixture in an autoclave bag; moisten (until saturation) with deionized or
distilled water and seal.
4. Autoclave for 1 hour. Repeat this step after 24 hours. Decant excess water from the
medium when necessary. The medium is now ready for spawning.
4. Inoculation and spawn running.

a. Place the cultivation mixture in layers of 15 cm deep into available sterile trays, beds,
bags or vegetable baskets held at 15 cm above ground. Sprinkle the spawn on the surface
of each layer. Nearly 0.5-kg spawn is used for 11 kg of cultivation mixture.
b. Spread a layer of the mixture on the surface of the last layer at a depth of 2 cm.
c. Cover the containers with black plastic sheets from all sides except from bottom to avoid
accumulation of CO2 and moisture.
d. Incubate at 25 ºC for 10 days or until the appearance of white layer of mycelium on the
surface of the cultivating mixture.
e. Lift the plastic cover and incubate at about 20 ºC for up to 14 days. Make sure that
temperature in the compost doesn’t exceed 28 ºC.
f. During the incubation period sprinkle the mixture with water twice a day (morning and
evening), light the incubation room for 2 hours twice a day and ventilate to lower carbon
dioxide content of the atmosphere.
5. Harvesting. Harvest over a two-week period commencing 14 days after the removal of
the plastic cover. Three to four crops may be obtained over a 2-month period.
6. Terminal disinfection. After cropping dispose of the culture and disinfect the trays.

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Questions:
1. How would you distinguish between edible and poisonous mushrooms?
2. What species of mushrooms are grown commercially?
3. How would you determine whether or not a given mushroom is self-incompatible or not?
EXERCISE 8. KINGDOM FUNGI: DEUTEROMYCETES: ASEXUAL

ASCOMYCETES AND OTHER ASEXUAL FUNGI
The classification of Deuteromycotina (Fungi Imperfecti) is artificial and based on the form of
the conidia and conidiophores produced by these fungi, and the absence of a sexual cycle. A few
representatives of these fungi will be discussed in this exercise. The description of the individual
taxa uses the terms form order, form family, form genus and form species corresponding to the
use of the term form class.
Materials needed:
Cultures on agar plates of: Epidermophyton floccosum, Trichophyton rubrum, T.
mentagrophytes, Microsporum canis, and Trichoderma sp.
Procedure:
Onygenales
Form genus Epidermophyton
Epidermophyton floccosum is a common cause of groin and foot infections and may occasionally
cause body and nail infections.
1. Examine a culture of E. floccosum on an agar plate.
2. Mount some of the mycelium on a slide and observe the different structures. Observe the
large, clavate, multi-septate, smooth, thin-walled conidia (macroconidia). The hyphae also
produce chlamydosporse, which you will recognize as swollen cells in the hyphae. Illustrate.
Onygenales
Form genus Trichophyton (Arthroderma)
Trichophyton rubrum causes groin, foot and nail infections; it can also cause body and facial
lesions. Trichophyton mentagrophytes causes small-spored non-florescent ectothrix infection in
domestic, farm and laboratory animals and a wide variety of wild animals, it also causes human
infections of the scalp, beard and skin.
1. Examine a culture of T. rubrum on agar plate. Cultures of this fungus are white and floccose
with red to brown pigment on the reverse side Mount some of the mycelium on a slide and
observe the different structures. Look for microconidia, which are small, tear-shaped and often
borne laterally on the hyphae. Macroconidia are usually few in numbers, multicelled, smooth-
walled, pencil-shaped and attached directly to hyphae. Illustrate.
2. Examine a culture of T. mentagrophytes. Cultures of this fungus are fluffy or granular, white
or cream in color often with radiate margins. A dark brown or red pigment often develops on the
reverse. Mount some of the mycelium on a slide and observe the different structures. Look for
microconidia which are spherical in shape and often produced in clusters. Macroconidia are
usually few in numbers, multi-celled, smooth-walled and cigar-shaped, with a definite narrow
attachment at the base. Illustrate.
Onygenales
Form genus Microsporum (Arthroderma)

Microsporum canis causes infection in kittens and puppies, which may be transmitted, to man
giving rise to scalp and body lesions. Infected hairs show a small- spored ectothrix infection with
bright green fluorescence.
1. Examine a culture of M. canis on agar plate. Cultures of this fungus show abundant white
aerial mycelium which, becomes buff in color, with bright yellow or orange pigment develops
on the reverse.
2. Mount some of the mycelium on a slide and observe the different structures. Macroconidia
are usually multicelled with roughened walls; spindle-shaped with terminal end sometimes
curved. Microconidia few or usually absent. Illustrate.
Form genus Trichoderma

T. viride
1. Examine a culture of Trichoderma sp. on an agar plate. Colonies are fast growing, wooly or
cottony, thin, white at first become green or yellow green or remaining white due to dense mats
of conidia on surface.
2. Mount some of the mycelium on a slide and observe the different structures. Look for the
septate hyphae with short-branched conidiophores with ultimate flask shaped phialides opposite
or in whorls. Single-celled conidia formed in rounded clusters at tips of conidiophores, colorless
to green. Illustrate.
Questions:
1. Why are the Deuteromycotina also called Fungi Imperfecti?
2. How do the genera Aspergillus and Penicillium relate to human affairs?
3. What types of nutrition occur among the Deuteromycetes?
4. What is the significance of predacious fungi?
5. What is the relationship between the Deuteromycetes and the Ascomycetes?
Basidiomycetes?
6. List some important animal and human diseases caused by members of the Deuteromycetes
and name the causal organism for each.
EXERCISE 9. KINGDOM PROTISTA: PHYLUM

PLASMODIOPHOROMYCOTA
Class Plasmodiophoromycetes
Order Plasmodiophorales
Family Plasmodiophoraceae
Genus Plasmodiophora
P. brassicae (causative agent of club-root disease of cabbage).
Materials needed:
Infected plant parts: Cabbage roots infected with Plasmodiophora brassicae.
Prepared slides: Cabbage roots infected with Plasmodiophora brassicae.
Procedure:
1- Examine cabbage roots infected with Plasmodiophora brassica and compare with normal
roots. Club-root disease or finger-and-toe disease is common in gardens and fields where
cabbages are frequently grown, especially if the soil is acid and poorly drained. Often infected
root hairs are hypertrophied, expanding at their tips to form club-shaped swellings, which are
sometimes lobed and branched. Illustrate.
2- With a very sharp razor blade cut a very thin section of a diseased rootlet. Mount in water and
examine for resting spores (RS) in a sporosorus. Swollen roots contain large numbers of
spherical resting spores, and when roots decay, the spores are released into the soil. The RS
germinates to give a single zoospore. Illustrate.
3- Study a prepared slide of an infected root. Find some infected cells containing plasmodia, and
some containing the mature spores of the fungus. Note how the tissues (especially the vascular
tissues) are disarranged. Compare infected and normal cells for size and contents. Illustrate.
Label the following diagram showing the life cycle of Plasmodiophora brassicae.
Questions:
1. During their growth both primary and secondary plasmodia in Plasmodiophora brassicae
are characterized by distinctive meitotic divisions (crusiform nuclear division). Explain.
EXERCISE 10. KINGDOM STRAMENOPILA: PHYLUM OOMYCOTA

Class Oomycetes
Order Saprolegniales (Water Moulds)
Family Saprolegniaceae
Genus Saprolegnia
Saprolegnia sp.
Materials needed:
Water and agar cultures of: Saprolegnia sp., Pythium aphanidermatum and Phytophthora
megasperma or Phytophthora parasitica (A1, A2 mating types).
Infected plant parts: Leaves of grape, crucifers, spinach, snapdragon, onion, or tobacco,
infected with downy mildews; Plant parts infected with white rust.
Prepared stained slide of: Saprolegnia; Sections of leaves of grapes and crucifers or spinach, or
snapdragon, or onion, or tobacco, infected with downy mildews; Sections of leaves of a
cruciferous plant infected with white rust.
Procedure:
1- Examine under the low power objective a culture of Saprolegnia sp. growing on a corn
kernel in water. Study the somatic hyphae, the oogonia and the sporangia. Search for zoospores,
encysted zoospores, and germinated zoospores with germ tubes. If you have enough time watch
the release of zoospores from sporangia. Illustrate.
2- Mount an excised small portion of the thallus in distilled water. Study the hyphae that have
sporangia at their tips. It is possible to observe the formation and release of zoospores in a
sporangium at the right stage of development. Observe the movement of the zoospores and the
germination of encysted zoospores under the high power objective. Search for a proliferated
zoosporangium (a new zoosporangium usually grows from the septum at the base of an old
zoosporangium following the discharge of the latter). Illustrate.
3- Study hyphae, which bear young and old sex organs. Search for young oogonia with
antheridial branches appressed to the walls of oogonia. Find some older oogonia in which the
protoplasts have differentiated into oospheres. Count the number of oospheres in several
oogonia. Find some intercalary and some terminal oogonia. After fertilization oospheres develop
into oospores. Observe the location of the oospore. Illustrate. Describe and name the type of
oospores.
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4- Study a prepared, stained slide of Saprolegnia and observe zoosprangia, oogonia, antheridia,
and oospores. Illustrate.
Label the following diagram showing the life cycle of Saprolegnia sp.
Questions:
1- How can you isolate members of the Saprolegniaceae?
2- Define monoplanetism, diplanetism.
3- What is the economic importance of the Saprolegniaceae?
4- What characteristics distinguish the Oomycetes from other fungi?
5- Describe the hormonal control mechanisms in Achlya. What is antherediol?
Order Peronosporales
Family Pythiaceae
Genus Pythium
P. aphanidermatum
Genus Phytophthora
Ph. parasitica
Procedure
1- Examine a culture of Pythium aphanidermatum, or other species growing on corn kernels in
water, by directly putting the Petri dish on the stage of the microscope and examining it with x 10
objective. Search for terminal and intercalary sporangia. Prepare a wet mount using a small
excised portion of the mycelium, and examine under x10 and x45 objectives. Search for oogonia,
antheridia, oospores, sporangia, and zoospores. Illustrate.
3. Examine a water culture of Phytophthora sp. (e. g., P. infestans, P. parasitica). Repeat the
same procedure used for Pythium for studying sporangial shape, zoospore discharge, sporangial
renewal, the relationships between oogonia and antheridia, fertilization tubes, numbers of
oospores and stages in maturation of oospores. Illustrate.
4. Using agar cultures of A1 and A2 mating types of Phytophthora parasitica cross between the
two mating types on cleared V8 agar medium or any other suitable media (e.g., lima bean agar,
CMA with beta-sitosterol at 0.01 g/L and thiamin-HCl at 0.001g/L). Illustrate and record your
observation.
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Label the following life cycle diagram for Pythium debaryanum.

Label the following life cycle diagram for Phytophthora infestans.
Questions:
1. Distinguish between Pythium and Phytophthora in relation to sporangial form, zoospore
formation and discharge, and relationships between oogonia and antheridia.
2. Name some important plant diseases caused by each of these two genera and give the name
of the causal organism for each disease.
Family Peronosporaceae (Downy Mildews)

Genus Plasmopara
Plasmopara viticola
Genus Peronospora
Peronospora parasitica
Procedure
Plasmopara viticola
1- Examine leaves of grapes infected with Plasmopara viticola. Look for the downy growth of
the fungus on the underside of the leaves. The downy growth consists of sporangiophores and
sporangia that emerge through the stomata. Scrape some of the downy growth on a slide and
mount in water under a coverslip. Look in the preparation for a typical sporangiophore and study
the method of branching (monopodial). Note the tiny sterigmata on which sporangia are borne.
Illustrate.
2- Examine prepared slides showing sections of infected leaves of grape. Study the
sporangiophores, the hyphae (intercellular), and the haustoria of the fungus. Study the oogonia,
the antheridia, and the oospores. Note the thick oospore wall. Illustrate.
Peronospora parasitica (downy mildews of crucifers)

1. Examine leaves of plant (snapdragon, onion, tobacco, crucifers, and spinach) infected with a
species of Perenospora, and note the downy growth on the underside of the leaves, which
consists of sporangiophores with sporangia. Illustrate.
2. Scrape some of the downy growth on a slide and mount in lactophenol under a coverslip.
Note the dichotomous branching of sporangiophores, which is typical of the genus. Note that the
branches taper to curved pointed tips on which sporangia are borne. Illustrate.
3. Examine prepared slides showing sections of infected leaves of a cruciferrous plant. Study
the sporangiophores, the hyphae (intercellular), and the haustoria of the fungus. Study the
oogonia, the antheridia, and the oospores. Illustrate.
4. Using the same procedure, study representative species of as many other genera
(Pseudoperonospora, Basidiospora, Sclerospora, and Bremia) as possible and familiarize
yourself with the characteristics of each. Illustrate.
Label the following life cycle diagram for Plasmopara viticola.
Questions:
1- Compare and contrast the Peronosporaceae and the Pythiaceae (in tabular form).
2- How do the sporangia of Plasmopara viticola and Peronospora germinate?
3- Distinguish between sympodial, monopodial, and dichotomous branching.
Family Albuginaceae (White Rusts);

Genus Albugo
Albugo candida
Procedure
1- Examine plants parts infected with white rust and note the white crust on the surface. Scrape
off some of this material and mount under a coverslip in lactophenol and examine. Illustrate.
2- Study prepared slides showing cross sections of leaves of a cruciferous plant infected with
Albugo candida. Look for the:
a- somatic hyphae; b- haustoria; c- sporangiophores; d- chains of sporangia; e-
antheridia and oogonia; f- oospores. Illustrate.
3- Examine prepared slides showing sections of infected leaves of a cruciferous plant. Look
for the different fungal structures mentioned in 2 above. Illustrate.
Label the following life cycle diagram for Albugo candida.
Questions
1. What are the main characteristics of the Albuginaceae?
2. What is the relation of the somatic hyphae to the cells of the host?
3. Of what material is the white, crust-like substance on the surface of the host composed?
4. Explain how the sporangia are formed and how they germinate?
5. How does the Albuginaceae overwinter?
EXERCISE 11. SOIL-BORNE FUNGI
Materials needed:
Plant material: Young citrus leaves; Cottony rot diseased cucumber fruits; Oat grains; Oat
grain-water agar medium (OGWA).
Culture media and chemicals: PDA; VP3 medium; 3P medium; 2P medium; Clorox.
Isolation Methods
Various methods have been used for the isolation and enumeration of fungi from soil. Plating
methods, including the soil-dilution-plate method, are the most widely used methods in
quantitative ecology of fungi.
Collection of Soil Samples

Soil samples from: (A) Permanent irrigated soils under vegetables; (B) Irrigated citrus orchards;
(C) Non-irrigated soils under vegetables; (E) Non-cultivated soils; (F) Waterlogged soil, may be
collected.
1. Collect soil from a depth of 0-10 cm with a trowel. Soil sample consists of 4 aliquots each
of approximately 250-g soil chosen randomly from an area of 4 m².
2. Mix the four aliquots thoroughly in a single plastic bag (a composite sample).
3. Weigh two 50-g aliquots of soil out of the composite sample and put into an oven to dry
overnight at 105 ºC to determine soil moisture content.
4. Divide the remaining composite sample into three equal parts, each of which is considered
as a true replicate sample.
A. Surface-Soil-Dilution Plates (SSDP)

Preparation of Soil Suspension
1. Prepare a soil suspension from each soil replicate sample by taking 50 g of fresh, wet soil, and
dilute in a graduated cylinder with 0.08% sterile water agar to give a total volume of 250 ml.
2. Transfer the suspension to a sterile 500-ml Erlenmeyer flask, close with aluminum foil and set
on a wrist shaker for about 20-30 minutes.
3. Remove the flask from the shaker, shake vigorously by hand, and then transfer 10 ml of the
suspension immediately to a sterile 100-ml measuring cylinder. Add sterile distilled water to
give a total volume of 100 ml. Repeat the same dilution procedure to give the desired
dilutions.
4. Use soil dilutions of 1:100, and 1:1000.
Inoculation of Medium Plates

1. Gently shake the desired soil dilution by hand, and by using a sterile pipette, transfer 1-ml
aliquot of the appropriate soil dilution on to the surface of an agar medium plate (PDA or VP3
selective medium) and spread over the surface using a sterile, bent glass rod.
2. Rotate gently the plate to give an even spread. Use five plates for each of the three
replicate samples from each soil and for each dilution used.
Label plates with soil, dilution, agar and your own name.
Determination of Inoculum Density of Fungi

1. After inoculation, incubate the plates in the dark at 23+1ºC for about 36 hours.
2. Mark colonies of fungi species on the back of the plate and count them.
3. Transfer fast-growing colonies to malt extract agar plates amended with 50 mg of
polymixin-B, and 50 mg of penicillin per liter.
4. Incubate the plates for an additional 24 hr to allow slower growing fungal species to
become visible.
5. After 72 hr, record final counts of fungal colonies as number of fungal colonies per plate.
The mean number of fungi colony forming units per gram dry weight (CFU g-¹ D.W) for
each replicate soil sample is calculated as follows: CFU g-1 D. W. = (Total number of fungal
colonies per replicate sample x dilution factor x soil sample fresh wt.) / (Soil sample dry wt. x
number of replicate plates).
1. Which dilution has produced between 20 and 40 fungal colonies (exclude yeast and
bacteria)?
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2. Is this the same dilution for each soil?

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3. How do your results compare with others in the class?

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4. Count the number of colonies per plate at this dilution, prepare a mean and calculate the
number of colony forming units per gm oven-dry weight of soil.
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5. Examine the plates for mycelia with different growth rates and growth habits. Can you
recognize any of the fungi?

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6. Can you see any signs of medium discoloration? Is this phenomenon associated with any
particular fungus?
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7. Can you see any evidence of antibiosis, due to the proximity of either bacteria or other
fungi?
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Record your results. Exchange and discuss the results with other groups in the class. Record
your conclusions.
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B. Soil Baiting Technique

Citrus leaf discs may be used for the isolation of Phytophthora spp from soil of citrus orchards as
follows:
1. Using a sterile cork borer, cut out 10-mm diameter discs from young citrus leaves, immerse
in Clorox for 30-60 seconds, wash three times in sterile distilled water, and then float the discs
on the surface of soil flooded with sterile distilled water in petri dishes or other sterile suitable
containers.
2. Incubate the plates for 3-4 days at room temperature.
3. Surface sterilize leaf discs by immersing them in 10 % Clorox for 30 seconds, wash three
times in sterile distilled water, blot dry on sterile paper towel, and place on the surface of suitable
agar medium. Place three to four of the leaf discs that show signs of infection (change in color
from green to olive-green) on each agar plate.
4. Subculture onto new agar plates and study fungi colonies that develop from leaf discs on the
agar plates.
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Sclerotia Bait Technique (SBT)

Sclerotinia sclerotiorum (Lib) de Bary may be isolated (as sclerotia) from cottony rot
diseased cucumber fruits and culture maintained on PDA plates.
1. Production of sclerotia: Sclerotia are produced on oat grain-water agar medium (OGWA)
as follows:
(a) Prepare OGWA by soaking the required amount of oat grains in sterile water at 4 ºC
overnight, then drain excess water, add 100-ml of grains to 500 ml Erlenmeyer flask, and
autoclave. Add 40 ml melted 0.75 % water agar medium to the flask and mix.
(b) Inoculate the flask with 4 sclerotia of Sclerotinia sclerotiorum and incubate the flask in
the dark at 25ºC for 14 - 21 days.
(c) Following incubation, remove mature sclerotia and air-dry overnight at room
temperature. Maintain mature sclerotia in a sterile flask until used.
2. Baiting:
(a) Weigh a soil sample of 60 g from the composite sample and divide into 3 sub-samples
each of 20 g.
(b) Place each sub-sample separately in a 90-mm diameter Petri dish and mix with 20 ml of
sterile distilled water.
(c) Inoculate each Petri dish with twelve air-dried sclerotia both on the surface of the soil
and buried in it.
To maintain the moisture level during 48-hour incubation period at 25 ºC, place 9-cm
diameter sterile wet filter paper in the lid of each petri dish.
(d) After incubation, transfer sclerotia to 3P medium plates.
(e) Transfer four sclerotia to each of three 3P medium plates for each sub-sample (i.e., thirty-
six sclerotia are used for each soil sample).
(f) Check plates for the presence of Pythium colonies, and transfer growing colonies on to
2P medium plates. Study fungi recovered in water culture.
Record your results. Exchange and discuss the results with other groups in the class. Record
your conclusions.
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Questions:
1. Explain soil fungistasis (mycostasis) and discuss its practical aspects.
EXERCISE 12. AQUATIC ‘FUNGI’
Materials needed:
Fungal cultures: Phytophthora parasitica or Pythium aphanidermatum on PDA medium plates.
Plant material: Surface-sterilized germinated lupine seeds.
1. Culture media and chemicals: Clorox; VP3 medium; PDA; CA; CMA amended with 250
mg ampicillin, 50 mg penicillin, 10 mg rifampicin, and 5 mg pimaricin.
Sterile soil extract.
Other materials: Sterile 2-L bottles; Sterile filtration units; 0.45 Millipore filters;
Haemocytometer.
Procedure:
a. Isolation of Zoosporic ‘Fungi’
Baiting:
1. Collect water samples in 2-L sterile containers from streams, and ponds.
2. Plate small samples of water in sterile Petri dishes or other suitable containers.
3. Add baits (e.g., surface sterilized germinated lupine seeds) on the surface of water.
4. Incubate at room temperature for 48 hr.
5. Subculture the colonies that may develop on the baits onto suitable agar medium (e.g.,
VP3, CMA amended with 250 mg ampicillin, 50 mg penicillin, 10 mg rifampicin, and 5 mg
pimaricin).
6. Prepare water cultures of the recovered isolates and examine for sporangia production and
zoospore release as mentioned in Exercise 3.
In the field, materials used as baits are usually placed in small-galvanized wire mesh
baskets or plastic-covered wire baskets. These baskets are suspended in the water by nylon
threads. The baskets can then be taken out from water at intervals (e.g., 3 days). Infected baits are
rinsed in sterile water, transferred to sterile water and then plated as mentioned in step 5.
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Filtration technique:
1. Collect two-liter samples of stream water from a nearby stream, using sterile 2L dark glass
bottles.
2. Using available sterile filtration units, separately filter two 100-ml aliquots of stream water
through 0.45 Millipore filters.
3. Remove the filter from the filtration unit, and re-suspend the supernatant in 10-ml sterile
distilled water.
4. For the SSDP technique, prepare a dilution of 1:100. Inoculate on VP3 and CMA agar
medium plates, incubate, and determine CFU ml-1 of stream water as mentioned in SSDP
technique (Exercise 10).
5. For the HBT, add further 20-ml sterile distilled water with antibiotics to the original 10-ml
supernatant suspension, to give a total volume of 30 ml.
6. Distribute the resultant suspension in 3 plates (10 ml/plate), and place 4 surface sterilized
germinated lupine seeds in each plate.
7. Incubate the plates at room temperature and proceed as in soil baiting technique (Exercise
10).
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b. Zoospore Counts
1. Prepare a culture of Phytophthora parasitica or Pythium aphanidermatum on PDA medium
plates.
2. Incubate in the light at 25 ºC for 3 days.
3. Transfer 5-mm disc from the growing edges of the colonies to CA plates and incubate under
continuous light until sporangia formed (4-6 days). To assess sporangia formation, examine the
cultures under the microscope through the bottom of the plates.
4. Flood the plates with a mixture of sterile soil extract and sterile distilled water (1:1) and re-
incubate in the light at 25 ºC for a further 3 days.
5. Wash the mycelium 3 times with SDW and then cover the agar surface with 10-ml of SDW.
6. Place the plates in the refrigerator at 2-8 ºC for 10-15 min, and then return to room
temperature. Zoospore release is completed within 30-40 min.
7. Count the swimming zoospores using a haemocytometer.

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Questions:
1. Discuss the role of Saprolegnia parasitica (an aquatic ‘fungus’) in fresh water environments.
2. Discuss the role of aquatic fungi in nutrient cycling in aquatic habitats.
EXERCISE 13. AIR-BORNE FUNGI
Materials needed:
Culture media: SDA, MEA, and PDA.
Procedure:
Expose a petri dish containing agar medium (e.g., SDA, MEA, PDA, or any other suitable
media) at each of the following locations, for 5, 10, and 30-minute periods.
1. Atmosphere in building.
2. Atmosphere outside building
3. Atmosphere in basement
4. Atmosphere in chicken house
5. Atmosphere in crowed-filled room
Count the number of colonies per plate.

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Transfer growing fungi to suitable agar medium plates and identify them using standard procedures.
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Compare types of fungi that grow from soil samples, with those in the plates that contain
airborne or aquatic fungi.
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Questions:
1. Discuss the role of airborne fungal spores in human health.
2. Devise a quantitative method for determination of airborne fungi concentrations.
EXERCISE 14. PLANT PATHOGENIC FUNGI
Materials needed:
Fungal cultures: Pythium ultimum on agar plates.
Infected plant parts: Potato leaves infected with late blight; Tomato leaves infected with early
blight; Stem or fruit infected with gray mold; Roots infected with Phytophthora or Pythium root
rot; Tomato stems infected with Fusarium wilt disease.
Plant material: Seeds of peas or other susceptible plant.
Culture media and chemicals: MEA, VP3, PDA; 10 % Clorox.
Other materials: Sterilized trays or flats; Peat moss; Fine sand; 1-% w/w ground rolled oats or
corn kernels.
Procedure:
A. Isolation of Plant Pathogenic Fungi:
a. Isolation from infected leaves (e.g., late blight of potatoes, early blight of tomatoes):
1. Cut out several 5 - 10 mm squares from the margin of the infected area (lesion) so as to
contain both healthy-looking and diseased tissues.
2. Immerse the squares in a surface sterilant solution (e.g., 10 % Clorox) for 15 - 30 seconds.
Aseptically take out the squares one by one and at regular 10 to 15-second intervals, so that each
one of the squares has been exposed for different times. In the correct immersion, e.g. 60
seconds, only the fungal pathogen survives in the square and grows out if placed on a suitable
nutrient medium.
3. Rinse the squares in three changes of sterile water and then blot dry on sterile paper towels,
and finally place on a suitable nutrient medium amended with (per liter) 50 mg penicillin (3 - 5
squares / plate).
4. Transfer growing fungi to suitable agar medium plates and identify them using standard
procedures.

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b. Isolation from stem and fruits (e.g., strawberry gray mold or fruit rot, stem of
Fusarium-wilted tomato plant)
Pathogens that have penetrated fairly deeply in infected stems, fruits, etc., can be isolated easily
as follows:
1. Split the stem and break the fruit from the healthy side and then tear apart toward and past the
infected margin, thus exposing tissues not previously exposed to contaminants.
2. Cut small sections of tissue from the exposed area of the advancing margin of the infections
with a sterile scalpel.
3. Place the sections in a plate containing a suitable nutrient medium.
4. Transfer growing fungi to suitable agar medium plates, incubate at 25 C for 7-14 days and
identify them using standard procedures.

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c. Isolation from plant parts in contact with soil (e.g., root rots)
Isolation of fungal pathogens from diseased plant parts in contact with soil, such as roots, tubers,
and vegetable fruits, requires the removal of the numerous contaminating saprophytic organisms.
1. Wash thoroughly diseased tissues to remove adhering soil and loose tissue in which most of
the saprophytic organisms are present.
2. Isolate pathogens from the infected plant parts that have been washed by using one of the
methods used for isolating pathogens from leaves.
3. Transfer growing fungi to suitable agar medium plates, incubate at 25 C for 7-14 days and
identify using standard procedures.

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B. Pythium Damping-off Disease – Inoculum Potential:

Plant disease establishment involves: (1) transfer of the inoculum from its source to an infection
court (inoculation), (2) penetration and colonization of a host tissue by the pathogen (incubation),
and (3) development of visible signs or symptoms of the disease (symptom inducement).
Pythium damping-off is a disease of the seedlings of any plant, caused by Pythium sp., in
which the seedlings wilt, collapse, and die. Pre-emergence damping-off occurs before the
seedlings have emerged through the soil and post-emergence damping-off occurs after
emergence. These diseases are generally most common in heavy soils and are favored by high
moisture levels and frequent wet periods.
The inoculum potential of soil-borne pathogens is an estimate of the population of
infectious propagules. The following experiment attempts to explain the relationship between the
number of pathogenic propagules per unit of soil and probable incidence of disease.
1. Preparation of initial inoculum.
a. Prepare the planting mix by mixing peatmoss and fine sand (1:1 v/v) and amend with 1-
% w/w ground rolled oats or ground corn kernels.
b. Add five 5-mm disks obtained from the margins of an actively growing colony of
Pythium ultimum to 1-L of plant mix in 2-L beakers covered with aluminum foil and
incubate under aseptic conditions at 25 ºC. Control preparations receive agar disks only.
c. After three weeks, determine the population density of each isolate in the inoculum
expressed as colony forming units per gram of soil (CFU g-¹), by the surface-soil-dilution
plate technique (SSDP) on VP3 selective medium.
2. Infestation of plant mix
a. Use the colonized mix preparation (inoculum), at calculated amounts, to infest the plant
mix placed in a series of suitable trays to reach the desired concentrations (0, 50, 100,
200, 400, and 500 CFU g-¹ plant mix of the phytopathogenic P. ultimum isolate).
b. Water to the dripping point each day for two days before use to provide the Pythium with
optimum growing conditions.
c. Plant each tray with 100 pea seeds as 10 rows of 10 seeds.
d. Keep the trays at room temperature and water thoroughly once each day or every other
day.
e. Count emergent seedlings and prepare a graph on a semi-log scale plotting percent
emergence as a function of inoculum size (CFU g-¹ plant mix). If percent damping-off is
plotted, the figure has to be corrected for the germinability of the seed lot.
Illustrate and record your observations (You may use a separate graph paper).
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C. Demonstration of Koch’s postulates with Pythium ultimum causing damping-off of pea

Koch’s postulates are used to establish fungi as disease causing organisms and can be
summarized as follows: (1) demonstration of the presence of the fungus in every host affected
by the disease, (2) the isolation growth and identification of the fungus in pure culture, (3) the
infection of a healthy host individual with the cultivated fungus, followed by development of
the specific disease symptoms, and (4) re-isolation of the same fungus from the infected host
(Stanier et al., 1987)
Procedure:
Using damped-off pea seedlings from the previous experiment, isolate, grow and identify the
fungus in pure culture.

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Using the isolated fungus and pea seeds, devise an experiment to demonstrate Koch’s postulates.
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Questions:
1. In a particular field, damping–off affects 10-15 % of the planted seeds. Would you
consider increasing planting rate as a solution for obtaining higher yields?
2. Is there a required minimum concentration for the occurrence of damping–off, or would
the disease occur regardless of how little inoculum there is in the soil?
3. Do you expect that the slope in the curve you drew will vary with environment,
susceptibility of the host and pathogenicity of the causal pathogen? Elaborate.
EXERCISE 15. INTER FUNGAL PARASITIC RELATIONSHIPS:

MYCOPARASITISM
Materials needed:
Fungal cultures: Pythium acanthicum, P. oligandrum, P. periplocum, and P. ultimum. All
fungal cultures are maintained on CMA or in water cultures.
Culture media and chemicals: CMA; VP3 medium; 2% water agar.
Plant materials: Cucumber seeds (Cucumis sativus L.); Ground rolled oats.
Other materials: Sterile glass coverslips; Nail varnish; Peatmoss; Fine sand; Aluminum foil;
200-ml plastic pots.
Procedure:
Hyphal Interactions on Thin Films of Agar (Laing & Deacon, 1991; Saleh, 1997)
Host-mycoparasite pairings are made on sterile glass coverslips coated with 2% water agar.
1. Dip a coverslip in autoclaved melted water agar at about 45 ºC for 3-4 seconds, hold with
forceps to allow excess agar to drain.
2. Place the coverslip on the surface of 2% solidified water agar in a 90-mm diameter Petri
dish, so that a thin film of agar set on the upper surface.
3. Take five mm disks from the edge of one week old growing colonies of each of host
fungus and mycoparasite, and place 3 cm apart on the agar surface, so that host and
mycoparasite colony margins would meet across the coated coverslip in less than 24 hours
later.
4. Incubate plates containing coated coverslips at 22-25 ºC and inspect daily for three
consecutive days for mycoparasitism.
5. Remove the coverslip carefully so as not to damage the mycelial interactions, just before
mycelial contact and invert on a sterile microscopic slide and seal by nail varnish to prevent
desiccation.
Observe under the microscopic throughout the coated coverslip at regular intervals.
Evaluation of host susceptibility to parasitism by mycoparasitic Pythium species is based
on Ribeiro & Butler’s rating system (1995):
Type 0. Immune: No effect noted.
Type 1. Resistant: Infected hyphae are less than 10 (across the entire zone of interaction).
Coiling by the mycoparasite weakly developed. No internal colonization.
Type 2. Slightly susceptible: Infected hyphae more than 10. Coiling is more intense than in
type 1. Infrequent internal colonization.
Type 3. Moderately susceptible: Infected hyphae abundant. Wide spread coiling, and abundant
internal colonization.
Type 4. Highly susceptible: Infected hyphae more intense than type 3. Hyphal coiling and
internal colonization formed throughout the entire host mycelium.
Record and discuss your observations regarding the mechanism of parasitism.

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Biological Control of Pythium Damping-off of Cucumbers with Mycoparasitic Pythium

Species:
Concern of conserving natural resources and reducing soil, water, and air pollution has lead to
increased emphasis on natural or biological control of fungal diseases. Biological control of
fungal disease is slow but can be long lasting, inexpensive and harmless to life. It does not
eliminate neither pathogen nor disease but bring them into natural balance.
Procedure:
1. Prepare initial inocula of P. ultimum and the three isolates of mycoparastitc Pythium
species as described in Exercise 13 (Paulitz & Baker 1987; Saleh, 1997).
2. Infestation of plant mix:

a. Use the colonized mix preparations (inocula), at calculated amounts, to infest large
quantities of the plant mix to reach the desired concentrations (500 CFU g-¹ of each of the
mycoparasitic isolates and 50 CFU g-¹ of the phytopathogenic P. ultimum isolate).
b. Incubate all preparations at 25±1 ºC in large plastic bags and keep until use.
3.Treatments:
a. Plant seven cucumber seeds (Cucumis sativus L. ‘Oscar’) in plastic pots (200 ml-
volume) after soil has been moistened. Grow plants in a constant-temperature growth
room at 26±2 ºC with a 12 hour light / dark cycle.
b. Use the following treatments: P. ultimum plus P. acanthicum; P. ultimum plus P.
oligandrum and P. ultimum plus P. periplocum. Control treatments: pots with sterilized
plant mix only, pots containing plant mix infested with P. ultimum alone, or only with the
mycoparasite.
c. Water all pots (twice daily) with distilled water.
d. Record percent emergence seven days after planting. Experiments are conducted in, two
replicate pots per treatment. Record and discuss your results.
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Questions:
1. Define mycoparasites, biotrophic and necrotrophic mycoparasites.
2. What are the main attributes that would confer on mycoparasites a high competitive
saprophytic ability?
EXERCISE 16. FUNGI AS HUMAN AND ANIMAL PATHOGENS
Materials needed:
Fungal culture: Candida albicans in agar plates
Culture media and chemicals: SDA; CMA; 70% alcohol; KOH; glycerine; dimethyle
sulfoxide (DMSO); Tween 80 (see Appendix A).
Antibiotics: Penicillin; Chloramphenicol; Cycloheximide.
Other materials: Rabbit or human serum; Durham’s tubes; McFarland No. 4 standard (see
Appendix A); Yeast fermentation tubes (glucose, maltose, sucrose, and lactose).
Dermatophytes:
1. Collection of cutaneous and infected nails specimens:
1. Swab the infected area with 70% alcohol to remove bacterial contaminants.
2. Scrape the site of infection gently with the side of a scalpel blade or the edge of a glass
microscope slide.
3. Collect scrapings either in a Petri dish or in a paper envelop for further processing.
2. Microscopic examination of clinical specimens:

Prepare a temporary mount as follows.
a. Mix a small portion of the specimen (scales, hair or nail) in a drop of water, physiologic
saline, or 10 - 20 % KOH containing 10 % glycerin on a microscopic slide. The incorporation of
dimethyle sulfoxide (DMSO) in KOH (DMSO 40 ml; distilled water 60 ml; KOH 30 g) may also
help to clarify specimens.
b. Add a coverslip over the drop.
c. Warm the slide on an electric hot plate or a very low Bunsen burner flame until the tissue has
cleared.
d. Gently press the coverslip to make the specimen almost transparent. Hairs should be handled
with particular care and allowed to soften without heat so that the arrangement of the spores will
not be destroyed.
e. Let the mount stand at room temperature for approximately 30 minutes if the specimen is
thick or viscous.
f. Examine microscopically for the presence of hyphae or other fungal structures.
Illustrate and record your results.

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3. Culturing of fungi:
a. Place skin scales, nail scrapings, or hairs on agar culture media (SDA supplemented with
chloramphenicol 0.005 %, and cycloheximide 0.05 %) and submerge portions beneath the
surface of the agar with an inoculating needle.
b. Incubate cultures at 25 – 30 ºC for 1-4 weeks.
c. Examine growing fungal colonies as outlined in Exercise 2 (for species identification see
Figure 2.2, and species descriptions in Rebell & Taplin (1978) and any other available
references).

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Trichophyton violaceum Trichophyton rubrum
Trichophyton schoenleinii Trichophyton mentagrophytes
Trichophyton tonsurans Trichophyton ajelloi

Figure 2.2 Main morphological features of some dermatophytes.
Microsporum canis Microsporum nanum
Epidermophyton floccosum
Microsporum gypseum
Figure 2.2 / Continued
Yeasts:
Yeasts are considered normal flora of oropharynx and gastrointestinal tract and may therefore
be recovered from sputum, throat swabs, bronchial washings, gastric washings, and stool
specimens. However, the repeated isolation of yeasts from a series of clinical specimens of
urine, nail scrapings, and vaginal washings, from the same patient usually indicates infection
with the organism recovered and identification of the isolates is necessary. Furthermore, the
presence of yeast in normally sterile body fluids such as blood, cerebrospinal fluid (CSF), or
fluids aspirated from the pleural cavity, or the pericardial sac, is also considered as a clinical
situation in which species identification is justified. Candida albicans is the species of yeast
most frequently cultured from clinical specimens.
1. Collection of clinical specimens:

1. Swab the infected area with 70% alcohol to remove bacterial contaminants.
2. Collect infected material (in sterile containers, as smears on slides or culture directly) as
follows: scraping from skin or nail; mucus patches from the mouth, vagina, or anus;
sputum, blood, or cerebrospinal fluid.
2. Culturing of specimens:
1. Culture infected material on media (e.g., SDA) supplemented with penicillin and
streptomycin or chloramphenicol and cycloheximide.
2. Incubate at room temperature or 37 ºC for 3-4 days.
By the end of incubation period colonies of C. albicans appear as cream colored, smooth
and pasty, and have a yeasty odor.
3. Identification of Candida albicans:

Microscopically a slide mount of C. albicans will show oval, budding cells 2.5-4 by 6 m in
size and some pseudomycelium if taken from submerged growth.
1. Germ tube test:
a. Suspend a very small portion of an isolated colony of the yeast to be tested in a test tube
containing 0.5 ml of rabbit or human serum.
b. Incubate the test tube at 37 ºC for no longer than 3 hours.
c. Place a drop of the yeast-serum suspension on a microscope slide, and cover with a
coverslip.
d. Examine under the microscope for the presence of germ tube (Figure 2.3).
Figure 2.3 Germ tube formation in Candida albicans

Note that the germ tube of Candida albicans has no constrictions at the point of origin, in
contrast to that of C. tropicalis.

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2. Production of chlamydospores on CMA-Tween 80:

a. Inoculate a portion of the yeast colony on CMA plates by making three parallel cuts
about 1 cm apart into the agar, holding the inoculating wire at about 45- degree angle.
b. Place a coverslip on the surface of the agar covering the inoculating streaks.
c. Incubate at 30 ºC for 24-48 h.
d. Examine under the microscope for the presence of chlamydospores (Figure 2.4).
Figure 2.4 Chlamydospores formation in Candida albicans: (a) terminal; (b) intercalary and terminal

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3. Carbohydrate fermentation test for yeast identification:

Yeast fermentation in appropriate culture media containing a single carbohydrate source is
detected by gas and acid production.
a. To each of the yeast fermentation tubes (glucose, maltose, sucrose, and lactose) add 0.2 ml of a
yeast suspension in saline equivalent to a McFarland No. 4 standard (see Appendix A).
b. Incubate at 37 ºC for 48 hours.
c. Leave all negative test in incubator for 6-10 days before discarding.
Note the presence of bubbles or a drop in the liquid level, in the inverted Durham’s tube,
which indicates the fermentation. The development of a yellow color is not a reliable indicator
of fermentation and should be ignored (Table B.1 & B.2 in Appendix B)

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Questions:
1. Classify the mycosis on the basis of tissues affected.
2. Define anthropophilic, zoophilic, and geophilic dermatophytes.
3. Name a human disease caused by Candida albicans.
EXERCISE 17. KERATINOLYTIC ACTIVITY OF FUNGI
Materials needed:
Fungal isolates: Arthroderma cuniculi; Chrysosporium keratinophilum; Microsporum
gypseum; Rhizopus stolonifer; Trichophyton ajelloi. All fungal isolates are maintained on
SDA slants.
Other materials: Sterile sand; Hairs from a blond-child, 1 cm long; Lactophenol cotton blue.
Procedure:
Keratinolysis test. The hair-soil method of English (1976) as modified by Filipello Marchisio
(1986) will be used to detect the keratinolytic activity. Culture the isolates on a sand-hair
substrate for determination of keratinolytic activity as follows:
1. Wash sand under tap water, dry and autoclave.
2. Place 25-ml sand in Petri dishes (9-cm diameter), and moisten with 10-ml sterile distilled
water.
3. Scatter hairs from a blond-child, 1 cm long, on the surface of the sand.
4. Inoculate the Petri dishes with aqueous spore suspension prepared by scraping off the surface
of 14-day old fungal colonies cultured on SDA, and then wash with 10 ml sterile distilled
water.
5. Incubate the cultures at room temperature for 20 days, and moisten with sterile distilled water
when necessary. Employ two replicate plates for each isolate.
6. Mount the inoculated hairs in lactophenol cotton blue for microscopic examination (10 X,
40 X, and 100 X).
7. Explain results (based on the examination of 10 hairs, 5 from each replicate plate for each
isolate) in light of model proposed by Filipello Marchisio (1986) (Figure 2.5). The term
surface erosion (SE) is used to indicate progressive destruction of the hair from the exterior or
inwards; this may occur either uniformly (U) along the length of the hair, or in localized areas
forming more or less extensive pockets (P). Random attack on the hair by more or less
specialized hyphae that penetrate the hair at right angles to the surface is termed radial
penetration (Rp). A boring hypha is used to describe fine hypha about 1 μm diam. which
penetrates the substratum at right angle to the layer of keratinized cells, while a perforating
organ is used to describe single column of up to 10 short hyphal cells, usually 3-4 times wider
than normal mycelial cells, that penetrates radially into the hair cortex, passing straight through
its keratinized cells irrespective of their boundaries. Wider boring hyphae (wbh) indicate
structure intermediate in diameter between boring hyphae and perforating organs. Swollen
boring hyphae (sbh) are structures that are similar to boring hyphae when penetrating the outer
cortex, but dilated in a series of balloons on reaching what are probably less compact regions
of the hair.
Figure 2.5 Invasive organs produced by keratinophilic fungi attacking hair: (1) uniform surface erosion; (2) erosion in
localized areas (pockets); (3) boring hyphae; (4) perforating organ; (5) wider boring hyphae; (6) swollen boring
hyphae. H, hair; M, mycelium; S, cuticular scales.

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8. Estimate the intensity of keratinolytic activity (IKA) as follows (Jamous, 1998). The activity is
based on a scale of 0 to 100, and is estimated by giving different weights to the presence of
various features of hair keratinolysis (i.e., invasive structures, hair degradation, etc.) as
follows: IKA= G + F + U + P + bh + sbh + wbh + po. Where G, fungal growth 0-5%; F,
fruiting 0-5%; U, uniform surface erosion 10%; P, pocket-like surface erosion 20%; bh, boring
hyphae 10%; sbh, swollen boring hyphae 10%; wbh, wider boring hyphae 20%; and Po,
perforating organ 20%. In case of complete degradation of hair, IKA is considered 100%.
Record and discuss your results.

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Questions:
1. What is the significance of keratinolytic fungi in the environment?
2. What is the relation between dermatophytes and other keratinolytic fungi?
EXERCISE 18. ANTIBIOTIC PROPERTIES OF FUNGI (PENICILLIN)
Materials needed:
Fungal isolates: Penicillium notatum or P. chrysogenum.
Bacterial isolates: Serratia marcescens (bright red colored, non-susceptible), and Sarcina sp.
(yellow colored, susceptible); both are harmless. Media containing sugar must not be used or
S. marcescens will not develop its red color.
Media: Beef extract agar (BEA); Nutrient agar (NA).
Procedure:
The best medium is Beef extract agar (BEA); nutrient agar may be substituted.
1. Prepare the medium, pour plates and allow to set.
2. Place a few drops of sterile water on a flamed microscope slide and make a suspension of P.
notatum or P. chrysogenum spores taken from a 2-wk old culture.
3. Smear this suspension tangentially across the plate with a flamed bent glass rod or loop needle.
As the mold grows, the penicillin will diffuse into the agar.
4. After three days, inoculate the bacteria as follows:
a. Take up some of the bacterial colonies from the culture on the tip of a sterile glass rod or
loop needle, and draw this across the agar at right angles to the line of the mold, and
moving towards the mould and right up to its edge.
b. Rinse and flame the rod, and repeat the operation with the other bacterium.
In two days, both colonies of bacteria will have developed well: Sarcina grows rather
more slowly than Serratia. The later will grow right up to the mould, but Sarcina will be
unable to develop near the mold, owing to the effect of the diffusing penicillin.
The operation can be preserved by placing in the dish, to one side, a small tuft of cotton
wool soaked in full-strength formalin. After a few days, the cotton wool can be removed, and
dish may be sealed with Sellotape.

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Questions:
1. Compare between antibiotic production and competition.
2. Name some commercially important antibiotics produced by fungi and mention their mode
of action.
EXERCISE 19. OBSERVATION OF MYCORRHIZAE

The aim of this exercise is to observe mycorrhizae on root of trees, and to differentiate
between roots with ectomycorrhizae and roots with VA (vesicular-arbuscular) mycorrhizae.
Materials needed:
Plant material: Seedlings (Pinus sp., and Acer sp.).
Chemicals and stains: Dilute hydrochloric acid; 10% KOH; 0.05% trypan blue in
lactophenol.
Procedure:
1. Remove the soil ball of the Pinus or Acer sp. seedlings provided to you from their containers.
2. Carefully and thoroughly wash the soil from around the roots.
3. Cut sections of the small secondary roots of each seedling and place them separately in Petri
dishes partially filled with water.
4. Observe the small feeder roots with dissecting microscope at a range of magnification and
compare with the drawings and photographs of mycorrhizae (Figure2.6).
Figure 2.6 Types of mycorrhizae: (a) ectotrophic mycorrhiza of forest trees, (b) vesicular-arbuscular
mycorrhiza of herbaceous plants, (c) endotrophic mycorrhiza of an orchid.
5. For staining of mycorrhizae, heat several sections of secondary feeder roots (approximately
2mm in diameter and 5 mm long) at 90 ºC in 10% KOH for 1-2 hours to clear the sections for
viewing.
6. Rinse the sections in water and acidify in dilute HCl.
7. Place in 0.05% Trypan blue stain in lactophenol and immerse for 5 minutes.
8. Move sections to clear lactophenol to remove the stain, mount on glass slide with coverslip,
and observe under a compound microscope.
This procedure may need to be modified for each species depending on the amount of
pigmentation of the roots, the thickness of the roots and the thickness of the sections used.

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Questions
1. Define the term mycorrhizae.
2. Name three main types of mycorrhizae.
3. Explain the role of mycorrhizae in mineral nutrition in plants.
EXERCISE 20. ANTIFUNGAL SUSCEPTIBILITY TESTING

In this exercise, nystatin (anticandidal drug), griseofulvin, and extracts of some medicinal plants
will be compared for their antifungal activity against some selected human-pathogenic fungi.
Materials needed:
Fungal isolates: Microsporum canis, Trichophyton mentagrophytes, T. violaceum, Candida
albicans, Saccharomyces cervisiae.
Culture media and chemicals: SDA; Mueller-Hinton broth; Sterile physiological saline;
Nystatin, Griseofulvin; Seed mill; 95% ethanol; Broth tubes; 10 % aqueous dimethylsulfoxide
(DMSO); MacFarland nephelometer tube no. 0.5 (see Appendices A & B for Preparation of
McFarland Nephelometer standards).
Plant parts: Micromeria nervosa; Juglans regia; Inula viscosa; Salvia fruticosa.
Other materials: Whatman filter paper no.1; Rotary evaporator; Spectrophotometer; Sterile
cotton applicators; Sterile forceps; Transparent rulers; Sterile 0.45 m membranes.
Procedure:
1. Collection of plant material
Some plant species, commonly used in folk medicine in Palestine for the treatment of fungal
skin infections, will be selected in this exercise. Mature plants can be collected, dried in the
shade, and ground into a powdered material using an appropriate seed mill.
2. Plant extracts (ethanolic extract) and discs preparation:

a) Soak a 20-g portion of the powdered plant material in 100 ml of 95% ethanol for 5 days
at room temperature, and stir the mixture daily for regular infusion.
b) After a five-day period filter the extract in muslin or Whatman filter paper no.1.
c) Dry the extracts using a rotary evaporator at 60 ºC, and store the final dried extract in
labeled sterile glass bottles and keep at –20 ºC
d) Reconstitute one gram of the dried extract using 5 ml of the solvent.
e) Sterilize paper discs and impregnate each with 50 l of the extract to give a final
concentration of 10 mg of dried extract per disc.
3. Preparation of inocula
a. Inoculate part of an isolated C. albicans colony into a 5ml Muller-Hinton broth tube and
incubate for 4-18 hrs at 37 ºC.
b. Adjust the growth turbidity in Muller-Hinton broth by further incubation or dilution with
sterile physiological saline, after comparison with that of a McFarland nephelometer tube
no. 0.5 (108 CFU/ml) using a spectrophotometer at 625 nm (optical density 0.08-0.1).
I. Anticandidal susceptibility test:

A. Disk diffusion method (Murray et al., 1995).
a. With a sterile cotton applicator swab 108 CFU/ml of C. albicans culture on the surface of
SDA agar by dipping the cotton applicator into the candidal suspension, rotate several
times and press against the inside wall of the tube to remove excess inoculum.
b. Streak the agar plate in three different directions and around the agar margin to ensure
even distribution of the inoculum.
c. Leave the plates to dry for 3-5 minutes.
d. Using sterile forceps, distribute the selected extract disks evenly on the surface of the
agar plates.
e. Use a positive control (nystatin, 10 mg / disc), and a negative control (solvent). Use three
replicate plates for each test.
f. Incubate the plates upside-down at 37 ºC for 48 hrs.
g. Measure the inhibition zone around each disk using a transparent ruler.

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B. Determination of minimum inhibitory concentration (MIC) - broth dilution

method
1. Preparation of media
For each isolate, 15 tubes, each with 9.9 ml Muller Hinton broth were prepared and autoclaved.
2. Preparation of the active ingredient dilutions

1. Reconstitute one gram of the dried plant extract using 5 ml of the solvent to give a final
concentration of 200 mg/ml (stock solution).
2. Prepare several dilutions of the stock solution as shown in (Table B.3).
3. Incorporation of active ingredients into media and reading the results:

1. Into broth tube 1, add 0.1 ml of stock solution. Into broth tube 2, add 0.1 ml of dilution 2
(Table B.3). Repeat the procedure for the remaining dilutions. The final concentrations of the
active ingredients in broth are shown in Table B.4.
2. For positive control, add 0.1 ml of reference antibiotic, while for negative control prepare
two tubes by adding 0.1 ml of solvent to the first tube and 0.1 ml of sterile water to the second.
3. Add 10 l of Candida albicans suspension of (108 CFU/ml) concentration to each of the 15
tubes of extract serial dilutions.
4. Incubate the tubes at 37 ºC for 24 hrs.
5. Prepare a positive control by adding 0.1 ml of nystatin to a 9.9-ml broth tube.
6. Prepare a negative control by adding 0.1 ml of sterile water to a 9.9-ml broth tube.
7. Examine turbidity after incubation. The lowest concentration of the extract that inhibits the
growth of the organism (visually clear) is designated MIC.

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C. Minimum fungicidal concentration (MFC)

Prepare subcultures from the visually clear tubes on SDA plates. The tube that has subcultured
and shows no growth on agar plate indicates the MFC.
II. Screening for antifungal activities against mycelial fungi - agar dilution method:
Antimycotic activity is carried out by the poisoned-food technique method, which involves the
cultivation of the test organism on a medium containing the test chemical, and then measuring
its growth. The use of poisoned agar is most common, but use of a shake culture in a liquid
media provides more precise results. The growth of fungi in poisoned liquid medium is
measured using dry weight. The following is an outline of the poisoned agar medium method
(Dikshit& Husain, 1984; see also Abu-Ghdaib, 1998; Ali-Shtayeh et al., 1998a).
A. Poisoned-food technique method:

1. Inoculate test isolates (see fungal isolates in Materials needed) onto SDA plates and
incubate at 25 ºC for 7-10 days to obtain young, actively growing cultures consisting of
mycelia and conidia.
2. Dissolve the required amount of the dried plant extract or reference antimycotic drug in
2 ml ethyl alcohol or 10 % aqueous dimethylsulfoxide (DMSO), and sterilize by filtration
through a 0.45 um membrane), and then mix in requisite amount of pre-sterilized SDA
medium to give a final concentration of 15 g/ml.

3. Cut out a mycelial disc of 5-mm diameter, from the periphery of the 7-10 day old
cultures, and aseptically inoculate onto the medium.
4. In controls, use sterile DMSO or distilled water in place of plant extract.
5. Incubate the inoculated plates at 25 ºC.
6. After 7 days measure and record colony diameter. Percentage of mycelial inhibition is
calculated as follows. % Mycelial inhibition = [(dc-dt)/dc] x 100; dc = colony diameter in
control, dt = colony diameter in treatment. Use three replicate plates for each treatment.

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B. Minimum inhibitory concentration determination is performed by a serial agar

dilution plate technique as follows:
1. Reconstitute extracts solutions (0.01mg - 3 mg/ml concentrations).
2. Incorporate into SDA sterilized pre-poured medium.
3. Pour the medium, and allow the agar in the plates to set.
4. Inoculate the plates with the test fungi and incubate as mentioned above. Control plates,
which contain no plant extracts, are also made with the test.
5. Determine the MIC of each plant after 7 days. This is the lowest concentration at which
no visible growth is observed.

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C. Minimum Fungicidal Concentration (MFC) determination is performed as follows:

1. Re-inoculate the inhibited discs of each fungal isolate on SDA medium separately.
2. Incubate as above.
3. Examine the plates after 7 days. Absence of mycelial growth on the seventh day
indicates fungicidal nature. MFC is regarded as the lowest concentration of the test
compound that prevented growth of the fungus, indicating > 99.5 % killing of the
original inoculum.
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Questions:
1. Name some of the antifungal antibiotics and explain their modes of action.
2. Name some local plants that are currently used for the treatment of fungal skin infections.
EXERCISE 21. SPORULATION AND SPORE GERMINATION
Materials needed:
Fungal cultures: Paecilomyces lilacinus; Penicillium griseofolvum; Acremonium strictum;
Gliocladium viride; Chrysosporium tropicum; Pythium aphanidermatum.
Culture media: 0.2% MEA; 2% MEA.
Asexual spores of most fungi germinate readily when provided with a suitable substrate
and compatible environment. Sexual spores however will not germinate even when provided
with a suitable substrate, temperature and humidity. They require a period of dormancy before
germination. The specific requirements for germination of these spores can be related to some
extent to their natural environment. The means of breaking dormancy and stimulating
germination include heat, cold, and chemical treatments.
Procedure:
A. Agar plate method
1. Prepare spore suspension as follows. Harvest the spores of a culture of one of the following
fungi (Paecilomyces lilacinus, Penicillium griseofolvum, Acremonium strictum, Gliocladium
viride, Chrysosporium tropicum, Pythium aphanidermatum) by adding sterile distilled water to
the surface of the culture. Scrape off the surface of the culture using sterile razor blade. Prepare a
series of dilutions as required. For the preparation of zoospore suspension of Pythium
aphanidermatum, see Exercise 11.
2. Alternatively, allow fungal spores to be discharged from a sporophore (basidiocarp) to give a
deposit of the appropriate density on the agar plate (0.2% MEA or any other suitable media).
3. By using a sterile pipette, transfer 1-ml aliquot of the appropriate spore dilution on to the
surface of an agar medium plate and spread over the surface using a sterile, bent glass rod.
4. Rotate gently the plate to give an even spread.
5. To follow germination of individual spores, cut out a 1-cm2 of agar on that spores have just
settled and transfer to sterile glass slide in a humid chamber (in a Petri dish) and cover with a
cover glass. Record stages in germination using camera Lucida drawings at intervals over a
period of 24 hours. Illustrate and discuss your results
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B. Glass slide method

1. Cover sterile glass slide with a film of an agar medium (MEA).
2. Seed the agar film with the spores of the test organism.
3. Incubate in a sterile moist chamber for 2-3 days.
4. Examine under the microscope every 2-3 hours and record germination stages as
mentioned above. Illustrate and discuss your results
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Questions:
1. Discuss in more detail the methods used for breaking dormancy in fungal spores.
EXERCISE 22: ENTOMOPATHOGENIC FUNGI IN SOILS
Materials needed:
Culture media and chemicals: Water agar; MEA; Sterile 0.05% aqueous (w/v) Triton X-100;
1% Sodium hypochlorite; Galleria artificial diet (see Appendix A for recipe).
Larvae of wax moth, Galleria mellonella.
Other materials: Glass rod; Tissue paper; Glass jar; 200 ml Plastic cups; Trowel; Plastic
bags; Sterile scalpel, Sterile inoculating needle; Filter paper.
Procedure:
1. Collection of soil samples (Chandler et al., 1997).
a. With a trowel collect 1 kg soil samples from a depth of 0-5 cm.
b. Place samples in clear plastic bags, seal the plastic bags with a rubber band, and
keep at 20 oC. Process the samples within 24 hr of collection.
2. Rearing of larvae of wax moth, Galleria mellonella (Lepidoptera: Pyralidae) susceptible

to insect pathogens (Vilcinskas & Wedde, 1997).
a. Rear G. mellonella larvae on an artificial at 25oC in darkness. Collect eggs on folded
tissue paper by placing the paper in the jar containing Galleria moths for 7-10 days. Remove
the tissue containing the eggs, cut into small pieces (1X1 cm) and place in a 500-ml jar,
immerse in 10% formalin solution for 1 hour, wash under running tap water for one hour,
remove the pieces of tissue with eggs and place them separately on tissue papers and allow to
dry. Transfer a suitable number of pieces of tissue with eggs to the bottom of a 1-L glass jar,
cover with artificial diet (5 cm high), and incubate at 25 oC in darkness for 2 weeks. Add a
further 5-cm layer of artificial diet and incubate for another 2 weeks.
b. For experiments use last instar larvae weighing between 250 and 350 mg.
2. Isolation (Galleria bait method (Zimmerman, 1986), and identification of fungi (Chandler et
al., 1997).
a. Remove coarse debris from soil samples, and mix thoroughly within the plastic bag.
b. Place about 180 ml of soil in 200-ml plastic cups.
c. Moisten the soil with sterile distilled water.
d. Place one fourth- instar larva of G. mellonella on to the surface of soil in each cup,
cover with aluminum foil and seal with a rubber band.
e. Turn the cups up side down and incubate at 20 oC.
f. Inspect larvae once every seven days for four weeks.
g. Remove dead intact larvae, surface sterilize in 1% sodium hypochlorite for three
minutes, wash three times in sterile distilled water, and cut the larva open using a
sterile scalpel.
h. Using a sterile inoculating needle, remove part of the larva haemolymph, streak on
the surface of MEA plate, and incubate at 20 oC for 7-14 days.
i. Identify growing fungi using available keys and references (Brady, 1979; Domsch &
Gams, 1980; Samson et al., 1988).
Illustrate, record and discuss your results.

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3. Preliminary testing for pathogenicity of suspected entomopathogenic fungi

1. Place a sterile filter paper in a 6-cm plastic Petri dish and moisten with sterile distilled water.
2. Transfer 3 fourth -instar larvae of G. mellonella to the dish. Use three replicate plates for
each fungus.
3. Using a sterile inoculating needle transfer a loop-full of fungal spores from a 10-12 day old
colony of the suspected fungus to the upper surface of each larva in the dish.
4. Incubate at 25 oC in the darkness and inspect daily for dead larvae.
5. Record number of dead larvae three and six days after incubation.
6. For dead larvae repeat step g, h, and i above.
7. Repeat this test three times to confirm the entomopathogenic activity of the fungus.

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4. Infectivity of entomopathogenic fungi on larvae of Galleria mellonella (Shimazu & Soper,

1986; Vestergaard et al., 1995):
1. Harvest a mixture of conidia and hyphae from 10-12 day old cultures of the fungus by
flooding the plates with sterile 0.05% aqueous (w/v) Triton X-100 and agitating with a
glass rode.
2. Filter the suspension through tissue paper and then centrifuge for 5 min at 3000 rpm to
separate conidia.
3. Wash the resulting pellet twice with sterile 0.05% Triton X-100 with intervening
centrifugation steps.
4. Adjust the concentration of the inoculum to 107 ml-1 with 0.05% Triton X-100.
5. Dip fourth-instar larvae of the wax moth for 15 sec into the conidial suspension. As
control dip the larvae into the solution without conidia.
6. Spread the spore suspension on water agar plate to check conidial germination.
7. After dipping, place the larvae on filter paper to drain, and place each larva in a plastic
Petri dish with a sterile piece of moistened filter paper.
8. Incubate at 24 oC and 16-hr photoperiod for two days with out diet in order to avoid the
influences of antimicrobial on conidial germination, after then feed with diet.
9. Examine the larvae daily for further seven days and for dead larvae repeat steps g, h, and i
above.

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Questions:
1. Outline field conditions required for the use entomopathogenic fungi as biological control
agents.
EXERCISE 23. DETECTION OF AFLATOXIN CONTAMINATION PRODUCED

BY ASPERGILLUS SPECIES IN FOOD PRODUCTS
Materials needed:
Grains: Maize; White corn; Wheat; Barley; Peanuts.
Culture media and chemicals: M2 agar plates; Coconut agar medium (CAM) plate; Sodium
hypochlorite containing 1000 p.p.m. available chlorine.
Antibiotics: chloramphenicol; Gentamycin sulfate.
Other materials: UV lamp; Sterile water.
Procedure:
a. Collect five 100-g samples of maize, white corn, wheat, barley, and peanuts.
b. Surface sterilize 20-g of each of the above-mentioned samples for 2-min with sodium
hypochlorite containing 1000 p.p.m. available chlorine, and wash twice with sterile water.
c. Place 25 seeds or kernels on M2 agar plates (Bauduret, 1990). containing 60 g/ml
chloramphenicol and 50 g/ml gentamycin sulfate (five per plate).
d. After 4-6 days of incubation at 25 oC count the infected seeds and identify the infecting fungi.
Express the results as percentage of mold-infected kernels or seeds.
Illustrate, record and discuss your results.

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e. When a sample contains several strains visually different, transfer one representative colony
per strain to a coconut agar medium (CAM) plate.
f. Incubate plates at 25 oC.
After 7 days of incubation examine the plates under a UV lamp at 365 nm for the presence or
absence of blue fluorescence in the agar surrounding the colonies (a good correlation between
presence of blue fluorescence and production of aflatoxin B1 by isolates of Aspergillus flavus
and A. parasiticus has been demonstrated by Davis et al. (1987).

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……………………………………………………………………………………………………
……………………………………………………………………………………………………
……………………………………………………………………………………………………
……………………………………………………………………………………………………
Questions:
1. Write a short account on methods used for the control of mycotoxins in food and food
products (See Beuchat, 1978).
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APPENDICES
MYCOLOGY MANUAL / APPENDICES 137
APPENDIX A
MEDIA AND REAGENTS
Acetic-Orcein: was filtered through 4 layers of cheesecloth, and the pH
Orcein ………………………………….………..….. 1% of the clear filtrate was adjusted to pH 7 with 2 N
Acetic acid …………………………..…….…….…60% NaOH. Agar was added and the mixture was heated to
Constituents dissolved and filtered. boiling and cooled to about 50 oC. The pH is checked
and adjusted to 7 when necessary. The mixture is then
Acridine Orange: autoclaved for 18 min, cooled to about 45 oC and
Acridine orange ……………………….………....2.5 mg poured while being stirred into sterile Petri dishes.
0.01 M HCl ………………………………..…… 74 ml
Sodium acetate ………………..……………...…0.971-g Corn Meal Agar (CMA): (Satisfactory for the
Soluble veronal (Na diethylbarbiturate) …………1.471-g growth and sporulation of many fungi)
Distilled water …………………………………...150 ml Ground maize kernels ………….…………………….60 g
The stain is prepared by mixing 2.5 mg acridine orange Agar…………………………………………….……15 g
with 100 ml of veronal acetate buffer (veronal acetate Water ……………………………………………1000 ml
solution 50ml, 0.01 M HCl 74 ml, and distilled water 100 Boil 60 g freshly ground maize kernels in 1-liter water for
ml). Veronal acetate solution is prepared by adding 0.971- 1 hour. Strain the suspension through two layers of muslin.
g sodium acetate and 1.471-g soluble veronal (Na Add the agar and heat until dissolved. Autoclave at 15 psi
diethylbarbiturate) to 50 ml distilled water. for 15 minuets.
Ascospore Agar: Czapek-Dox Agar (CzA): (suitable for growing

Potassium acetate………………………….10 g Aspergillus, Penicillium and Nocardia spp.)
Yeast extract…………………………….….………2.5 g NaNO3 ……..……………...…………………………2 g
Glucose…………………………….….………….…...1g K2HPO4 ……………………………………………....1g
Agar……………………………….….………….… 30 g
Distilled water …………………………………...1000 ml KCl ………………...……………...…..………….....0.5 g
Heat to dissolve and autoclave for 15 minutes. MgSO4. 7H2O …………..……..……..………….....0.5 g
FeSO4. 7H2O ……………………………..…….....0.01 g
Beef Extract Agar (BEA): Sucrose ……………………………..…………...…..30 g
Beef extract……………………………….….……… 5 g Agar …………………………….………….…….…15 g
Peptone……………………………….………..……10 g Distilled water …………………………...……...1000 ml
Common salt ……………………..………….….……5 g Sucrose is added just before sterilization in order to reduce
Agar………………………….…………….…….… 20 g contamination.
Distilled water……………..…………………… 1000 ml
Mix peptone and salt to a paste with a little water at 60oC; Dermatophyte Test Medium ( DTM) :
add the beef extract, agar and water. Used for isolating dermatophytes. This selective medium
excludes most bacteria and many contaminant fungi,
Carrot Agar (CA): gives a yellow red H indicator, Positive growth and color
Whole carrot ………………..…………………..…. 200g change on (DTM) diagnosed dermatophytes infection of
Agar………………………………………...………...20g man.
Distilled water ………………..…………………..1000ml Phytone (BBL)………………..…………....……10 gm
Lightly cook carrot until tender in 500 ml distilled water, Dextrose ……………………..……..……….10 gm
macerate, and autoclave for 30-min. Add 30-g agar and Agar ………………………………….…..……...20 gm
dissolve. Add distilled water to reach 1000 ml and Phenol Red solution ……………………..………40 ml
autoclave for 15 min. HCl (0.8 N) ………………………………..……..6 ml
Cycloheximide ……………………………….....0.5 gm
Coconut Agar Medium (CAM): Distilled water………………………………….1000 ml
Shredded coconut ……………………………..……100g Gentamicine sulfate…. 0.1gm of active drug (= 100 g /
Agar ……………………………………….….……..20g ml)
Shredded coconut was homogenized for five minutes Chlortetracycline HCL…….… 0.1 gm (= 100 g / ml)
with 200-300 ml of hot distilled water. The homogenate1. Add phytone, dextrose and agar to 1000 ml distilled
water, boil to dissolve. Yeast …….…………….…………….……….………2 g

2. Add 40 ml of phenol red solution while stirring Agar …….…………….…………………….………20 g
[phenol red solution, 0.5 gm phenol red (Difco Water …….…………….……………….………1000 ml
Bacto) dissolved in 15 ml 0.1 N NaOH, made up to
100 ml with distilled water]. Hemp-seeds:
3. Add 6 ml 0.8 N HCL with stirring. Certain types of aquatic fungi, namely members of the
4. Dissolve 0.5 gm cycloheximide in 2 ml Acetone , Saprolegniales, Leptomitales, and many of the
add it to hot medium while stirring Peronosporales, are commonly cultured on halves of
5. Dissolve Gentamicin sulfate powder in 2 ml Distilled boiled hemp seeds placed in water from ponds or streams.
water. Corn, wheat, or dead insects may also be utilized.
6. Autoclave at 121 C for 10 minutes, cool to 47 C.
7. Dissolve 0.1 g chlortetracycline HCL in 25ml of Hemp seed Agar (HSA):
sterile distilled water, then added to the medium Hemp seed ……………………………………….… 20 g
while stirring. Agar …………………………………………………15 g
8. Pour in sterile vials (as slants) or in plates, cool. Water ……………………………………………1000 ml
9. Store under refrigeration. Wash 20 g of non-denatured seeds in distilled water. Boil
10. After inoculation leave caps loose to allow adequate in 1 liter of distilled water for 30 minutes. Filter through
growth and to develop color change. Incubation is cotton wool. Add agar and heat until dissolved, and fill up
at 28 C. the liquid with distilled water to 1 liter. Autoclave at 15
1. Color change of indicator (in case of dermatophyte psi for 20 minutes. The pH should be adjusted to about 6 -
growth) should be interpreted not later than two 7. Remember that the pH will be lowered by autoclaving.
weeks after inoculation. A change in pH produced Autoclave at 15 psi for 20 minutes.
by the proteolytic activity of dermatophytes is
demonstrated by Phenol red indicator that is added
to the DTM, changing from yellow to red indicates Hoyer’s medium:
dermatophyte presence. Gum Arabic ………………………………….. …30g
Chloral hydrate ………………………………….200g
Glycerin ………………………………….. ……..20 g
Galleria Artificial diet: Water ………………………………….. ……...50 ml.
Maize meal ………………….………………..… 22g
Wheat meal ………….……………………….… 22 g Soak gum Arabic in the water and chloral hydrate.
Dried milk …………. ………………………..… 11 g Leave the mixture to stand and occasionally agitate
Dry yeast …………………………………….…5.5 g
over several days until the material is dissolved. Then
Bees wax ….…………………………………..17.5 g
add glycerin. Wetting with absolute ethanol for 1 min
Honey ……….………………………………..…11 g
followed by a 1-min treatment in 2% KOH and
Glycerine …………………………………….…...11 g
washing in 70% ethanol may be necessary for certain
Autoclave honey, maize meal, wheat meal and bees wax
specimens before mounting in this medium.
separately. Mix glycerine, yeast, dried milk, bees wax
and honey. Add wheat meal and maize meal and mix
thoroughly. Lactophenol (Aman's medium):
Lactic acid……………..……………………… 20 ml
Phenol crystals…………..……………………… 20 g
Galleria Artificial diet (Alternative): Glycerol…………………..…………………… 40 ml
Wheat bran (or dried milk) ……………………….320 g Water………………………..………………… 20 ml
Dry yeast ……………………….………………….47 g Lactophenol can be prepared by warming the phenol
Honey ………. ………………….………………..200 g with the water until dissolved, and then adding lactic
Glycerine ………………………………………… 183 g acid and glycerol. The refractive index is 1.45. A stain
Nipagene ………………………………………….. 4 g may be added to this medium, e.g. 0.05-g aniline blue
Autoclave honey and wheat bran separately. Mix (cotton blue), or trypan blue per 100 ml.
glycerine, yeast, nipagene and honey. Add wheat bran
and mix thoroughly.
Lima beans agar:
Lima beans…….…………….………...…………… 50 g
Glucose Yeast Extract (GYE): Agar …….…………….………...………………..…15 g
Glucose …….…………….………...……….………20 g Water …….…………….………...……………..1000 ml
Peptone…….…………….………….……….……… 5 g Blend 50-g lima beans, previously soaked for 10 hours in
500ml of water. Add agar to the mixture; adjust the final Mueller Hinton Agar:
volume to 1 L, and autoclave for 15 min. Beef infusion……….…………….………………300 ml
Casein hydrolysate……….…………….…………17.5 g
Malt Extract Agar (MEA): Starch……….…………….…………….….………..1.5g
Useful for the growth of wood destroying and many other Agar……….…………….…………….…………….10 g
fungi Dissolve 35-g powder in 1000 ml distilled water, boil
Malt extract ………………………..………………...25 g until completely dissolved. Autoclave at 121 oC for 15
Agar ………..………………………………………..15 g min.
Water ……………………………………………1000 ml
Heat the malt extract in water until dissolved. Add agar Mueller Hinton Broth:
and dissolve. Fill up the liquid with distilled water to 1 Beef infusion……….…………….………………300 ml
liter. Adjust the medium to a final pH of 6.5 by NaOH. Casein hydrolysate……….…………….………….17.5 g
Starch……….…………….…………….…………...1.5g
McFarland Nephelometer standards: Dissolve 21-g powder in 1000 ml distilled water,
Determination of the number of microorganisms in a autoclave at 121 oC for 15 min.
liquid medium by using the turbidity method:
The number of microorganisms in a liquid Nutrient Agar:
medium can be determined by visually comparing the Meat extract ………………………..………………..1.0g
turbidity of the liquid medium to a standard that Yeast extract ..……………………………………….2.0 g
represents a known number of microorganisms in Peptone ..…………………………………………...5.0 g
suspension. Sodium chloride …………………..………………...5.0g
Turbidity standards can be prepared by mixing
Agar…………………..…………………………...15.0g
chemicals that precipitate to form a solution of
Water ……………………………………………1000 ml
reproducible turbidity. Such solutions, using barium
Mix the required amount of the powdered medium in 1
sulfate, were developed by McFarland to approximate
liter distilled water. Autoclave for 15 min.
numbers of bacteria in solutions of equal turbidity, as
determined by colony counts (Table B.2).
Preparation of McFarland Nephelometer standards: Nutrient Broth:
Principle: A chemically induced precipitation reaction Meat extract ………………………..………………..1.0g
can be used to approximate the turbidity of a bacterial Yeast extract ..……………………………………….2.0 g
suspension. Peptone ..…………………………………………...5.0 g
Method: Sodium chloride …………………..………………...5.0g
1. Set up 10 test tubes or ampoules of equal size and Water ……………………………………………1000 ml
of good quality. Use new tubes that have been Mix the required amount of the powdered medium in 1
thoroughly cleaned and rinsed. liter distilled water. Distribute into tubes. Autoclave for 15
2. Prepare 1 % chemically pure sulfuric acid. min.
3. Prepare a 1.175 % aqueous solution of barium Oat grain-water agar medium (OGWA):
chloride (BaCl2 . 2 H2O). Oat grains 1000-ml
4. Slowly, and with constant agitation, add the 0.75 % water agar medium 400ml
designated amounts of the two solutions to the tubes as
shown in Table 1 to make a total of 10 ml per tube. Soak the required amount of oat grains in sterile
5. Seal the tubes or ampoules. The suspended barium water at 4 ºC overnight, then drain excess water. Add
sulfate precipitate corresponds approximately to 1000-ml of grains to 1500 ml Erlenmeyer flask, and
homogenous E. coli cell densities per millimeter autoclave. Add 400 ml sterile 0.75 % water agar
throughout the range of standards as shown in Table medium to the flask and mix.
B.2.
6. Store the McFarland standard tubes in the dark at Oatmeal Agar (OA):
room temperature. They should be stable for 6 months. Oatmeal …………………………..…………………60 g
Agar …………………………………..…………….12 g
M2 Agar: Distilled water …………………………….……...1000ml
Malt extract …………………………….………...….5% Heat the oatmeal in water until dissolved. Add agar and
Yeast extract ………………………………….……0.2% dissolve. Fill up the liquid with distilled water to 1 liter.
Agar ………………………………………….…….1.8% Autoclave for 15 min.
Potassium Hydroxide (KOH) Solution: Treacle medium:

KOH crystals………….…...………………….….10 gm Solution 1
Glycerin ……………….………………………..…10 ml Treacle (from sugar beet) …………..…………5 g
Distilled water……………………...……………. 80 ml Tween 80………………………………..…1 drop
10% to 20% solution. Use of KOH is one of the most Distilled water …………………………….. 40ml
rapid methods for direct examination of clinical Adjust to pH6, autoclave for 15 min
specimens. The rate of clearing can be increased by gently Solution 2
heating the slide. Penicillin G …………………………….…12 mg
Streptomycin sulfate……………………… 12 mg
Potato Carrot Agar (PCA): Distilled water ………………………….… 40 ml
Suitable for conservation of fungi Sterilize the solution by membrane filtration. Before use,
Potato* ……………….………………….…………20 g mix the two solutions
Carrot* ……………….……………………….……20 g For the germination of teliospores of Ustilaginales.
Agar …………………………………..……………20 g
Water ………………………………..…………. 1000 g Tween 80:
* Washed, peeled and grated Tween 80 ……………………….………………200 ml
Chop and then boil the carrots and potatoes for 10 minutes Distilled water …………………..………………800 ml
in 1 liter water. Filter the suspension through a cloth. Add
agar and heat until dissolved. Fill up the liquid with water 2P Medium:
to 1 liter. Autoclave at 15 psi for 20 minutes. Cornmeal agar …………………….…….………….17 g
Polymixin-B …………………….…….….………50 mg
Potato Dextrose Agar (PDA): Penicillin ……………………………..…….….…50 mg
Useful for the growth of many fungi, especially Distilled water …………………………..……....1000 ml
phytopathogens, some bacteria
Potato (peeled and diced) ……………..………….. 200 g Urease Test Medium:
Dextrose (glucose) ………………………..……….. 20 g Useful for separation of Trichophyton mentagrophytes
Water ………….……………………………..…1000 ml from Trichophyton rubrum. Within seven days
Rinse potato under running water, and then add to water. Trichophyton mentagrophytes strains should hydrolyze
Boil for 1 hour. Filter through a cloth, squeezing through urea and the medium becomes deep red, while action of
as much pulp as possible. Autoclave at 15 psi for 30 Trichophyton rubrum is less rapid.
minutes. Peptone ….………………………..…………...…….1 g
Nacl…..………………………………..……...……..5 g
Sabouraud’s Dextrose Agar (SDA): KH2PO4…………………………..………….……….2 g
Dextrose …..………………………………..………. 40 g Glucose …………………………..…..…………...….5 g
Peptone ………..………………..………………….. 10 g Agar ………………………………………………...20 g
Agar …………………………………………………20 g Distilled water ………………………….….…….1000 ml
Mix the ingredients, boil to melt the agar, adjust pH to 5.6 Dissolve by heat, add 5 ml of phenol red solution (0.2% in
and sterilize. Autoclave for 10 min. 50% ethanol). Autoclave at 121 C for 15 minutes, cool,
and add 100 ml of urea (20% aqueous solution, sterilized
Sterile Soil Extract: by filtration). Tube and slant were prepared. Place a small
Soil ………………………..……..………………… 50 g amount of colony in the medium and incubate at 24-26 C.
Distilled water…………………………..……… 1000 ml Read result in seven days. A deep red color through the
Add soil to water and shake well and allow to settle for 48 medium is a positive reaction.
hours. Filter through Whatman no. 1 filter paper and
autoclave for 20 min. V-8 juice agar medium (V-8 A):
V-8 juice…………………….………..….………. 200 ml
3P Medium: CaCO3…………………….………..….………..… 3g
Cornmeal agar ………………………………..……17 g Agar…………………….………..….…………… 20g
Pimaricin ………………………………………….5 mg Distilled water…………………….………..…. 1000ml
Polymixin-B ……………………………………..50 mg Mix ingredients and autoclave for 15 min.
Penicillin……..……….………………………… 50 mg
Distilled water …………………………………1000 ml
V-8 agar (Clarified) (CV-8A):

V-8 juice…………………….………..….……… 200 ml
CaCO3………………..…………………….……… 3g
Agar …………………….……………….…………20g
Distilled water 1000ml
Dissolve CaCO3 in 200ml V-8 juice, centrifuge, and
remove supernatant. Dissolve agar in 600ml water, adjust
pH to 7-7.5, adjust the final volume to 1 L, and autoclave
for not more than 10 min.
VP3 Medium:
Agar ………………………….…………………….23 g
Cornmeal agar ……………………………………..17 g
Sucrose …………………………………………….20 g
CaCl2 …………………………………………….10 mg
MgSO4. 7H2O ……………..……………………10 mg
ZnCl2 …………………………..…………………1 mg
CuSO4.5 H2O …………………….…………..0.02 mg
MoO3 ………………………………….……....0.02 mg
MnCl2……………………………………..……0.02 mg
FeSO4.7H2O …………………………..………0.02 mg
Thiamin HCl ……………..…………………..…100 µg
Pimaricin ……………………………………….…5 mg
Vancomycin ……………………….…………….75 mg
PCNB…………………………………………..100 mg
Rose Bengal ……………………………………2.5 mg
The basal medium is made to 1000 ml with deionized
water in a 2-L flask and autoclaved for 15 minutes.
Vancomycin, penicillin, and pimaricin are added to the
medium when it has cooled to 50-55°C. After addition,
the medium is mixed thoroughly by gentle rotation of the
flask. The plates are then poured, and when the medium
in the plates has solidified, the plates are stored in the
dark because pimaricin is sensitive to light.
APPENDIX B
Table B.1. Characteristics of Candida species most often isolated from clinical specimens.
Assimilations Fermentations Other reactions
Species of candida
India ink capsule

Growth at 37 oC
Pseudohyphae
Germ tubes
Cellobiose
Melibiose
Galactose
Trehalose
Galactose
Trehalose
Raffinose
Dulcitol
Glucose
Glucose
Maltose
Maltose
Sucrose
Sucrose
Lactose
Lactose
Inositol
Xylose
Urease
KNO3
C. albicans +* + + - + - - - + - + - AG A† AG - AG or A AG or A† - - + + + -
C. stellatoidea‡ + + - - + - - - + - +† - AG - AG - - - - - + + + -
C. parapsilosis + + + - + - - - + - + - AG or A - - - AG or A AG or A† - - + + - -
C. tropicalis + + + - + - +† - + - + - AG AG AG - AG AG - - + + - -
C. pseudotropi-calis + - + + + - + - +† + - - AG - AG AG AG AG or A† - - + + - -
C. krusei + - - - - - - - - - - - AG - - - - - +† - + + - -
C. guillermindii + + + - + + + - + + + + AG - AG - AG or A† AG or A† - - + + - -
C. rugosa + - - - - + - - + - - - - - - - - - - - + - - -
* + = Assimilation, growth density greater than 1+ turbidity by the Wickerham card; AG= acid and gas; A= acid only produced in fermentation broth; -= negative.
† Strain variation. ‡ Report as C. albicans when C. stellatoidea is identified.
Table B.2. McFarland Nephelometer standards

Tube number
0.5 1 2 3 4 5 6 7 8 9 10
Barium chloride (ml) 0.05 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Sulfuric acid (ml) 9.95 9.9 9.8 9.7 9.6 9.5 9.4 9.3 9.2 9.1 9
Approximate bacterial cell 1.5 3 6 9 12 15 18 21 24 27 30
density (x 10 8/ml)
Approximate candidal cell
density (x 10 6/ml) 1-5
Table B.3. Dilutions of active ingredients
Tube Stock solution (ml) Solvent Final concentration

no 200 mg/ml (ml) mg/ml mg/0.1ml
1 4 ---- 200 20
2 2 2 100 10
3 1.5 2.5 75 7.5
4 1 3 50 5
5 0.8 3.2 40 4
6 0.6 3.4 30 3
7 0.5 3.5 25 2.5
8 0.4 3.6 20 2
9 0.3 3.7 15 1.5
10 0.2 3.8 10 1
11 0.1 3.9 5 0.5
12 0.02 3.98 1 0.1
Table B.4. Final concentration of active ingredients in the medium.
Tube no. 1 2 3 4 5 6 7 8 9 10 11 12
Conc. (X102g/ml) 20 10 7.5 5 4 3 2.5 2 1.5 1 0.5 0.1

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Book · January 1998

Mohammed saleem Ali-shtayeh Rana M. Jamous

SEE PROFILE SEE PROFILE

The user has requested enhancement of the downloaded file.

Mohammed S. Ali Shtayeh

Rana Majid Jamous

Reem M-R. Yaghmour

Authors, Nablus, Department of Biological Sciences, An-Najah University

Copyright  Ali-Shtayeh M. S., Jamous R. M., &Yaghmour R. M-R. 1998.

PART ONE: GENERAL MYCOLOGICAL LABORATORY PROCEDURES…………1

1.1 CULTIVATION OF FUNGI…………………………………………………………… 1

PART TWO: EXERCISES

EXERCISE 1. Preparation of Media, Pouring of Plates, and Preparation of Agar Slants…….13

The manual includes two parts: General Mycological Laboratory

GENERAL MYCOLOGICAL LABORATORY

GENERAL MYCOLOGICAL LABORATORY

EXERCISE 1: PREPARATION OF MEDIA, POURING OF PLATES, AND

What a complete medium should provide a fungus with?

When preparing a medium its necessary to adjust its pH. Why?

2. Prepare agar slants (slopes) in screw-capped bottles or in tubes as follows:

3. Pour the media into 9-cm plates as follows:

Agar plates are usually stored up side down. Why?

EXERCISE 2: CULTIVATION AND MICROSCOPIC EXAMINATION OF

B. Slide culture technique

Record and illustrate your observations.

C. Single spore cultures

Record and illustrate your observations.

Record and illustrate your observations.

c. Measurement of microscopic fungal structures:

EXERCISE 3. MORPHOLOGY OF FUNGI

Can you see any cytoplasmic movement?

b. Septate mycelium: Examine microscopic preparations of excised portions of

2- Fungal tissues (plectenchyma)

L.s. in the stroma of Claviceps purpurea showing prosenchyma.

b. Pseudoparenchyma: Examine a cross section (c.s.) of a sclerotium of Claviceps

C.S. of a sclerotium of Claviceps purpurea showing Pseudoparenchyma.

3- Main reproductive structures

b- Buds: These can be seen in actively growing yeast. Illustrate.

Yeast buds in actively growing yeast

c- Sporangiospores (formed inside sporangia)

Sporangia and sporangiospores (aplanospores, non-motile spores) of Rhizopus

How do you think these spores disseminate?

Sporangia, zoospores (planospores, motile spores), and zoospores discharge of … …. …………

Oospores of Pythium ultimum

b. Zygospores (result of gametangial copulation): Examine under the high powers of a

Stages of Zygosporangium and zygospores formation in Rhizopus stolonifer zygospores.

c. Ascospores (spermatization): Examine the ascocarps of Claviceps purpurea.

Ascospores, asci and ascocarps (embedded in the stroma) in Claviceps purpurea

d. Basidiospores: Examine a permanent slide of Puccinia graminis. Observe the

teliospores which give rise to basidia and basidiospores. Illustrate.

Teliospores in Puccinia graminis.

EXERCISE 4. KINGDOM FUNGI: PHYLUM CHYTRIDIOMYCOTA

Label the following diagram of the life cycle of Synchytrium endobioticum

EXERCISE 5. KINGDOM FUNGI: PHYLUM ZYGOMYCOTA

EXERCISE 6. KINGDOM FUNGI: PHYLUM ASCOMYCOTA

Ascospores, Asci, and Ascocarps:

Label the different types of ascocarps shown in the following diagram

Illustrate various stages of ascospores development in Saccharomyces cervisiae.

Other Filamentous Ascomycetes

EXERCISE 7. KINGDOM FUNGI: PHYLUM BASIDIOMYCOTA

Label the following diagram showing life cycle of Puccinia graminis.

1. Main morphological features of Agaricus bisporus

Construct a life cycle diagram of Agaricus bisporus.