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Practical Mycology

Safety Rules in the Microbiology laboratory:

1. Lab coats must be worn at all times in the lab

1. Treat all microorganisms as potential pathogens, all cultures should be

properly labeled

2. Use a disinfectant soap to wash your hands before and after working with

microorganisms

3. Do not pipette by mouth to avoid accidental ingestion of cultures or

chemicals, use pipettes with rubber bulbs or mechanical manipulators.

4. When flaming an inoculating loop or needle, always place the needle, loop

or cork borer near the base of the flame (to incinerate any M.O. on the loop).

5. Autoclave or disinfect all waste material.

6. Clean up spills with care. Cover any spills or broken culture tubes with a

70% ethanol or 10% bleach solution; then cover with paper towels. After

allowing the spill to sit with the disinfectant for a short time, carefully clean

up and place the materials in a biohazard autoclave bag to be autoclaved

Sterilization Methods:

Sterilization is the kill and destruction of living microorganisms

A. Physical methods:-

1. Heating:

i. Dry heat:

Hot air oven used for sterilizing of glassware at 180ºC for 1 hour (150ºC for 1.5

hour, 200ºC for 30 minutes).

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ii. Wet heat:

Moist heat under pressure use for sterilizing of culture media, autoclave used for

this purpose at 121ºC for 17 minutes, 1.5 bar.

iii. Flame:

Used for sterilization of needle, loop, cork hole, forceps by heating to redness.

2. Radiation:

Both ultraviolet and ionized radiation used for sterilization by using hood.

3. Filtration:

This technique used for separation of micro organisms from liquids, as Seitz filter,

chamber land filter.

4. Incineration:

Incineration is a waste treatment process that involves the combustion of organic

substances contained in waste materials. Incinerator converts the waste into ash,

flue gas and heat.

2. Chemical methods:

a. Disinfectants:

Chemical agents used for surface sterilization as chloroform, formaldehyde, detol,

mercuric chloride.

b. Gaseaous sterilization:

This method is used for soil sterilization and usually used for vaporing of

agricultural products, as methyl bromide, ethylene oxide.

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Preparation of media for microbial isolation

Types of Media according to chemical structure

1. Natural media:

Consist of Natural complex substances of unknown structure and

concentration (for morphological study).

2. Semi synthetic media:

Consist of 2 parts of known structure and concentration and other of

unknown structure and concentration (for morphological study).

3. Synthetic media:

Consist of known structure and concentration (for physiological study).

Types of Media according to texture

1. Solid media:

These media contain solidified substance (%2 agar) or gelatin, agar is used

more than gelatin, because agar is complex carbohydrate substances, and not

lysis by microorganisms and solidified at 4°C while gelatin is protein

substances, lysis by microorganisms and become liquid at 25°C (used for the

study of structure and morphology of fungal culture).

2. Semi-solid media:

Consist of 1% of agar that produces semi solid texture to the media (used for

studying of zoospores).

3. Broth media:

These media lack of agar (used for studying of nutrition and metabolism of

microorganisms).

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Preparation of media

1. Potato Dextrose Agar (PDA)

Chemical compound Quantity

Dextrose………………………20 g.

Potato………………………...200 g.

Agar…………………………..20 g.

Distill water (D.W.)………….1 L.

Procedure:

1. Scrub the potatoes, clean then cut into small cubes, weight out 200 gm and place

in 500 ml of D.W., and boil for 20 minutes until potato tissue become soft then

filtered through 3-4 layers of fine gauze.

2. Add the sugar to another 500 ml of D.W., then add the agar gradually and boil,

then mixed with potato extract.

3. The volume make-up to 1000ml by D.W. then distributes into 4 flasks and

sterilized by autoclave at 121ºC, 1.5 bar for 15-20 minutes.

2. Sabouraud Dextrose Agar (SDA):

For isolation and cultivation of dermatophytes

Chemical compound Quantity

Peptone ……………………….…10 g.

Dextrose ………………………....40 g.

Agar……………………………....20 g.

Cyclohexamide ……………….…0.5 g.

Chloramphenicol…………….…..0.05g.

D.W……………………………...1 L.

pH………………………………..6.5

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Soak all ingredients in 100ml water and boil remaining water and boil to dissolve

then autoclave it. It provides basic nutrition that will support the growth of any

pathogenic fungi; also Chloramphenicol and Cycloheximide were added to the

medium.

Note: Chloramphenicol (Anti bacterial) + Cycloheximide (Antifungal-

Antisaprophyte).

3. Czapek (Dox) Agar

For physiological studies of fungi.

Chemical compound Quantity

NaNO3…………………………2g.

KH2PO4…………………….….1g.

MgSO4.7H2O………….…….0.5g.

KCl……………………….....0.5g.

FeSO4.7H2O……………......0.01g.

Sucrose……………………....30g.

Agar……………………….....20g.

D.W…………………………..1L.

Soak the entire ingredient in small amount of water, bring the remaining water

to boil and bring to the boil again, stirring continuously, and

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Isolation of fungi

Fungi grow in many different habitats and have such varied ecological

requirements such as sources of nitrogen, carbohydrate and also need a range of

elements which required for normal growth. They found in soil especially

containing organic mater, water and even in air. Fungi have important role in

decomposing and recycling of compound substances in ecosystem.

Fungi isolate from its natural habitat and cultured on PDA in order to identify the

fungal species depending on its morphology of colony and characters of spores,

fungi isolated from:

1- isolation of fungi from air: By opening Petri dish that contains sterilized PDA in laboratory air for about

5 minutes, then closed and incubates at 25oC for 5-7 days.

2- isolation of fungi from water: a- Tap water: 1ml of tap water pour into a Petri dish contains sterilized PDA then

incubates at 25oC for 5-7 days

b- Sewage water: The fungal spores which was found in sewage water can be isolate by

pouring 1ml of sewage water into a Petri dish contains sterilized PDA,

then incubates at 25oC for 5-7 days.

3- Isolation of fungi from soil: a- Directly: 1gm of soil takes randomly from 5 different places mixed well then

spread some of soil into a Petri dish contains sterile PDA then incubate at

25oC fro 5-7 days.

b- In Directly: soil fungi can be isolated by serial dilution, for this purpose 1gm of soil

transfer to 9ml of sterile distilled water (SDW) after shaking well 1ml of

this suspension transfer to a tube containing 9ml SDW by using sterile

pipette, after shaking well serial delusion continued to 10-6, then 1ml of

each dilution transfer to a Petri dish that contains sterile PDA then

incubate at 25oC for 5-7 days.

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4- isolation of fungi from seeds:

a- endo seeds: The seeds submerge in the sodium hypochloride 6% concentration for 1-

2 minutes, and then the seeds washed by distill water 3 times for 1.5- 2

minutes to removing the toxic activity of the chemical agent on the seeds,

then transfer by using sterilized forceps into Petri dish contain sterilized

PDA then incubate for 5-7 days at 25oC.

b- exo seeds: The seeds washed by SDW, then transfer by using sterilized forceps into

Petri dish contain sterilized PDA then incubate for 5-7 days at 25oC.

5- Isolation of fungi from infected plant tissue: a- outside infected tissue: Plant pathogenic fungi can be isolated from infected tissue by taking a

part of the tissue a round infected zone by 10cm because the fungi are

usually found in the healthy tissue near the infected zone.

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The sample is transfer to a Petri dish or beaker then cuts into small cube

pieces, after washing it by SDW, and then the pieces transfer to a Petri

dish contains PDA by using forceps then incubates for 5-7 days at 25oC.

b- inside infected tissue: The sample is transfer to a Petri dish or beaker then cuts into small cube

pieces, after that submerged in sodium hypochloride 6% for 1-2 minutes,

then washed by SDW three times each for 1-2 minutes, to removing the

toxic activity of chemical agent on the tissue, then the pieces transfer to a

Petri dish contains PDA by using forceps then incubates for 5-7 days at

25oC.

6- Isolation of fungi from infected human: The infected area cleans with 70% ethanol, and then the specimens were collect

as the followings:

1- Skin scales: Collect by scraping the surface of the margin of the lesion by using a clean

slide.

2- Crusts: Collected by removing part it on healthy skin, by using sterilized scissors or

forceps.

3- Nail pieces: Collect by taking a part the infected nail by using sterilized scissor.

4- Hairs: Collect by removing broken hairs from the margin of the lesion using

sterilized forced.

Fungal Identification

• Eukaryotic heterotrophic microorganisms

• They are saprotrophic living on dead organic material or parasitic on

plants or humans

• They may be unicellular (yeast) or multicellular (mold).

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Fungi are reproducing sexually and asexually, the most common method is

asexually by production of spores, the spores vary on:

1. Color: Hyaline, Yellow, Orange, Red, Brown or Black.

2. Size: minute or large

3. Shape: spherical, oval, oblong, needle shape….etc.

4. Number of cells: unicellular or multicellular and

5. varies in their arrangement

1- Direct microscopic examination:

part of the specimen used for direct microscopic examination, by adding a drop

of KOH 20%, put a cover slide on it, then allow for about 15 minutes then

diagnosed by light microscope directly.

2- Cultural examination:

Transfer the specimen to a Petri dish contains sterilized SDA (Sabouraud

Dextrose Agar + Antibiotic (Cycloheximide and Chloramphenicol) then

incubate at 30oC for 10-15 days

Microscopic examination:

By preparing slide as followings:

1. Place a drop of fungal satin on a clean slide

2. Transfer a small piece of the fungal hyphae onto the drop using a flamed

cooled needle.

3. Place a cover glass over the drop gently to avoid producing air bubbles in

the satin.

4. Examine under low power then at 40X.

Note: For direct examination of pathogenic fungi, the specimen were

placed in 20% KOH on the slide then put a cover and let for 15 minutes

at 30ºC then examined under light microscope.

Fungal Stain:

1. Lactophenol cotton blue

2. KOH: for pathogenic fungi

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Slide culture: The microscopic examinations of molds usually break the arrangement of spores

that the identification becomes very difficult. For this purpose the slide culture

technique were made.

Procedure 1:

1. Pour sterilized PDA into a sterilized Petri dish, after solidify take a square pieces of 1cm2, and transfer it to another Petri dish containing V- shaped

glass or metal on the wet filter paper and a slide over it.

2. Inoculate the center of square medium with a mycelium of fungi then cover the square with a cover slip.

3. Incubate the plate at 25oC for 5-7 days 4. Take a cover slip carefully and put it on the clean slide at a drop of

lactophenol cotton blue and examined.

5. Remove the template from the slide carefully and put a drop of stain on it and covered with a cover slip then examined by microscope.

Procedure 2:

1. Pour sterilized PDA into a sterilized Petri dish and after become solid inoculate the medium with a fungus by streaking method

2. Take 4 pieces of cover slip and sink it in the alcohol and dried by a burner 3. Place a cover slip on the media in a slant situation on opposite side, and then

incubate it at 25oC for 5-7 days

4. Put each cover slide carefully on a clean slide contain a drop of stain at the center and then examined

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Preservation and maintenance of fungal culture:

1- Oil covered slants: This is simple economical method for preserving fungi where they remain viable

for several years at refrigerator, in this method sterile liquid paraffin is poured over

a slop culture of the fungi and store upright at room temperature, the layer of

paraffin prevent dehydration of the medium and by ensuring anaerobic condition

the fungi remain in a dormant state.

2- Lyophilization (Freeze- Drying): In this technique the culture is rapidly frozen at -70oC and then dehydrated by

vacuum and the tubes containing freeze-dried cultures are stored in dark at 4oC in

refrigerators, the fungi preserved for over 30 years, for sub culturing the fungi, the

fungi powder is dissolved in 1ml of sterile water then streaked on agar plates.

3- Liquid nitrogen: Freezing in liquid nitrogen at temperatures of -196oC, the cell suspension in the

presence of stabilizing agent such as glycerol that prevents the formation of ice

crystals which may kill frozen cells is sealed into small ampoules and stored in

liquid nitrogen refrigerator -196oC, the cell remain viable for 10 years.

4- Storage by dehydration: Widely used specially for molds, the dehydration of media leads to reducing

metabolic activity and have 2 types:

a- preservation by using soil: the soil sterilized then inoculate by the fungus at optimum temperature for

several day then allow to be dry at room temperature and preserved at

refrigerator.

b- storage in silica gel: The same method of soil using silica gel.

Classification of fungi: There are several classifications of fungi due to different data collected by

mycologists on the basis of reproduction method, structure, physiology and

development. The following classification is based upon that was patterned by

Alexopoulos and Mims. The name of classificational level have standard ending:

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Fungal Reproduction

Is the formation of new individuals having all the characteristics typical of the

species, fungi are reproducing sexually and asexually, the asexual reproduction is

common in fungi.

These asexual spores are formed on a phase of the fungal life cycle termed the

mitosporic, or anamorphic phase. The sexual stage of the fungus can be termed the

teleomorph, and the characteristics of this phase of the life cycle are much more

stable and reliable for taxonomic purposes.

1- Asexual reproduction: Also called somatic reproduction, the sexual reproduction includes:

a- fragmentation:

Segmentation of the hypha into a number of fragments each growing into a new

individual; many fungi can reproduce by fragmentation. Any mycelium that is

fragmented can grow into a new colony. Many fungi are sub-cultured using this

hyphal fragment technique.

b- Fission:

The simple splitting of a cell into two daughter cells, it is characteristic of some

yeast.

c- Budding:

Is the production of a small outgrowth (bud) from a parent cell, the bud increase in

size and breaks off and forms a new individual, chain of buds forming a short

pseudo mycelium.

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d- production of spores (sporulation):

The most common method of a sexual reproduction in fungi is by means of spores.

It is responsible for the production of large numbers of spores throughout the year;

spores vary in color, size, shape, number of cell, arrangement of the cells and the

way in which the spores are borne. There are many types of spores:

1- sporangiospore:

spores are borne in sporangia called sporangiospores, a sporangium is a sac like

structure whose entire content converted into spores, a motile spores called

zoospores, non motile spores called aplanospores.

2- Chlamydospore:

A hyphal cell enveloped by a thick cell wall, which eventually becomes

separated from the parent hypha and behaves as a resting spore.

3- Arthrospore:

A spore resulting from the fragmentation of a hypha also called oidium.

4- Conidia:

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Spores are usually born at the tips or sides of hyphae. Conidia are generally

produced on conidiophore, which may be produced free from each other, or

they may be organized into definite fruiting bodies, the most common fruiting

bodies are:

i- Pycnidium:

Is a globose or flask shaped structure its wall are lined with conidiophores

ii- Acervulus:

A mat of hyphae usually formed below the epidermis of parasitic fungi,

conidiophores are short and closely packed together.

iii- Sporodochia:

A cushion shaped structure covered with conidiophores.

iv- Synnema:

A group of conidiophores semented together and forming an elongated spore- bearing structure.

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Classification of fungi:

There are several classifications of fungi due to different data collected by

mycologists on the basis of reproduction method, structure, physiology and

development. The following classification is based upon that was patterned by

Alexopoulos, Mims and Blackwell (1996). The name of classificational level have

standard ending:

Kingdom: Fungi (Myceteae)

Phylum: ...………mycota

Class: ...…….……mycetes

Order: …………...ales

Family: ………….aceae

Alexopoulos, Mims and Blackwell (1996).

Kingdom: Fungi (Myceteae)

• Phylum: Chytridiomycota • Phylum: Zygomycota • Phylum: Ascomycota • Phylum: Basidiomycota

Phylum: Chytridiomycota

Class: Chytridiomycetes

Order: Chytridiales

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Family: Synchytriaceae

Synchytrium endobioticum

(Black warts disease of potato)

These are endobiotic, holocarpic fungi (Holocarpic: the whole thallus converted to

zoosporangium), the thallus divided into sporangia to produce zoospores which

posses single posteriorly whiplash flagella that may be asexual zoospores or sexual

cells gametes depending on the presence of water, the zoospore penetrate host cell,

grow in size and secretes thick wall then called (Prosorus) causing hypertrophy

(increasing in size of host cell) and hyperplasia (increase in number of host cell),

after maturing the prosorus divided by mitotic division producing sorus that consist

of 4-9 segments each contain about 200-300 nuclei, that releasing as zoospores that

swims and infect new host.

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Phylum: Zygomycota

Class: Zygomycetes

Saprophytes or facultative parasites of plants and specialized parasites of animals,

the mycelium usually non-septate. Asexual reproduction by means of

sporangiospores or conidia, sexual reproduction by fusion of two multinucleate

gametangia of different strains which are morphologically in distinguishable

(isogametes) that forming thick walled resting spore ( Zygospore).

Order: Mucorales

Family: Mucoraceae

Mucor sp.

Produce only one sporangia on sporangiophore at each point, rhizoid is absent but

poses absorptive hyphae, the sporangium of Mucor sp. is smaller in size than that

of Rhizopus sp.

Family: Absidiaceae

Absidia sp.

Have stolon and rhizoid, attack human internal nervous system with fatal

consequences.

Rhizopus sp.

Common bread mold, causing soft rot of sweet potatoes in storage, characterized

by brown sporangiophores simple or branched, and arises from a cluster of rhizoids,

adjacent group of rhizoids and sporangiophore connected by stolon.

Zygomycosis (mucormycosis)

Commonest isolates belong to Mucor, Rhizopus, Absidia, Rhizomucor and

Cunninghamella genera, acquired mainly by inhalation of spores; occasionally

inoculated directly to skin or ingested. Clinical manifestations include

rhinocerebral, pulmonary, cutaneous, gastrointestinal and disseminated infections.

Patients with diabetic, immunosuppressant, burns, leukemia are the most

commonly infected with mucormycosis they may invade the walls of blood vessels

producing thrombosis.