Lab 1

By:
Fatimah lafi Al.Ghamdi
Health and Safety in
microbiology
laboratories
 Don't

• Do not play in the laboratory.

• Do not use any laboratory equipment until you
are told to do so.
• Do not perform any laboratory procedure until
you are told to do so.
• Do not bring food or drinks into the laboratory.
• Do not take culture out of the laboratory.
• Never pipette by mouth.
 Do
• Wear lab coat.
• Wear gloves.
• Tie long hair neatly at the back of the neck.
• Wear shoes that enclose your entire feet.
• Wash your hands with disinfectant soap when you arrive at
the lab and again before you leave.
• Disinfect work areas before and after use.
• Label everything clearly.
• Inoculating loops and needles should be flame sterilized in a
Bunsen burner before you lay them down.
• Sterilize equipment and materials.
• Dispose of all solid waste material in a biohazard bag and
container.
Laboratory Safety Signs
 Instruments and tools used
in microbiology laboratories
2. Safety Cabinet

Microbiological safety cabinets

are designed to capture and
retain infected airborne
particles released in the work
and to protect the laboratory
worker from inhaling them.
3- Autoclave
Hot air oven
Cork borer
 Introduction
The habitats of fungi

Fungi is classified as Eukaryotic and contain cell walls and

produce spores.

Three major groups of fungi are recognized as the molds,

the yeast and the mushrooms depending the size.

Fungi are found all around the world.

Many of fungi are symbiotic with plants, animals, or other

fungi.
- Eukaryotic.

-heterotrophic
Nutrition in fungi:
They are saprophytes, or parasites or
symbionts.
Three major groups of fungi

Mold

Yeast

Mushrooms
Mushrooms
 Mold

Aspergillus sp.

Rhizopus sp.
Penicillium sp.
Yeasts
Filamentous fungi ; Molds ,Yeast

Each filament grows mainly at the tip, by

extension of the terminal cell.

Molds are multinucleated, filamentous fungi

composed of hyphae.
 hyphae (singular; hypha)

Threadlike filaments forming the mycelium of a

fungus.
Hypha
Mycelium (plural mycelia)

The total mass of hyphae is termed a mycelium.

The portion of the mycelium that anchors the mold

and absorbs nutrients is called the vegetative
mycelium ; the portion that produces asexual
reproductive spores is termed the aerial mycelium.
Mycelium
A) Non Septet hypha.(Coenocytic)

B) Septet hypha.
(a) Septet hypha.
(b) Non Septet hypha.
 Pores in cross walls

In septate hyphae, the structures allow

cytoplasmic to distribute nutrients, synthesized
compounds throughout the hyphae
-Mononucleote
-Binucleote
-Multinucleote
-Obligate aerobes.
-Fungi are acidic, PH is (4-5)
They are typically non-motile
-Mesophilic fungi.
Optimum Temperature (25-28) °c.
-Osmophilic fungi.
-Halophilic fungi.
 Lab 2

By:
Fatimah lafi Al.Ghamdi
Isolate Fungi from different habitats

Materials:
-Potato dextrose Agar

Samples:

Air, soil, water and grains

Incubate all plates at 25°c for 5 to 7 days

 Media
PDA

Air Soil Water Grains

Mycoflora isolated from raisins after 7
day of growth at 28 °c on PDA medium.
Fungi quantitative estimation numbers
in soil
Introduction:

Soil is considered as a natural habitat of several groups of

microbes.
In order to isolate fungi from complicated environment like
soil, special Medias are required.

Fungi selective media has a ph of 4.8 that’s allow the

growth of fungi faster for its acidic base, by adding
hydrochloric acid (HCL).

Adding antibiotic like streptomycin (rose Bengal agar) will

stop the bacterial growth
Materials:

-Potato dextrose Agar ph (4.5 - 6.5)

-Rose Bengal Agar

-Soil sample
-Test tubes contains 9ml distilled water
-Sterilize pipettes
-Sterilize Petri dishes
Methods:
Rose Bengal Agar
Incubate all plates at 25°c for 5 to 7 days
 Using the equation:

Median x Inverted dilution

Number of colonies in 1g = ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Soil weight
1/10 1/1000
Isolate fungi from food:
1-Alcohol 70%.
2-Sterile distilled water.
Purification of the isolated
fungi from food.
Materials:

-Cork Borer
-PDA media

-Inoculums needle
-Fungi cultures
Methods:
Incubate plate at 25°c for 5 to 7 days
Cork borer
 Prepare slides of fungi purification from
food :

Materials:

-Pure fungi cultures

-Inoculums needle

• The lactophenol cotton blue (LPCB)

-Covers
Methods:
Reproduction of Fungi:
Introduction:

Reproduction is the formation of new members of its typical

qualities of the same type.

Fungal
Reproduction

Sexual Asexual
reproduction reproduction
 Asexual reproduction

Fragmentation
Binary Budding Asexual spores
fission
Oidium Chlamydospore Endospores Exospores
1-Fragmentation:
A) Oidium:
B) Chlamydospore:
 Chlamydospore

A chlamydospore is the thick-walled large resting spore of

several kinds of fungi.

It is the life-stage which survives unfavourable conditions,

such as dry or hot seasons.
2-Binary fission
Shizosaccharomyces sp.
3-Budding:
Saccharomyces sp.
Saccharomyces cerevisiae
4- Asexual spores:
A) Endospores:
-Zoospores:
-Aplanospores (Sporangiospores):
Rhizopus sp.
B) Exospores:

It various forms and are found on

the surface of mycelium.
Aspergillus sp.
Penicillium sp.
 Sexual
reproduction

Oospores Zygospores Ascospores Basidospores

1- Reproduction by Oospores.
Albugo sp.
Albugo sp.
2- Reproduction by Zygospores.

Rhizopus sp.
3- Reproduction by Ascospores.

An ascospore is a spore contained in an ascus or that

was produced inside an ascus.

This kind of spore is specific to fungi classified as

ascomycetes (Ascomycota).

A single ascus will contain eight ascospores.

3- Reproduction by Ascospores.

Ascus
Ascus
 Ascospores

Naked asci Ascocarps

Cleistothecium

Perithecium

Apothecium
1- Naked asci:
Taphrina sp.
Taphrina sp.
Taphrina sp.
2-Ascocarps:
A) Cleistothecium.
Erysiphe sp.
Erysiphe sp.
B) Perithecium:

Claviceps sp.
Claviceps sp.
Claviceps sp.
Claviceps sp.
C) Apothecium.
Peziza sp.
4- Reproduction by Basidiospores.
4- Reproduction by Basidiospores.

Basidiospores found on the installation of special

called Basidium.

Basidium :
Club shaped organ involved in sexual reproduction
in basidiomycete fungi (mushrooms, toadstools
etc.). Bears four haploid basidiospores at its tip.
Agaricus sp.
 Lab 5

By:
Fatimah lafi Al.Ghamdi
 Linear extension Method
(Agar Plate):
Materials:

- PDA media
- Inoculums needle
- Pure fungi culture
-Cork Borer
Cork borer
Method:
Incubate plate at 25°c for 5 to 7 days
 ‫‪C‬‬

‫‪B‬‬
‫‪A‬‬

‫‪A+B+C‬‬
‫ـــــــــــــــــــــــــــ=‪Mean‬‬
‫‪3‬‬
 Materials:
- Solution flasks (liquid Sabouaraud’s)
- Pure fungi cultures
- Inoculums needle
- Filter paper
- Heat oven at 80°c
- Glass Suppression
- Glass flasks
Materials

scale

drying oven

flasks
 cork borer
filter paper

fungal growth
Method:
Incubate it at 25°c for different period
time (3-5-7 days)
Dry weight= filter weight of fungi- filter weight
 Physiological factors

The ideal temperatures to grow fungi

- Optimum temperature
- Maximum temperature
- Minimum temperature
Materials:

- PDA media
- Pure fungi cultures
- Inoculums needle
- Cork Borer
Method:
10°c 25°c 30°c 37°c 55°c

Incubate (5-7) days

 ‫‪C‬‬

‫‪B‬‬
‫‪A‬‬

‫‪A+B+C‬‬
‫ـــــــــــــــــــــــــــ=‪Mean‬‬
‫‪3‬‬
 The best media to grow fungi
Materials:
- Malt extract Agar
- Potato dextrose Agar
- Sabouaraud’s Agar
- Czapeck’s Agar
- Cork Borer
- Pure fungi cultures
- Inoculums needle
Method:
 media

PDA CZ Sabu Malt

Incubate plate at 25°c for 5 to 7 days

 ‫‪C‬‬

‫‪B‬‬
‫‪A‬‬

‫‪A+B+C‬‬
‫ـــــــــــــــــــــــــــ=‪Mean‬‬
‫‪3‬‬
 The best Ph to growth fungi
Materials:

- Potato dextrose Agar ph (5, 7, 9)

- Cork Borer

- Pure fungi cultures

- Inoculums needle
Method:
 media

PDA PDA PDA

5 7 9

Incubate plate at 25°c for 5 to 7 days

 ‫‪C‬‬

‫‪B‬‬
‫‪A‬‬

‫‪A+B+C‬‬
‫ـــــــــــــــــــــــــــ=‪Mean‬‬
‫‪3‬‬
 Lab 6

By:
Fatimah lafi Al.Ghamdi
 Classification of Fungi
Fungi

Mastigomycota Amastigomycota Gymnomycota

Haplomastigomycotina Zygomycotina Plasmodiogymnomycotina

Ascomycotina
Diplomastigomycotina

Basidiomycotina

Deutormycotina
Kingdom: Fungi

Division: Mastigomycota

Subdivision: Diplomastigomycotina

Class: Oomycetes

Ex1: Saprolegnia sp.

Ex2: Pythium sp.

Ex2: Phytophthora sp.

Saprolegnia sp.

Saprolegnia is a genus of water molds.

Sexual Reproduction : by Oospores

Asexual Reproduction : by Zoospores

Ex1: Saprolegnia sp.
Ex1: Saprolegnia sp.
Ex1: Saprolegnia sp.
Ex1: Saprolegnia sp.
Kingdom: Fungi

Division: Amastigomycota

Subdivision: Zygomycotina

Class: Zygomycetes

Ex1: Rhizopus sp.

Ex2: Mucor sp.

Ex2: Cunninghamella sp.

Rhizopus sp.

Sexual Reproduction : by zygospore

Asexual Reproduction : by sporangiospores

Ex1: Rhizopus sp.
Ex1: Rhizopus sp.
Ex1: Rhizopus sp.
Ex2: Mucor sp.
Ex2: Mucor sp.
Rhizopus sp. Mucor sp.
Rhizopus sp. Mucor sp.
What are the differences between Rhizopus and
Mucor?

The main difference between Mucor and Rhizopus

is that the Mucor does not have rhizoids and
stolons while Rhizopus has both rhizoids and
stolons.
Ex3: Cunninghamella sp.
Ex3: Cunninghamella sp.
 Lab 7

By:
Fatimah lafi Al.Ghamdi
 Classification of Fungi
Fungi

Mastigomycota Amastigomycota Gymnomycota

Haplomastigomycotina Zygomycotina Plasmodiogymnomycotina

Ascomycotina
Diplomastigomycotina

Basidiomycotina

Deutormycotina
Kingdom: Fungi

Division: Amastigomycota

Subdivision: Ascomycotina

Class: Ascomycetes

Sexual Reproduction : by Ascospores

Asexual Reproduction : by Conidospores

Class: Ascomycetes
Ex1: Taphrina sp.

Ex2: Saccharomyces sp.

Ex3: Shizosaccharomyces sp.

Ex4: Aspergillus sp.

Ex5: Penicillium sp.

Ex6: Erysiphe sp.

Ex7: Uncinula sp.

Ex8: Claviceps sp.

Ex9: Peziza sp.

Ex1: Taphrina sp.
Ex1: Taphrina sp.
Ex2: Saccharomyces sp.
Ex2: Saccharomyces sp.
Ex3: Shizosaccharomyces sp.
Ex3: Shizosaccharomyces sp.
Ex4: Aspergillus sp.
Ex4: Aspergillus sp.
Ex5: Penicillium sp.
Ex5: Penicillium sp.
Aspergillus sp. Penicillium sp.
Ex6: Erysiphe sp.
Ex6: Erysiphe sp.
Ex6: Erysiphe sp.
Ex7: Uncinula sp.
Ex7: Uncinula sp.
Ex7: Uncinula sp.
Ex8: Claviceps sp.
Ex8: Claviceps sp.
Ex8: Claviceps sp.
Ex9: Peziza sp.
Ex9: Peziza sp.
Ex9: Peziza sp.
 Lab 8

By:
Fatimah lafi Al.Ghamdi
 Classification of Fungi
Fungi

Mastigomycota Amastigomycota Gymnomycota

Haplomastigomycotina Zygomycotina Plasmodiogymnomycotina

Ascomycotina
Diplomastigomycotina

Basidiomycotina

Deutormycotina
Kingdom: Fungi

Division: Amastigomycota

Subdivision: Basidomycotina

Class: Basidomycetes

Sexual Reproduction : by Basidiospores

Asexual Reproduction : by Conidospores

Class: Basidomycetes

Ex1: Gymnosporangium sp.

Ex2: Uromyces sp.

Ex3: Puccinia graminis

Ex4: Ustilago zeae

Ex5: Agaricus sp.

Ex1: Gymnosporangium sp.
Ex1: Gymnosporangium sp.
Ex1: Gymnosporangium sp.
Ex2: Uromyces sp.
Ex2: Uromyces sp.
Ex2: Uromyces sp.
Ex3: Puccinia graminis
Ex3: Puccinia graminis
Ex3: Puccinia graminis
Aecidium
Aecidium
Pycnium
Pycnium
Uredium
Uredium
Ex3: Puccinia graminis
(teleutospores)
Ex3: Puccinia graminis
(teleutospores)
Teleutospores
Ex4: Ustilago sp.
Ex4: Ustilago sp.
Ex4: Ustilago sp.
Ex4: Ustilago sp.
Ex5: Agaricus sp.
Ex5: Agaricus sp.
Ex5: Agaricus sp.
Ex5: Agaricus sp.
 Lab 9

By:
Fatimah lafi Al.Ghamdi
 Classification of Fungi
Fungi

Mastigomycota Amastigomycota Gymnomycota

Haplomastigomycotina Zygomycotina Plasmodiogymnomycotina

Ascomycotina
Diplomastigomycotina

Basidiomycotina

Deutormycotina
Kingdom: Fungi

Division: Amastigomycota

Subdivision: Deutormycotina

Class: Deutormycetes

Sexual Reproduction : unknown

Asexual Reproduction : by Conidospores

Class: Deutormycetes

Ex1: Alternaria sp.

Ex2: Helminthosporium sp.

Ex3: Botrytis sp.

Ex4: Fusarium sp.

Ex5: Sclerotium sp.

Ex6: Rhizoctonia sp.

Ex1: Alternaria sp.
Ex1: Alternaria sp.
Ex1: Alternaria sp.
Ex2: Helminthosporium sp.
Ex2: Helminthosporium sp.
Ex3: Botrytis sp.
Ex3: Botrytis sp.
Ex3: Botrytis sp.
Ex4: Fusarium sp.
Ex4: Fusarium sp.
Ex5: Sclerotium sp.
Ex6: Rhizoctonia sp.
Ex6: Rhizoctonia sp. (Hyphae)
 Classification of Fungi
Fungi

Mastigomycota Amastigomycota Gymnomycota

Haplomastigomycotina Zygomycotina Plasmodiogymnomycotina

Ascomycotina
Diplomastigomycotina

Basidiomycotina

Deutormycotina
Kingdom: Fungi

Division: Gymnomycota

Subdivision: Plasmodiogymnomycotina

Class: Myxomycetes

Ex1: Physarum sp.

Ex: Physarum sp.
Ex: Physarum sp.
Ex: Physarum sp.
A) Stemonites sp. B) Dictydium sp.
C) Physarum sp. D) Arcyria sp.
Ex: Stemonites sp.
Ex: Dictydium sp.
Ex: Arcyria sp.
Ex: Arcyria sp.
 Lab 3

By:
Fatimah lafi Al.Ghamdi
Somatic structure:
A) Single hypha:
1-Rhizomorphs:
(Rhizoid)

Rhizopus sp.
 Rhizoid

❖ Single hyphae grow to be like root, its usefulness

install fungi on the media, absorption of food and
carring sporangium.

❖ For example: Rhizopus sp.

2- Appressorium:

Is a flattened, hyphal "pressing" organ, the tubes

enters the host puts out branches between the
cells of the host and forms a mycelial network
within the invaded tissue.
2- Appressorium:
3-Haustoria:

Consists of obligate parasitic fungi, mycelium

fungus grows between the cells and haustorium
extends inside the cells in order to get the fungus
food.

Haustorium in form depending on the fungus some

spherical, barrel and branched tubular.
3-Haustoria:
Albugo sp.
Albugo sp.
B) Aggrgates hypha:
1- Sclerotium:
1- Sclerotium:
Sclerotium consists of aggregates hyphae combine
with each other to be tight.

The external cell wall thickness to be a thick crust

protects the inner cell.

Different sclerotium size they may be large or

small.

Also vary in shape and some are cylindrical, round

or irregular shape and are filled with cells of the
sclerotium with food and this makes them bear the
store as a member of the environmental conditions
inappropriate.
Sclerotium sp.
Ergot
2- Stroma:
2- Stroma:

Function of the stroma is to carrying the structures

fruit and germ.

Claviceps sp.
Claviceps sp.
 Summary:
1. heterotrophs - digest food with secreted enzymes
“exoenzymes” (external digestion).

2. have cell walls made of chitin.

3. most are multicellular, with slender filamentous units

called hyphae

Septate hyphae and Coenocytic hyphae

hyphae may be divided into cells by cross walls called
septa; typically, cytoplasm flows through septa.
hyphae can form specialized structures for things such
as feeding, and even for food capture.
 Summary:

4. Mycelium - interwoven mat-like network of

hyphae

5. Haustoria are specialized hyphae that

penetrate cells (for feeding or other purposes)