Phylum: Ascomycota

Class: Hemiascomycetes: Yeast

&

Class: Plectomycetes

Class: Hemiascomycetes:

Yeast

Yeast - Major Characteristics

Unicellular Fungi

Eukaryotic

Facultative anaerobes

Capable of forming colonies on solid culture media.

Occur worldwide

Over 1,500 species described

They reproduce either asexually (most common) or sexually.

•Asexual reproduction is through budding or binary fission.

•Sexual reproduction (if any) results in the formation of the

appropriate spore structure.

Budding

Yeast - Reproduction

Fission

Spores Saccharomyces cerevisiae

Schizosaccharomyces octosporus

Budding Fission

Budding Fission

Yeast Significance

Food Industry • Fermentation of bread, and alcoholic beverages.

E.g. Saccharomyces cerevisiae (also called baker’s yeast or sugar

yeast) used in baking.

Medical

• E.g. Candida albicans - common in the human mouth, but can

become pathogenic and cause Candidiasis (oral and/or genital

infection).

Biofuel Industry •Production of ethanol for car fuel.

Principal Criteria and Tests for Identifying Yeasts

1. Culture characteristics - Colony color, shape,

texture

2. Asexual structures

a. Shape and size of cells

b. Fission, bipolar, multipolar or unipolar

budding.

c. Absence or presence of germ tubes, hyphae,

or pseudohyphae.

3. Sexual structures - Arrangement, cell wall, number, shape and size of Ascospores.

4. Physiological studies

a- Carbohydrate Assimilation.

b. Fermentation.

c. Nitrogen utilization.

d. Urea hydrolysis.

API 20 c

Filamentous Ascomycotina

Class: Plectomycetes

The ascocarp in this class is a

- Spherical.

- Complete closed structure called

cleistothecium.

- It has no opening to release asci. The class

included two orders:

• Include two Orders:

•Eurotiales (Saprophytic Plectomycetes).

•Erysiphales (Parasitic Plectomycetes).

Order: Eurotiales:

Saprophytic plectomycets

Asci globos and scattered within

cleistothecium

Aspergillus spp. are examples of this

order

Order: Erysiphales:

* Parasitic plectomycetes on vascular plant.

* Cause a disease called powdery mildew.

* Cleistothecia formed on superficial

mycelium and asci are cylindrical.

Identification the genera of Erysiphales

(Powdery Mildew Fungi)

- Powdery mildews are caused by several genera of Ascomycotina

- They are host specific, biotrophic, and obligately parasitic.

- Fungus produces hyphae and conidia usually at the upper surface, or on stems, flowers, or fruit.

- They can be identified most easily by the formation of ascocarp with no natural opening, called a cleistothecium.

Powdery Mildew Cleistothecia.mp4

MATERIALS

Fresh and dried plant materials with powdery mildew signs

Dissecting needles/ single-edge razor blades

Microscope slides

Coverslips

PROCEDURES

- Asexual stage

- Sexual stage

Key to Genera of Powdery Mildew Fungi

Appendages coiled or hooked at tip – Uncinula Appendages simple and straight with bulb-like base – Phyllactinia

Appendages branching dichotomously at tip Cleistothecium contains a single ascus –

Podosphaera Cleistothecium contains several asci –

Microsphaera Appendages simple or irregularly branched, often

entwined Cleistothecium contains a single ascus – Sphaerotheca Cleistothecium contains several asci – Erysiphe

Ascomycotina

1- Yeast

2- Mycelial Ascomycotina

These species have septate, mycelium.

Produce asci and ascospores that are borne in

an ascocarp.

There are four types of ascocarps recognized;

cleistothecium, perithecium, apothecium

and ascostroma.

Species that produce cleistothecia, perithecia

and apothecia have unitunicate asci

while those that produce ascostroma have

bitunicate asci.

A unitunicate-operculate ascus has a:

"lid", the Operculum, which

breaks open when the spores

ripen and in this way sets

them free.

Unitunicate-operculate

asci only occur in those ascocarps

which have apothecia, 'Unitunicate' means 'single-walled'.

Mechanism of ascospore discharge from Unitunicate Ascus

A bitunicate ascus is enclosed in a double wall.

When the spores are ripe

the shell splits open, so

that the spores can escape

into free air. Bitunicate asci

Examples: Venturia inaequalis

(apple scab) and Guignardia sp.

(Brown Leaf Mold of Horse Chestnut).

Mechanism of ascospore discharge from Bitunicate Ascus

Prototunicate asci

are mostly spherical in shape and they have

no active dispersal

mechanism at all.

Asci of this type can

be found both in

perithecia and in

cleistothecia.

Class: Plectomycetes (Cleistothecia)

Class: Pyrenomycetes (perithecium) Formation of an ascocarp called a perithecium. This ascocarp is typically flask-shaped or

globose Have a small ostiole through which the

ascospores are released. Asci are unitunicate and are arranged, in a

single fertile layer throughout the base of the perithecium (hymenium).

Ascospores are often forcibly ejected from the ascus and perithecium when mature.

Order: Sordariales

The perithecia are usually dark or pallid, flask-shaped ascocarps with an ostiole

paraphyses are absent when ascospores are mature.

Sordaria fimicola: This is an example of a species that does not produce a stroma.

In nature, this species grows on dung.

Sordaria fimicola

Order: Xylariales

This order includes a very large and diverse group of Pyrenomycetes that typically produce their perithecia in stromata.

Xylaria sp. and Penzigia sp. Their stromata are externally black, and can be seen to be mostly white in section.

The perithecia are entirely immersed in the stromata with only the ostioles opened to the surface.

The stromata of Xylaria are long and tapering while those of Penzigia sp. are hemisphaerical to globose that usually occur in clusters.

Stromata of Xylaria

Stromata of Penzigia globosum

Class: Discomycetes

The series Discomycetes is characterized by the formation of an ascocarp called an apothecium.

Typically an apothecium is cup-shaped which is why Discomycetes are sometimes called "cup fungi".

The asci are unitunicate and forcibly eject the ascospores.

Paraphyses are generally present in apothecia. Morchella sp. a species in which the apothecium has

now formed depressions that are fertile with sterile ridges in between;

Leotia sp. a species in which the "cup" of the apothecium is interpreted as being folded back,

Gyromitra sp. a species in which the apothecium is said to be "saddle-shaped

Sarcoscypha mesocyatha, apothecium

Sarcoscypha mesocyatha, typical cup

apothecium

Morchella esculenta Gyromitra californica

Leotia lubrica apothecium

General characteristics of Zygomycotina

•The name comes from zygosporangia.

•They are mostly terrestrial in habitat living in soil or on

decaying plant or animal material.

•Zygomycete hyphae are coenocytic, forming septa only

where gametes are formed

•Some are parasites of plants, insects, small animals and

other fungi, while others form symbiotic relationships

with plants.

Mycoparasitic fungus

Spinellus fusiger

Aseptate hyphae

Types of sporangium

True sporangium

e.g. Mucor sp.

Sporangiola e.g. Thamnidium sp. Merosporangium e.g. Syncephalastrum racemosum

Sporangiole: one or few spores per sporangia

Sexual and A Sexual Reproduction

• Gametangial copulation

• Conjugation by two morphologically similiar

gametangia

• Production of zygospores (thick-walled resting

spores) within zygosporangia that are formed

by fusion of gametangia

• Produce a zygosporangium

• Homo- & heterothallic species

Zygospore

Suspensor

Meeting of two different mating type

Zygospores

How to differentiate between Mucor and Rhizopus

Macroscopic Features of Mucor

- Colonies of Mucor grow rapidly at 25-30°C and quickly

cover the surface of the agar.

- Its cottony appearance.

- From the front, the color is white initially

and becomes grayish brown in time.

Macroscopic Features Rhizopus

- Colonies of Rhizopus grow very rapidly at 40-45°C

fill the Petri dish, and mature in 4 days.

- The texture is typically cotton-candy like.

- From the front, the color of the colony is

white initially and turns grey to

yellowish brown in time.

Microscopic Features of Mucor

- Nonseptate, with broad hyphae.

- Sporangiophores, sporangia, and sporangiospores are

visualized.

- Apophysis, rhizoid and stolon are absent.

- Sporangiophores are short, erect and may

form short sympodial branches.

- Columella are hyaline or dematiaceous

- Sporangia are round, 50-300 µm in diameter,

gray to black in color, and are filled with sporangiospores.

- The sporangiospores are round (4-8 µm in diameter)

or slightly elongated.

Microscopic Features Rhizopus

- Nonseptate broad hyphae

- Sporangiophores, rhizoids, sporangia, and

sporangiospores are visualized.

- Sporangiophores are brown in color and unbranched

and they can be solitary or form clusters.

- Apophysis, rhizoids stolons is present.

- Rhizoids are located at the point where

the stolons and sporangiophores meet. - Sporangia (40-350 µm in diameter).

- Sporangiospores (4-11 µm in diameter)

are unicellular, round to ovoid in shape,

hyaline to brown in color.

Genus Best

growth

Sporangi-

ophore

Apophysis Columella Sporan-

gium

Rhizoid

Mucor <37°C

Branched or

unbranched,

hyaline

- +, in varying

shapes Spherical -

Rhizopus 45°C

Unbranched

and brown

mostly

Not

prominent

Spherical or

elongated Spherical +

Soft rot caused by Mucor

Soft rot of fruit caused by Rhizopus

Rhizopus stolonifer

Mucor Rhizopus

Apophysate

sporangiospores

columella

Mucor

Rhinocerebral Zygomycosis

Rhinocerebral mucormycosis: is a rare opportunistic infection of the sinuses, nasal passages, oral cavity, and brain which commonly caused by Rhizopus oryzae. The infection can rapidly result in death.

Subcutaneous zygomycosis

Basidiobolus meristosporus

Homework

What is the function of columella?

What is Azygospore?

General characteristics There is no sexual reproduction in life cycle or the

reproduction occurs by nonsexual stages.

Nonsexual reproduction may occur by cellular division or by germination of mycelial fragments or spores of various types.

The spores may be chlamydospores or conidia.

Chlamydospores are formed by transformation of an intercalary somatic cell or group of cells by rounding up of the cells and deposition of a thick wall.

http://www.fungionline.org.uk/6asexual/7thallic.html

Classes of Deuteromycotina:

Blastomycetes.

Hyphomycetes.

Coelomycetes.

Blastomycetes:

Members of this class include yeast like fungi. which fail to produce ascospores.

Others appear to have affinities with Basidiomycotina.

These yeasts may be isolated from a variety of habitate, including water, the soil, or plants.

Many are pathogenic on animals or human, such as Candida.

Hyphomycetes:

Include those deuteromycetes that form a mycelium but lack a sporocarp (Conidiomata).

In some species the conidiophores may be tightly clustered together to form a pulvinate mass, the Sporodochium or Synnema.

Development of conidia from phialid: a basipetal succession of conidia is formed from a fixed locus on the conidiogenous cell (phialide)

Conidiogenous Cells

A hyphal compartment or cell from which, or on which, a conidium is formed

Conidiogenous cell may be formed directly from a hypha, or on a specialized simple or branched hypha called a conidiophore.

Conidiophore

Hyphae bearing conidiogenous cells.

1. Macronematous

2. Micronematous

Conidiophores and Phialids of Aspergillus sp

Conidiophores and Phialids of Penicillium sp.

Sporodochium of Fusarium sp.

Sporodochium: A compact, cushion-like aggregation of hyphae on which conidiophores are formed in a dense layer

Synnema in Arthrobotryum sp

Synnema: •Conidiophores united at base •Conidiogenous cells at apex

Coelomycetes:

In this class the conidiophores are borne on or within a multicellular sporocarp, called Conidiomata, which has two types, Pycnidium and Acervulus.

Pycnidium is a closed sporocarp (Conidiomata) bearing its conidiophores, conidiogenous cells and conidia within a cavity.

The pycnidium may be discoid, globose or flask like. Pycnidia may be entirely closed or may open to the outside by an ostiole.

Pycnidium of Septoria sp.

Acervulus is an open mass of closely packed conidiophores and conidiogenous cells that may form a flat discoid cushion of conidia.

Acervuli are usually formed on a plant host and are often erumpent from the epidermis of the host, pushing aside flaps of host tissue as they emerge.

Acervulus of Cryptocline betularum

Home work What is Ontogeny?

What are the differences between Perithecium and Pycnidium?

The type and quality of specimens submitted to the mycology laboratory are an initial factor in determining the success of isolating and identification of fungi.

Important steps for successful isolation of etiological agents of mycoses are:

1. Proper collection of the specimens.

2. Rapid transport of the specimens to the laboratory.

3. The correct processing of the specimens.

4. Inoculation of specimens onto appropriate culture media and incubation at suitable temperatures.

COLLECTION AND TRANSPORT OF SPECIMENS:

A- Specimens should be collected aseptically, placed in

sterile humidified, leak-proof container, delivered to

the laboratory within 2 hours, processed, and then

inoculated to primary isolation media within a few

hours of collection.

.

COLLECTION AND TRANSPORT OF SPECIMENS:

Dermatologic specimens, however, should be

transported in a dry container. Transport medium

should not be used unless the specimen can be easily

and completely retrieved from the medium.

B - The effect of refrigeration on fungal specimens has not

been well-studied.

- If processing is to be delayed for more than several

hours, it is recommended that specimens be stored under

refrigeration at 4 ̊C.

- Exceptions: blood and cerebrospinal fluid are stored at

30- 37 ̊C.

C- Swabs are not encouraged; however, specimens from

the environment or certain body sites such as the ear

canal, nasopharynx, and throat are not readily collected

by other means.

2. SPECIMENS:

A. Sputum (tracheal lavage, and bronchial lavage)

1. Sputum should be fresh and collected in the early morning??

2. Sputum should be the result of a deep cough (not saliva).

3. Collect 5-10 ml in sterile container.

B. Respiratory specimens other than sputum, such as tracheal

aspirates, and lung biopsy material

C. Blood

1. Blood is collected aseptically to avoid microbial contamination.

2. Use sodium polyanethol sulfonate (SPS, Liquoid) as an

anticoagulant.

D. Pus, Exudates - Using a sterile needle and syringe.

E. Tissue

1. Tissue is aseptically collected from the center and edge

of the lesion.

2. Place between moist gauze squares, add a small amount

of sterile water to keep tissue from drying out.

F. Bone Marrow - Aspirate approximately 3-5 ml of bone

marrow and place it in a sterile container. SPS or heparin

can be added as an anticoagulant.

G. Cerebrospinal Fluid - spinal fluid as possible is

collected and placed in a sterile container.

H. Urine

1. The urine specimen most suitable for making a

diagnosis of mycoses of the urinary tract.

2. Early morning specimens are aseptically collected in

sterile containers. Twenty-four hour collections have no

value. Urine may be stored at 4 ̊C for up to 12-14 hours

I. Body Fluids (pleural, synovial, and peritoneal).

J. Hair

1. Select infected areas and with forceps, and take at least 10 hairs.

2. Place hairs between two clean glass slides or in a clean envelope

labeled.

K. Nail

1. Clean nail with 70% alcohol.

2. Scrape outer surface and discard.

3. Collect whole nail or nail clippings.

4. Place all material in a clean envelope labeled with the patient's

data.

L. Skin and Interspaces

1. Clean the lesions and interspaces between the toes

with alcohol sponge or sterile water.

2. Scrape the entire lesion(s) and both sides of

interspaces with a sterile scalpel.

3. Place scrapings between two clean glass slides or

place in a clean envelope labeled with the patient's data.

EXAMINATION OF SPECIMENS

MACROSCOPIC AND MICROSCOPIC EXAMINATION:

1. MACROSCOPIC EXAMINATION:

Before inoculating a specimen to the appropriate

isolation media, the specimen is examined

macroscopically for bloody areas, and necrotic

material.

Specimens from cases of mycetoma are

examined with the dissecting microscope for the

presence of granules before proceeding

Mycetoma

1.Potassium Hydroxide Procedure (KOH Examination)

KOH may be used to examine hair, nails, skin scrapings, fluids, or

biopsies. The fungal structures such as hyphae, large yeast

(Blastomyces), and sporangia may be distinguished.

2. MICROSCOPIC EXAMINATION:

1. Potassium Hydroxide Procedure (KOH Examination)…continued

Specimens placed in a drop of 15% KOH will

dissolve at a greater rate than fungi because fungi have

chitinous cell walls.

Fluids such as CSF generally do not need to be

treated with KOH.

The clearing effect can be accelerated by gently

heating the KOH preparation.

Visualization of fungi can be further enhanced by the

addition of Parker Superquink permanent black ink

to the preparation.

2- India Ink Examination:

India ink can be added to specimens such as spinal fluids or exudates to provide a dark background that will highlight hyaline yeast cells and capsular material.

Objectives

What is Candida albicans ?

Applying Germ tube test, Chlamydospore production, Temperature tolerance techniques to differentiate Candida albicans from other Candida species.

Using API 20 Candida and CHROMagar Candida to differentiate between Candida species.

Aim

To understand the clinical significance of Candida albicans.

To explain some practical approaches for identification of

Candida albicans.

Also applying some techniques to differentiate between

Candida species.

What is Candida albicans?

• Candida albicans is a yeast growth present in all of us

and is normally controlled by bacteria in the intestines.

• But when something destroys helpful bacteria, the yeast

begins to invade and colonize the body tissues.

What is Candida albicans? continued..

• These yeast colonies release powerful chemicals into the

bloodstream, causing such varying symptoms as lethargy,

chronic diarrhea, bladder infections, muscle and joint pain,

and severe depression.

• The medical term for this yeast overgrowth is candidiasis

Isolation of Candida albicans:

•Insert the cotton end of each swab into 0.5 ml of

sterile water in a micro centrifuge tube.

•Mix the tube for 30s with a laboratory tabletop

vortex mixer.

•Spread 0.15 ml of the wash onto plates containing

Sabouraud dextrose agar or (PDA).

•Incubate plates at 37C for 48 h.

.

Identification of Candida albicans:

1- Germ Tube Test: This is a rapid test for the

presumptive identification of C. albicans

- Reagents / Materials / Media

•Bovine or human serum - A small volume to be used as a

working solution may be stored at 2 to 8 0C .

•Stock solution can be dispensed into small tubes and stored at -

20 0C . •Clean glass microscope slides

•Glass cover slips

•Glass tubes

•Pasteur pipettes

- Procedure

Put 3 drops of serum into a small glass tube.

Using a Pasteur pipette, touch a colony of yeast and gently

emulsify it in the serum. The pipette can be left in the tube.

Incubate at 350C to 370C for up to 3 hours but no longer.

Transfer a drop of the serum to a slide for examination.

Coverslip and examine microscopically using x 40 objective.

Positive test: presence of short lateral filaments (germ tubes) one

piece structure

Negative test: yeast cells only (or with pseudohyphae) always two

pieces

Germ tube test

Germ tube positive C. albicans

2- Analytical profile index (API 20 Candida):

• API is a classification of bacteria and fungi based on

experiments, allowing fast identification.

• The API 20E/C fast identification system combines some

conventional tests and allows the identification of bacteria and

fungi.

• The test systems are stored in limited small reaction tubes, which

include the substrates.

CHROMagar Candida appears to be a medium well-suited

for medical mycological use.

It is a new differential culture medium that is claimed to

ease the isolation and presumptive identification of

some clinically important yeast species.

It is also use as an adjunctive differential medium for the

identification of yeasts isolated on other media.

3- CHROMagar

CHROMagar Candida components:

peptone (10 g/liter),

glucose (20 g/liter),

agar (15 g/liter),

the medium contained chloramphenicol (0.5 g/liter)

chromogenic mix (2 g/liter).

The medium was prepared according to the

manufacturer’s instructions and dispensed into petri

dishes (20 ml into 100-mm-diameter dishes).

CHROMagar Candida

4- Chlamydospore production

Corn Meal Agar (CMA) is a culture medium used as

presumptive test for identification of Candida albicans.

Chlamydospore production on CMA is a characteristic feature

of Candida albicans.

Procedure

17 g of Corn Meal Agar incorporated with Tween 80 is

prepared according to manufacture’s instruction.

Yeast colonies would then be inoculated on the CMA.

Plates will then incubate at 25 ̊C for 72 hrs.

Chlamydospore production would be examined after staining

with lactophenol cotton blue under microscope.

Chlamydospore production

5- Temperature tolerance

Several Candida species can be differentiated by the ability

to grow at different temperatures.

C. albicans can grow at 37 °C and 45 °C, while C.

dubliniensis is only able to grow at 37 °C.

By: Azad S. Abdul

Objectives What is mycotoxin?

What are the types, toxicity and mode of action

mycotoxins?

How to remove mycotoxin?

Aims To provide a basic background about mycotoxins, their

types, toxicities, and mode of actions.

To explain how to prevent cereal from contamination by

mycotoxins.

What is mycotoxin? The term mycotoxin is a combination of Greek

word “mykes” means fungus and Latin word “toxicum” meaning by poison.

Mycotoxins are secondary metabolites of moulds that exert toxic effects on animals and humans.

The toxic effect of mycotoxins on animal and human health is referred to as mycotoxicosis.

Some mycotoxins were heat stable up to as much as

400°C.

Consumption of a mycotoxin contaminated diet may result

in teratogenic, carcinogenic, and/or immune-suppressive

effects.

Outbreak

Outbreaks occur in groups because of a shared

contaminated food supply and the optimal weather

conditions for Aspergillus growth

First recorded outbreak was in England in 1962, where

100000 turkeys died.

Common mycotoxins Zearalenone

Aflatoxin

Fumonisins

Ochratoxin

Trichothecenes

Nivalenol

Zearalenone

Source: F. graminearum, F. culmorum, F. equiseti,

and F. crookwellense.

Toxicity:

It causes of a reproductive disorder in pigs known as vulvo-vaginitis.

It has carcinogenic effect.

The mode of action: it is an estrogenic mycotoxin which is involved in reproductive disorders and hyperestrogenicity in farm animal because of its structural similarity with estradiol.

Aflatoxin Source: Aspergillus flavus A. parasiticus, A. bombycis, A.

ochraceus, A. nomius and A. Pseudotamari.

Toxicity: pulmonary carcinogen, acute aflatoxicosis.

Mode of Action:

Cytochrome P450 enzymes convert aflatoxins to the reactive 8, 9-epoxide form which is capable of binding both DNA (inducing point mutation and DNA strand break) and proteins.

Fumonisins Source: Fusarium verticillioides (formly F. moniliforme),

F. proliferatum, F. nygamai and Alternaria.

Toxicity: toxicosis in swine, equine leukoencephalopathy

Mode of Action:

disruption of sphingolipid because of structural similarity

with sphingoid bases (sphinganine and sphingosine)

Free sphingoid bases are toxic to the most cells by

affecting cell proliferation and inducing apoptosis or

necrotic cell death.

Ochratoxin Source: A. ochraceus, A. alliaceus, A. auricomus, A.

carbonarius, A. glaucus, A. melleus and A. niger.

Toxicity: nephrotoxic, hepatotoxic, teratogenic in laboratory animals.

Mode of Action: inhibiting the phenylalanine-tRNA ligase complex which involves in the synthesis of phenylalanine and inhibits mitochondrial ATP production.

Trichothecenes Source: Fusarium, Trichoderma, Trichothecium,

Myrothecium, Stachybotrys.

Toxicity: weight loss, vomiting, bloody diarrhea, severe dermatitis, hemorrhage, decreased egg production, abortion, and death in animals.

Mode of Action: it is a potent inhibitors of protein, DNA, RNA synthesises and interact with cell membrane.

Nivalenol Source: F. cerealis and F. poae , F. culmorum and F. graminearum.

Toxicity: embryo toxic and fetotoxic.

The mode of action: inhibitor of protein, RNA, DNA

synthesis in mammalian cells, necrosis of the

proliferating cells in vivo,

Food contamination by mycotoxin Cereal plants may be contaminated by mycotoxins in two ways:

Fungi growing as pathogens on plants.

Or fungi may grow saprophytically on stored plants.

How to control mycotoxicosis? Three methods can be used to remove mycotoxin from

contaminated diets:

1. Physical method (using adsorbents such as activated carbon and

bentonite).

2. Chemical method (calcium hydroxide, ozone or ammonia).

3. Biological method (conversion of aflatoxin B1 (particularly by

Flaobacterium auranticum) to harmless degradation products).