3. Yeast - Major Characteristics
Unicellular Fungi
Eukaryotic
Facultative anaerobes
Capable of forming
colonies on solid culture
media.
Occur worldwide
Over 1,500 species
described
4. 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
7. 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.
8. 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.
9. 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.
12. 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).
14. Order: Erysiphales:
* Parasitic plectomycetes on vascular plant.
* Cause a disease called powdery mildew.
* Cleistothecia formed on superficial
mycelium and asci are cylindrical.
15. 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.
16.
17. MATERIALS
Fresh and dried plant materials with powdery mildew
signs
Dissecting needles/ single-edge razor blades
Microscope slides
Coverslips
PROCEDURES
- Asexual stage
- Sexual stage
18. 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
24. 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.
25. 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'.
27. 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).
29. 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.
30. 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.
31. 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.
33. 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.
36. 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
42. 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.
47. 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
53. 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.
54. 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.
55. 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.
56.
57. 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 +
65. 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.
71. 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.
74. 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.
75.
76. 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)
77. 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.
84. 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.
86. 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.
88. Home work
What is Ontogeny?
What are the differences between Perithecium and
Pycnidium?
89.
90.
91. 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.
92. 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.
93. 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.
.
94. 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.
95. 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.
96. 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.
97. 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.
98. 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
99. 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.
100. 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.
101. 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
103. 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:
104. 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.
105. 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.
106.
107. 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.
108. 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.
109. 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.
110. 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
111. 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.
.
112. 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
113. - 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
115. 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.
116.
117.
118. 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
119. 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).
122. 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.
123. 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.
125. 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.
127. Objectives
What is mycotoxin?
What are the types, toxicity and mode of action
mycotoxins?
How to remove mycotoxin?
128. 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.
129. 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.
130. 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.
131. 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.
133. 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.
134. 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.
135. FumonisinsSource: 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.
136. 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.
137. 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.
138. 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,
139. 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.
140. 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).
