Mycology & Viro - 05

Corn Meal Agar
Infusion from Corn Meal is the source of carbon, nitrogen, and

vitamins required for organism growth in Corn Meal Agar. Agar
is the solidifying agent.
Composition
Ingredients
g/l
Corn meal infusion
50.000
Agar
15.000
Final pH ( at 25C)
6.00.2
Suspend appropriate amount in distilled water. Heat to boiling

to dissolve the medium completely. If desired add 1%
polysorbate 80. Sterilize by autoclaving at 15 lbs pressure
(121C) for 15 minutes. Mix well and pour into sterile Petri
plates.
Principle
- Chlamydospore production is an accepted criterion for the
identification of Candida species. Corn Meal Agar is a well
established mycological medium used for the cultivation
of fungi and to study chlamydospores production of
Candida species. Corn Meal Agar is a general purpose
medium used for the cultivation of fungi and for the study
of Candida species for chlamydospore production.
- Walker and Huppert modified this medium by adding
polysorbate 80, which then stimulated faster and plenty of
chlamydospore formation of Candida species.
- This is a very simple formulation containing only cornmeal
infusion and agar. However this infusion has enough
nutrients to enhance the growth of fungi. Polysorbate 80
is a mixture of oleic esters, which activates the production
of chlamydospore by Candida albicans, Candida
stellatoides and Candida tropicalis.
- Some Candida species lose their ability of chlamydospore

formation by repeated sub culturing. Pick a suspected colony
from Sabouraud Dextrose Agar using a straight wire, and make a
deep cut in the Corn Meal Agar plate. Repeat for each colony.
Place a flamed sterile coverslip over the line of inoculum. After
incubation for 24-48 hours at 25-30C, the streaks are examined
microscopically, through the coverslip, using low and high power
objectives. C. albicans produces mycelium bearing ball-like
clusters of budding cells and characteristics thick walled round
chlamydospores.
- The addition of glucose (0.2% w/v) to Corn Meal Agar enhances
the
chromogenesis
of
some
species
ofTrichophytone.g.Trichophyton rubrum.
- The addition of `Tween 80 (e.g. 1%) to Corn Meal Agar greatly
enhances the development of chlamydospores on the medium.
RICE EXTRACT AGAR

Rice Extract Agar is recommended for differentiation of yeasts by
means of their typical chlamydospores and on basis of
micromorphological criteria.
Composition:
Ingredients
g/l
Concentrated rice
extract
0.700
Agar
14.300
Final pH ( at 25C)
5.80.2
- It was developed by Taschdjian to aid in the identification of

chlamydospore producing species of Candida. This medium can
be used for culturing yeasts and differentiating them on basis of
micromorphological characteristics particularly for differentiation
of C. albicans and C. stellatoidea on basis of formation of
chlamydospsores.
- Rieth had demonstrated that this medium can be used for
mycological diagnostic procedures. Rice extract in the medium
serves as sole nutrient source. A small inoculum of suspected
Candida colony can be inoculated by streaking (very thinly) on
- If the specimen is heavily infected with Candida it can be

streaked directly on agar. On incubation for approximately 96
hours at 22-25C culture can be directly examined under
microscope through cover glass. Culture may be confirmed
further as suggested by Ajello et al. Typical morphologies can be
revealed as under:
Fungal structures
Fungi
Chlamydospores (diameter 6-12 m,

thick, refractile cell wall with double
contours), pseudomycelium,
blastospores (diameter 3-6 m)
C.albicans Very
occasionally C.
stellatoidea
Pseudomycelium, some times also true

mycelium, usually balstospores. No
chlamydospores
Other Candida species,
Arthrospores, blastospores, true

mycelium, occasionally also
pseudomycelium
Trichospore species
Blastospores, no chlamydospores, no
pseudomycelium
Other yeast species
Ascospores in the asci
Perfect yeasts
- The lack of nutrients together with the oxygen-deficient culture

conditions create an environment which induces the formation
of specific morphological forms (chlamydospores and
pseudomycelia in particular) in some yeasts. Obtain oxygendeficient culture conditions by covering the inoculated plates
with a cover glass.
- Vaginal smears obtained using cotton wool swabs can be
directly smeared on the surface of the culture medium.
- The addition of polysorbate 80 further stimulates
chlamydospore formation due to its content of oleic acids.
- The Addition of Tween 80 solution enhances the formation of
chlamydospores in most Candida species.
- Rice Extract Agar with 2% dextrose may be used to promote
chromogenesis (pigment formation) and, therefore, is helpful in
distinguishing Trichophyton rubrum from Trichophyton
mentagrophytes.
Limitations:
1. A germ tube is not constricted at the point of origin with the
blastoconidium.
2. Candida dubliniensis also produces germ tubes and chlamydospores.
Growth at elevated temperature and morphology on different media
has been shown to facilitate differentiation of C. albicans and C.
dubliniensis.
3. The test is only part of overall scheme for identification of yeasts.
Further tesing is required for definitive identification.
Birdseed Agar or Staibs Medium :

Bird Seed Agar is used for selective isolation and differentiation
of Cryptococcus neoformans from other Cryptococcus species
and yeasts.
Composition (HiMedia):
Ingredients
g/l
Guizotia abyssinica
seeds
70.000
Creatinine
0.780
Dextrose
10.000
Chloramphenicol
0.050
Agar
20.000
Final pH ( at 25C)
6.70.2
Directions:
Suspend 10.08 grams in 99 ml distilled water. Heat to boiling to
dissolve the medium completely. Sterilize by autoclaving at 15 lbs
pressure (121C) for 15 minutes. Cool to 45C and add 100 mcg
diphenyl per ml of medium (1 ml of sterile 1% w/v aqueous
solution of dipehnyl). Mix well and pour into sterile Petri plates.
Principle:
Cryptococcus neoformans is an encapsulated yeast-like fungus
that can live in both plants and animals. This species, also known
by its teleomorph name, Filobasidiella neoformans, belongs to the
broad class of organisms called club fungi or division
Basidiomycota. C. neoformans usually grows as a yeast
(unicellular) and replicates by budding.
The seeds of Guizotia abyssinica contains caffeic acid which serves
as a substrate for detection phenoloxidase, and enzyme produced
by Cryptococcus neoformans. The action of phenoloxidase results
in the production of melanin which is absorbed by the yeast cell
wall forming a tan or dark brown pigment. Chloram phenicol is
added to inhibit the growth bacteria and fast growing fungi.
Ingredients:
Guizotia abyssinica(niger seed) 50 g
Glucose 1 g
KH2PO4 (potassium dihydrogen orthophosphate) 1 g
Creatinine 1 g
Agar 15 g
Distilled water 1000 ml
Additives: to each 500 ml bottle.
Penicillin G (20 units/ml) 0.5 ml
Gentamicin (40 mg/ml) 0.5 ml
Method:
1. Grind seeds of Guizotia abyssinica as finely as possible with an
electric mixer and add to 1000 ml distilled water in a stainless steel
jug.
2. Boil for 30 minutes, pass through filter paper and adjust volume to
1000 ml.
3. Add remaining ingredients except Agar to filtrate and dissolve.
4. If required: Cool to room temperature and pH to 5.5.
5. Dispense into 500 ml bottles.
6. Add 7.5 g Agar to each 500 ml reagent bottle.
7. Autoclave at 121C for 20 minutes.
8. Cool to 48C and add 0.5 ml Penicillin G and 0.5 ml Gentamicin to
each 500 ml of Bird Seed Agar.
Interpretation of the test

Positive test Growth with tan to golden brown pigmented
colonies
Negative test No pigmentation of colonies
Limitations:
- It has been reported that no single medium supports
pigmentation of C. neoformans within a 72 hrs incubation
period. False negative reactions may occur.
- Organisms that produce pigmented colonies on Birdseed agar
should be compared with growth of the same organism or
another medium such as SDA to determine if organism is
naturally pigmented.

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Corn Meal Agar

Infusion from Corn Meal is the source of carbon, nitrogen, and

Corn meal infusion

Suspend appropriate amount in distilled water. Heat to boiling

- Some Candida species lose their ability of chlamydospore

RICE EXTRACT AGAR

- It was developed by Taschdjian to aid in the identification of

- If the specimen is heavily infected with Candida it can be

Chlamydospores (diameter 6-12 m,

Pseudomycelium, some times also true

Other Candida species,

Arthrospores, blastospores, true

Other yeast species

Ascospores in the asci

- The lack of nutrients together with the oxygen-deficient culture

Birdseed Agar or Staibs Medium :

Interpretation of the test

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