MYCOLOGYFUNGAL CULTURE

CUTURE METHOD:

All specimens should be inoculated onto a general purpose fungal medium

fungi will grow very well on culture media used to isolate bacteria

Temperature:

Most MOLDS grow best at: 25 – 30 OC

Most YEAST grow best at 35 – 37 OC

Most pathogenic fungi grow best: 30 to 32°C

EXCEPT: Sporothrix schenckii : 25 to 27°C than 30°C

Incubation period: 14 days: general incubation period 7 days: to detect presence of yeast in the mouth, throat, or

vagina 21 days: tissues and sterile body fluids other than blood 28 days: respiratory, bone marrow, blood specimens, and

specimens in which dimorphic fungus are suspected Plates should be checked at least TWICE during the first week,

when rapidly growing isolates may appear, weekly hereafter.

CHROMagar A selective medium for the isolation and presumptive identification of

yeast and filamentous fungi and differentiation of Candida albicans, C. tropicalis and C. krusei.

Due to the differences in morphology and colors of the yeast colonies, this medium facilitates the detection of mixed yeast cultures in specimens.

It may also be used as a selective isolation medium for other yeasts and for filamentous fungi instead of Sabouraud Dextrose Agar or similar media.

CHROMagarFORMULA IN GRAMS PER LITER

Glucose ....................................... 20.00 Peptone …….................................. 10.00 Chloranphenicol ............................ 0.50 Chromogenic Mixture.............. ........ 0.40 Bacteriological Agar ....................... 15.00

Final pH 6.1 ± 0.2 at 25°C

CHROMagarPreparation Suspend 45.9 grams of the medium in one liter of

distilled water. Mix well and heat with frequent agitation until

complete dissolution. Distribute into adequate containers.

CHROMagarApproximate Formula* Per Liter Purified Water Chromopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g Chromagen Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g Chloramphenicol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g

CHROMagar Candida albicans- green Candida tropicalis - steel

blue Candida krusei - rose,

fuzzy

C. krusei

C. tropicalis

C. albicans

CHLAMYDOSPORE AGAR Used for differentiating Candida albicans from other species

of Candida on the basis of chlamydospore formation. Candida albicans always form chlamydospore on this medium. The medium contains trypan blue to visualize the

chlamydospore under microscopic evaluation. Biotin and polysaccharide are growth factors which stimulate

chlamydospore formation. Potassium phosphate function as a buffer in the medium

Components (g/L)Ammonium Sulphate…………………………… 1.00Monopotassium Phosphate…………………… 1.00Purified Polysaccharide………………………… 20.00Trypan Blue………………………………………… 0.10Biotin…………………………………………………. 0.000005Agar…………………………………………………… 15.00

Final pH (at 25°C) 5.1 ± 0.2

CHLAMYDOSPORE AGAR

Organisms Growth Chlamydospores

Candida albicans luxuriant (+) Candida tropicalis luxuriant

(-) Candida krusei luxuriant (-) Candida minosa luxuriant (-)

CHLAMYDOSPORE AGAR

LEVINE’S EMB AGAR For the isolation and differentiation of Escherichia coli and

Enterobacter The dyes contained in this medium inhibit the growth of many

accompanying Gram-positive microorganisms LEVINE EMB Agar can be used to identify Candida albicans in

clinical specimens, if chlorotetracycline hydrochloride is added to inhibit the entire accompanying bacterial flora

LEVINE EMB Agar can also be utilized for the identification of coagulase-positive staphylococci which grow characteristically as colorless "pin-point" colonies and which show good agreement with the results of the coagulase test

LEVINE’S EMB AGARTypical Composition (g/liter) Peptone …………………………………………………………….10.0 Lactose ……………………………………………………………..10.0 Di-potassium hydrogen phosphate ………………………..2.0 Eosin, yellowish …………………………………………………..0.4 Methylene blue …………………………………………………...0.065 Agar-agar …………………………………………………………..13.5 If cultivating Candida, add 100 mg tetracycline hydrochloride/litre after

autoclaving and mix homogeneously. The culture medium then is blue

LEVINE’S EMB AGAR To obtain a primary culture of Candida, incubate the plates containing chlorotetracycline in a 10 % carbon dioxide atmosphereAppearance: "Spidery" or "feathery“ - Candida albicans Yeast-like, round, smooth - Other Candida species; Sometimes Nocardia

SABHI AGAR Used for the cultivation of pathogenic and nonpathogenic

fungi from a variety of clinical and nonclinical sources Sabouraud Dextrose Agar is a general purpose medium

devised by Sabouraud for the cultivation of dermatophytes Brain Heart Infusion (BHI) Agar has proven to be effective in

the cultivation of a wide variety of microorganisms and is recommended for the primary recovery of fungi from clinical specimens

SABHI AGAR SABHI Agar combines the

ingredients of these two formulations to provide a medium which was found to yield greater recovery of pathogenic fungi than either medium individually

It is recommended for the recovery of fungi from clinical specimens

SABHI AGARFormulation: SABHI Agar contains two

peptones and brain heart infusion solids as sources of amino acids, nitrogen, sulfur, carbon and trace ingredients

Dextrose is an energy source for the metabolism of microorganisms. Sodium chloride provides essential electrolytes

SABHI AGARApproximate Formula* Per Liter Purified Water Brain Heart, Infusion from (Solids) . . . . . . . . . . . . . . . . . 4.0 g Peptic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . . . 5.0 g Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . 10.5 g Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.0 g Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25 g Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g

Sabhi agar with Chloramphenicol and Cycloheximide

Selective medium for use in the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sources

Chloramphenicol Gram positive and Gram negative organisms

Cycloheximide Most saprophytic molds

CZAPEK’S AGAR Czapek's Solution Agar is a synthetic medium widely used in

mycological laboratories Many moulds produce very characteristic colonies on it and may also

exude pigmented substances Aerial growth is often suppressed and sporulation may be enhanced Some moulds, however, grow poorly on this medium and may even

fail to sporulate altogether, often because of their inability to synthesize vitamins

As noted above, the addition of agar to this medium makes it, in reality, a semi-synthetic one

CZAPEK’S AGARFormulation: Sucrose . . . . . . . . . . . . . . . . 30

g NaNO3 . . . . . . . . . . . . . . . . 3.0

g K2HPO4 . . . . . . . . . . . . . . . 1.0

g MgSO4.7H2O . . . . . . . . . . .0.5

g KCl . . . . . . . . . . . . . . . . . . . . 0.5

g FeSO4.7H2O . . . . . . . . . . . 0.01

g Agar . . . . . . . .. . . . . . . . . . . . 15

g Distilled water. . . . . . . . . . 1

liter

Immunodiffusion

27

Immunoelectrophoresis

28

Complement Fixation

29

Enzyme-linked Immunosorbent Assay

30

Latex Agglutination

31

Radioimmunoassay

32

Immunoblotting

33

ENDGood evening!