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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 OCMost YEAST grow best at 35 – 37 OCMost pathogenic fungi grow best: 30 to 32°C EXCEPT: Sporothrixschenckii : 25 to 27°C than 30°C

Incubation period: 14 days: general incubation period7 days: to detect presence of yeast in the mouth, throat, or vagina21 days: tissues and sterile body fluids other than blood28 days: respiratory, bone marrow, blood specimens, and specimens in which dimorphic fungus are suspectedPlates should be checked at least TWICE during the first week, when rapidly growing isolates may appear, weekly hereafter.

CHROMagarA 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 LITERGlucose ....................................... 20.00 Peptone …….................................. 10.00Chloranphenicol ............................ 0.50Chromogenic Mixture.............. ........ 0.40Bacteriological Agar ....................... 15.00Final pH 6.1 ± 0.2 at 25°C

CHROMagarPreparationSuspend 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 WaterChromopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 gGlucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 gChromagen Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 gChloramphenicol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 gAgar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g

CHROMagarCandida albicans- green Candida tropicalis - steel blue Candida krusei- rose, fuzzy

C. kruseiC. tropicalisC. albicans

CHLAMYDOSPORE AGARUsed 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

CHLAMYDOSPORE AGARComponents (g/L)Ammonium Sulphate…………………………… 1.00Monopotassium Phosphate…………………… 1.00Purified Polysaccharide………………………… 20.00Trypan Blue………………………………………… 0.10Biotin…………………………………………………. 0.000005Agar…………………………………………………… 15.00Final pH (at 25°C) 5.1 ± 0.2

CHLAMYDOSPORE AGAROrganisms Growth ChlamydosporesCandida albicans luxuriant (+)Candida tropicalis luxuriant (-)Candida krusei luxuriant (-)Candida minosa luxuriant (-)

LEVINE’S EMB AGARFor the isolation and differentiation of Escherichia coli and EnterobacterThe dyes contained in this medium inhibit the growth of many accompanying Gram-positive microorganismsLEVINE EMB Agar can be used to identify Candida albicans in clinical specimens, if chlorotetracycline hydrochloride is added to inhibit the entire accompanying bacterial floraLEVINE 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 …………………………………………………………….10.0Lactose ……………………………………………………………..10.0Di-potassium hydrogen phosphate ………………………..2.0Eosin, yellowish …………………………………………………..0.4Methylene blue …………………………………………………...0.065Agar-agar …………………………………………………………..13.5If 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 AGARUsed for the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sourcesSabouraud Dextrose Agar is a general purpose medium devised by Sabouraud for the cultivation of dermatophytesBrain 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 AGARSABHI 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 individuallyIt 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 ingredientsDextrose is an energy source for the metabolism of microorganisms. Sodium chloride provides essential electrolytes

SABHI AGARApproximate Formula* Per Liter Purified WaterBrain Heart, Infusion from (Solids) . . . . . . . . . . . . . . . . . 4.0 gPeptic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . . . 5.0 gPancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . 10.5 gDextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.0 gSodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 gDisodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25 gAgar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g

Sabhi agar with Chloramphenicol and CycloheximideSelective medium for use in the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sourcesChloramphenicolGram positive and Gram negative organismsCycloheximideMost saprophytic molds

CZAPEK’S AGARCzapek's Solution Agar is a synthetic medium widely used in mycological laboratoriesMany moulds produce very characteristic colonies on it and may also exude pigmented substancesAerial growth is often suppressed and sporulation may be enhancedSome moulds, however, grow poorly on this medium and may even fail to sporulate altogether, often because of their inability to synthesize vitaminsAs noted above, the addition of agar to this medium makes it, in reality, a semi-synthetic one

CZAPEK’S AGARFormulation:Sucrose . . . . . . . . . . . . . . . . 30 gNaNO3 . . . . . . . . . . . . . . . . 3.0 gK2HPO4 . . . . . . . . . . . . . . . 1.0 gMgSO4.7H2O . . . . . . . . . . .0.5 gKCl . . . . . . . . . . . . . . . . . . . . 0.5 gFeSO4.7H2O . . . . . . . . . . . 0.01 gAgar . . . . . . . .. . . . . . . . . . . . 15 gDistilled water. . . . . . . . . . 1 liter

Enzyme-linked Immunosorbent Assay30

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