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Mycology lab

ü introduction

mycology laboratory is involved in the study of fungi causing human disease.

Diagnosis involves microscopic examination of infected tissue and culture to identify

the disease-causing fungi. Accurate diagnosis enables the clinician to prescribe

appropriate antifungal treatment.

ü Type of samples:

• Respiratory: BAL, sputum, ETA, esophageal brushes, lung biopsy, etc.

• Tissues and biopsy

• Body fluids, CSF, urine, abscess aspirations.

• Superficial samples: skin, nails, hair and swabs.

• Blood and bone marrow.

• Environmental samples

ü Types of media used in mycology:

1- Potato Dextrose Agar (PDA): It is a relatively rich medium for growing a wide range of fungi.

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2- sbouraud’s dextrose agar (SDA): o Sabouraud’s agar is sufficient for the recovery of

dermatophytes from cutaneous samples and yeasts from vaginal cultures.

o Not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi.

o Sabouraud’s dextrose agar (2%) is most useful as a medium for the subculture of fungi recovered on enriched medium to enhance typical sporulation and provide the more characteristic colony morphology.

3- Brain-heart infusion (BHI) agar: It is a non-selective fungal culture medium that permits the growth of virtually all clinically relevant fungi. It is used for the primary recovery of saprophytic and dimorphic fungi.

4- Neutral sabouraud dextrode agar (SABNS). For primary isolation

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5- sabouraud dextrode agar with cyclohexamide and Chloramphenicol For primary isolation.

6- Dermatophytes test media (DTM) . for Dermatophytes differentiation.

7- Corn meal agar (CMA) for yaest spores production.

ü Types of stains used in mycology:

• India Ink - demonstrates the capsule of Cryptococcus neoformans in CSF specimens.

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• Diff KWIK stain. Screening stain

• KOH for superficial samples, cutaneous sampels and tissue.

• Lactophenol Cotton Blue - very popular for quick evaluation of fungal structures; stains chitin in cell walls of fungi.

• Gomori Methenamine Silver Stain - silver nitrate outlines fungi in black due to the silver precipitating on the fungi cell wall.

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ü Tests:

• Germ Tube Test

Germ Tube Test is a screening test which is used to differentiate Candida albicans from other yeast. Germ tube (GT) formation was first reported by Reynolds and Braude in 1956. When Candida is grown in human or sheep serum at 37°C for 3 hours, they forms a germ tubes, which can be detected with a wet KOH films as filamentous outgrowth extending from yeast cells. It is positive for Candida albicans and Candida dubliniensis. Approximately 95 – 97% of Candida albicans isolated develop germ tubes when incubated in a proteinaceous media.

• API 20 C

All yeast identification procedures were conducted in accordance with the manufacturer’s instructions. Portions of growth of each isolate were aseptically transferred from a freshly inoculated stock culture to an ampule of API 20C basal medium and then emulsified to give a density of 1+ on a Wickerham card. Each well of the API 20C strip was inoculated with the suspension, and the strip was placed in the incubation tray provided by the manufacturer, covered loosely with a lid, and incubated at 30°C for 72 h. Reactions were visually examined at 72 h and determined to be positive or negative based on the presence or absence of turbidity in the carbohydrate wells