Saturday, October 12, 2013

Yeasts Identifications

Yeast-like fungi may be basidiomycetes, such as Cryptococcus neoformans or ascomycetes such as Candida albicans.
1. Ensure that you start with a pure culture; streak for single colony isolation if necessary.
2. Germ Tube Test: lightly inoculated 5 ml of serum, containing 0.5% glucose and incubated at 35oC for 2-3 hours.
Positive = Candida albicans or Candida dubliniensis.
Negative or from HIV positive patient = perform assimilation tests.
3. For the identification of germ tube negative yeasts, morphological (Dalmau plate culture), physiological and biochemical tests are essential.
Dalmau Plate Culture: To set up a yeast morphology plate, dip a flamed sterilized straight wire into a light inoculum (sterile distilled water suspension) and then lightly scratch the wire into the surface of a cornmeal/tween 80, rice/tween 80 or yeast morphology agar plate, then place a flamed coverslip onto the agar surface covering the scratches. Dalmau morphology plates are examined in situ using the lower power of a microscope for the presence of pseudohyphae which may take up to 4-5 days at 26oC to develop. C. albicans also produces characteristic large, round, terminal, thick-walled vesicles (often called chlamydoconidia). The key features to remember are to use a light inoculum and to scratch the surface of the agar with the wire when inoculating.
Physiological and biochemical tests: including fermentation and assimilation studies should be performed. Reliable commercially available yeast identification kits are the API 20C AUX, ATB 32C, MicroScan and Vitek systems
Identification of Common Dermatophytes.
Microscopic morphology of the micro and/or macroconidia is the most reliable identification character, but you need a good slide preparation and you may need to stimulate sporulation in some strains. Culture characteristics such as surface texture, topography and pigmentation are variable and are therefore the least reliable criteria for identification. Clinical information such as the site, appearance of the lesion, geographic location, travel history, animal contacts and race is also important, especially in identifying rare non-sporulation species like M. audouini, T. concentricum and T schoenleinii etc. Note: mating experiments are not practical for the clinical mycology laboratory.
Three genera are recognized:
Smooth thin-walled Macroconidia only present, no microconidia, colonies a green-brown to khaki colour.
Macroconidia with rough walls present, microconidia may also be present.
Microconidia present, smooth-walled macroconidia may or may not be present.
Lactophenol Cotton Blue.
For the staining and microscopic identification of fungi
This stain is prepared over two days.
1. On the first day, dissolve the Cotton Blue in the distilled water. Leave overnight to eliminate insoluble dye.
2. On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. place on magnetic stirrer until the phenol is dissolved.
3. Add the glycerol.
4. Filter the Cotton Blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at room temperature.
Sabouraud's Dextrose Agar for Dermatophytes
Sabouraud's Dextrose Agar with Cycloheximide, Chloramphenicol, Gentamicin and Yeast Extract.
For the primary isolation and cultivation of dermatophytes.

1. Soak all ingredients, except Gentamicin, in 100 ml water.
2. Boil remaining water, add to soaking ingredients, and bring to boil to dissolve, stirring well to prevent from charring.
3. Add the Gentamicin. Mix well.
4. Dispense for slopes if required.
5. Autoclave at 121C for 10 minutes. Remove and slope, or pour for plates as required.

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