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Fungal Descriptions

Laboratory Methods

	Antifungal Susceptibility Testing

	Culture Techniques and Media

	Microscopy Techniques and Stains

	Specimen Collection and Processing

School of Molecular & Biomedical Science
THE UNIVERSITY OF ADELAIDE
AUSTRALIA 5005

Contact:
Dr David Ellis

Email

Telephone:
+61 8 8161 6459
Facsimile:
+61 8 8161 7589

Calcofluor White with 10% KOH.

For the direct microscopic examination of skin scrapings, hairs, nails and other clinical specimens for fungal elements.

Calcofluor white (M2R powder from Polysciences) or Blankophor BA (from Bayer) are used as whitening agents by the paper industry and selectively bind to cellulose and chitin. The dye fluoresces as it is exposed to ultraviolet light. This as a very sensitive method, however, a fluorescence microscope with the correct ultraviolet filters is required.

Method:

Solution A Potassium hydroxide reagent.

Dissolve the KOH in water and add glycerol.

Potassium hydroxide 10 g

Glycerin 10 ml

Distilled water 90 ml

Solution B Calcofluor white reagent.

Dissolve Calcofluor white powder in the distilled water by gentle heating.

Calcofluor white 0.1 g

Distilled water 100 ml

Method for making microscopic mounts:

1. Mix one drop of each solution on the centre of a clean microscope slide.

2. Place the specimen in the solution and cover with a coverslip, squash the preparation with the butt of the inoculation needle and then blot off the excess fluid.

3. Gently heat the slide and examine microscopically for the presence of fungal elements that fluoresce a chalk-white or brilliant apple green colour, depending on the filters used.

Comments: This is a very rapid and sensitive method, however a fluorescence microscope fitted with filters to give an excitation with ultraviolet light below 400 nm wavelength is required.

Further reading: Hageage and Harrington, Laboratory Medicine 1984,15:109-112