Direct Microscopic Examination

KOH PREPARATION

acts as a clearing agent that digests keratin, cellular debris, and proteinaceous material in hair, skin, nails, and tissue, leaving intact chitinous fungal cell walls intact.

Procedure for KOH preparation

Sample are added to the drop of 10-20% of KOH solution, and a coverslip is added on top of the slide

Slide is gently heated and then allowed to cool for approximately 15 mins

Dimethyl Sulfoxide (DMSO)

It can be added to clear spx rapidly without heating

Calcofluor White Stain

Dye binds to polysaccharides present in the chitin of the fungus or to cellulose.

Procedure for Calcofluor White Stain

A drop of calcofluor white is added to the KOH preparation before the addition of the coverslip

Fungal elements fluoresce apple green or blue-white.

Positive results for Calcofluor White Stain

Lactic Acid, Phenol, Cotton Blue, Glycerol

Components of LACTOPHENOL COTTON BLUE (LPCB) MOUNT

Lactic Acid

for preservation of fungal agent

Phenol

Killing Agent

Cotton Blue

Stain

Glycerol

Prevent spx from drying out

India Ink

Ink particles cannot penetrate the large polysaccharide capsule surrounding certain fungi, thus outlining the fungi

Purpose of India Ink

Detect presence of Cryptococcus on CSF sample.

Procedure of India Ink

The CSF sample is centrifuged and separate the sediment from the supernatant.

A drop of sediment is added with a drop of India ink.

GRAM STAIN (HUCKER’S MODIFICATION)

Crystal Violet + Ammonium Oxalate

PERIODIC ACID SCHIFF & GOMORI’S METHENAMINE

For detection of fungi in histologic / tissue specimens.

ACID FAST STAIN

Detects Blastomyces dermatitis.

GIEMSA OR WRIGHT’S STAIN

To demonstrate the yeast if H. capsulatum.

MASSON FONTANA & H&E STAIN

To determine the hyphal pigmentation.

MAYER’S MUCICARMINE

To determine the capsule of C. neoformans.

ACRIDINE ORANGE

For detection of Tinea versicolor (M. furfur)

WOOD’S LAMP

To detect fluorescence.