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KOH Preparation Test: Principle, Procedure, Results and Uses  

Image 1: Left: Fungal hyphae in a (KOH) preparation of skin scales as seen with the 10x objective. Right: Hyphae and arthroconidia as seen with the 40x objective. (Image source-Reference-1)

Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal elements in clinical specimen, as it clears the specimen making fungal elements more visible during direct microscopic examination.

KOH is a strong alkali. When specimen such as skin, hair, nails or sputum is mixed with 20% w/v KOH (preparation of KOH is posted at the end of this post), it softens, digests and clears the tissues (e.g., keratin present in skins) surrounding the fungi so that the hyphae and conidia (spores) of fungi can be seen under microscope.

Uses:

In diagnostic laboratories, KOH mount is one of the main methods of investigating fungal infections. It is used as a primary screening tool, it detects fungal elements present but may not necessarily identify the species of the fungi. (Note: To identify the fungal isolate, specimen must be cultured in either general purpose fungal culture media such as Sabouraud Dextrose Agar (SDA) or specific media based on the type of anticipated isolate.)

KOH preparation is recommended in the following suspected conditions (this is not the exclusive lists);

Suspected conditions Specimen Diagnostic characteristics
Aspergillus infection Sputum Septate hyphae with V-shaped branching
Dermatophytes (ringworm fungi) skin scrapings, nails or hair Septate hyphae or spherical yeast cells depending on the nature of etiologic agents involved.
Blastomyces dermatitidis infection Pus, sputum or skin specimens Yeast cells (large budding yeast cells with distinct broad base) of Blastomyces dermatitidis. B.dermatitidis is a dimorphic fungus with yeast cells in tissue.
Mucormycosis Exudates from infected lesions or tissue Aseptate hyphae of fungi causing mucormycosis.

Procedure of KOH Preparation

  1. Place a drop of KOH solution on a slide.
  2. Transfer the specimen (small pieces) to the drop of KOH, and cover with glass. Place the slide in a petri dish, or other container with a lid, together with a damp piece of filter paper or cotton wool to prevent the preparation from drying out.
    Note: To assist clearing, hairs should not be more than 5 mm long, and skin scales, crusts and nail snips should not be more than 2 mm across.
    Image 2: Malassezia furfur yeast cells and hyphae in KOH blue–black ink preparation. (Image source: Reference 1)

    (Image source-Reference-1)

  3. As soon as the specimen has cleared, examine it microscopically using the 10X and 40X objectives with the condenser iris diaphragm closed sufficiently to give a good contrast.
    If too intense a light source is used the contrast will not be adequate and the unstained fungi will not be seen.

Disadvantages of KOH preparation method

Procedure to make 100 ml of KOH 20% w/v solution:

  1. Weigh 20 g potassium hydroxide (KOH) pellets.
  2. Transfer the chemical to a screw-cap bottle.
  3. Add 50 ml distilled water, and mix until the chemical is completely dissolved, add remaining distilled water and make the volume 100 ml.
  4. Label the bottle and mark it corrosive. Store it at room temperature. The reagent is stable for up to 2 years.

Caution: Potassium hydroxide is a highly corrosive deliquescent chemical, therefore handle it with great care and make sure the stock bottle of chemical is tightly stoppered after use.

Modification in KOH Preparation method 

References and Further Readings:

  1. District Laboratory Manual in Tropical Countries, Part 2; Cambridge University Press
  2. Bailey & Scott’s Diagnostic Microbiology, Elsevier