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Ringworm Causing fungus

Potassium hydroxide, KOH preparation of a skin scale, showing ringworm causing fungus with branching hyphae and conidia too as shown above picture.

Principle of potassium hydroxide preparation

As you know, KOH is a strong alkali. When specimens such as pus, skin, hair, nails, or sputum mix with, it softens, digests, and clears the tissues i.e. keratin present in the skin surrounding the fungi so that the fungal elements( yeast, a cell with pseudohyphae, budding, hyphae, granules, conidia, etc.) of fungi can be seen under a microscope.

Requirements

1. Equipment

• Microscope

2. Reagent and laboratory wares

• Glass Petri dishes
• clean and grease-free glass slide
• Cove slip
• Straight wire or bent wire
• Needle
• Bunsen burner
• 20% KOH

3. Specimen

It may vary according to the site of infections such as pus from draining sinus, aspirate from nasal sinuses, respiratory specimen, skin scrapings, nail clipping, hair, corneal scraping, material from ear discharge, etc. But here, we received a skin scrapping specimen from the patient having ringworm.

Procedure of KOH mount

1. Emulsify the specimen in a drop of 20% KOH on a glass slide with the help of inoculating loop. 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.
2. Apply gentle heat by passing the slide over a Bunsen burner 3-4 times in case of 10% KOH or hard specimens like nail clipping or hair. (Note: This step can omit by using a high concentration of KOH or KOH with DMSO or treating the specimen with KOH for a longer duration.)
3. Cover the smear with the coverslip and leave for 5-10 minutes. (generally, but when using nail clipping or hair, there is a need for a longer duration.)
4. As soon as the specimen has cleared, examine the preparation microscopically focusing the 10X and finally observation using  40X objectives with the condenser iris diaphragm closed sufficiently to give a good contrast. If you are using too intense a light source the contrast will not be adequate and the unstained fungi will not be seen. Examine the preparation carefully for the demonstration of shining fungal elements.

Observation

Observe fungal elements in microscopy of the clinical specimens.

Result and Interpretation

1. If there is the presence of any fungal elements either yeast cells, cells with pseudohyphae, budding, septate hyphae, aseptate hyphae, branching hyphae, conidia, or granules, etc. during the examination, KOH mount is positive i.e. fungal elements seen.
2. No fungal elements were seen during in microscopy of the clinical specimen, KOH preparation is negative.
3. Interpretation of results should do by critical analysis of the type, size, and color of the fungal elements that will be different for different fungi. A fungal culture is necessary for the isolation of etiological fungal agents.

Application of KOH preparation

1. The uses of KOH wet mount preparation are as follows-
2. It is used for the rapid detection of fungal elements in clinical specimens, as it clears the specimen making fungal elements more visible during direct microscopic examination.
3. It is very useful for the presumptive diagnosis of fungal infections.

Further Readings

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. A Textbook of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.