KOH Wet Mount Preparation: Introduction, Principle, Procedure, Result Interpretation and Uses

KOH mount of ear discharge showing septate hyphae and conidia of Aspergillus fumigatus

Introduction of KOH Wet Mount

The  KOH Wet Mount is very important for presumptive diagnosis of the type of fungal infections whether ringworm, aspergillosis, dermatomycosis, blastomycosis, mucormycosis, otomycosis, and so on. It is also helpful in the selection of appropriate culture media for the isolation of etiological fungal agents.

Principle of KOH Wet Mount Preparation

As you know, potassium hydroxide (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 for KOH Wet Mount Preparation

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.

Procedure of KOH Wet Mount Preparation

  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, bur 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.

Quality control (QC)

It can maintain using the following points-

  •  The right concentration of  20% KOH should be prepared.
  • Emulsification of specimen should be homogenous in potassium hydroxide (KOH) preparation.

Observation

Observe fungal elements in microscopy of the clinical specimens.

Result and Interpretation of KOH Wet Mount 

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.

No fungal elements were seen during in microscopy of the clinical specimen, KOH preparation is negative.

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.

Uses of KOH Wet Mount Preparation

The uses of KOH wet mount preparation are as follows-

  1. 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.
  2. It is very useful for the presumptive diagnosis of type fungal infections.
  3. The recommendation of KOH wet mount preparation is very useful in the following suspected clinical conditions as shown in this table.

Keynotes on KOH  Mount 

  1.  Potassium hydroxide(KOH) reagent is highly corrosive therefore handle it with great care.
  2. Experience is necessary for reporting because background artifacts are often confusing.
  3. Clearing off some specimens such as nail clipping, hair may require an extended time.

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. 3rd 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.
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