Potassium hydroxide (KOH) Wet Mount Introduction

The KOH Wet Mount is a very simple and important technique in mycology for presumptive diagnosis of the type of fungal infections whether ringworm, aspergillosis, dermatomycosis, blastomycosis, mucormycosis, otomycosis, etc. It is the most common assay ordered by clinicians, particularly by dermatologists. It is also helpful in the selection of appropriate culture media for the isolation of etiological fungal agents. Here you can see in the image fungal elements (hyphae-septate/aseptate, yeast cells, budding, and pseudohyphae) from a variety of clinical specimens like skin scraping, aural discharge, blood sputum, and urine.

Principle of KOH Wet Mount Preparation

As you know, potassium hydroxide (KOH) is a strong alkali. When clinical samples such as pus, skin, hair, nails, or sputum mix, 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-septate/aseptate, granules, conidia, etc.) of fungi can be observed under a microscope.

Requirements for KOH Wet Mount Preparation

1.  Equipment

• Compound 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
• Potassium hydroxide (Generally 20% KOH but depending on the nature specimen its concentration can be decreased or increased)
• Tissue paper roll

3. Clinical Specimen/sample-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 applying inoculating loop. To assist in 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. Use 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 exclude by using a high concentration of KOH or KOH with Dimethylsulfoxide(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 as shown above image.

Quality control (QC)

Quality control can maintain using the following points-

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

Observation of KOH Mount Footage

Observe fungal elements in microscopy of the clinical samples.

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, and 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 while conformation biochemical tests and molecular tests are needed.

Uses of KOH Wet Mount Preparation

The applications of KOH wet mount preparation are as follows-

1. KOH Mount 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.

Suspected Conditions	Etiological Agents	Specimens	Diagnostic Characteristics
Mucormycosis	Mucor species	Samples from infected tissue or lesion	Aseptate hyphae
Aspergillosis	Aspergillus flavus, A. fumigatus, A.niger, etc	According to the site of infection e.g. respiratory system-sputum while in ear-aural discharge	Septate hyphae with V-shaped/acute-angle/dichotomous branching
Blastomycosis	Blastomyces dermatitidis (Dimorphic fungus)	Sputum, pus, or skin	Large budding yeast cells have a distinct broad base and due to being a dimorphic fungus, it may be with yeast cells in tissue
Ringworm	Dermatophytes (Trichophyton, Microsporum, Epidermophyton)	Skin, hair, and nail	Septate hyphae or spores (microconidia or macroconidia) depending on the nature of the etiological agent involved

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, and hair may require an extended time.

Collection of KOH Mount Footages

KOH preparation of a skin scale, showing ringworm-causing fungus structures

Nail Dermatophytes in Nail Clipping KOH mount and Growth on SDA

KOH Mount of Sputum Showing Fungal Elements-Hyphae and yeast cells

Fungal pseudohyphae, yeast cells, and budding in KOH mount of Sputum Microscopy

KOH mount of aural discharge showing fungal elements-Septate hyphae with V-shaped or acute-angle or dichotomous branching

Fungal hyphae in KOH mount of Ear discharge of CSOM Patient showing septate hyphae with dichotomous branching-Tentative organism-Aspergillus

Nail Dermatophytes in Nail Clipping KOH mount showing thin-walled macroconidia in the cluster and lacking microconidia

Skin scrapping in KOH mount showing yeast cells and an elongated cigar-shaped morphology

KOH mount of ear discharge showing fungal elements-Septate hyphae with V-shaped or acute-angle or dichotomous branching

Aspergillus fumigatus fungal elements in KOH mount of aural discharge microscopic footage

Fungal hyphae and conidia in KOH mount of ear discharge

Ochroconis gallopava in 0.05% Tween 80 wet mount of culture microscopy exhibiting conidia, conidiophores, hyphae, and hyphae are brown with relatively thick walls

Urine under the Microscope showing hyphae indicates Fungal UTI

Fungal Hyphae in Urine Microscopy

Heavy load of fungal elements in KOH mount of Sputum sample

KOH mount of sputum under the Microscope showing fungal elements- yeast cells and fungal hyphae

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.