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Fungal hyphae in KOH Preparation of Pus Discharge

Fungal hyphae in KOH preparation of pus discharge at 40X objective under the microscope as shown above picture.

Principle of KOH Preparation

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

Requirements for KOH 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 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 got pus discharge for KOH mount.

4. Quality assurance
Fungal spores or hyphae may contaminate the KOH solution kept in the
laboratory and may give false positive results. A negative control should
therefore be put up every day.

Procedure of KOH Preparation 

1.  Emulsify the specimen in a drop of 20% KOH on a glass slide with the help of  inoculating loop.
2. Apply gentle heat by passing the slide over a Bunsen burner for 3-4 times in case of 10% KOH.
3. Cover the smear with the cover slip  and leave for 5-10 minutes.
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.

Observation

Observe for fungal elements in microscopy of the clinical specimens. Examine the clear specimen under low power (10X objective). Scan the entire cover slip from end to end in a zigzag fashion. If any fungal elements are suspected, examine under high power(40X objective). Reduce the light coming into the condenser while examining at high power. Look for budding yeast cells, branching hyphae, type of branching, the colour, septation and thickness of  fungal hyphae.

Result and Interpretation of KOH Preparation 

1. If there is presence of any fungal elements either yeast cells, cells with peudohyphae, budding, septate hyphae, aseptae hyphae, branching hyphae, conidia or granules etc. during examination, KOH mount is positive i.e. fungal elements seen.
2. No fungal elements 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. Fungal culture is necessary is necessary for the isolation etiological fungal agents.
4. Above pictures has fungal hyphae in KOH preparation.

Application of KOH Preparation

The uses of KOH wet mount preparation are as follows-

1. It  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.
2. It is very useful for the presumptive diagnosis of the type fungal infections.

Advantages of KOH Test

• It is simple.
• It is also economical and rapid.

Dermatophytes in KOH preparation

Disadvantages

1. Sputum and pus may contain artifacts, which may superficially resemble hyphal and budding forms of fungi. These artifacts may be from cotton or wool fibers, starch grains (in pleuritis) or cholesterol crystals.
2.  KOH test gives an idea about the presence of hyphal element, but can not distinguish different fungi.
3.  Preparation can not be kept for too long because of drying; but drying can be
   prevented by keeping the slides in a moist chamber (covered Petri dish with a wet filter paper on which a triangular glass rod is placed).

Modification of KOH Test

For more distinction, stains like methylene blue or Parker blue-black
fountain ink may be used along with KOH. This will impart a colored
background and fungal elements, if present, will show as prominent
refractile objects as shown below.

Precautions

Following precautions should be taken during KOH preparation.

1. The drop of reagent (KOH) should not be so large that the cover slip floats.
2. Do not leave KOH slide outside a moist chamber, the KOH dries and crystals form that restrict the visibility of the fungus or ignore this step using KOH modification with DMSO.
3.  After clearing,  gently  apply pressure on the top of the cover slip with a fold of filter paper or the handle of a teasing needle. This ensures even spreading of the material onto the slide.
4. KOH should be kept in a closed container in small aliquots ready to use on the workbench.

References

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 Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. A Text Book of Medical Mycology. Editor: Jagdish Chandar.  Publication Mehata, India.
5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.