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KOH Preparation 

KOH preparation  of sputum showing fungal elements in sputum of patient admitted in medical ICU on the way of diagnosis as shown above image. KOH ( potassium hydroxide)  mount preparation is direct microscopic examination of the clinical material  like sputum, pus, urine sediment, homogenate from biopsy tissue, nail, hair, etc. provides valuable information about the fungal aetiology.

Principle of KOH Mount

KOH can be used on clinical samples to clear cellular material and for better visualization of fungal elements like  yeast cells, hyphae, conidia , etc. KOH preparation is a commonly used method for the diagnosis of superficial fungal infections and for the rapid detection of fungal elements in a clinical specimens like hair, skin, nail,etc. KOH is a strong alkali which  separates the fungal elements from intact cells as it digests the protein debris and dissolves cement substances that hold the keratinized cells together surrounding the fungi as clearing agent so that the  fungal elements of fungi can be seen clearly  under the microscope as shown above picture.

Requirements for KOH Mount Preparation 

• A clean and grease free slide and cover slip
• Microscope
• Glass Petri dish ( optional)
• Pasteur pipette or dropper
• 20% KOH (  10-20% KOH is usually used depending on the specimen; occasionally 40% is also used when not cleared by 10%-20% KOH and  it uses  for specimen such as sputum, pus, urine sediment, homogenate from biopsy tissue, nail, hair, etc., to clear cell debris.)
• Scalpel or inoculating loop
• Specimen ( We used sputum from MICU patient.)

Procedure of KOH Mount

• Take a clean grease-free glass slide.
• Place a large drop of KOH solution with a Pasteur pipette.
• Transfer small quantity of the specimen with a loop or the tip of a scalpel into the KOH drop.
• Put a clean cover slip over the drop gently so that no air bubble is trapped.
• Place the slide in a moist chamber ( wet tissue paper in Petri disk), and keep at room temperature.
• Tissue usually takes 20-30 minutes; sometimes overnight contact with KOH is useful for getting a positive result.
• Clearing can be hastened by gentle heating of the slide, but it is best avoided.

Observation of KOH Preparation

1. Examine the clear specimen under low power (10X or 20X
   objective) and then scan the entire cover slip from end to end in a zigzag
   fashion.
2. If any fungal elements are suspected, examine under high power
   (40X) objective.
3. Reduce the light coming into the condenser while examining at
   high power.
4. Look for budding yeast cells, branching hyphae, type of
   branching, the color, septation and thickness of hyphae.

Result of our KOH Preparation

Hyphae of fungi  were seen as shown above picture.

Bibliography

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