KOH Preparation: Introduction,Preparation and Resullt Showing Septate Hypha and Conidia of Aspergillus fumigatus in Ear Discharge

KOH preparation showing septate hypha and conidia of Aspergillus fumigatus in ear discharge

KOH Preparation 

KOH preparation showing septate hypha and conidia of Aspergillus fumigatus in ear discharge from a patient of chronic suppurative otitis media as shown above image. The organism was later confirmed on the basis of culturing on SDA, LPCB preparation from growth, and biochemical tests. KOH ( potassium hydroxide)  mount preparation is a 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 etiology.

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 clinical specimens like hair, skin, nail, etc. KOH is a strong alkali that 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 a 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 coverslip
  • 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 specimens such as sputum, pus, urine sediment, homogenate from biopsy tissue, nail, hair, etc., to clear cell debris.)
  • Scalpel or inoculating loop
  • Specimen ( We used ear discharge from CSOM patient.)

Procedure of KOH Mount

  • Take a clean grease-free glass slide.
  • Place a large drop of KOH solution with a Pasteur pipette.
  • Transfer a small quantity of the specimen with a loop or the tip of a scalpel into the KOH drop.
  • Put a clean coverslip 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 coverslip 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

Septate hyphae and conidia of fungi were seen as shown above picture.

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 Vol. I & II, 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.
  6. Topley & Wilsons Medical Mycology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  7. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  8. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  9. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
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