KOH Test- Principle, Procedure, Results, Uses, Limitations

Objective of Potassium hydroxide (KOH) Test

  • To visualize the fungal element in the clinical specimens.
  • To examine skin scrapings or flakes and hair for the presence of hyphae and arthroconidia in suspected dermatophyte infections.

Principle of Potassium hydroxide (KOH) Test

Potassium hydroxide (KOH) can be used on clinical specimens to clear cellular material and for better visualization of fungal elements. 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 specimen. KOH is a strong alkali. KOH 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 so that the hyphae and conidia (spores) of fungi can be seen under the microscope. The specimen is placed in a few drops of 10% to 20% KOH and incubated for 5 to 10 minutes where gentle heating can clear samples more quickly. A coverslip is placed over the KOH-digested sample, and the slide is examined microscopically without staining.  Different fungal elements like hyphae, pseudohyphae, yeast cells, spores, spherules, and sclerotic bodies can be seen clearly in a KOH wet mount. In dermatophytosis, arthrospores develop and form as hyphae break apart and appear as a linear chain of small, round to rectangular, highly refractile structures. In potassium hydroxide preparations of sputum, the fungus appears as non-pigmented septate hyphae, 3–5 µm in diameter, with characteristic dichotomous branching and an irregular outline.

Potassium hydroxide (KOH) test

Some modifications in KOH Preparation method 

  • Use of dimethylsulphoxide-KOH reagent: The addition of dimethylsulphoxide (DMSO) to KOH enables specimens to be examined immediately or after only a few minutes.
  • KOH with blue-black fountain pen ink added: The ink is not specific for fungi as it stains cells and other components in the skin. The addition of ink is recommended when Malassezia furfur is suspected.

Procedure of Potassium hydroxide (KOH) Test

Slide method

  1. Place the specimens like epidermal scales, nail, hair, skin scraping or tissue on a clean glass slide.
  2. Pour a drop of 10% KOH on the specimen and place a coverslip over it.
  3. Heat the slide gently over the flame.
  4. Leave the slide for 5-10 minutes or place the slide in a petri dish, or other containers with a lid, together with a damp piece of filter paper or cotton wool to prevent the preparation from drying out.
  5. Examine the slide under the microscope using 10X and 40X objectives.

Tube test

  1. Place the homogenized tissue material in a test tube and add 10% KOH.
  2. Incubate the tube overnight at 37°C.
  3. Following incubation, place a drop of suspension in the clean slide and cover with a coverslip.
  4. Examine the slide under the microscope in 10X and 40X objectives.

Note: This procedure can also be used for nail clippings and skin biopsies which dissolve with difficulty and the concentration of KOH may be increased.

Uses of Potassium hydroxide (KOH) Test

  1. KOH preparation is used for the diagnosis of ringworm infection. Laboratory diagnosis of tinea rests on the identification of an organism by microscopic examination of skin or nail scrapings with 10% to 20% KOH on wet mount examination.
  2. KOH with blue-black ink preparation is recommended if Malassezia furfur is suspected.
  3. Calcofluor White–Potassium Hydroxide Preparation can also be used for the examination of Fungal infection because CW is a nonspecific stain, an appreciation for fungal element morphology on direct examination is crucial for adequate specimen interpretation.

Limitations of Potassium hydroxide (KOH) Test

  1. Potassium hydroxide is a highly corrosive deliquescent chemical; therefore it should be handled with great care.
  2. Experience required since background artifacts are often confusing.
  3. Clearing of some specimens like biopsy material, nail clippings may require extended time.


  1. Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen editions, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
  2. Bilge Fettahlıoğlu Karaman B.F, Topal S.G,  Aksungur V.L, Ünal L,  İlkit M. 2017. Successive Potassium Hydroxide Testing for Improved Diagnosis of Tinea Pedis. 100(2):110-114.
  3. Cheesbrough M. 2006. Medical laboratory manual for tropical countries part 2. Second edition. Cambridge University Press.

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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