KoH Prepration, CLASSIFICATION OF FUNGAL DISEASES

KOH (potassium hydroxide) preparation is a diagnostic test for fungal infections that involves placing a specimen from the affected area (like skin, nails, or hair) on a glass slide, adding a drop of 10-20% KOH solution, and examining it under a microscope. The KOH dissolves skin and other cells, leaving fungal elements like hyphae or yeast to be viewed and identified.

1. Preparation of KoH Mahesh Chandra Ph.D. Medical microbiology, Adesh University Bathinda Punjab

2. GENERAL MYCOLOGY Medical mycology - branch of medical science that deals with the study of medically important fungi ‘fungus’ is derived from Greek ‘ mykes ’ meaning mushroom (a type of edible fungus) Essentials of Medical Microbiology

3. Fungi differ from bacteria & other eukaryotes Eukaryotic and possess eukaryotic cell organelles Possess a rigid cell wall, composed of chitin, β- glucans and other polysaccharides Cell membrane contains ergosterol instead of cholesterol May be unicellular or multicellular Lack chlorophyll and divide by asexual and/or sexual means by producing spores Essentials of Medical Microbiology

4. CLASSIFICATION OF FUNGAL DISEASES Superficial mycoses: These are the fungal infections involving the skin, hair, nail and mucosa Subcutaneous mycoses: These are the mycotic infections of the skin, subcutaneous tissue and sometimes bone, resulting from inoculation of saprophytic fungi of soil. Systemic mycoses: They involve multiple organs. Mosdy Liley are caused by the saprophytic fungi, which spread by inhalation of spores leading to pulmonary infection. From lungs, they disseminate to cause various systemic manifestations. Opportunistic mycoses: they are caused by the fungi that are normally found as human commensals or in environment, but can act as human pathogen in presence of opportunistic such as low immunity.

5. Morphological Classification of Fungi 1. Yeast : Round to oval cells that reproduce by budding Cryptococcus neoformans (pathogenic) Saccharomyces cerevisiae (non-pathogenic) 2. Yeast-like : Yeasts forming pseudohyphae (e.g. Candida) - Differentiated from true hyphae as they have constrictions at septa 3. Molds : long branching filaments called hyphae - Hyphae - septate or nonseptate Essentials of Medical Microbiology

6. Morphological forms of fungi

7. Classification of Fungi Based on the growth pattern in culture medium Aerial mycelium: It is the part of the mycelium which projects above the surface of culture medium Vegetative mycelium: It is the part of the mycelium that grows on the surface of the culture medium

8. LABORATORY DIAGNOSIS OF FUNGAL DISEASES Specimen Collection Depends on site of infection - skin scraping, hair, nail, sputum, etc. Systemic mycoses - blood sample, Cerebrospinal fluid (CSF), etc Essentials of Medical Microbiology

9. Preparation of KoH Potassium hydroxide (KOH) preparation: Keratinized tissue specimens such as skin scrapings and plucked hair samples are treated with 10% KOH which digests the keratin material so that the fungal hyphae will be clearly seen under the microscope. 1O% is the usual concentration of KOH used. 20-40% KOH is needed for the specimens such as nail and biopsy tissues that take longer time to dissolve. Glycerol (10%) can be added to prevent drying DMSO (dimethyl sulfoxide) - help in tissue digestion

10. Collect the specimen:   Gently scrape the affected area to collect a sample of skin, nails, or hair. The scraping should include material from the active margin of a lesion.  Place the specimen on a slide:   Put the sample in the center of a clean glass slide.  Add KOH solution:   Add a few drops of 10% or 20% KOH solution to the specimen. For added detail, a calcofluor white stain can be added to the KOH solution to make fungal elements fluoresce under a UV microscope. Cover the specimen:   Place a coverslip over the mixture, pressing down gently to remove air bubbles and spread the sample.  Heat the slide (optional):   Gently heat the underside of the slide for a few seconds. This can help speed up the dissolving of human cells, making the fungal structures more visible.  Wait for it to dissolve:   Allow the solution time to dissolve the non-fungal cells. This may take 10-15 minutes.  Examine under a microscope:   Look for fungal structures like hyphae or yeast under low power first, then switch to high power for confirmation.  How to prepare for a KOH test

11. Microscopy Demonstration of Fungal elements in the specimen Potassium hydroxide (KOH) preparation: Keratinized tissue specimens treated with 10% KOH  digests keratin  fungal hyphae clearly seen 20–40% KOH - nail & hair Essentials of Medical Microbiology

12. Morphological forms of fungi Essentials of Medical Microbiology

13. Safety precautions Wear gloves:  Always wear gloves when handling KOH, as it is a corrosive chemical.  Avoid contact:  Prevent contact with skin, eyes, and clothing.  Ventilate the area:  Perform the preparation in a well-ventilated area. Handle with care:  Handle the specimen and the KOH solution with care to avoid spills and accidents.  Label properly:  Label all slides clearly with patient identifiers and specimen type to prevent mix-ups. Dispose of properly:  Dispose of the slides and contaminated materials in a biohazard bin.