Koh-Calcofluor White Stain is used to detect the presence of Acanthamoeba fungal elements and cysts in clinical specimens.
Principle of Koh-Calcofluor White Stain
The alkali potassium hydroxide (10 percent KOH) aids in the digestion of tissue debris while also acting as a cleaning agent and disinfectant. The fungal elements become more visible as the debris is cleared away.
Calcofluor stains the cell walls of fungi (filamentous fungi and yeasts) and Acanthamoeba cysts.
The yield may be reduced if the specimen is stored for an extended period of time. As a result, a fresh specimen is always preferred for examination.
Type of Sample
Clinical specimens such as corneal scrapings, vitreous aspirate, aqueous aspirate, pus material, eviscerated material, corneal button, skin scrapings, sputum, nail clippings, hair, or any biopsy specimen.
The specimen must be collected in a sterile container. If staining is not done immediately, store the specimen at 2–8 °C.
Reagents And Consumables
- Clinical specimen
- Micro slides
- Cover slip
- 4 mm Nichrome wire-loop
- Sterile Pasteur pipette.
Solution A (10% Potassium Hydroxide)
One gram of potassium hydroxide is dissolved in ten milliliters of distilled water and stored in a plastic dropper bottle.
0.01 gm Calcofluor White (0.1 percent )
0.005 gms Evans Blue (0.05 percent )
Distilled water – 10 mL
Dissolve by thoroughly mixing, filter, and store in a dropper bottle.
- Biological safety Cabinet (Class II)
- Fluorescence microscope with violet filter.
- Cytospin instrument
Koh-Calcofluor White Stain Procedure
- Mark the area of the smear on the slide and label it with the specific lab number.
- Depending on the type of clinical specimen, the smear is prepared using one of the following methods: cytospin, bacteriological loop, drop method with syringe and needle, or Pasteur pipette.
- The slide is then covered with a drop of 10% KOH (solution A) and one drop of Calcofluor stain (solution B), followed by a cover slip.
- The slide is left in this position for 5 minutes to allow the tissues to digest at room temperature.
- Examine under a fluorescence microscope with a violet filter set to 410 nm. Initially at 10 X magnification, the magnification is gradually increased to 20X, 40X, and dry objectives.
Under the fluorescence microscope, the fungi (filamentous and yeast) absorb the stain and fluoresce bluish white.
Acanthamoeba are bluish white cysts with double walls that are hexagonal or polygonal in shape.
Cysts of Pneumocystis carinii and spores of Microsporidia Cryptosporidium oocysts and Cryptococcus species are also bluish white.
Quality Control Procedure
Whenever new batches of stain are used, test their reliability with known Fungi/Acanthamoeba/ATCC strains of Yeast.
- Take all precautions when handling biological fluids.
- Only handle the specimen inside the biosafety cabinet.
Variability’s Potential Source
Process specimens before they dry up.
Forbes B, Sahm D F, Weissfeld AS. Laboratory Methods in Basic Mycology. In Bailey Scott’s Diagnostic Microbiology 1998;10:958.