Obtaining the sample

The scraping should be taken with a #15 scalpel blade or the edge of a glass slide. The scraped scale should fall onto a microscope slide or into a test tube. False-negative KOH preparations often result from inadequate scrapings.

A tinea capitis sample for KOH preparation can be taken by scraping the black dots (hairs broken off at the skin line).

For suspected onychomycosis, consider a periodic acid–Schiff stain of nail clippings instead of KOH preparation.

Because the scrapings will easily blow off the slide, shield it from drafts or apply KOH preparation to the slide before transport.

Preparing the slide

Place two drops of 10% or 20% KOH on the scrapings, followed by a coverslip. Alternatively, place a coverslip over the dry scrapings and a drop or two of KOH next to the coverslip and allow it to run under the coverslip. KOH dissolves squamous cells but leaves the fungal elements intact.

Heat the slide with a match or alcohol lamp. The match may leave a smoky deposit on the slide. Avoid boiling the KOH, but the slide should be hot enough to be uncomfortable to the dorsum of the hand, usually three to four seconds over the flame. Skin scrapings and hair can be examined under the microscope immediately. Toenail curettings should wait at least 10 minutes to several hours before examination.

After heating the slide, tap down the coverslip to compress the sample and separate the hyphae from the squamous cells.

Examining the slide under the microscope

Adjust the light filter and drop the condenser to achieve a low light level and increased refraction.

Scan the slide under low power, and use high power to confirm hyphae in suspicious areas.

False-negative results on KOH preparations are common and are usually caused by inadequate material on the slide. False-positive results can occur from misinterpretation of hair shafts or clothing fibers, which are often larger than hyphae, not segmented, and not branching. The borders between squamous cells can also be mistaken for hyphae.

The shelf life of a bottle of KOH is at least five years. KOH can damage microscope lenses. Therefore, use an old microscope, and avoid spills and excess KOH on the slide.