The potassium hydroxide or KOH exam or preparation. This video describes the correct procedure for performing a potassium hydroxide preparation. The potassium hydroxide or KOH exam is a method for microscopically preparing and examining a specimen from the skin, hair, or nails for the presence of fungal elements. It is a relatively fast, simple, and inexpensive test that can easily be performed in the clinic setting. Applying the alkaline potassium hydroxide solution causes separation and gradual destruction of the stratum corneum cells. This makes it easier to see foreign materials such as hyphae and spores. Fungal elements take much longer to be affected by the alkaline solution, however they will eventually be destroyed so it is important to analyze a prepared specimen promptly. Indications A KOH prep is appropriate virtually any time a patient presents with a scaling rash, which may or may not have central clearing. Tinea infections are sometimes itchy. Red scaly rashes that don't respond to topical steroids or are persistent despite the use of topical steroids should be considered suspect for fungal infections. For more information about the presentation of fungal infections, review the basic dermatology curriculum modules on adult fungal infections, pediatric fungal infections, the red scaly rash, blisters, and hair loss. Before starting, it is important to gather all the equipment you will need. This includes rubbing alcohol wipes, a scalpel such as a 15 blade, or a toothbrush or cotton swab if you're swabbing the scalp of a child, a nail clipper if you suspect nail infection, as well as a curette to remove subungual debris. Glass microscope slides, cover slips, and potassium hydroxide solution are needed to prepare the specimen. Potassium hydroxide, 10%, is usually used for skin samples, although 20% may be used for nail specimens. Potassium hydroxide containing a fungal stain, such as chlorazole black or Chicago sky blue, is helpful because it highlights the fungal walls and makes them easier to see. This is especially helpful with nail specimens. You will need a fungal culture plate or slant or sterile specimen container if you are submitting a fungal culture. Finally, you will need access to a microscope. Make sure the patient is seated in a well-lit room. Choose an appropriate site for collecting your specimen. We will present three discussions of how to collect specimens from three body sites. First, we will demonstrate specimen collection from the skin. Scales should be collected from the border of the lesion, adjacent to normal appearing skin, and rub the area with alcohol to remove debris. Then collect loosely adherent scale by gently scraping perpendicular to the skin surface with your scalpel blade. Do not scrape hard enough to draw blood. Hold the microscope slide just underneath as you are scraping to collect the scales. If vesicles are present, as in Bullis tineapetus, scrape on the blister roof for fungal elements. If the scales adhere to the blade, gently wipe the blade on the edge of the slide to transfer the scale. Using a plain glass coverslip, heap the fine skin scale scrapings into an area small enough to fit under the coverslip. Place the coverslip over the piled up scales and proceed to the microscope work area. Putting the coverslip over the specimen, even without the liquid, will keep the scales from flying off the slide. Some providers prefer to apply the solution in an exam room and put the coverslip on to avoid losing specimen in transit. For nails, clipping part of the nail and sending it to the pathology lab to be evaluated with PAS or other fungal stains is the most sensitive method of diagnosing nail fungus, but it is also the most expensive. KOH preparation in the clinic is the least expensive option that can be done immediately. First, wipe the area clean with an alcohol wipe. The best quality specimens come from beneath the nail plate, sampling the subungual debris. Cut back a portion of the affected nail. This could be saved in case you decide to send it for PAS or other fungal stains. Now, the nail bed can be more easily accessed. Gently scrape out debris from the nail bed with a 15-blade scalpel or a small, sharp curette. Catch the debris on a glass slide or weighing paper. Now process it as you would a skin specimen. Consider using KOH with a fungal stain such as chlorazole black or Chicago sky blue. Scalp specimens. Usually we are swabbing the heads of children we suspect might have tinea capitis due to a patch of hair loss or scaling. First, wipe the area clean with rubbing alcohol. Next, remove a few broken pieces of hair from the scalp and place them on your slide. Then scrape scale from the scalp or with your sterile cotton swab or new toothbrush moistened with tap water. This technique is especially helpful in younger children who may become frightened by the use of a scalpel. KOH exam is a useful in-office test for quick diagnosis, but a fungal culture is also recommended in cases of tinea capitis to direct the therapy. Rubbing a sterile cotton swab in the scan amount of fluid on top of a fungal plate or slant or with a small amount of water and then rubbing it vigorously on the affected area of the scalp and transferring it back to the fungal slant will help improve the yield of the fungal culture. Be sure to include some broken hairs on the fungal agar. After collecting the specimen from the skin, under the nail, or the scalp, apply several drops of potassium hydroxide with or without fungal stain immediately adjacent to the edge of the coverslip. The solution will be drawn under the coverslip by capillary action. Tilt one end up slightly, about 10 degrees, and gently put pressure on one end with the end of a pen for a few seconds to loosen the tiny bubbles that form under the coverslip to create a thin layer of skin cells. Some providers prefer to apply the solution, then place the coverslip. Either way works. Slides do not need to be heated to be examined. However, gentle application of heat from a lighter or other source can help accelerate dissolving the keratinocytes. You can also just leave the slide sitting while you see the next patient, then come back to it. 10 minutes is plenty of time to dissolve the keratinocytes enough. Longer may be needed for nail specimens. The use of fungal stains with your KOH also helps better visualize and may obviate the use of open flame or heat sources. If you will be doing a fungal culture, inoculate the plate with part of the specimen onto the fungal plate or slant. If you use the toothbrush or cotton swab to collect specimens, then you can gently touch this to the surface of the culture plate. Position the slide on the microscope stage and start examining under low power, or 10X, using low intensity light. Use the coarse focus knob to look at a cluster of epithelial cells or hair debris. Next, use the fine focus knob to focus in and out while viewing the specimen. High power, or 40X, is very rarely needed. To diagnose a fungal infection, you're looking for hyphae, which will appear as long, straight or curved, filamentous structures. They have uniform thickness and parallel walls throughout and may be divided along their length by septations. They may be branching. They will always cross the cell membranes of the remnant ghost-like epithelial cells when you adjust the fine focus knob on low power. Hyphae are refractal, meaning that when you adjust the fine focus knob up and down, you may note that the hyphae shift from looking darker to looking lighter with dark edges. Start at one corner of the coverslip and progress in an orderly search pattern to look at the entire specimen carefully. True fungal hyphae may need to be distinguished from artifacts like small bubbles, which are actually lipid drops that surround the outside of stratum corneum cells. These form a non-uniform mosaic pattern like a chicken wire fence or irregular polygons with lines that do not cross cell membranes. Fibers can also be distinguished from hyphae by their non-uniform diameter, non-parallel walls, lack of septae, and the fact that they do not cross cell membranes. Tinea versicolor. When diagnosing tinea versicolor, you're looking for short, linear, hyphae-like structures and clusters of uniformly sized round spores. This finding has a characteristic spaghetti and meatballs appearance. When diagnosing a fungal infection of the hair, you're looking for the presence of spores rather than hyphae in the hair specimen. These may be inside the hair shaft, an endothrix, or outside the hair shaft, an ectothrix. There are several reasons your KOH might be falsely negative. They include recent use of topical antifungals, examining too little specimen, or inexperience of the person performing the examination. If you don't have enough experience or supervision to feel confident in the results of your KOH exam, you may collect a specimen and submit it to your microbiology laboratory to perform a KOH exam for you. Collect about an eighth of a teaspoon of fine scale into a sterile specimen cup. Properly label it and submit an order for a KOH exam. Most labs that have microbiology services, fungal culture for instance, should be able to perform a KOH exam on submitted skin scrapings. It is important to submit an adequate sample. You have now learned how to conduct a KOH examination. As you've seen, KOH preparation is a simple, fast, and inexpensive test that can diagnose a fungal infection during a clinic appointment. If a patient presents with a scaling rash, do a KOH prep. Gather all your supplies ahead of time. Collect the skin sample from the edge of a lesion with a scalpel or another microscope slide. For a nail, collect the sample from under the nail with a scalpel or a curette. After applying KOH to your slide, look for hyphae, which cross cell membranes. If you have any doubt about the results, ask a more experienced provider to help you. The best way to improve your confidence in doing KOH exams is to do them repeatedly in the presence of mentors who can assist you in reading them. The more KOH exams you perform, the more confident you will be. This simple test in clinic is highly sensitive and cost effective and a great service to our patients. For more information, visit www.fema.gov

potassium hydroxide

KOH preparation

fungal elements

specimen collection

fungal infections