Provided - CSB Fungal Stain 12ml as the contrast stain

Required but not provided - 10-30% Potassium hydroxide (KOH) as the clearing agent

Method of staining:

Clean area well with alcohol swab to remove all traces of cream and reduce surface bacteria that might otherwise make reading of the slide difficult. Scrape area with a no. 15 scalpel blade, taking care to obtain an adequate sample. In cases of dermatophytosis (ringworm), scrape the outer margin of the ring, if present. Otherwise scrape areas that are scaly. Hair specimens should include hair stubs, the contents of plugged follicles and skin scales. Hair may also be plucked from the scalp with epilation forceps. Take nail clippings as far proximal as is tolerable to include subungual debris and take scrapings from their undersurface for staining. Place the specimen in the center of a clean microscope slide and proceed as follows:

1. Add a drop of acetone to fix the slide and leave to dry completely.

2. Add a drop of the Clearing Agent (KOH) and let slide sit for about 5 - 20 minutes for skin scales and hair. 20 minutes to 2 hours may be required for thicker nail specimens. Slides for suspected Malassezia furfur (tinea versicolor) can be stained immediately.

3. Add a drop of the CSB Stain and mix with an orange stick.

4. Place the slide in a humidifying chamber (any covered plastic container lined by moist paper towel) for at least 20 minutes at room temperature.

5. Apply a cover slip and press gently to flatten the specimen and remove air bubbles. Blot off excess stain. Grip the cover slip and slide with the thumb and index fingers and use the other hand to twist the slide to and fro to crush and further flatten the specimen.

6. Scan the slide under x10 magnification to identify fungal elements -M furfur and dermatophytes stain light blue against the purplish background of cellular debris. Increase to x40 magnification to confirm the fungal spores and hyphae. Short blue angular hyphae and variable sized spores in a "spaghetti and meatballs" arrangement suggests tinea/pityriasis versicolor; blue filamentous, septate, hyphae with or without arthrospores suggests dermatophytosis. Blue staining artifacts such as clothing fibres can be differentiated by their spiral configuration and absence of septa.

7. Candida species take up the stain less well and more slowly. After checking for M furfur and dermatophytes, lower the condenser to make candidal yeasts refractile against the puplish cellular background. Candida is confirmed on the finding of refractile oval budding yeast cells and pseudohyphae.

8. Oil immersion (x100 magnification) may be used for more detailed study of fungal morphology and to better differentiate artifacts such as clothing fibres.

9. Return the slide to the humidifying chamber and re-examine it the following day. Candida spp should by then have taken up enough of the stain to be seen without lowering the condenser. Other fungi will also appear more intensely stained.

Note:
Fungal cell walls take up the blue stain to different degrees:

· Cell walls of M. furfur and dermatophytes take up the blue stain well and can easily be seen against the purplish cellular background.
· Cell walls of candida species take up the blue stain more slowly and less intensely. Lower the microscope condenser to make the hyphae refractile. Additionally, examine the slide again after sitting in the humidifying chamber overnight.

Caution:
CSB Fungal Stain is non-hazardous but can stain skin and clothes. KOH is a known skin irritant. Wear gloves and overalls to protect skin and clothes. Rinse thoroughly with water in case of accidental contact.

References
1. Lim SL, Lim CSH. New contrast stain for the rapid diagnosis of pityriasis versicolor. Arch Dermatol. 2008; 144: 1058-9.
2. Lim CSH, Lim SL. New contrast stain for the rapid diagnosis of dermatophytic and candidal dermatomycoses. Arch Dermatol. 2008; 144: 1228-9.

For more information or distribution rights, please email: csbstain@gmail.com