The Role of Trypan Blue as a Conventional and Fluorescent Dye for the Diagnosis of Superficial Mycoses by Potassium Hydroxide (KOH) Testing

Direct microscopic examination of samples using potassium hydroxide (KOH) is a fast, simple, and inexpensive method to confirm clinical suspicion of superficial mycosis. However, the lack of color contrast in this test makes it difficult to separate any fungal structures from artifacts. The sensitivity of the KOH mount technique may be enhanced using both fluorochromes and conventional stains that highlight the fungal structures when observed under fluorescence microscopy and bright-field, respectively. Here we study the possibility of using Trypan Blue (TB), an azo dye which is often used as a live/dead marker, in the diagnosis of superficial mycoses by KOH testing. TB at 0.01% displayed a fluorescent staining pattern similar to that of Calcofluor White (CFW), the conventional cell wall fluorophore. Furthermore, by adjusting the TB concentration to 0.1–0.3%, in addition to maintaining the fluorescent staining pattern, the fungal elements were stained in blue under bright-field microscopy. Thus, we demonstrate for the first time that TB has the unique property as a fungal stain that can be used in both bright-field and fluorescence microscopy for diagnosis of superficial mycoses by direct microscopic examination.