Dermatology Online Journal

Practical tip: Chicago Sky Blue (CSB) stain can be added to the routine potassium hydroxide (KOH) wet-mount to provide a color contrast and facilitate the diagnosis of dermatomycoses
Christopher Seng-Hong Lim MBBS, Siew-Lin Lim MBBS
Dermatology Online Journal 17 (8): 11

Central Manpower Base, Ministry of Defence, Singapore, Singapore

---

Abstract

Rapid confirmation of dermatomycoses is desirable because it allows the clinician to initiate appropriate therapy without delay. The routine potassium hydroxide (KOH) wet-mount is cheap and rapid to use but this method lacks a color contrast. We offer a simple practical tip of adding Chicago Sky Blue (CSB) stain to KOH to highlight fungal elements and provide a color contrast that makes reading and interpretation simple, even for the novice.

---

Most laboratories, especially those outside the developed world, routinely use the KOH wet-mount for the rapid diagnosis of dermatomycosis. However, the KOH wet-mount lacks a color contrast and considerable skill is required in its interpretation. Parker blue-black ink has been added to KOH to provide a color contrast but this method only works well for Malassezia furfur [1]. Additionally, current batches of Parker blue-black ink do not seem to work well with KOH, possibly because of some change in the formula. The Swartz-Lamkins stain highlights both M furfur and dermatophytes blue and has been found to be useful by many laboratories [2]. Chlorazole-KOH is 60 percent sensitive and 71.9 percent specific, but may be a potential carcinogen. KOH-acridine orange staining has a sensitivity of 61.7 percent and specificity of 67.2 percent [3]. Calcofluor White with KOH is 92 percent sensitive and 95 percent specific [4] and is clearly superior, but it requires a fluorescent microscope, which is not available in most mycology labs. Skin surface and nail biopsies followed by periodic acid-Shiff (PAS) stain [5,6] is more complicated, less suitable for obtaining samples from interdigital webs, and impractical for most mycology laboratories. Polymerase chain reaction (PCR) methods offer the possibility of rapid species identification but such molecular approaches are still developing and not available for general use [7]. CSB stain contains Chicago Sky Blue as one of its constituents. It can be added to KOH to highlight fungal elements and provide a color contrast that makes reading and interpretation simple, even for the novice [8, 9]. We report on the methodology and utility of adding CSB stain to KOH for the rapid diagnosis of dermatomycoses.

The method of staining is very simple. Areas of skin to be scraped are first cleaned with an alcohol swab to remove traces of creams and reduce surface bacteria. Scrapings are taken from the affected skin or nail surfaces with a blunt scalpel and subungual debris is obtained with an eye curette. Nail clippings are taken to include subungual debris as far proximal as is tolerable and scrapings taken from their undersurface. The latter is useful for reducing the digestion time normally required for full nail clippings. Collected samples are placed on clean microscope slides and fixed by adding a drop of acetone and left to dry. A drop of 20 percent KOH is then added and the slides are left to sit at room temperature for 20-30 minutes before adding a drop of CSB stain. The slides are examined after 10 minutes or more with an ordinary light microscope at x10 and x40. We used an Olympus BX41 for this purpose. Oil immersion at x100 magnification can be used for studying the morphology in greater detail.

Figure 1	Figure 2
Figure 1. Pityriasis versicolor: CSB stain shows the short bluish hyphae and flask-shaped yeasts of Malassezia furfur (original magnification x400). Figure 2. Tinea corporis related to Trichophyton rubrum: CSB stain of skin scrapings show bluish septate hyphae against a pink cellular background (original magnification x400).

M. furfur can be easily visualized as short bluish angular hyphae and spores in a spaghetti and meatballs arrangement against a pink-purplish background of cellular debris (Figure 1). Uptake by M. furfur is extremely fast and we found that M. furfur slides can be read almost immediately after adding CSB stain.

Dermatophytes can be detected as blue hyphae against the pink-purplish background of cellular debris at x10 magnification and confirmed on the finding of septate hyphae at x40 magnification (Figure 2). Septate hyphae are more clearly seen under oil immersion (Figure 3).

Figure 3	Figure 4
Figure 3. Same slide as depicted in Figure viewed under oil immersion (original magnification x1000). Figure 4. Candidiasis of the groin related to Candida albicans. CSB stain shows the blue and pink budding yeasts and pseudohyphae (original magnification x400).

Candida albicans takes up the stain lightly and stains pink or light blue (Figure 4). More intense staining can be observed if the slides are left overnight in a humidifying chamber (covered plastic container lined by moist paper towel).

Figure 5	Figure 6
Figure 5. Toenail onychomycosis due Fusarium spp. CSB stain shows bluish septate branching hyphae (original magnification x400). Figure 6. Same slide as depicted in Figure 5 viewed under oil-immersion: CSB Stain shows septate hyphae with dichotomous branching (oil immersion, original magnification x1000).

The septate hyphae of non-dermatophyte molds appear to be wider, more variable in diameter and more contorted and branched than dermatophytes (Figure 5), especially when viewed under oil immersion (Figure 6).

In conclusion the addition of CSB stain to KOH provides a color contrast, making interpretation easy. CSB highlights dermatopytes well and is an advantage over the Parker blue-black ink, which is better for highlighting M. furfur than dermatophytes [1]. CSB is a useful alternative to the Swartz-Lamkins stain [2]. The CSB stain is inexpensive and requires an ordinary light microscope for reading as opposed to Calcofluor White, which incurs the extra expense of an immunofluorescence microscope. The cost for a 12cc bottle of CSB stain, which stains about 200 specimens, is only $25.00 (US). CSB stain also allows for a detailed study of morphology under oil-immersion. Whether this is sufficient for genus identification requires further study by experienced mycologists.

References

1. Crespo-Erchiga V, Florencio VD. Malassezia yeasts and pityriasis versicolor. Curr Opin Infect Dis. 2006 Apr;19 (2):139-47. [PubMed]

2. Swartz JH, Lamkins BE. Rapid, Simple Stain for Fungi in Skin, Nail Scrapings, and Hairs. Arch Dermatol. 1964 Jan; 89(1):89-94. [PubMed]

3. Panasiti V, Borroni RG, Devirgiliis V, Rossi M, Fabbrizio L, Masciangelo R, Bottoni U, Calvieri S. Comparison of diagnostic methods in the diagnosis of dermatomycoses and onychomycoses. Mycoses. 2006 Jan; 49 (1): 26-9. [PubMed]

4. Haldane DJ, Robart EA. Comparison of calcofluor white, potassium hydroxide, and culture for the diagnosis of superficial fungal infection. Diagn Microbiol Infect Dis. 1991 Jul-Aug; 13 (4): 337-9. [PubMed]

5. Lousberg D, Buntinx F, Pierard G. Diagnosis dermatomycosis in general practice. Family Medicine. 1999 Dec; 16 (6): 611-5. [PubMed]

6. Weinberg JM, Koestenblatt EK, Tutrone WD, Tishler HR, Najarian L. Comparison of diagnostic methods in the evaluation of onychomycosis. J Am Acad Dermatol. 2003 Aug; 49 (2): 193-7. [PubMed]

7. Bontems O, Hauser PM, Monod M. Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis. Br J Dermatol. 2009 Oct. 161 (4): 791-6. [PubMed]

8. Lim SL, Lim CSH. New contrast stain for the rapid diagnosis of pityriasis versicolor. Arch Dermatol. 2008 Aug; 144 (8): 1058-9. [PubMed]

9. Lim CSH, Lim SL. New contrast stain for the rapid diagnosis of dermatophytic and candidal dermatomycoses. Arch Dermatol. 2008 Sep; 144 (9): 1228-9. [PubMed]

© 2011 Dermatology Online Journal