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| Laboratory \ Chlorazol |
Chlorazol Fungal Stain Information / Comments
| A Simple Stain for Rapid Office Diagnosis of Fungus Infections
William A. Burke, MD, Billy E. Jones, MD
Archives of Dermatology, Vol 120, pages 1519 - 1520, 1984.
Direct microscopy using potassium hydroxide as a clearing agent is a diagnostic procedure commonly used for the detection of fungal hyphae in suspected dermatophytoses, tinea versicolor, and candidiasis. Although this method is quick, simple and inexpensive, it lacks sensitivity (especially when hyphae are sparse in the specimen), and often the physician must rely on culture results.
A practical, more sensitive method for the detection of fungal infections of the skin would be useful to the office practitioner. Routine histopathologic stains for fungi (eg PAS, silver methenamine) and fluorescent techniques are time-consuming, complex, and certainly not amenable to routine office settings. Many investigators have attempted to find an improvement on the routinely used potassium hydroxide wet mount.
Early investigators used Parker's blue ink in an alkaline solution for staining of fungal hyphae. (Delasco Note: Delasco's Swartz-Lamkin stain is an adaptation of this formula, using actual Parker Pen Ink in the concentrated powdered form as the main ingredient, along with KOH.)
Swartz and Medreck described a rapid-staining procedure using Parker's ink and rose bengal, which provided for a contrast between fungi and background cells. (Delasco Note: Bengal Red Counterstain is available from Delasco). Using a different approach, other researchers used cyanoacrylate adhesives followed by Gram's and PAS staining. A later publication reported good results using Parker's ink and potassium hydroxide with this method of "biopsy".
The present communication reports a practical, more sensitive modification of the potassium hydroxide preparation using chlorazol black E stain.
METHODS
Chlorazol black E stain was prepared by first dissolving the dye in dimethyl sulfoxide (DMSO) (100 mg of chlorazol black E in 10 mL of DMSO). This was then added to 90 mL of water containing 5 g of
potassium hydroxide. (Delasco Note: This is the method and proportions used for Delasco Chlorazol Black Fungal Stain.) The solution was then used unfiltered.
The preparation of a microscopic mount with this stain was identical to a routine potassium hydroxide wet mount. Two to three drops of the staining solution were added to the specimen on a clean glass slide and a coverslip applied. The slide was then gently heated by passing through a flame several times for 15-30 s. The cover glass and slide were pressed between pieces of filter paper to remove excess bubbles and then examined microscopically under low ("scanning") power with "high-dry" power used for confirmation of the fungus.
RESULTS
Microscopic examination of dermatophyte-infected skin scrapings stained by this method showed hyphae that stained green against a cellular background. The mycelial elements and spores of Candida and tinea versicolor stained as well as dermatophytes. Photomicrographs of tinea unguium are shown (Figs 1 to 3). Although the stain was immediately taken up by the fungal cell wall in the scrapings, the staining usually became much brighter after five to ten minutes.
In addition, preparation of thick scales (eg, palms or soles) sometimes required five to ten minutes before a definitive diagnosis could be made. When there was inadequate clearing of the cellular background, repeating the heating step was necessary. Heating seemed to enhance the staining qualities. Although boiling is not recommended, it did not seem to impair staining. Unlike the routine potassium hydroxide preparation, we found that bright light was best when scanning the slide. The use of DMSO in the stain mixture was not necessary, but provided for a much clearer stain solution by increasing the solubility of the dye.
COMMENT
Among the previously reported improvements on the routinely used potassium hydroxide wet mounts, various problems were encountered. Parker's blue ink in an alkaline solution was not entirely selective for fungi. Difficulty was encountered with thicker sections due to blue staining of the cellular background and blue staining of clothing fibers.
The rapid staining procedure using Parker's ink and rose bengal described by Swartz and Medreck provides a contrast between fungi and background cells. Although this stain worked well in our trials, it was difficult to prepare (especially for the office practitioner). (Delasco Note: Now preprepared as Delasco's Swartz-Lamkin Fungal stain) and was not effective for thicker specimens e.g., nails.)
Skin surface biopsies using cyanoacrylate adhesives followed by Gram's and PAS staining and using Parker's ink in potassium hydroxide were frequently painful for the patient. Another drawback to this method is the inability to obtain material from the interdigital areas, nails, palms, or soles. Gram's staining with this technique was usually inadequate in our experience, primarily due to a positive staining of epithelial cell "granules" (thought to be desmosomes).
The method using skin surface biopsies stained with Parker's ink and potassium hydroxide was less sensitive in detecting isolated fungal hyphae. The alkaline solution dissolved the cyanoacrylate adhesive (especially if heated) and was usually inadequate in clearing the relatively thick (three to five cells) sections.
Chlorazol black E is an acid tris-azo dye that was originally described by Cannon who later reviewed its uses. Armitage described the compatibility of chlorazol black E with lactophenol, a commonly used mycologic mounting medium. However, the stain is much more soluble in an alkaline solution, and in our trials gave a much better result with potassium hydroxide. This dye has been primarily used as a botanic stain, with only occasion use in animal tissues.
The stain is selective for chitin, which it stains green against a cellular background of colorless to gray-black. As with most mycological stains, young hyphae are stained much more readily than old. Since the stain is selective for chitin, few artifacts are noted.
The dye is compatible with the addition of glycerin for those who prefer glycerin in their potassium hydroxide mounts. We have used this staining preparatin on skin, hair and nails with good results. The mycelial elements and spores of Candida and tinea versicolor also stain well.
We propose that the alkaline chlorazol black E Solution gives a substantial increase in sensitivity over the routinely used potassium hydroxide preparation. This is especially true for the inexperienced observer. The stain requires no mordant or differentiation, and gives a clear-cut picture of fungal nuclei and cytoplasmic structures. The hyphae are readily visible under low power (using bright light) so that an entire slide may be rapidly scanned.
The green, gray, and black tones are well-suited for photomicroscopy. We have been using a mixture of the dye for several months without loss of staining characteristics. Amber bottles or protection from light are, however, necessay. Although we have not made permanent mounts using this stain, Cannon reports no fading after one year in bright light.
Figures:
Fig 1.--Stained scrapings from tinea unguium of feet. Mycelial elements are readily apparant under low "scanning" power. (X200)
Fig 2.--Stained scrapings from tinea unguium of feet. Cellular detail is readily apparant at higher power for easy identification(X400)
Fig 3.--Stained scrapings from tinea unguium of feet. STain is selective for chitin in fungal cell wall (X1,000)
End Article
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Delasco Chlorazol Fungal Stain
Supplied in plastic squeeze opaque white dropper bottles, 1/2 oz and 1 oz. Should be stored in a dark place. The Chlorazol stain deteriorates over time, and should usually be replaced at intervals of 6 to 9 months.
If kept in a dark place, one years service may be obtainable. The correct color is very bluish-black to black color. When it develops a coffee-like to slightly lavenderish color it should be discarded.
It is supplied ready to use, as described in the above article. No mixing is required.
Chlorazol Black fungal stain is also a good stain to use for the Durotak fungal touch preparations which is a variation of the cyanoacrylate preparations described above, but designed for easy and rapid office use.
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