In our studies concerning the ability of some yeasts and fungi to hydrolyse chromogenic peptide substrates, among them aminoacid naphthylamides, a diazonium reaction was used in order to couple the enzymically liberated naphthylamine to get a coloured azo dye. In the system employed, the substrate was brought into contact with the yeast and some hours later diazonium salt was added. It was found that the colonies of some species of yeasts were constantly stained more intensely than the others. In control experiments it was found that the same colonies were stained also in the absence of the substrates, and consequently the colour had to be due to a direct reaction of the diazonium salts with some structural groupings or with metabolic products of the yeasts. This suggested a wider study of the diazonium staining of the yeasts commonly met in dermatologic yeast diagnostic. It might even be possible to find a test to be used as a tool in routine diagnostic. This report presents the methods used, some of the results obtained and suggests an explanation to the observations made.
Material and MethodsR e a g e n t s. Stabilized diazonium salts Blue B (0-dianisidine), Garnet GBC (4-amino-3:1-dimethyl azobenzene), Red B (5-nitro-o-anisidine), Red RC (4-chloro-o-anisidine), Red TR (4-chloro-o-toluidine) and Violet B (2-benzoyl-amino-4-methoxy toluidine) were from Imperial Chemical Corporation, Ltd., England, or from Verona Dyestuffs, New Jersey, USA. The diazonium salts ware dissolved in 0.1 M Tris-HC1 buffer pH 7.0 (1 mgiml) just before use in order to prepare the diazonium reagent used. Sodium salts of the keto acids, pyruvic, a-ketoglutaric and oxalacetic acid as well as the usual laboratory reagents (analytical grade) were from commercial sources. Ketoacid salts were dissolved in distilled water at a concentration of 2 mg/4 ml. Y e a s t c u 1 t u r e s. The yeasts were collected in the daily routine diagnostic in our clinic. They were isolated and grown on G r ii t z -K i m m i g ' s test agar plates (a modified Sabouraud-agar containing Penicillin and Streptomycin) or exceptionally on rice, corn meal, glucose or maltose agar. Identification of the yeasts was based on the outlook of the colonies, microscopic study, fermentation, assimilation and other characteristics, as described by L o d d e r and K r e g e r -v a n R i j (1952), F r a g n e r (1958) and R i e t h u. S c h 6 n f e 1 d (1959). The age of the colonies used for diazonium tests varied from 2 to 2,O days, usually 10-12 days. Yeasts were tested just after isolation as well as after cultivation of several generations. T h e d i a z o n i u m t e s t . A drop of the freshly prepared diazonium salt reagent was applicated on the yeast colony. The result was read 30 seconds later. The test was considered positive if the colony was stained red. A negative test showed only a yellowish staining of the agar proteins.Ex p e r i m e n t s w i t h k e t o a c i d s. About 4 ml of the solutions of the ketoacids were mixed with 0,5 ml of the solutions of the various d...
