Rapid Diazonium Blue B Test to Detect Basidiomycetous Yeasts

Abstract

More than 100 known eucaryotic microorganisms were examined for capacity to form red to violet pigments with diazonium blue B after alkaline hydrolysis and an ethanol wash. All 50 basidiomycetes gave rapid and unequivocal positive reactions when 1-to 6-day-old cultures were tested, whereas all ascomycetes and algae gave negative reactions. Extracellular enzymes generally considered to be typical of basidiomycetous yeasts were detected in several ascomycetous taxa.A variety of physiological and morphological characteristics distinguish the anamorphs of ascomycetous and basidiomycetous yeasts. Ascomycetous yeasts have two-layered cell walls that are attacked by (1,3)-P-glucanases to release protoplasts, have deoxyribonucleic acid guanineplus-cytosine contents of about 30 to 60%, frequently are fermentative, usually lack extracellular urease and deoxyribonuclease (DNase), and have respiratory enzymes of coenzyme Q types 6 through 9 (2,3,11,14,20,25). Basidiomycetous yeasts have laminar cell walls that resist (1,3)-P-glucanase activity, have deoxyribonucleic acid guanine-plus-cytosine contents of about 50 to 7056, with rare exception are strictly oxidative, generally have extracellular urease and DNase, have respiratory enzymes of coenzyme Q types 7 through 10, and give a red to violet color reaction with diazonium blue B Sonck and E. Tunnela, Antonie van Leeuwenhoek J. Microbiol. Serol. 35(Suppl.) :E27, 1969). Except for the cell wall properties, these characteristics overlap among the species of these two groups.The DBB reaction appears to be the most practical test for establishing basidiomycetous relationships in laboratories, but unfortunately the DBB test has not been standardized. A variety of different media and incubation times of several weeks have been employed, with some species giving questionable results. Therefore, the use of the DBB test is not widespread. This report describes an improved DBB test to detect basidiomycetous relationships of yeasts and yeastlike organisms and correlates the results of the DBB test with extracellular enzyme production.(DBB) (2,3,6,8,9,11,14,17,(20)(21)(22) 26; C. E. MATERIALS AND METHODSOrganisms. All strains used in this study were authenticated cultures obtained from the freeze-dried culture collection at Georgia State University or from other laboratories specializing in the taxonomy of yeasts (Table 1). Routinely, cultures were maintained by monthly transfers on Sabouraud dextrose agar; the cultures were incubated for 3 days at 25°C and were then stored at 4OC. DBB reagent. The DBB reagent was prepared by adding 15 ml of cold 0.25 M tridhydroxymethy1)aminomethane buffer (pH 7.0) to 15 mg of DBB (o-dianisidine, tetrazotized; practical grade; 20% pure; Sigma Chemical Co.). DBB is unstable in warm and moist conditions, but 15-mg portions of the dye can be stored in well-sealed test tubes at 4OC for several weeks. The dissolved reagent was maintained in an ice bath and was used either before it turned dark yellow or within about 30 min.Alkali-ethanol-DBB test. Culture...

Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences

“…lusitaniae and C . krusei is supported by a negative diazonium blue B staining reaction (Van der Walt & Hopsu-Havu, 1976;Hagler & Ahearn, 1981) and a negative urease test (Hagler & Ahearn, 1981).…”

A colorless variant derived from Rhodosporidium toruloides strain YK 212 (IAM 13509, IFO 1236) and its taxonomic implication.

“…The activity of extracellular deoxyribonuclease (DNase) was determined by the method of SEN and KoMAGATA (19). The Diazonium Blue B (DBB) color test was performed according to the methods of VAN DER WALT and HoPsu-HAVV (20) and HAGLER and AHEARN (21). Carotenoid pigments were detected by the method of HASEGAWA et al (22).…”

Distribution of pyrophosphatidic acid in yeast species.

“…The chilled DBB (1 to 2 drops) was applied directly to the surface of colonies, of Pyrophosphatidic Acid in Yeast Species 167 and the color reaction was recorded. The alkaline-ethanol procedure was applied to the red yeast strains according to the method described by HAGLER and AHEARN (6). The strains were grown in 5.0 ml of Yeast Nitrogen Base with O.5° glucose for 5 days at 24°C.…”

“…The DBB test (1,(3)(4)(5)(6)12), in conjunction with the cell wall structure, was a practical procedure for discrimination between ascomycetous and basidiomycetous yeasts in which a sexual stage was not demonstrated. However, the simple procedure on solid medium often gave indecisive results, particularly in the coloration of red yeasts such as Rhodosporidium, Rhodotorula, and Sporobolomyces.…”

“…Ascomycetous and basidiomycetous yeasts have been discriminated by characteristics of cell wall structure (1)(2)(3)(4), Diazonium Blue B (DBB) staining (4)(5)(6), urease activity (7,8) and the GC content of DNA (9)(10)(11)(12). Ascomycetous yeasts have twolayered cell walls, are not stained by DBB, usually lack extracellular urease, and have about 30 to 50 mol% GC content.…”