Isolation of tremelloid yeasts on glucuronate medium

D-Glucuronate-containing agar is suggested for evaluating the population density and diversity of tremelloid yeasts in natural cenoses. This medium is superior to the commonly used wort agar on which many representatives of tremelloid yeasts cannot be revealed.     

COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Evaluation of Media for Selective Isolation of Yeasts from Oral, Rectal, and Burn Wound Specimens

Six media were evaluated to determine their ability to isolate yeasts and inhibit bacteria. The media included the following: Snyder, Snyder tellurite, Sabouraud tellurite, Littman-gentamicin, molybdate, and Mycosel (BBL). Doses of mixed intestinal gram-negative bacilli and enterococci were most effectively inhibited by Snyder tellurite agar. Klebsiella pneumoniae was the most common bacterial contaminant of the other media. All six media were comparable in isolating yeasts while preventing the growth of the oral bacterial flora. The selection of a basal fungal growth medium for tellurite incorporation to inhibit bacteria but permit growth of yeasts was affected by pH. The bacteriostatic effect of tellurite was decreased with increasing pH of media while fungistatic action was increased. The arbitrary selection of Snyder and Littman agars to isolate yeast from burn wound cultures demonstrated the need to include a selective medium for these specimens. Blood, phenylethyl alcohol blood agar, and Columbia CN blood agar were all inadequate for isolating yeasts from burns. Growth of a variety of filamentous saprophytic and pathogenic dimorphic fungi grew adequately on four of five selective media tested.     

A visual method for rapid screening of xylose-fermenting ethanolic strains of Klebsiella pneumoniae.

A simple and rapid screening method for selecting hyper-ethanolic strains of Klebsiella pneumoniae is described. The method involves a novel biological screening marker, namely, the yeast Candida ethanothermophilum. The screening marker was seeded on an agar plate to the surface of which agar blocks, each containing a colony of K. pneumoniae, were subsequently fixed. This seeded plate lacked sources of carbon and energy. Ethanol formed in the agar blocks by the K. pneumoniae colonies diffused into the seeded medium and served as a carbon and energy source for the ethanotrophic yeasts. Colonies of yeasts appeared around the agar blocks in regions of ethanolic diffusion. Hyper-ethanolic strains of K. pneumoniae were thus selected on the basis of the number of colonies of the screening marker that appeared around the blocks containing the ethanolic colonies of K. pneumoniae.     

The Identification of Wild Yeast Colonies on Lysine Agar

The most common wild yeasts infecting pressed baker's yeast in Great Britain are Candida tropicalis, C. krusei, C. mycoderma, Trichosporon cutaneum, Torulopsis candida and Rhodotorula mucilaginosa. Wild yeasts are readily detected and quantitatively estimated by plating infected baker's yeast on lysine agar, which permits of only limited growth of baker's yeast.
Morphology of wild yeast colonies on lysine agar is affected by duration of incubation, location in the agar plate, and sometimes by temperature of incubation, density of infection and numbers of baker's yeast cells present. It is therefore possible to identify each species by at least one characteristic type of colony produced under specified conditions. Ability to grow at 30° and 37° serves to distinguish further between certain species.     

Influence of composition of diluent on populations of yeasts and moulds recovered from raw fruits

AIMS: The aims of this study were (i) to determine the retention of viability of mycoflora removed from raw fruits, and how this affected diluents used to prepare samples for enumeration of propagules, and (ii) to evaluate the performance of recovery media for supporting colony development. METHODS AND RESULTS: Yeasts and moulds removed from seven types of raw fruit were held in seven diluents for 1 h before plating on dichloran rose bengal chloramphenicol (DRBC) agar and plate count agar supplemented with chloramphenicol (100 micro g ml-1) (PCAC). Significant reductions (P=0.05) in populations of yeasts, moulds, and yeasts plus moulds occurred within the 1 h holding period, regardless of diluent composition. Overall, retention of viability was not influenced by diluent composition, and neither DRBC agar nor PCAC were superior in supporting colony development. CONCLUSIONS: The composition of diluents used to prepare food samples for mycological analysis has little affect on the number of yeasts and moulds recovered from seven types of naturally contaminated raw fruit. Both DRBC agar and PCAC are suitable as enumeration media. SIGNIFICANCE AND IMPACT OF THE STUDY: Diluents and media most often recommended for enumerating yeasts and moulds in foods are appropriate for raw fruits.     

Isolation and clonal pre-selection of enological Saccharomyces

The aim of the present study was to perform a fast pre-selection from a great number of wine yeasts using a simple phenotypic-based methodology that allows many different strains to be simultaneously tested. A total of 150 elliptic yeasts, isolated from must and wine from black grapes of a distinctive Italian variety, were studied. Yeasts were identified to genus level by assessing their ability to ferment glucose and their production of spores on acetate agar. The Saccharomyces strains were seeded on BiGGY agar to determine their H(2)S production, on calcium carbonate agar to test their acetic acid production, and on grape-skin agar and on grape-seed agar to assess their interaction with phenolic compounds. The Saccharomyces strains were also examined for fermentative vigor after 2 d or 7 d both with and without the addition of 100 mg L(-1) of SO(2) in must at 20 degrees brix and pH 3.20. At the end of fermentation, the wines produced by the 18 best yeasts were analyzed and the strains were studied for additional biochemical and technological characteristics. The resistance of the strains to simultaneous acid-stress and osmotic-stress was studied carrying out in duplicate winemaking tests in must at 30 degrees brix and pH 2.60. A remarkable heterogeneity among the 150 autochthonous yeasts studied was demonstrated. The phenotypical biodiversity is particularly interesting for several technological characteristics useful in winemaking, such as fermentation vigor, acetic acid production and malic acid content of the wines. The vast majority of the elliptic wine yeasts isolated did not show suitable characteristics, so only 18 strains, 12% of the total, remained for the final tests. Many of the strains that had passed the preliminary screenings revealed some defects when they were studied for fermentation performance, both in standard winemaking and under stressors. Two strains exhibited particularly interesting performances: one strain for winemaking of normal musts and the other for winemaking of musts from dried grapes or under stressful conditions.     

A note on an improved molybdate agar for the selective isolation of yeasts from tropical fruits

R ale , V.B. & V akil , J.R. 1984. A note on an improved molybdate agar for the selective isolation of yeasts from tropical fruits. Journal of Applied Bacteriology 56 , 409–413.
Molybdate agar was fortified with 0.125% calcium propionate and used for routine isolation and differentiation of a variety of yeasts from mixed floras including large numbers of fungi and actinomycetes inhabiting tropical fruits. The results suggest that this technique could be usefully incorporated in yeast isolation and identification procedures.     

RAPID METHOD FOR SCREENING THE TOLERANCE OF YEASTS TO ZINC (II) AND CHROMIUM (III)

Rapid screening of the tolerance of yeasts to zinc (II) and chromium (III) was performed by an agar diffusion test. A rapid and reliable procedure for the determination of metal ion concentration gradients on agar plates was developed. Different species of yeasts from the following genera were investigated: Arthroascus, Bulera, Dekkera, Debaryomyces, Dipodascopsis, Eremothecium, Candida, Hansenula, Kluyveromyces, Hormoacus, Geotrichum, Lipomyces, Pachysolen, Pichia, Saccharomyces, Schizosaccharomyces, Schizoblastosporion, Schwan-niomyces, Sporobolomyces, Yarrowia, Torulaspora, Zygosaccharomyces and Williopsis. The experimental conditions were defined as a constant volume of malt agar 32 cm³, and a temperature of 29C. After periods of 24, 47 and 72 h, the concentration intervals of growth inhibition were determined, and the yeasts investigated were grouped in different classes according to their tolerance to Zn (II) and Cr (III). Yeasts were found to be tolerant to significantly higher zinc (II) concentrations in the malt agar medium (5.9 mM to 20 mM) in comparison to chromium (III) (1.5 mM to 6.9 mM). Yeasts showed inter- and intra-generic differences in zinc (II) and chromium (III) tolerance.     

Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses

AIM: To test a total of 15 strains belonging to four species of yeasts by different in vitro methods against propolis and itraconazole (ITC). METHODS AND RESULTS: Three methods were compared for susceptibility testing of yeast isolates to propolis: disc diffusion method, agar dilution method and National Committee for Clinical Laboratory Standards (NCCLS, M27A) broth microdilution method. ITC was selected as the antifungal agent for comparison study. Using the broth microdilution method, the geometric mean for MIC (microg ml(-1)) with regard to all isolates was < or =0.06 for propolis and < or =0.35 for ITC. The broth microdilution and the agar dilution methods were in good agreement (75%) for propolis against yeasts isolated from patients with superficial mycoses. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (24 or 48 h). An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. A favourable correlation was found between MIC and inhibition zone around the disc for propolis sample and the correlation coefficient was: r = -0.626 (P < 0.01). CONCLUSIONS: This study suggests the potential value of the agar dilution and disc diffusion method as a convenient alternative method for testing of yeasts to propolis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that propolis and ITC were very active against yeasts from patients with superficial mycoses. The other prominent finding in this study is that RPMI 1640 with L-glutamine was the available broth for the in vitro susceptibility testing of yeasts.     

WL营养琼脂对葡萄酒相关酵母的鉴定效果验证

利用WL营养琼脂对采自葡萄园和葡萄汁发酵过程中的35株酵母菌进行了分类鉴定,同时进行了5.8S-ITS和26S rDNA D1/D2区的扩增与测序。结果表明利用WL营养琼脂的鉴定结果与测序结果基本符合。WL营养琼脂是一种较为有效的葡萄酒相关酵母菌的分类鉴定培养基。     

Antifungal Activity of Sodium Bicarbonate Against Fungal Agents Causing Superficial Infections

Although sodium bicarbonate—NaHCO₃ (SB) has many domestic and medical, traditional and empirical uses, only little scientific documentation of its activity is available. The aims of this study were to investigate the antifungal activity of SB on the three fungal groups (yeasts, dermatophytes and molds) responsible for human skin and nail infections. We first evaluated the in vitro antifungal activity of SB on 70 fungal strains isolated from skin and nail infections: 40 dermatophytes, 18 yeasts and 12 molds. A concentration of 10 g/L SB inhibited the growth of 80 % of all the fungal isolates tested on Sabouraud dextrose agar. The minimal inhibitory concentration 90 (MIC90) of SB measured on Sabouraud dextrose agar, Sabouraud dextrose broth and potato dextrose broth was 5 g/L for the yeasts, 20 g/L for the dermatophytes and 40 g/L for the molds. In a second step, we prospectively evaluated the ex vivo antifungal activity of SB on 24 infected (15 dermatophytes, 7 yeasts and 2 molds) clinical specimens (15 nails and 9 skin scrapings). The fungal growth was completely inhibited for 19 (79 %) specimens and reduced for 4 (17 %) specimens after 7 days of incubation on Sabouraud dextrose–chloramphenicol agar supplemented with 10 g/L of SB as compared to Sabouraud dextrose–chloramphenicol agar without SB. In conclusion, we documented the antifungal activity of SB on the most common agents of cutaneous fungal infection and onychomycosis, and we specified the effective concentrations for the different groups of pathogenic fungi. The mechanism of action of SB has yet to be explored.     

In vitro comparison of antifungal activity of oxiconazole and econazole against yeast.

Oxiconazole was compared in vitro with econazole in tests with 400 yeasts strains. Tests were performed by measurement of growth inhibition by both microdilution in Sabouraud broth and a Shadomy' agar diffusion systems. The sensitivity/resistance percentages were similar in Candida albicans strains. Oxiconazole showed a higher activity than that of econazole in Candida spp. and yeasts other than Candida.     

Modification of Cycloserine Cefoxitin Fructose Agar to Suppress Growth of Yeasts from Stool Specimens

Cycloserine cefoxitin fructose agar is a widely used selective isolation medium for Clostridium difficile from stool specimens. Yeasts often colonize in the intestine of C. difficile disease patients and, if colonized heavily, pure culture of C. difficile can be delayed. The aim of this study was to modify cycloserine cefoxitin fructose agar to suppress the growth of yeasts. Antimicrobial activities of three commonly available antifungal agents were tested against recent clinical isolates of Candida species. Amphotericin B was most active in inhibiting all isolates by ≤0.5 mg/L concentration. Cycloserine cefoxitin fructose agar was modified by adding 2 mg/L of amphotericin B. Serial ten-fold dilution of stool specimens from 126 suspected C. difficile -associated diarrhea patients were cultured both on cycloserine cefoxitin fructose agar plates and modified agar plates. Yeasts grew from 60 specimens on cycloserine cefoxitin fructose agar, but none grew on the modified medium. Growth of C. difficile was detected from 37 and 39 of 126 specimens on cycloserine cefoxitin fructose agar and modified medium, respectively. The number of C. difficile colonies was similar on both media. In conclusion, 2 mg/L of amphotericin B supplementation to cycloserine cefoxitin fructose agar can facilitate the isolation of C. difficile from stool specimens which are densely colonized with yeasts.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

Analysis of non‐Saccharomyces yeast populations isolated from grape musts from Sicily (Italy)

Aims: The aim of this study was to identify the non‐Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Methods and Results: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5·8S‐internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. Conclusions: We isolated non‐Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. Significance and Impact of the Study: This investigation is a first step in understanding the distribution of non‐Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non‐Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.     

Rapid method for detection of urea hydrolysis by yeasts.

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.     

Preservation of microorganisms

Cultures of bacteria, yeasts and fungi were grown on common agar media in normal culture tubes. About 1 week after maximum growth the cotton plugs of the tubes were replaced by sterile rubber seals. The cultures were stored in the dark at room temperature. Tests for viability of cultures were made after periods between 1 and 10 years. The results of this simple method show long survival periods of many bacteria and yeasts.     

Rapid method for detection of urea hydrolysis by yeasts.

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.