BiGGY Agar Research Articles

Clonal selection of wine yeasts with differential adsorption activities towards phenolics and ochratoxin A

ABSTRACT To design a rapid, simple, and low-cost procedure for yeast selection with differential adsorption activities toward phenolics and ochratoxin A, 284 yeast strains were screened. This was done by evaluating the type of growth during grape must fermentation, acetic acid production on Chalk agar, H2S production on BiGGY agar, and spore-formation on acetate agar. After that initial step, 84 strains were pre-selected and further studied by Petri plate tests and to determine their wine-making ability in trials and evaluating their differential adsorption activities toward phenolics and ochratoxin A. Three yeast strains were selected based on the above evaluations. After confirming that they belonged to Saccharomyces cerevisiae species and were diploids, a spore clonal selection was performed. The strain Sc1741A_1D was selected and used in winemaking at six Calabrian wineries and found to be suitable as wine starter to improve quality and safety of red wines.

Identification of Candida species by direct inoculation of cornmeal tween™ 80 agar from blood culture bottles: Rapid and cost-effective approach

Background: This study aimed to compare the accuracy of Candida species identification by direct inoculation of positive blood culture specimens into CornMeal Tween™ (CMT) 80 agar with the conventional methods. Methods: This study was performed in two phases: Phase 1: Inoculation of direct CMT from artificially spiked blood culture bottles with identified Candida species. Phase 2: Direct inoculation of smear-positive bottles on CMT agar for identification. Routine identification method was carried out using urease production, sensitivity to cycloheximide, CMT inoculation, and colony morphology on BiGGY agar. All isolates were also evaluated for sugar assimilation on the biochemical test panel Analytical profile index (API) 20C AUX (BioMerieux). Statistical Analysis Used: The correlation between both methods was determined as percentage agreement rate using kappa score. Results: In the first phase 50 artificially spiked blood culture bottles were included in the study. The sample set consisted of 24% C albicans, 38% C tropicalis, and 22% C glabrata isolates. There was 100% agreement between the directly inoculated and CMT inoculated from colonies at 48 hours. Phase 2: The overall agreement between directly inoculated CMT and conventional method of identification at 72 h was 92%. Conclusion: Direct inoculation of CMT agar can easily differentiate common Candida species microscopically on the basis of chlamydospores, blastopores, and arrangement of pseudo hyphae. However, identification of rare species from specimens containing two different species using direct method is challenging. Therefore, conventional methods including macroscopic/microscopic characteristics and biochemical profile with the aid of API with API 20 AUX would be a better choice for such cases.

Identification of MET10-932 and characterization as an allele reducing hydrogen sulfide formation in wine strains of Saccharomyces cerevisiae.

A vineyard isolate of the yeast Saccharomyces cerevisiae, UCD932, was identified as a strain producing little or no detectable hydrogen sulfide during wine fermentation. Genetic analysis revealed that this trait segregated as a single genetic determinant. The gene also conferred a white colony phenotype on BiGGY agar (bismuth-glucose-glycine-yeast agar), which is thought to indicate low basal levels of sulfite reductase activity. However, this isolate does not display a requirement for S-containing amino acids, indicating that the sulfate reduction pathway is fully operational. Genetic crosses against known mutations conferring white colony color on BiGGY agar identified the gene leading to reduced H(2)S formation as an allele of MET10 (MET10-932), which encodes a catalytic subunit of sulfite reductase. Sequence analysis of MET10-932 revealed several corresponding amino acid differences in relation to laboratory strain S288C. Allele differences for other genes of the sulfate reduction pathway were also detected in UCD932. The MET10 allele of UCD932 was found to be unique in comparison to the sequences of several other vineyard isolates with differing levels of production of H(2)S. Replacing the MET10 allele of high-H(2)S-producing strains with MET10-932 prevented H(2)S formation by those strains. A single mutative change, corresponding to T662K, in MET10-932 resulted in a loss of H(2)S production. The role of site 662 in sulfide reduction was further analyzed by changing the encoded amino acid at this position. A change back to threonine or to the conservative serine fully restored the H(2)S formation conferred by this allele. In addition to T662K, arginine, tryptophan, and glutamic acid substitutions similarly reduced sulfide formation.

Survey of Hydrogen Sulfide Production in Wine Strains ofSaccharomyces cerevisiae

The level of variation in hydrogen sulfide formation as a function of yeast genetic background was evaluated for 170 strains, comprised of 169 commercial strains and one native isolate, using six different media: BiGGY agar (assess basal levels of sulfite reductase), synthetic grape juice media with differing nitrogen and vitamin content, and Syrah grape juice. Statistical and cluster analysis placed the strains into 15 clusters, ranging from 1 to 43 members. Sulfide production was not well correlated with the nitrogen content of the medium. In general, sulfide formation was highest in the Syrah juice, in both total number of strains producing the compound and in level of production. There was no correlation between colony color on BiGGY agar and level of production of H₂S in any of the liquid media. Strains producing high levels of H₂S in Syrah also tended to produce H₂S in at least one of the synthetic media. Many moderate to low level producers of H₂S in Syrah produced trace to undetectable levels of H₂S in synthetic media. Thus, synthetic media can be used to identify strains with the potential to produce high levels of H₂S during fermentation. However, a low level of sulfide production in synthetic juices does not indicate low level of production in juice, and strain evaluation in actual juices is still necessary.

Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

A screen of the Saccharomyces cerevisiae deletion strain set was performed to identify genes affecting hydrogen sulfide (H(2)S) production. Mutants were screened using two assays: colony color on BiGGY agar, which detects the basal level of sulfite reductase activity, and production of H(2)S in a synthetic juice medium using lead acetate detection of free sulfide in the headspace. A total of 88 mutants produced darker colony colors than the parental strain, and 4 produced colonies significantly lighter in color. There was no correlation between the appearance of a dark colony color on BiGGY agar and H(2)S production in synthetic juice media. Sixteen null mutations were identified as leading to the production of increased levels of H(2)S in synthetic juice using the headspace analysis assay. All 16 mutants also produced H(2)S in actual juices. Five of these genes encode proteins involved in sulfur containing amino acid or precursor biosynthesis and are directly associated with the sulfate assimilation pathway. The remaining genes encode proteins involved in a variety of cellular activities, including cell membrane integrity, cell energy regulation and balance, or other metabolic functions. The levels of hydrogen sulfide production of each of the 16 strains varied in response to nutritional conditions. In most cases, creation of multiple deletions of the 16 mutations in the same strain did not lead to a further increase in H(2)S production, instead often resulting in decreased levels.

Evaluation of Albicans ID2 and Biggy agar for the isolation and direct identification of vaginal yeast isolates

In this study, 250 vaginal samples from patients with vulvovaginal candidosis were inoculated onto two chromogenic media, Albicans ID(2) and Biggy agar, as well as onto Sabouraud chloramphenicol agar, yielding a total of 63 yeast (25.2 %) on all three media. These strains were identified as Candida glabrata in 20 (31.8 %) samples, Candida albicans in 15 samples (23.8 %), Candida tropicalis in 10 samples (15.9 %), Candida krusei in five samples (7.9 %), Candida kefyr in five samples (7.9 %), Candida dubliniensis in four samples (6.3 %), Candida parapsilosis in two samples (3.2 %) and Candida guilliermondii in two samples (3.2 %). Mixed fungal cultures and bacterial growth or filamentous fungi were not detected on any of the selected media. The sensitivity and specificity of the Albicans ID(2) and Biggy agar with regard to the identification of C. albicans were 80.0 and 64.6 %, and 86.7 and 56.3 %, respectively. This study showed these two chromogenic media to be as effective as Sabouraud chloramphenicol agar with respect to fungal detection. However, neither Albicans ID(2) nor Biggy agar was sufficient for reliable differentiation of yeasts to the species level.

Comparison of three differential media for the presumptive identification of yeasts

This study evaluated three differential media, CHROMagar Candida, BiGGY agar and Albicans ID2 agar, for the presumptive identification of yeast species. In total, 215 yeast isolates were included in the study. The sensitivity and specificity of CHROMagar Candida, BiGGY agar and Albicans ID2 agar for the detection of Candida albicans were 100% and 100%, 91% and 92.7%, and 99.2% and 92.7%, respectively. CHROMagar Candida was a reliable tool for the presumptive identification of C. albicans, Candida tropicalis, Candida krusei and Candida glabrata. Albicans ID2 agar was useful for the detection of C. albicans.

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species

BackgroundThe importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species.MethodsA total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37°C.ResultsThe sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively.ConclusionsIt can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar.

Survey of Hydrogen Sulphide Production by Wine Yeasts

Twenty-one strains of commercial wine yeasts and 17 non-Saccharomyces species of different provenance were surveyed for their ability to produce hydrogen sulphide in synthetic grape juice medium indicator agar with different nitrogen sources, as well as in natural grape juice. Bacto Biggy agar, a commercially available bismuth-containing agar, was used to compare our results with others previously reported in the literature. Under identical physiological conditions, the strains used in this study displayed similar growth patterns but varied in colony color intensity in all media, suggesting significant differences in sulphite reductase activity. Sulphite reductase activity was absent for only one strain of Saccharomyces cerevisiae. All other strains produced an off-odor to different extents, depending significantly (P <0.05) on medium composition. Within the same species of some non-Saccharomyces yeasts, strain variation existed as it did for Saccharomyces. In natural musts, strains fell into three major groups: (i) nonproducers, (ii) must-composition-dependent producers, and (iii) invariable producers. In synthetic media, the formation of sulphide by strains of S. cerevisiae results from the reduction of sulphate. Therefore, this rapid screening methodology promises to be a very useful tool for winemakers for determining the risk of hydrogen sulphide formation by a given yeast strain in a specific grape juice.

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.

Control of Candida albicans vaginitis in mice by short-duration butoconazole treatment in situ.

A short-duration treatment for candidal vaginitis applying butoconazole in situ was tested in an experimental mouse model. One week after artificial induction of an oestrus state (by oestradiol benzoate injection), mice were inoculated intravaginally with 1.5 x 10(7) blastospores of Candida albicans (strain ATCC 44858). Treatment consisted of butoconazole solutions (1%, 2.5% and 5%) administered intravaginally. The development of the infection was monitored daily for 12 days and then three times a week by local samples plated on BiGGY agar. In parallel, twice a week, the vaginas of three mice in each group (control and treated groups) were isolated in order to estimate organ invasion by C. albicans. This was assessed by anatomopathological studies on a fixed and stained part of the organ and by serial dilutions of a homogenate of the remaining part plated on Sabouraud glucose chloramphenicol agar. After a 48-h incubation period the number of colony forming units per gram of tissue was counted. For each treatment, a remission of 10-15 days was observed, but was followed by a recurrence for the lower dose of butoconazole; only the higher butoconazole concentrations tested (2.5% and 5%) gave an apparent full cure for most of the mice tested. The use of the mouse model of candidal vaginitis confirms that short-duration treatment is possible when elevated doses of butoconazole are used.

Quantification of vaginalCandida albicansinfections in rodents

Estradiol-treated mice and estradiol-treated ovariectomized rats support vaginal infections with Candida albicans for several months; low-grade uterine infections occur in around half the animals. A comparison has been made and the relative advantages discussed of quantifying these infections by sampling with a wire loop and plating on BiGGY agar, sampling by vaginal washing, or removal and homogenization of the vagina followed by dilution and plate counting.