A convenient microtitre tray procedure for yeast identification

Media for yeast identification tests were incorporated into the wells of a microtitre tray. The tests included fermentation and assimilation of carbohydrates, assimilation of nitrogen compounds, growth in vitamin-free medium, resistance to cycloheximide, and observations for cell morphology and sporulation. Results of tests conducted in the trays showed very good agreement with those obtained by conventional methods. Eighteen reference yeasts were correctly identified from tests conducted in the trays. The trays of media could be stored, and provided a convenient system for yeast identification.     

Yeast identification: reassessment of assimilation tests as sole universal identifiers

Aims: To assess whether assimilation tests in isolation remain a valid method of identification of yeasts, when applied to a wide range of environmental and spoilage isolates. Methods and Results: Seventy‐one yeast strains were isolated from a soft drinks factory. These were identified using assimilation tests and by D1/D2 rDNA sequencing. When compared to sequencing, assimilation test identifications (MicroLog?) were 18·3% correct, a further 14·1% correct within the genus and 67·6% were incorrectly identified. The majority of the latter could be attributed to the rise in newly reported yeast species. Conclusions: Assimilation tests alone are unreliable as a universal means of yeast identification, because of numerous new species, variability of strains and increasing coincidence of assimilation profiles. Assimilation tests still have a useful role in the identification of common species, such as the majority of clinical isolates. Significance and Impact of the Study: It is probable, based on these results, that many yeast identifications reported in older literature are incorrect. This emphasizes the crucial need for accurate identification in present and future publications.     

Identification of yeasts present in sour fermented foods and fodders

This paper deals with rapid methods for identification of 50 yeast species frequently isolated from foods and fodders that underwent a lactic acid fermentation. However, many yeast species present in olive brine, alpechin, and other olive products were not treated. The methods required for identification include light microscopy, physiological growth tests (ID32C system of BioMérieux), assimilation of nitrate and of ethylamine as sole nitrogen sources, vitamin requirement, and maximum growth temperature. An identification key to treated yeast species is provided. In another table characteristics of all yeast species treated are listed.     

Evaluation of the Biolog system for the identification of food and beverage yeasts

The inconvenience of conventional yeast identification methods has resulted in the development of rapid, commercial systems, mainly for clinical yeast species. The Biolog system (Biolog Inc., Hayward, CA, USA) is a new semi-automated, computer-linked technology for rapid identification of clinical and non-clinical yeasts. The system is based around a microtitre tray and includes assimilation and oxidation tests. This paper evaluates the Biolog system for the identification of 21 species (72 strains) of yeasts of food and wine origin. Species correctly identified included Saccharomyces cerevisiae , Debaryomyces hansenii , Yarrowia lipolytica , Kluyveromyces marxianus , Kloeckera apiculata , Dekkera bruxellensis and Schizosaccharomyces pombe. Zygosaccharomyces bailii and Zygosaccharomyces rouxii were identified correctly 50% of the time and Pichia membranaefaciens 20% of the time.     

Development and evaluation of a rapid identification test for candida albicans

Summary A new technique for the rapid identification ofC. albicans has been developed and evaluated. This yeast can be identified in one hour by the formation of germ tubes after inoculation in 1/2 ml of human or animal plasma, and commercial plasma substitutes.C. albicans also forms germ tubes within 2 to 4 hours after inoculation in human serum and incubation at 37° C.Filamentation ofC. albicans in these blood derivatives is a reliable method for the identification of this yeast. It is more rapid than the assimilation and fermentation sugar tests and chlamydospore formation.Assimilation and fermentation sugar tests are used to identify those isolates ofCandida that fail to produce filaments in plasma or serum.     

Reproducibility of three microdilution systems for identification of enterobacteriaceae,compared with API 20E and micro-ID test systems

Five systems for identifying Enterobacteriaceae were evaluated in terms of their ability to provide reproducible results. Frozen microdilution panels (Micro-Media Systems), prepared media ready for dispensing into microdilution trays (Dynatech laboratories), media prepared in our laboratories and dispensed into microdilution trays (UC media), the Micro-ID system (General Diagnostics), and the API 20E system (Analytab Products, Inc.) were all evaluated. Fifty selected isolates were tested with each system on three separate days. Although biotypes were some-what variable, the final identification was rarely affected. The API 20E system was the most variable and microdilution tests, with UC media, were the least variable. Precision of tests performed in microdilution trays was as good, if not better than, that obtained with the other two commercial products (Micro-ID and API 20E).     

Characterization of a new xylitol-producer Candida tropicalis strain

A xylitol-producer yeast isolated from corn silage and designated as ASM III was selected based on its outstanding biotechnological potential. When cultivated in batch culture mode and keeping the dissolved oxygen at 40% saturation, xylitol production was as high as 130 g l(-1) with a yield of 0.93 g xylitol g(-1) xylose consumed. A preliminary identification of the yeast was performed according to conventional fermentation and assimilation physiological tests. These studies were complemented by using molecular approaches based on PCR amplification, restriction-fragment length polymorphism analysis and sequencing of the rDNA segments: intergenic transcribed spacer (ITS) 1-5.8S rDNA-ITS 2, and D1/D2 domain of the 26S rRNA gene. Results from both the conventional protocols and the molecular characterization, and proper comparisons with the reference strains Candida tropicalis ATCC 20311 and NRRL Y-1367, led to the identification of the isolate as a new strain of C. tropicalis.     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.     

Rapid identification of yeasts by serological methods: a combined serological and biological method.

A total of 387 yeasts from the contents of the digestive tracts of domestic animals and poultry were identified by slide agglutination tests using factor antisera and urease tests. The results of this serological test were very satisfactory with respect to accuracy and rapidity, particularly when performed in combination with concomitant physiological tests only for assimilation of inositol and potassium nitrate. It may be concluded that such a combination of serological and biological tests is very useful for identifying yeast strains from various sources.     

Screening yeast artificial chromosome libraries with robot-aided automation

Screening of collections of yeast artificial chromosomes utilizing the polymerase chain reaction (PCR) requires large numbers of reactions in parallel. Four steps were implemented to reduce the labor involved: (a) The number of initial samples for DNA extractions was decreased by compressing libraries up to 12-fold. (b) DNA extraction from yeast clones was robot assisted. (c) A Biomek 1000 station was adapted to pipette samples for PCR assays. (d) Sample preparation was integrated with a temperature cycler constructed to carry out up to 576 reactions in six 8 × 12-well trays. The implementation of these steps increases the number of reactions per person per day by an order of magnitude. In tests with X-chromosome-specific probes, the robot-aided screening recovered all of the clones detected by slower manual methods.     

A taxonomic and ecological study of candidosis

A total of 450 yeast isolates were obtained from up to 34 sites on 59 human subjects. The yeasts were characterized using morphological features and assimilation tests. Ten species were identified but, of these,Candida albicans andCandida parapsilosis were the most common and accounted for 84% of the isolates. An examination of the biotypes of the various species indicated a much greater diversity in the yeast microflora than that detected by species identifications alone. Fifty-five biotypes were differentiated and it is suggested that these could be regarded as distinct taxonomic or ecological entities.     

Preliminary identification and classification of five new yeast strains isolated from oil-polluted environment

Bioremediation is a very interesting alternative for restoring the oil-polluted ecosystems. Many studies concerning the possibility of using microorganisms (bacteria and yeasts) in the degradation of oil compounds have as starting point the isolation and taxonomical identification of new species and strains with degradative abilities. Our study focusses on the preliminary classification of five yeast strains (D1, D2, D3, D4 and D6) isolated from oil-polluted environments. The strains were characterized by conventional taxonomical techniques: microscopical and macroscopical appearance, fermentation abilities, assimilation of various carbon or nitrogen compounds, growth under stress conditions (non-permissive temperatures, high glucose concentration) and urea degradation. According to these tests, D1, D2 and D4 showed great similarity to Rhodotorula glutinis, D3 to Candida parapsilosis and D6 to Candida tropicalis. Further supplementary tests were performed in order to establish their ability to degrade hydrocarbons, by observing growth in media with n-alkanes (n-decane, n-dodecane, n-tetradecane, n-hexadecane). Thus, D1, D2 and D4 were the best alkane-consuming strains, presenting possible similar degrading abilities and pathways, which correlates well to our identification as Rhodotorula strains. For D3 and D6 the growth was not so spectacular as for D1, D2 and D4, but continuous along the entire experiment. The resemblance between the curves profiles confirms the idea that both belong to the same genus, Candida.     

The necessity of adjusting tests of protein category enrichment in discovery proteomics

MOTIVATION: Enrichment tests are used in high-throughput experimentation to measure the association between gene or protein expression and membership in groups or pathways. The Fisher's exact test is commonly used. We specifically examined the associations produced by the Fisher test between protein identification by mass spectrometry discovery proteomics, and their Gene Ontology (GO) term assignments in a large yeast dataset. We found that direct application of the Fisher test is misleading in proteomics due to the bias in mass spectrometry to preferentially identify proteins based on their biochemical properties. False inference about associations can be made if this bias is not corrected. Our method adjusts Fisher tests for these biases and produces associations more directly attributable to protein expression rather than experimental bias. RESULTS: Using logistic regression, we modeled the association between protein identification and GO term assignments while adjusting for identification bias in mass spectrometry. The model accounts for five biochemical properties of peptides: (i) hydrophobicity, (ii) molecular weight, (iii) transfer energy, (iv) beta turn frequency and (v) isoelectric point. The model was fit on 181 060 peptides from 2678 proteins identified in 24 yeast proteomics datasets with a 1% false discovery rate. In analyzing the association between protein identification and their GO term assignments, we found that 25% (134 out of 544) of Fisher tests that showed significant association (q-value ≤0.05) were non-significant after adjustment using our model. Simulations generating yeast protein sets enriched for identification propensity show that unadjusted enrichment tests were biased while our approach worked well.     

Superior molasses assimilation, stress tolerance, and trehalose accumulation of baker's yeast isolated from dried sweet potatoes (hoshi-imo)

Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker's yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker's yeast for frozen dough and high-sugar dough.     

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.     

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.     

丛生丝孢酵母菌的生物学特性观察

探讨了丛生丝孢酵母菌的生物学鉴定特性。采用真菌培养法观察丛生丝孢酵母菌的培养特性、同化试验;耐受试验测定对放线菌酮的耐受性;体外双相形态转换试验确定霉菌相及酵母相;微量稀释法进行抗真菌药物敏感试验;温度耐受性试验、干燥试验、紫外线照射试验测定其抵抗力;小鼠腹腔接种法观察其侵袭力。本菌为双相型真菌,经分离培养该菌呈现真菌所特有的菌落特征,在沙氏肉汤中呈菌膜和沉淀生长;玉米粉琼脂小培养,在镜下可见到大小不等圆形和卵圆形孢子。对10种糖类同化试验阳性,不分解尿素,还原硝酸盐。对氟康唑等4种抗真菌药均敏感;对温度、干燥、紫外线有一定耐受力;侵袭力较弱,组织内酵母相呈细胞内感染。结论观察丛生丝孢酵母菌的生物学鉴定特性,对丛生丝孢酵母菌的实验室鉴定奠定基础和临床诊断提供依据。     

三种性信息素诱捕器对烟田棉铃虫的诱捕效果比较

比较了笼罩、水盆、盘式粘胶诱捕器对烟田棉铃虫的诱捕效果。结果表明,3种诱捕器的诱蛾量变化趋势基本一致,其中笼罩诱捕器的诱蛾量最大,显著高于另外两种诱捕器,且诱捕效果比较稳定。水盆、盘式粘胶诱捕器诱蛾量差异不显著。笼罩诱捕器更适用于烟田棉铃虫成虫的防治及监测。