A solid agar overlay method for recovery of heat-injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58 degrees C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.     

Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii)

Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type, there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp.     

A rapid and sensitive non‐radioactive method applicable for genome‐wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome‐wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analysing small RNAs from Saccharomyces cerevisiae wild‐type (wt) and mutants cells in the deletion and temperature‐sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96‐deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive non‐radioactive northern method for RNA detection. The RNA isolation procedure requires only 4 h for processing 96 samples, is highly reproducible and yields RNA of good quality and quantity. The non‐radioactive northern method employs digoxigenin (DIG)‐labelled DNA probes and chemiluminescence. It detects femtomole levels of small RNAs within 1 min exposure time. We minimized the processing time for large‐scale analysis and optimized the stripping and reprobing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe and cost‐effective for genome‐wide screens of novel genes involved in the biogenesis, subcellular trafficking and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. Copyright © 2013 John Wiley & Sons, Ltd.     

Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis

The biocontrol activity of Rhodotorula glutinis on green mold decay of oranges caused by Penicillium digitatum was investigated in vitro and in vivo. Significant control was achieved with a washed cell suspension and an unwashed cell culture mixture of R. glutinis. Treatment of wounds with autoclaved cell cultures or cell-free culture filtrate did not prevent decay. The protection provided by the washed yeast cells was dose-dependent. The higher the concentration of R. glutinis, the better the effect of the biocontrol capacity. At concentrations of yeast of 1×10⁹ colony-forming units per milliliter or higher and pathogen spore suspensions of 5×10⁴ spores per milliliter, green mold was almost inhibited after 4-days incubation at 20 °C. The interval between the pathogen inoculation and the antagonist application significantly influenced the biocontrol ability. The biocontrol efficacy of R. glutinis applied before the pathogen was better than that of applied after the pathogen. Surprisingly, R. glutinis was also effective in controlling green mold at low temperature (4 °C). Rapid colonization of the yeast in wounds was observed during the first 3 days at 20 °C, and remained stable after 5-days incubation. On fruits stored at 4 °C, even after 21 days, the population of R. glutinis in wounded fruits was more than 1,600-fold of what it was just prior to storage. In the test on potato dextrose agar plates, agar disks of R. glutinis nutrient yeast dextrose agar cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. digitatum. Spore germination of pathogens in potato dextrose broth was greatly controlled in the presence of living cell suspensions.     

Extracellular levansucrase of Bacillus subtilis produced in yeast remains in the cell in its precursor form

Levansucrase, a Bacillus subtilis extracellular enzyme, was not secreted in the culture medium when produced in yeast. The protein accumulated inside the cell in its precursor form which represented 0·3% of total proteins. The absence of any post-translational modifications, such as signal sequence cleavage or addition of N-linked sugars, indicated that this protein did not enter the reticulum secretion pathway. Direct observation of the cells by confocal laser scanning microscopy showed that levansucrase was associated with the cytoplasmic membrane. Subcellular fractionation experiments revealed that levansucrase precursor form is associated with membranes through weak ionic interactions. The purified precursor displayed the same catalytic properties as levansucrase secreted by B. subtilis. Thus yeast could be used as a source of levansucrase precursor allowing its isolation as a pure form on a milligram scale.     

Comparison of recovery of airborne microorganisms in a dairy cattle facility using selective agar and thin agar layer resuscitation media

Thin agar layer (TAL) medium was developed at Kansas State University to improve the resuscitation of injured cells and has been shown to result in higher recovery than is obtained with selective media alone for cold-, heat-, salt-, and acid-injured cells. The experiment presented here was designed to determine the effectiveness of the TAL method for the recovery of possibly injured organisms from air. Eleven agar media were used for the experiment: tryptic soy agar (TSA), MacConkey sorbitol agar (MSA), TAL-MSA, Baird-Parker (BP) agar, TAL-BP agar, modified Oxford (MOX) agar, TAL-MOX agar, xylose lysine sodium desoxycholate (XLD) agar, TAL-XLD agar, Yersinia-selective (CIN) agar, and TAL-CIN agar. The TAL plates were prepared by pipetting 6 ml of selective agar into a BBL Rodac plate (65 by 15 mm). Selective agar was allowed to solidify, and then each plate was overlaid with 6 ml of TSA. Selective agar plates were prepared by pipetting 12 ml of agar into BBL Rodac plates and allowing the agar to solidify. Samples were taken at an indoor cattle facility at five separate locations with a BioScience SAS air-sampling instrument. For each plate, 60 liters of air was sampled. Three replications of the experiment were performed. The TAL method resulted in higher counts of microorganisms on all media tested. In addition, 175 isolates were selected randomly and identified in order to test the selectivity of TAL and the selective media for target organisms. The data obtained in this study show that the TAL resuscitation method is effective and necessary for the recovery of airborne organisms that may be injured.     

Evaluation of Potassium‐Clavulanate‐Supplemented Modified Charcoal‐Cefoperazone‐Deoxycholate Agar for Enumeration of Campylobacter in Chicken Carcass Rinse

Potassium‐clavulanate‐supplemented modified charcoal‐cefoperazone‐deoxycholate agar (C‐mCCDA) that was described in our previous study was compared with original mCCDA for the enumeration of Campylobacter in pure culture and chicken carcass rinse. The quantitative detection of viable Campylobacter cells from a pure culture, plated on C‐mCCDA, is statistically similar (P > 0.05) to mCCDA. In total, 120 chickens were rinsed using 400 mL buffered peptone water. The rinses were inoculated onto C‐mCCDA and mCCDA followed by incubation at 42 °C for 48 h. There was no statistical difference between C‐mCCDA (45 of 120 plates; mean count, 145.5 CFU/mL) and normal mCCDA (46 of 120 plates; mean count, 160.8 CFU/mL) in the isolation rate and recovery of Campylobacter (P > 0.05) from chicken carcass rinse. The Pearson correlation coefficient value for the number of Campylobacter cells recovered in the 2 media was 0.942. However, the selectivity was much better on C‐mCCDA than on mCCDA plates (P < 0.05). Significantly fewer C‐mCCDA plates (33 out of 120 plates; mean count, 1.9 CFU/mL) were contaminated with non‐Campylobacter cells than the normal mCCDA plates (67 out of 120 plates; mean count, 27.1 CFU/mL). The C‐mCCDA may provide improved results for enumeration of Campylobacter in chicken meat alternative to mCCDA with its increased selectivity the modified agar possess.     

Vacuole Segregation in the Saccharomyces cerevisiae vac2-1 Mutant: Structural and Biochemical Quantification of the Segregation Defect and Formation of New Vacuoles

The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10, 1741–1748] shifted to non-permissive temperature (37°C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37°C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10–15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37°C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding. © 1997 by John Wiley & Sons, Ltd.     

Campylobacter fetus ssp. jejuni: Recovery Methodology and Isolation from Lamb Carcasses

Campylobacter fetus ssp. jejuni has increasingly been implicated as the causative agent in human cases of gastroenteritis. The first account of C. jejuni isolation from red meats—lamb carcasses—is reported. Recovery of Campylobacter from meat surfaces was tested as follows: selective agar plates consisted of tryptic soy agar, 5% lysed defibrinated horse blood, 10 mg/L vancomycin, 2500 units/L polymyxin B, 5 mg/L trimethoprim lactate, and 15 mg/L cephalothin (Keflin; VPTK: plates). These VPTK plates were incubated at 35°C for 72 hr in a gas atmosphere of 5% O₂, 10% CO₂, and 85% N₂. With these selective elements, recovery from meat surfaces inoculated with a calculated 32 Campylobacter cells per cm² was accomplished in 5 of 5 replicate tests. At levels of 3.2 and 0.3 cells of Campylobacter per cm², recoveries were accomplished in 2 of 5 replicate tests at each inoculum level.     

The Potential of Confocal Imaging for Measuring Physiological Changes in Brewer's Yeast

This study focused on the implementation of fluorescence optical methods and laser scanning confocal microscopy for monitoring brewing yeast performance. Physiological parameters and cell compounds in yeast cells (glycogen, neutral lipids, trehalose, bud scars, DNA and intracellular proteinases) have been successfully visualised with the aid of highly specific fluorochromes. The expression and sub cellular localisation of proteinase A during fermentation has been studied employing a Saccharomyces cerevisiae green fluorescent protein clone. This novel approach to monitoring brewing yeast performance provides new insights into physiological events that occur during wort fermentation.     

Detection and Isolation of Low Levels of E. coli O157:H7 in Cilantro by Real-Time PCR, Immunomagnetic Separation, and Cultural Methods with and without an Acid Treatment

Abstract: Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real‐time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (～0.02 CFU/g) and slightly higher (～0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow^®agar, R&F^®E. coli O157 Chromogenic medium, TC‐SMAC and CHROMagar? 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. Practical Application: Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.     

RECOVERY LIMITS OF HEAT-INJURED LISTERIA MONOCYTOGENES FROM ENRICHMENT BROTH AND ENRICHED CULTURES OF INOCULATED FOODS

Recovery of heat-injured Listeria monocytogenes strain LM82 was evaluated quantitatively in Listeria enrichment broth (LEB) and in enriched cultures of cooked shrimp and Brie cheese. LM82 cells [10⁸ colony forming units (CFU)/ml] were heated for 60 min at 52C in phosphate-buffered saline. After 24 and 48 h enrichment, injured LM82 (6 replicates at each of 5 inoculation levels) were isolated on 3 selective media: lithium chloride-phenylethanol-moxalactam agar (LPMA), modified McBride agar (MMA) and Oxford agar (OXA). The recovery limit was expressed as a 50% end point value (RL₅₀), which is the calculated inoculation value necessary to recover LM82 on half of the replicates of each type of isolation agar plate after streaking from the enrichment of measured inoculum. The RL₅₀ values for injured cells were comparable to those of uninjured cells after 48 h enrichment in LEB without food. The type of isolation agar did not affect the RL₅₀ value, although with food, MMA gave consistently but not significantly higher values, i.e., recovery inferior to that of LPMA and OXA. RL₅₀ values were higher in Brie and cooked shrimp, presumably because of the competitive microflora in those foods. Addition of lactose or pyruvate to LEB improved recovery but had little or no effect when foods were present .     

Enhancement of plasmid DNA transformation efficiencies in early stationary‐phase yeast cell cultures

Chemical‐based methods have been developed for transformation of DNA into log‐phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary‐phase cells, e.g. cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log‐phase cells but is simpler and more adaptable to high‐throughput projects. In this study we have tested different approaches for transformation of early stationary‐phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulphoxide (DMSO) as the most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40‐fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving > 50 000 transformants/µg DNA in most backgrounds tested. Copyright © 2013 John Wiley & Sons, Ltd.     

Improvement of Karmali Agar by Addition of Polymyxin B for the Detection of Campylobacter jejuni and C. coli in Whole‐Chicken Carcass Rinse

The Karmali agar was modified by supplementation with a high concentration of polymyxin B. The goal of the study was to evaluate the effect of a high concentration of polymyxin B on the ability and selectivity of the modified Karmali agar to isolate Campylobacter jejuni and Campylobacter coli from whole chicken carcass rinse. A total of 80 whole chickens were rinsed with 400 mL of buffer peptone water. The rinsed samples were incubated with 2× blood‐free modified Bolton enrichment broth for 48 h, and then streaked onto unmodified Karmali agar and modified Karmali agar supplemented with 100000 IU/L polymixin B (P‐Karmali agar). The suspected colonies were finally confirmed by colony PCR. The P‐Karmali agar exhibited a significantly better (P < 0.05) isolation rate than the unmodified Karmali agar (P‐Karmali agar, 73.8%; unmodified Karmali agar, 33.8%). Moreover, the selectivity of the P‐Karmali agar was also better (P < 0.05) than that of the other selective agar when comparing the number of contaminated plates (P‐Karmali agar, 68.8%; unmodified Karmali agar, 87.5%) and growth index of competing flora (P‐Karmali agar, 1.4; unmodified Karmali agar, 2.7). The improved selective agar excluded competing flora resistant to antibiotic agents in unmodified Karmali agar, increasing isolation rate and selectivity for C. jejuni and C. coli.     

Bacterial populations associated with poultry processing in a South African abattoir

Bacteria were isolated from yeast extract supplemented tryptone soya agar (TSAYE) plates (357 isolates) and violet red bile glucose agar (VRBGA) plates (311 isolates) of micro- biological surveys in a South African Grade B poultry abattoir. Bacterial populations from TSAYE plates of carcasses (neck skin) were dominated byAcinetobacterspp.,Escherichia coli, Micrococcusspp. andFlavobacteriumspp., which collectively comprised 75.4% of isolates, while isolates from VRBGA plates showed dominance ofEscherichia coli(80.0%). Environmental isolates dominant on TSAYE plates wereMicrococcusspp. from equipment surfaces and air samples of the defeathering area of the abattoir (48.5 and 68.7%, respectively) andBacillusspp. from air samples of the packaging area (37.4%). Isolates from TSAYE plates of scald tank water were dominated byE. coli(47.8%) andMicrococcusspp. (39.1%), while isolates from immersion chiller water were predominantlyE. coli(54.1%). Environmental isolates dominant on VRBGA plates wereE. coliin water samples and air of the defeathering and packaging areas (from 93.7 to 100%).E. coli, Enterobacterspp. andSerratiaspp. were dominant on VRBGA plates of equipment surfaces, collectively comprising 77.8% of isolates.     

Isolation of Salmonella typhimurium from outbreak-associated cake mix

During May and June of 2005, 26 persons in several states were infected by a single strain (isolates indistinguishable by pulsed-field gel electrophoresis) of Salmonella enterica serotype Typhimurium after eating cake batter ice cream. The cake mix used to prepare the cake batter in the ice cream was implicated by epidemiologic investigation as the source of Salmonella contamination. Initial tests did not detect Salmonella in cake mix collected during the outbreak investigation. The objective of this study was to evaluate different procedures to isolate Salmonella from the implicated cake mix, cake, and ice cream. All outbreak-associated food samples (14 samples) were collected during the outbreak investigation by health departments of several of the states involved. Different combinations of Salmonella isolation procedures, including sample size, preenrichment broth, enrichment broth, enrichment temperature, and isolation medium, were used. Salmonella Typhimurium was isolated from two cake mix samples; the food isolates were indistinguishable from the outbreak pattern by pulsed-field gel electrophoresis subtyping. Universal preenrichment broth was substantially better than was lactose broth for preenrichment, and tetrathionate broth was better than was Rappaport-Vassiliadis broth for isolating Salmonella from the two positive cake mix samples. Although more typical Salmonella colonies were observed on plates from enrichment cultures grown at 35 degrees C, more confirmed Salmonella isolates were obtained from plates of enrichment cultures grown at 42 degrees C. Brilliant green agar, xylose lysine tergitol 4 agar, xylose lysine desoxycholate agar, Hektoen enteric agar, and bismuth sulfite agar plates were equally effective in isolating Salmonella from cake mix. The best combination of preenrichment-enrichment conditions for isolating the outbreak strain of Salmonella was preenrichment of cake mix samples in universal preenrichment broth at 35 degrees C for 24 h, followed by enrichment in tetrathionate broth at 42 degrees C for 24 h.     

豉香型白酒酒饼微生物的分离

通过平板稀释法微生物分离试验，认为分离豉香型白酒酒饼微生物时，在麦芽汁琼脂培养基中加入质量浓度为500mg/L的去氧胆酸钠，在高氏合成1号琼脂培养基中加入质量浓度为100mg/L的重铬酸钾后，有利于酒饼微生物的生长和分离，豉香型白酒大小酒饼的微生物分离结果表明，大酒饼中的细菌含量较小酒饼中的明显升高，小酒饼提供的根霉和酵母等微生物来源在大酒饼中得到扩大培养，而大小酒饼中放线菌含量变化则不大。     

Biotechnological potential of non-Saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.)

Non-Saccharomyces yeast species assume an important role in wine flavor. Notwithstanding, the chemical basis for the flavor characteristics of wines from some grape varieties is not yet defined. The value of this work lies in the use of Malvar white grape, an autochthonous variety from Madrid (Spain) winegrowing region to conduct spontaneous fermentations. This is the first time that a comparative characterization of a wide range of non-Saccharomyces species and a comprehensive analysis of these yeast-derived volatiles has been carried out in this grape variety. β-glucosidase and pectinase (polygalacturonase) extracellular activities were tested on agar plates as primary selection criteria among the 504 non-Saccharomyces isolated from Malvar spontaneous fermentations during four consecutive harvests. Analysis of the wines obtained after fermentation using the selected yeast strains indicates that non-Saccharomyces yeasts isolated along the fermentative process seem that could have a positive impact, showing a high variability in the volatile compounds contributing to the organoleptic characteristics of Malvar wines. Torulaspora delbrueckii CLI 918 was defined as the yeast strain with potential interest for its contribution to the aromatic wine profile with flowery and fruity aromas and could be used in mixed starter cultures with Saccharomyces cerevisiae. However, Hanseniaspora guilliermondii increased the volatile acidity and ethyl acetate, but this species along with the genus Pichia and Candida seem to provide a high quantity of extracellular enzymes which may be beneficial in wine making.     

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC?), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC?) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations—concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate—induced different degrees of transience from VC+ to VC? states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting.     

Rapid and reliable protein extraction from yeast

The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1. It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates.