Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk.

Monitoring the quality and safety of milk requires careful analysis of microbial and somatic cell loading. Our aim was to demonstrate proof of the principle that flow cytometry (FCM), coupled with fluorescence techniques for distinguishing between cell types, could potentially be employed in a wide variety of biological assays relevant to the dairy industry. To this end, we studied raw milk samples and ultraheat-treated milk, into which known numbers of bacteria or mouse cells were inoculated. For bacterial analyses, protein and lipids were removed, whereas only centrifugal lipid clearing was needed for somatic cell analyses. Cleared samples were stained with fluorescent dyes or with bacterial-specific fluorescent-labeled oligonucleotides and analyzed by FCM. A fluoresceinated peptide nucleic acid probe enabled efficient enumeration of bacteria in milk. Dual staining of samples with fluorescent dyes that indicate live (5-cyanol-2,3-ditolyl tetrazolium chloride, CTC or SYTO 9) or damaged cells (oxonol or propidium iodide, PI) enabled determination of viable bacteria in milk. Gram-positive and -negative bacteria were distinguished using hexidium iodide and SYTO 13 in dual staining of cleared milk samples. An FCM-based method gave a good correlation (r=0.88) with total microscopic counts of somatic cells in raw milk. The FCM method also correlated strongly (r=0.98) with the standard Fossomatic method for somatic cell detection. We conclude that FCM, coupled with fluorescence staining techniques, offers potentially diverse and rapid approaches to biological safety and quality testing in the dairy industry. Potential application of flow cytometers to a broad range of assays for milk biological quality should make this instrumentation more attractive and cost effective to the dairy industry and indeed the broader food industry.

Application of the novel fluorescent dye Beljian red to the differentiation of Giardia cysts.

Beljian red (BR) is a novel long Stokes shift fluorescent dye that fluoresces orange when illuminated with UV or blue light. Due to its long Stokes shift, and the fact that it is excitable at 488 nm, BR has particular utility in multi-colour applications with short Stokes shift fluorophores such as fluorescein. Here we have demonstrated that BR can be used to discriminate Giardia cysts seeded into water samples from those naturally present in the sample. We show that the dye does not interfere with other staining methods such as DAPI, and is compatible with mAb-FITC staining in a multi-colour fluorescence technique. This should be useful in determining the specific recovery of protozoan parasites from environmental samples.

Generation of a novel Saccharomyces cerevisiae strain that exhibits strong maltose utilization and hyperosmotic resistance using nonrecombinant techniques.

A yeast strain capable of leavening both unsugared and sweet bread dough efficiently would reduce the necessity of carrying out the expensive procedure of producing multiple baker's yeast strains. But issues involving the use of genetically modified foods have rendered the use of recombinant techniques for developing yeast strains controversial. Therefore, we used strong selection and screening systems in conjunction with traditional mass mating techniques to develop a strain of Saccharomyces cerevisiae that efficiently leavens both types of dough.

Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae.

Knowledge of gene expression and cellular responses in microorganisms is derived from analyses of populations consisting of millions of cells. Analytical techniques that provide data as population averages fail to inform of culture heterogeneity. Flow cytometry and fluorescence techniques were used to provide information on the heterogeneity of stress-responsive gene expression and stress tolerance in individual cells within populations. A sequence of DNA encoding the heat shock and stress response elements of the Saccharomyces cerevisiae HSP104 gene was used to express enhanced green fluorescent protein (EGFP). When integrated into the genome of yeast strain W303-1A, intrinsic expression of EGFP increased about twofold as cells progressed from growth on glucose to ethanol utilization in aerobic batch cultures. Staining of cells with orange/red fluorescent propidium iodide (PI), which only enters cells that have compromised membrane integrity, revealed that the population became more tolerant to 52 degrees C heat stress as it progressed from growth on glucose and through the ethanol utilization phase of aerobic batch culture. Exposure of cultures growing on glucose to a mild heat shock (shift from 25 degrees C to 37 degrees C) resulted in significantly increased expression of EGFP in the population. However, there was heterogeneity in the intensity of fluorescence of individual cells from heat-shocked cultures, indicating variability in the strength of stress response in the clonal population. Detailed analysis of the heterogeneity showed a clear positive trend between intensity of stress response and individual cell resistance, measured in terms of PI exclusion, to heat stress at 52 degrees C. Further experiments indicated that, although the mean gene expression by a population is influenced by the genetic background, the heterogeneity among individual cells in clonal populations is largely physiologically based.

Comparison of fermentative capacities of industrial baking and wild-type yeasts of the species Saccharomyces cerevisiae in different sugar media.

AIMS: To compare the fermentative capacity of wild and domesticated isolates of the genus Saccharomyces. METHODS AND RESULTS: The fermentative capacity of yeasts from a variety of wild and domesticated sources was tested in synthetic dough media that mimic major bread dough types. Domesticated yeast strains were found to have better maltose-utilizing capacity than wild yeast strains. The capacity to ferment sugars under high osmotic stress was randomly distributed amongst wild and baking strains of Saccharomyces. CONCLUSION: The domestication of bakers' yeast has enhanced the ability of yeasts to ferment maltose, without a similar impact on the fermentative capacity under high osmotic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, combined with molecular studies of both wild and domesticated yeast, showed that domestication of bakers' yeast has resulted in improved maltose utilization, apparently via the duplication and mutation of the MAL genes.

Hyperosmotic stress response by strains of bakers' yeasts in high sugar concentration medium.

Four strains of bakers' yeast were analysed for their hyperosmotic responses when in media that mimic conditions occurring in bread doughs. Two of the strains produced strong fermentative activity in medium with low osmotic stress, but produced considerably less ethanol in high sucrose concentration medium. Two other strains produced more similar fermentation activities across the range of media tested. The strains that were inhibited by high sucrose concentration were unable to produce significant amounts of glycerol under hyperosmotic conditions. By contrast, the yeasts that were not inhibited significantly by high sucrose produced a considerable amount of glycerol. The strains that produced significant glycerol exhibited efficient expression of the glycerol-3-phosphate dehydrogenase gene GPD1. These novel data on the molecular responses of industrially relevant strains of bakers' yeasts are prerequisite to designing strategies for improving the performance of industrial yeasts in high sugar concentration media.

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts.

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.

Fluorescence staining and flow cytometry for monitoring microbial cells.

Large numbers of microbiological samples are analysed annually using traditional culture-based techniques. These techniques take hours to days to yield a result, are tedious and are not suitable for non-culturable microorganisms. Further, culture-based techniques do not provide real-time information on the physiological status of the organism in situ which is important in the industrial manufacture of many microbial products. Flow cytometry offers the prospect of real-time microbial analysis of individual microorganisms, without dependency on microbial culture. However, flow cytometry has not been extensively used as a tool for routine microbial analysis. This has been mainly due to the high cost and complexity of instrumentation, the need for trained flow cytometrists and the lack of assay kits with appropriate biological reagents for specific applications. Many modern instruments are now relatively simple to operate, due to improvements in the user-interface, and no longer need a specialist operator. However, most cytometers are still reliant on analogue technology first developed 20-30 years ago. The incorporation of modern, solid state opto-electronics combined with micro-fabrication and digital signal processing technology offers the prospect of simple to use, low cost and robust instruments suitable for microbial analyses. Advances are being made in the development of a range of biological reagents and these are now being formulated into simple to use kits for microbiological applications. Currently, these kits are largely restricted to simple analyses, for example to assay for total or viable numbers of microorganisms present. However, technologies are available to selectively label specific types of microorganisms. For example, fluorescent antibodies can be used to label microorganisms according to expression of particular antigens, fluorescent in situ hybridisation to label according to phylogeny and fluorogenic enzymatic substrates to label according to expression of specific enzyme activities. Reagents are also available that stain viruses sufficiently brightly to enable their direct detection in environments such as sea water. Microorganisms need to be detected in a variety of different matrices (e.g., water, mud, food, and beverages) and these matrices may be highly variable in nature (e.g., tap water compared to river water). Many matrices have high background autofluorescence (e.g., algae and minerals in water samples) or may bind non-specifically to the fluorescent biological reagents used (e.g., protein micelles in milk). Formulation of biological reagents and sample pre-treatments are critical to the development of suitable microbiological assays. Here, developments in instrumentation and biological reagents for microbiological applications are reviewed with specific examples from environmental or industrial microbiology. The broader considerations for the development of microbial assays for flow cytometry are also considered.

A flow cytometry method for rapid detection and enumeration of total bacteria in milk.

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (/= 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (P < 0.01) and a good agreement (r = 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was

Facilitating functional analysis of the Saccharomyces cerevisiae genome using an EGFP-based promoter library and flow cytometry.

A promoter library was generated to facilitate identification of differentially regulated promoters in Saccharomyces cerevisiae. The library was constructed in a vector containing two reporter genes (EGFP and lacZ) divergently arranged about a unique cloning site. Approximately 2x10(5) clones were obtained and a flow cytometer was used to screen the library for copper-induced EGFP expression. A DNA fragment conferring copper-inducible expression of EGFP was rapidly identified. This DNA fragment, which contained several motifs associated with copper and oxidative stress homeostasis, lies upstream of two 'orphan' genes of unknown function. Further studies comparing expression from episomal vs. integrative vectors showed that construction of a similar library using an integrative vector would further enhance rapid identification of genes that are differentially regulated in S. cerevisiae. The ability to identify regulated promoters rapidly should facilitate the functional analysis of the yeast genome by identifying genes induced by specific physiological conditions.

Leu343Phe substitution in the Malx3 protein of Saccharomyces cerevisiae increases the constitutivity and glucose insensitivity of MAL gene expression.

To utilise maltose as a carbon source Saccharomyces cerevisiae needs one or more functional MAL loci that contain the MALx1 gene encoding maltose permease, MALx2 encoding maltase, and MALx3 encoding a transcriptional activator. Maltose causes a rapid MALx3-dependent induction of MAL gene transcription, and glucose represses this activation via Mig1p. A MALx3 gene conveying high MAL gene expression in the absence of maltose in a malx3 laboratory mutant strain has been isolated from baker's yeast. The construction of hybrid genes between the isolated gene and a highly regulated MALx3 gene showed that constitutivity was the result of multiple amino-acid alterations throughout the structural gene. The combined effect of these amino-acid alterations was shown to be stronger than the sum of their individual effects on constitutivity. Analysis in glucose-repressed conditions confirmed that increased MALx3 transcript levels increased the glucose insensitivity of MAL gene expression but did not affect constitutivity. Analysis of four mutations between aa 343 and 375, lying within a proposed negative regulatory domain, showed that the single mutation of Leu343Phe increased the glucose insensitivity of MAL gene expression by 30-fold. These results demonstrate that not only Mig1p modulation of MALx3 expression, but also the MALx3 protein structure, is involved in the glucose-insensitive expression of the MAL genes.

Genetic evidence that high noninduced maltase and maltose permease activities, governed by MALx3-encoded transcriptional regulators, determine efficiency of gas production by baker's yeast in unsugared dough.

Strain selection and improvement in the baker's yeast industry have aimed to increase the speed of maltose fermentation in order to increase the leavening activity of industrial baking yeast. We identified two groups of baker's strains of Saccharomyces cerevisiae that can be distinguished by the mode of regulation of maltose utilization. One group (nonlagging strains), characterized by rapid maltose fermentation, had at least 12-fold more maltase and 130-fold-higher maltose permease activities than maltose-lagging strains in the absence of inducing sugar (maltose) and repressing sugar (glucose). Increasing the noninduced maltase activity of a lagging strain 13-fold led to an increase in CO2 production in unsugared dough. This increase in CO2 production also was seen when the maltose permease activity was increased 55-fold. Only when maltase and maltose permease activities were increased in concert was CO2 production by a lagging strain similar to that of a nonlagging strain. The noninduced activities of maltase and maltose permease constitute the largest determinant of whether a strain displays a nonlagging or a lagging phenotype and are dependent upon the MALx3 allele. Previous strategies for strain improvement have targeted glucose derepression of maltase and maltose permease expression. Our results suggest that increasing noninduced maltase and maltose permease levels is an important target for improved maltose metabolism in unsugared dough.

A flow cytometric method for rapid selection of novel industrial yeast hybrids.

We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 x 10(6) cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.

The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway.

The ability of cells to survive freezing and thawing is expected to depend on the physiological conditions experienced prior to freezing. We examined factors affecting yeast cell survival during freeze-thaw stress, including those associated with growth phase, requirement for mitochondrial functions, and prior stress treatment(s), and the role played by relevant signal transduction pathways. The yeast Saccharomyces cerevisiae was frozen at -20 degrees C for 2 h (cooling rate, less than 4 degrees C min-1) and thawed on ice for 40 min. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing is the main determinant of freeze-thaw damage. Regardless of the carbon source used, the wild-type strain and an isogenic petite mutant ([rho 0]) showed the same pattern of freeze-thaw tolerance throughout growth, i.e., high resistance during lag phase and low resistance during log phase, indicating that the response to freeze-thaw stress is growth phase specific and not controlled by glucose repression. In addition, respiratory ability and functional mitochondria are necessary to confer full resistance to freeze-thaw stress. Both nitrogen and carbon source starvation led to freeze-thaw tolerance. The use of strains affected in the RAS-cyclic AMP (RAS-cAMP) pathway or supplementation of an rca1 mutant (defective in the cAMP phosphodiesterase gene) with cAMP showed that the freeze-thaw response of yeast is under the control of the RAS-cAMP pathway. Yeast did not adapt to freeze-thaw stress following repeated freeze-thaw treatment with or without a recovery period between freeze-thaw cycles, nor could it adapt following pretreatment by cold shock. However, freeze-thaw tolerance of yeast cells was induced during fermentative and respiratory growth by pretreatment with H2O2, cycloheximide, mild heat shock, or NaCl, indicating that cross protection between freeze-thaw stress and a limited number of other types of stress exists.

Involvement of CIF1 (GGS1/TPS1) in osmotic stress response in Saccharomyces cerevisiae.

The transcriptional responses of the osmotically induced genes ALD2, CTT1, ENA1, GPD1, HSP12 and HSP104, were studied in Saccharomyces cerevisiae strains differing in CIF1 gene function following application of osmotic stress. The CIF1 gene (allelic to GGS1 and TPS1) encodes a subunit of the trehalose synthase complex that affects trehalose synthesis. Recent work has implicated this gene in various signalling events in the cell, including transcriptional response to heat-shock treatment. Because many genetic factors can influence S. cerevisiae osmoresponse, we have compared the expression of osmotically induced genes and glycerol production in isogenic strains differing only in functionality of CIF1, growing logarithmically on galactose medium. When cultures were exposed to 0.8 M NaCl or 1.5 M sorbitol the cif1 strain showed greatly reduced transcription of osmotically induced genes compared to the wild type. These treatments did not affect viability of the yeast strains. Treatment with 0.3 M NaCl produced no significant differences in transcription of these genes in CIF1 or cif1 strains. Treatment with 0.6 M sorbitol induced small but reproducible differences, with gene expression higher in the CIF1 strain compared to the cif1 mutant. When cultures were treated with 0.3 M NaCl or 0.6 M sorbitol for 1 h, glycerol production was similar for both strains, but after 3 h of the same treatment, total glycerol production was higher in the CIF1 strain. When cultures were treated with 0.8 M NaCl for 3 h, the wild type strain produced more glycerol than the mutant strain. Both strains produced similar amounts of glycerol following exposure to 1.5 M sorbitol for 3 h, although the wild type strain showed enhanced ability to retain glycerol inside the cell. The results are discussed in the context of the possible role that the CIF1 gene product has in response to osmotic stress.

Influence of invertase activity and glycerol synthesis and retention on fermentation of media with a high sugar concentration by Saccharomyces cerevisiae.

In the past, the fermentation activity of Saccharomyces cerevisiae in substrates with a high concentration of sucrose (HSuc), such as sweet bread doughs, has been linked inversely to invertase activity of yeast strains. The present work defines the limits of the relationship between invertase activity and fermentation in hyperosmotic HSuc medium. Fourteen polyploid, wild-type strains of S. cerevisiae with different invertase levels gave a similar ranking of fermentation activity in HSuc and in medium in which glucose and fructose replaced sucrose (HGF medium). Thus, invertase is unlikely to be the most important determinant of fermentation in sweet doughs. Yeasts produce the compatible solute-osmoprotective compound glycerol when exposed to hyperosmotic environments. Under low sugar concentrations (and nonstressing osmotic pressure), there was no correlation between glycerol and fermentation activities. However, there was a strong correlation between the ability of yeasts to ferment in HSuc or HGF medium and their capacity to produce and retain glycerol intracellularly. There was also a strong correlation between intracellular glycerol and fermentation activity of yeasts in a medium in which the nonfermentable sugar alcohol sorbitol replaced most of the sugars (HSor), but the ability to produce and retain glycerol was greater when yeasts were incubated in HGF medium under the same osmotic pressure. The difference between the amounts of glycerol produced and retained in HSor and in HGF media varied with strains. This implies that high fermentable sugar concentrations cause physiological conditions that allow for enhanced glycerol production and retention, the degree of which is strain dependent. In conclusion, one important prerequisite for yeast strains to ferment media with high concentrations of sugar is the ability to synthesize glycerol and especially to retain it.

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae.

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52 degrees C for 4.5 min), ethanol (20% v/v for 30 min), H2O2 (0.3 M for 60 min), rapid freezing (-196 degrees C for 20 min, cooling rate 200 degrees C min-1), slow freezing (-20 degrees C for 24 h, cooling rate 3 degrees C min(-1)), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H2O2 stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H2O2 and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H2O2 tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast.

Stress tolerance: the key to effective strains of industrial baker's yeast.

Application of yeasts in traditional biotechnologies such as baking, brewing, distiller's fermentations, and wine making, involves them in exposure to numerous environmental stresses. These can be encountered in concert and sequentially. Yeast exhibit a complex array of stress responses when under conditions that are less than physiologically ideal. These responses involve aspects of cell sensing, signal transduction, transcriptional and posttranslational control, protein-targeting to organelles, accumulation of protectants, and activity of repair functions. The efficiency of these processes in a given yeast strain determines its robustness, and to a large extent, whether it is able to perform to necessary commercial standards in industrial processes. This article reviews aspects of stress and stress response in the context of baker's yeast manufacturing and applications, and discusses the potential for improving the general robustness of industrial baker's yeast strains, in relation to physiological and genetic manipulations.

Evidence that the Saccharomyces cerevisiae CIF1 (GGS1/TPS1) gene modulates heat shock response positively.

The CIF1 gene (also called GGS1/TPS1) encodes a protein of the trehalose synthase complex that affects trehalose accumulation and general glucose sensing by Saccharomyces cerevisiae cells. There is considerable debate as to whether CIF1-dependent trehalose accumulation is a determinant in heat shock-acquired thermotolerance. Thermosensitivity of cif1 mutants could alternatively, or also, be related to gene expression-signalling defects in such strains. Because many signal-dependent factors are involved in stress protection and repair in yeast, we have compared the expression of various stress response and heat shock genes in 'isogenic' CIF1 and cif1 strains growing exponentially in galactose medium. Transcription of CTT1, CIF1, HSP26, HSP82, HSP104, SSA4 and UB14 was notably lower in the cif1 mutant following heat shock. Moreover, a single copy of chromosomally integrated HSP104-lacZ fusion gave up to 5.5-fold more heat shock induction in the CIF1 strain compared to the cif1 mutant. We conclude that reduced heat shock-acquired thermotolerance in cif1-deletion mutants growing exponentially on galactose is more likely to result from a general reduction in expression of stress response and heat shock genes, than simply or solely through deficiency of trehalose accumulation. The possible role of CIF1 in modulating stress response is discussed.

Concomitant appearance of intrinsic thermotolerance and storage of trehalose in Saccharomyces cerevisiae during early respiratory phase of batch-culture is CIF1-dependent.

Strains of Saccharomyces cerevisiae that exhibit varied capacities for accumulation of trehalose were tested for intrinsic thermotolerance. Yeast that accumulated trehalose rapidly in early respiratory phase showed equally rapid attainment of thermotolerance, whereas a strain unable to accumulate trehalose at this stage of culture showed markedly delayed appearance of thermotolerance. These results were obtained using closely related but non-isogenic diploids and so it is possible that variable factors other than trehalose were responsible for the observed thermotolerance effects. Therefore, a pair of isogenic diploid S. cerevisiae strains was generated to facilitate further testing of whether trehalose functions in intrinsic stress tolerance. Both isogenic strains inherited a partially reverted cif1 phenotype, designated CPR, from the trehalose-deficient progenitor that had been used in construction of the non-isogenic strains. The CPR phenotype permitted growth on glucose but not accumulation of trehalose, indicating that not all cif1-related deficiencies were suppressed in the CPR strains. However, one of the isogenic CPR pair was cif1/cif1 and failed to accumulate trehalose, whilst the other was cif1/CIF1 and was able to accumulate this sugar. The trehalose-proficient strain showed intrinsic stress tolerance whereas the trehalose-deficient strain was sensitive to heat stress during early respiratory growth. These results suggest that one or more functions of CIF1, not operating in the cif1/cif1(CPR) strains, are important for intrinsic thermotolerance of yeast in early respiratory phase.(ABSTRACT TRUNCATED AT 250 WORDS)