Paul V. Attfield

A Flow Cytometry Method for Rapid Detection and Enumeration of Total Bacteria in Milk

ABSTRACT 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 (≤1-h) FCM assay based on enzymatic clearing of milk to determine total bacteria in milk. When bacteria were added to ultra-heat-treated milk, a good correlation (r ≥ 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 ≤104 bacteria ml of milk−1. This limit is below the level of detection required to satisfy legislation in many countries and states.

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.

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.

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.

Inducible Gene Expression by Nonculturable Bacteria in Milk after Pasteurization

ABSTRACT The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5°C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.

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°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°C to 37°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°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.

Generation of a Novel Saccharomyces cerevisiae Strain That Exhibits Strong Maltose Utilization and Hyperosmotic Resistance Using Nonrecombinant Techniques

ABSTRACT 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 bakers 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 cerevisiaethat efficiently leavens both types of dough.

Genetics and classical genetic manipulations of industrial yeasts

Yeasts represent highly important and valuable organisms for research and industrial applications. Although Saccharomyces cerevisiae has historically been a key species, other yeast genera and species are increasingly important. The rapid emergence and expansion of genomic and functional genetic information on various yeasts indicates a high level of complexity amongst these organisms. It is apparent that the S. cerevisiae genome was duplicated some 100 million years ago and that several of the other important yeast species diverged prior to this duplication, whilst others diverged post-polyploidization of the ancestral yeast genome. Here we present an overview of the various yeast genomes that serves to highlight the complexity of yeasts, especially within the context of industrially relevant strains. The challenges faced by industrial yeast geneticists are discussed and key issues concerning strategies of mating, mutagenesis, spheroplast fusion and cytoduction are presented. Examples of strain improvements achieved via classical genetics are presented, along with an analysis of molecular genetics of maltose metabolism in industrial yeasts, which serves to highlight the usefulness of combined molecular and classical genetics in strain improvement.

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 2×105 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. Copyright

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.