Search

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

Dysregulation of the plasminogen activation cascade is a prototypic feature in many malignant epithelial cancers. Principally, this is thought to occur through activation of overexpressed urokinase plasminogen activator (uPA) concomitant with binding to its high specificity cell surface receptor urokinase plasminogen activator receptor (uPAR). Up-regulation of uPA and uPAR in cancer appears to potentiate the malignant phenotype, either (i) directly by triggering plasmin-mediated degradation or activation of uPA's or plasmin's proteolytic targets (e.g., extracellular matrix zymogen proteases or nascent growth factors) or indirectly by simultaneously altering a range of downstream functions including signal transduction pathways ( Romer, J. ; Nielsen, B. S. ; Ploug, M. The urokinase receptor as a potential target in cancer therapy Curr. Pharm. Des. 2004, 10 ( 19), 235976 ). Because many malignant epithelial cancers express high levels of uPAR, uPA or other components of the plasminogen activation cascade and because these are often associated with poor prognosis, characterizing how uPAR changes the downstream cellular "proteome" is fundamental to understanding any role in cancer. This study describes a carefully designed proteomic study of the effects of antisense uPAR suppression in a previously studied colon cancer cell line (HCT116). The study utilized replicate 2DE gels and two independent gel image analysis software packages to confidently identify 64 proteins whose expression levels changed (by > or =2 fold) coincident with a moderate ( approximately 40%) suppression of cell-surface uPAR. Not surprisingly, many of the altered proteins have previously been implicated in the regulation of tumor progression (e.g., p53 tumor suppressor protein and c-myc oncogene protein among many others). In addition, through a combination of proteomics and immunological methods, this study demonstrates that stathmin 1alpha, a cytoskeletal protein implicated in tumor progression, undergoes a basic isoelectric point shift (p I) following uPAR suppression, suggesting that post-translational modification of stathmin occur secondary to uPAR suppression. Overall, these results shed new light on the molecular mechanisms involved in uPAR signaling and how it may promulgate the malignant phenotype.

Ensuring that proteolytic digestions are complete before submitting samples for downstream proteomic analyses is important, as failure or partial digestion can waste valuable instrument time and make results difficult to interpret. Conversely, overdigestion can also be problematic, such as when removing affinity tags from recombinant proteins or using nonspecific proteases. The techniques of HPLC, circular dichroism, SDS-PAGE, and MS have each been used to assess protein digestion. These techniques are slow, may require expensive instrumentation, can be inaccurate, and/or are unsuitable for real-time monitoring. Epicocconone is a natural fluorophore that reacts reversibly with proteins to form a highly fluorescent adduct and has previously been used to quantify proteins in 1D and 2D gels and in solution. Here, we describe a new method for the real-time monitoring of protein digestion based on epicocconone. This unique in situ fluorescent assay can tracelessly follow proteolysis of samples, at low microgram levels, destined for proteomics analysis or purification.

A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.

Epicocconone is a heterocyclic natural product from the fungus Epicoccum nigrum that fluoresces weakly in the green (520 nm). However, cells exposed to epicocconone rapidly absorb the dye and become bright orange fluorescent because the natural product reacts reversibly with proteins. The orange fluorescence is enhanced in lipophilic environments, allowing the visualization of membranous organelles and lipid rafts but does not stain oligonucleotides. As the unconjugated dye has no orange fluorescence, there is no need to wash out the excess fluorophore. Epicocconone is a neutral, non-toxic, small molecule that appears to diffuse readily into live of fixed cells without the need for permeabilization. These features enable the real-time imaging of live cells and the study of organelle movements. Cells stained with epicocconone are excitable by common lasers (UV, 405, 488, and 532 nm) and its long Stokes' shift allows multiplexing applications with more common short Stokes' fluorophores using a single light source.

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays.

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 x 10(6) oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum "bovine" genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium "marsupial" genotype I or "marsupial" genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.

Escherichia coli has long been used as an indicator organism for water quality assessment. Recently there has been an accumulation of evidence that suggests some strains of this organism are able to proliferate in the environment, a characteristic that would detract from its utility as an indicator of faecal pollution. Phenotypic and genotypic characterization of E. coli isolated from blooms in two Australian lakes, separated by a distance of approximately 200 km, identified that the blooms were dominated by three E. coli strains. A major phenotypic similarity among the three bloom strains was the presence of a group 1 capsule. Genetic characterization of a conserved region of the cps gene cluster, which encodes group 1 capsules, identified a high degree of genetic variation within the bloom isolates. This differs from previously described encapsulated E. coli strains which are highly conserved at the cps locus. The phenotypic or genotypic profiles of the bloom strains were not identified in 435 E. coli strains isolated from vertebrates. The occurrence of these encapsulated strains suggests that some E. coli have evolved a free-living lifestyle and do not require a host in order to proliferate. The presence of the same three strains in bloom events in different geographical regions of a temperate climate, and at different times, indicates that free-living E. coli strains are able to persist in these water reservoirs. This study provides further evidence of circumstances where caution is required in using E. coli as an indicator organism for water quality.

Molecular characterisation of Cryptosporidium oocysts isolated from faeces collected from eastern grey kangaroos Macropus giganteus inhabiting an Australian water catchment revealed that this host was susceptible to three types of Cryptosporidium. Nucleotide sequence analysis of the 18S rDNA, Cryptosporidium oocyst wall protein (COWP) and a 70kDa heat shock protein (HSP70) identified an isolate identical to the described Cryptosporidium 'marsupial' genotype. A second isolate had less than 0.5% variation, compared to the described Cryptosporidium 'marsupial' genotype, within the sequences of the 18S rDNA, COWP and HSP70 and 10% variation in the internal transcribed spacer 1 (ITS1). Multilocus analysis of the third Cryptosporidium revealed a novel genotype that had a degree of genetic variation, at the four loci characterised, which was greater than or equivalent to that used to discriminate between currently recognised Cryptosporidium species. These findings have increased our current understanding on the molecular epidemiology of Cryptosporidium in Australian wildlife and have provided information on the types of Cryptosporidium marsupials may shed into the environment.

Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon-arc lamps; 488 nm and 457 nm Argon-ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid-state lasers; 543 nm helium-neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one-dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G-250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI-MS and Edman-based sequencing chemistry.

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.

Abstract Pseudomonas corrugata, strain 2140, a biological control agent of take-all disease of wheat, was originally isolated from an acidic red-brown earth soil in New South Wales, Australia. A spontaneous rifampicin-resistant mutant of this bacterium was marked with the disarmed transposon, Tn7::lacZY. This marked strain (2140RlacZY) was introduced into a calcareous sandy loam soil (pH 8) in South Australia. Up to 4 years after its release, P. corrugata 2140RlacZY cells were re-isolated, single colony purified and stored at -80 degrees C. Re-isolated bacteria, including re-isolates obtained 3 (22 re-isolates) and 4 (3 re-isolates) years after release, were examined for stability of the lacZY insert site and for gross chromosomal changes. Hybridization of a cloned lacZY fragment to DNA extracted from the soil re-isolates did not reveal any major changes to the lacZY insert site. Gross chromosomal changes were further examined by restriction endonuclease fingerprinting and PCR based on repetitive sequences (repetitive extragenic palindromic-, enterobacterial repetitive intergeneric consensus- and BOX-PCR). MspI digests distinguished the lacZY-marked strain from the parental strain. None of the genetic techniques used revealed any polymorphisms between the original 2140RlacZY-marked strain and the soil re-isolates. The results demonstrated that the chromosomal landscape within and around the insertion site of the lacZY construct had not altered in the re-isolated bacteria during the 4 years the organism had been in the field.

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.

A combined immunomagnetic separation (IMS) and flow cytometry (FC) technique was developed for the sensitive detection of Cryptosporidium in faecal samples. The IMS/FC technique was found to be approximately 50-fold more sensitive than formol-ether concentration, which is commonly used for Cryptosporidium epidemiological investigations. Of 31 faecal samples from captive animals 16 were found to contain Cryptosporidium oocysts when analysed using the IMS/FC compared to four when using formol-ether concentration (FEC). In a wild population of eastern grey kangaroos Macropus giganteus 66.3% of infected animals were shedding <500oocysts/gfaeces when analysed using IMS/FC. This is below the detection limit for the FEC method. The dispersal of Cryptosporidium in host populations is aggregated, with many individuals shedding low numbers of oocysts and few individuals shedding numbers of oocysts sufficiently high to be detected by FEC. This research demonstrates that the prevalence and oocyst shedding intensity of Cryptosporidium in animal populations will be significantly underestimated using standard detection methods.

Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum = 80% and G. lamblia = 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITC-conjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of human-infectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.

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.

Aims: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk., Methods and Results: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products., Conclusions: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk., Significance and Impact of the Study: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.

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 degrees 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.

Aims: Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia., Methods and Results: Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully., Conclusion: Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period., Significance and Impact of the Study: Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.

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.

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.

The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG(1) monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.

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.

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

The biphenyl degradation pathway of Sphingomonas paucimobilis BPSI-3 was investigated using a degradation-deficient mutant generated by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) mutagenesis. The mutant, designated AN2, was confirmed as originating from BPSI-3 through the use of ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR and by detection of the diagnostic pigment, nostoxanthin, in cellular methanol extracts. Mutant AN2 produced a yellow followed by red extracellular substance when grown in the presence of biphenyl. In the presence of 2,3-dihydroxybiphenyl, yellow followed by red then yellow compounds were formed over time. This colour change was consistent with the characteristics of a quinone, 1-phenyl-2,3-benzoquinone, which could arise from the oxidation of 2,3-dihydroxybiphenyl. A quinone was synthesised from 2,3-dihydroxybiphenyl and compared to the red compound produced by mutant AN2. Gas chromatography-mass spectrophotometry (GC-MS) confirmed that a similar quinone (4,5-dimethoxy-3-phenyl-1,2-benzoquinone) compared to the structure of the proposed biogenic compound, had been formed. This compound was also found after GC-MS analysis of mutant AN2 culture extracts. Spectrophotometric analysis of the quinone synthesised and the red product produced revealed almost identical spectral profiles. A likely inference from this evidence is that the mutant AN2 is blocked, or its activity altered, in the first gene cluster, bphA to C, of the biphenyl degradation pathway.

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.

A bacteria inducible antibacterial protein, P2, was isolated from the old world bollworm Helicoverpa armigera. Fifth-instar larvae were injected with live Escherichia coli NCTC 8196. P2 was isolated by HPLC using reversed-phase and size-exclusion columns. In addition, P2 was isolated by an alternative method of sequential cation-exchange and reversed-phase HPLC. The structure of P2 was determined by N-terminal Edman degradation and mass spectrometry. P2 had similar mass (14.1 kDa) structure and activity to gloverin, an inducible glycine-rich antibacterial protein isolated from Hyalophora gloveri [Axén, A.; Carlsson, A.; Engström, A.; Bennich, H. Eur. J. Biochem. 247:614-619; 1997]. At the N-terminus P2 had approximately 60% identity with gloverin. P2 is basic, heat stable, and displayed rapid antibacterial action. P2 was active against the Gram-negative bacteria tested and was inactive against the Gram-positive bacteria, Candida albicans, a bovine turbinate cell line, and pestivirus.

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.

Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline ( approximately 30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.

A simple method is described for the evaluation and quality control of Cryptosporidium-specific antibodies used in monitoring environmental water samples. Purified oocysts were fluorescently labelled with a test antibody at the appropriate concentration. Labelled oocysts were analysed using flow cytometry and a region was defined on a bivariate dotplot of fluorescence versus light scatter that enclosed all oocysts. Concentrates of environmental water samples that did not contain oocysts were then incubated with the test antibody and analysed using flow cytometry. The number of particles that appeared in the region defined for oocysts was recorded and was a measure of non-specific binding. The technique provides a simple, rapid and quantitative tool for both evaluating the binding specificity of test antibodies and optimizing sample staining conditions.

In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.

A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types. DNA was extracted from 100 g of soil using direct lysis with glass beads and sodium dodecyl sulphate (SDS) followed by polyethylene glycol precipitation, potassium acetate precipitation, phenol extraction and isopropanol precipitation. The crude extract could be used in PCR directed at high-copy number (bacterial small subunit rRNA) and single-copy (fungal beta-tubulin) genes.

Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates.

Sphingomonas paucimobilis BPSI-3 was previously isolated from a mixed microbial consortium growing on biphenyl as the sole source of carbon and energy. Transformation of 4-chlorobiphenyl (4CBP) was demonstrated by this strain, although little or no growth was observed. In minimal salts medium supplemented with 4CBP or bromobiphenyl and dextrose, yellow coloured product(s) were rapidly formed. Gas chromatography-mass spectrometry (GC-MS) revealed single-ring N-heterocyclic compounds that were identified as halopicolinic acids. We believe this to be the first report of such compounds being formed via biological transformation of halobiphenyls. A mechanism is proposed for their formation.

Secretions from exocrine metapleural glands of Myrmecia gulosa (Australian bull ant) exhibit broad-spectrum antimicrobial activity. Treatment of the yeast Candida albicans with metapleural secretion resulted in the rapid and total leakage of K+ ions from cells within 10 min. Ultrastructural analysis of the bacteria Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa, and cells and protoplasts of Candida albicans demonstrated gross damage of the cell membrane and aggregation of the cytoplasmic matrix of treated cells. Degradation of membrane-bound organelles was also observed in Candida albicans. The antimicrobially active components of metapleural secretions were nonpolar and interacted with the phospholipid bilayer, causing damage to the structural integrity of liposomes and the release of carboxyfluorescein. The data suggest that the antimicrobial agents in metapleural secretion act primarily by disrupting the structure and function of the phospholipid bilayer of the cytoplasmic membrane.

A spectrophotometric method has been developed for the rapid measurement of the antimicrobial activity of natural products, including crude extracts or pure materials. The assay depends on the measurement of non-specific esterase activity using fluorescein diacetate (FDA) hydrolysis in broth cultures of microbes after they have been treated with test compounds. The assay is accurate, reproducible and economical in both time and materials. The speed and economy of the method make it suitable for the rapid screening of many samples and the bioassay directed purification of antimicrobial substances. The assay can also be used with a wide variety of micro-organisms since most micro-organisms are FDA positive. Applications are described in the fields of marine natural products chemistry and essential oils research.

Myrmecia gulosa (Australian bull ant) produce secretions from their metapleural exocrine glands which have broad spectrum antimicrobial properties. Such secretions are probably of importance in disease control in bull ant communities. These antimicrobial secretions are stable at 100 degrees C, resistant to proteolytic enzymes and are active over a wide pH range. Of the organisms tested only endospores of Bacillus cereus were found to be resistant. The antimicrobial agent(s) are absorbed by cells and result in cell lysis. The secretions do not interfere with any growth-related processes. These observations demonstrate that insects may be a source of novel antimicrobial agent(s).