Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

Statistical approach for comparison between methods of bacterial enumeration in drinking water

A general statistical procedure based on the likelihood ratio test is presented for the purpose of comparing estimates of mean bacterial density derived from different sets of data. This approach is much more appropriate than the conventional ways of analyzing bacteriological results (e.g., analysis of variance) which usually require previous transformation of the data. An illustrative application of the method compares three distinct titration techniques for enumerating heterotrophic bacteria in drinking water at 20°C incubation temperature. It was shown that both the standard plate count (SPC) and the membrane filter (MF) procedures supplied substantially the same information, whereas the microplate technique using the most probable number (MPN) for total bacterial enumeration could yield considerably different estimates: MPN values were significantly lower in three cases and significantly higher in one case out of a total of five experiments. The results consistently indicate a strong interaction between the technique used and the sample analyzed. Three different media (nutrient agar, R-2A low nutrient agar and m-SPC agar) were then evaluated for enumerating heterotrophic bacteria, using the MF technique at 48, 72 and 96 h of incubation time at 20°C. Although the media recovered approximately the same numbers of bacteria after 96 h of incubation, statistically significant discrepancies occurred after intermediate periods of incubation, perhaps because the relative rates of bacterial growth differed among media.     

Epifluorescence microscope methods for bacterial enumeration in a 4-chlorophenol degrading consortium

Epifluorescence microscope methods, namely BacLight, direct epifluorescence filter technique and Rhodamine 123, consistently underestimated plate bacterial counts in a 4-chlorophenol degrading consortium. Cells capable of passing through 0.2 microm filters, referred as 'ultramicrocells', were found. Although cell counts were higher when traditional methods were used, BacLight and direct epifluorescence filter technique were convenient techniques for the systematic monitoring of bacteria involved in biodegradation processes, as results were consistent and available within a short time.     

Comparison of intracellular and extracellular mitogenic activity

Endothelial cell-derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat-stable and trypsin-sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen-secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56 degrees C) and were not inactivated by trypsin. Similar to platelet-derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.     

A review of conventional detection and enumeration methods for pathogenic bacteria in food

With continued development of novel molecular-based technologies for rapid, high-throughput detection of foodborne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. In fact, a number of unique approaches and variations on existing techniques are currently on the market or are being implemented that offer ease of use, reliability, and low cost compared with molecular tools. Approaches that enhance recovery of sublethally injured bacteria, differentiation among species using fluorogenics or chromogenics, dry plate culturing, differentiation among bacteria of interest using biochemical profiling, enumeration using impedence technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are summarized here and discussed in relation to their specific advantages or disadvantages when implemented in a food microbiology setting.     

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.     

Comparison of methods for enumeration of selected coliforms exposed to ozone

mT7 medium performed no better than m-Endo medium in enumerating cells of Escherichia coli and Citrobacter freundii exposed to ozone. Also, there was no difference in the plate count of heterotrophic bacteria in ozonated raw water determined on modified Henrici agar or R2A agar. Statistically significant differences were seen between bacteria and the type of water in which they were suspended during ozonation.     

Induction of intracellular and extracellular beta-galactosidase activity in Phycomyces blakesleeanus

The inductive effect of different sugars on beta-galactosidase synthesis in Phycomyces blakesleeanus has been studied. The enzyme was inducible by galactose and fructose. When grown on these sugars the enzyme level was 10-20 times greater than when grown on glucose. We have detected both intra- and extracellular beta-galactosidase activity when Phycomyces blakesleeanus was grown on galactose, but only extracellular beta-galactosidase activity when grown on fructose plus lactose.     

Quality control of bacterial enumeration.

Standard bacterial suspensions can be used to assess test method performance, via control charts, and inhibition of recovery when analyzing water samples. Variability in standard suspensions prepared from different strains and species and the use of frozen environmental samples for quality control for spore and bacteriophage analyses are also discussed.     

Comparison of methods for enumeration of selected coliforms exposed to ozone.

mT7 medium performed no better than m-Endo medium in enumerating cells of Escherichia coli and Citrobacter freundii exposed to ozone. Also, there was no difference in the plate count of heterotrophic bacteria in ozonated raw water determined on modified Henrici agar or R2A agar. Statistically significant differences were seen between bacteria and the type of water in which they were suspended during ozonation.     

Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria

The genotype of a mouse influences whether or not it will survive infection with the agent of murine typhoid, Salmonella typhimurium. The best-characterized murine salmonella response gene is a Chromosome 1 locus designated Ity. Inbred strains of mice that express the Itys allele are unable to contain the net growth of Salmonella typhimurium within their spleens and livers, and usually die early in the infection. By contrast, mice homozygous or heterozygous for the Ityr allele are able to control the net multiplication of Salmonella typhimurium within these organs. The Ity gene also appears to regulate the extent of replication within murine reticuloendothelial cell tissues of the obligate intracellular parasite Leishmania donovani, as well as the facultative intracellular bacteria Mycobacterium bovis and Mycobacterium lepraemurium. Previous studies from our laboratory strongly suggested that Ityr mice are more resistant to S. typhimurium infection than are Itys mice, because resident Ityr macrophages kill salmonellae more efficiently than do Itys macrophages. In this study, we used an in vitro macrophage assay to assess the specificity of the enhanced killing capacity of Ityr macrophages. We found that Ityr macrophages were better able than Itys macrophages to kill both intracellular bacteria (Salmonella typhi) and extracellular bacteria (Escherichia coli, Staphylococcus aureus, Corynebacterium diphtheriae). Thus, the diversity of organisms affected by Ity expression suggests that the product of this gene may play a key regulatory role in the initial interaction of mice with a variety of microbial agents.     

Differential requirement of P2X7 receptor and intracellular K+ for caspase-1 activation induced by intracellular and extracellular bacteria

Interleukin-1beta (IL-1beta) is a pro-inflammatory cytokine that plays an important role in host defense and inflammatory diseases. The maturation and secretion of IL-1beta are mediated by caspase-1, a protease that processes pro-IL-1beta into biologically active IL-1beta. The activity of caspase-1 is controlled by the inflammasome, a multiprotein complex formed by NLR proteins and the adaptor ASC, that induces the activation of caspase-1. The current model proposes that changes in the intracellular concentration of K(+) potentiate caspase-1 activation induced by the recognition of bacterial products. However, the roles of P2X7 receptor and intracellular K(+) in IL-1beta secretion induced by bacterial infection remain unknown. Here we show that, in response to Toll-like receptor agonists such as lipopolysaccharide or infection with extracellular bacteria Staphylococcus aureus and Escherichia coli, efficient caspase-1 activation is only triggered by addition of ATP, a signal that promotes caspase-1 activation through depletion of intracellular K(+) caused by stimulation of the purinergic P2X7 receptor. In contrast, activation of caspase-1 that relies on cytosolic sensing of flagellin or intracellular bacteria did not require ATP stimulation or depletion of cytoplasmic K(+). Consistently, caspase-1 activation induced by intracellular Salmonella or Listeria was unimpaired in macrophages deficient in P2X7 receptor. These results indicate that, unlike caspase-1 induced by Toll-like receptor agonists and ATP, activation of the inflammasome by intracellular bacteria and cytosolic flagellin proceeds normally in the absence of P2X7 receptor-mediated cytoplasmic K(+) perturbations.     

Culture dependent methods for enumeration of sulphate reducing bacteria (SRB) in the Oil and Gas industry

The group of anaerobic microorganisms collectively referred to as Sulphate Reducing Bacteria (SRB) is a major concern in the Oil and Gas industry primarily because of this group’s ability to generate substantial amounts of hydrogen sulfide and insoluble ferrous sulfide in the presence of iron. Traditionally, the Oil industry has relied on two recommended standard practices i.e. API RP-38 and NACE TM0194 for the detection and enumeration of culturable sulphate reducing bacteria for routine field monitoring. API RP-38 has now been withdrawn without any replacement. Data generated by nonstandard molecular microbiological methods which are still in the developmental stage cannot be compared with the accepted control levels for SRBs in oil field systems, monitored over the years with viable culture methods. Culture based methodologies are still important tools for the study of SRB, as they help in understanding the physiological characteristics which may be similar or different across phylogenetically similar bacteria. This review article therefore tries to highlight the continued importance of culture dependent methods for detection and enumeration of SRB in Oil field systems and the need for further development of an universal standard culture based method for studying SRB in the Oil and Gas industry.     

Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites

Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.     

Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands

Aims:  The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique.
Methods and Results:  Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts.
Conclusions:  The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types.
Significance and Impact of the Study:  Method standardization will improve the understanding of how sands affect surface water quality.     

The enumeration of proteolytic bacteria in foods

Summary For the enumeration of proteolytic bacteria in foods, regularly or sometimes obtained by microbial fermentation, a surface plating technique, using Frazier's gelatin/agar, incubated for two days at 31±1°C. and thereupon developed with sublimate solution, appeared useful.     

A method for the enumeration of bacterial adhesion to epithelial cells using image analysis

Abstract Computerised image analysis was utilised to enumerate the attachment of Staphylococcus epidermidis to HEp2 cell monolayers. A differential staining technique was employed such that individual staphylococcal cells stood out in sharp contrast against the uneven cell surface and granular contents of the epithelial cells. The primary image analysis operation involved subtracting an out-of-focus image from an in-focus image of the bacteria on the monolayer, thereby accentuating the bacterial image. Enumeration, using a particle counting routine, was rapid and reproducible, facilitating counting in excess of 700 bacteria per field at ×500 magnification. The computerised programme compared favourably with manual counting and would provide a rapid, objective and morphologically discriminatory method for evaluating bacterial attachment to various tissues.