RAPID ENUMERATION OF CLOSTRIDIAL SPORES IN RAW MILK SAMPLES USING AN IMPEDIMETRIC METHOD

Late spoilage of cheese is due to gas formation during lactic acid fermentation by spore-forming, gram-positive, anaerobic clostridia of the species Ciostridium tyrobutyricum, Clostridium butyricum and Clostridium sporogenes. Since small numbers of such clostridial spores readily cause considerable losses in cheese production, spore numbers of fewer than 100 spores/liter must be determined reliably. Until recently, the only reliable method available was the time-consuming (7 days) and cumbersome Most Probable Number Method (MPN). The objective of this study was to examine the feasibility of using impedance technology as an alternative method for the enumeration of clostridial spores. Three to fifteen replicates of 7.5–12.0 mL samples were tested using an impedimetric method with and without the addition of Oxyrase to generate anaerobic conditions within the impedance measurement cells. Results were obtained in less than 48 h. Data derived from the rapid impedance method were statistically comparable to those obtained using the reference method (MPN).     

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

AIM: Calibration of impedance measurement was performed vs the Association Fran?oise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.     

Quantitative analysis of ammonia oxidising bacteria using competitive PCR

Culture-based methods for enumeration, such as most probable number (MPN) methodologies, have proved inefficient due to difficulties in the isolation and cultivation of ammonia oxidising bacteria in the laboratory. Biases are associated with the isolation of bacteria in selective media and organisms cultivated in the laboratory may not be truly representative of those in the environment. In this study, we developed a competitive PCR (cPCR)-based method based on the amplification of 16S rRNA genes specific for the beta-subgroup proteobacterial ammonia oxidising bacteria for enumeration of these organisms. Populations in both agricultural soils and estuarine sediments were quantified by traditional MPN and by cPCR. The numbers of ammonia oxidisers for both sample types were significantly underestimated by conventional MPN and were 1-3 orders of magnitude lower than those obtained by cPCR. Higher numbers of ammonia oxidisers found in fertilised plots in agricultural soils by the cPCR technique were not observed in MPN estimates. It was necessary to construct a separate standard curve for each sample type as differences in DNA extraction, quantity and purity had a significant bearing on the ease of PCR of both competitor and target DNA.     

Most-probable-number technique for the enumeration of aromatic degraders in natural environments

A most-probable-number (MPN) method is described for the enumeration of heterotrophic populations capable of utilizing chlorinated and nonchlorinated benzoates and phenols as sole carbon sources. A correlation coefficient of 0.91 was obtained between the numbers determined by the MPN technique and the standard plate count. The MPN method gave realistic cell counts when population densities were low, and the presence of oligocarbophiles did not give spurious results.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

Enumeration of High Numbers of Bacteria Using Hydrophobic Grid-Membrane Filters

Printing a wax grid on a conventional membrane filter yields a device functioning as a most probable number apparatus (MPN), used at a single dilution but with a very large number of growth compartments (e.g., 3,650). By restraining the lateral spread and confluence of colonies, the hydrophobic grid-membrane filter (HGMF) allows growth- or colony-forming units (GU) to be resolved at levels far above those which produce an uncountable lawn on a conventional membrane filter. It also eliminates the size variation of normal bacterial colonies. As a result, the HGMF can give more accurate estimates of the concentration of GU. The method by which grid-cell count observations can be used to obtain MPN estimates of the number of GUs is described, and estimates obtained using the MPN method on the HGMF are compared with those resulting from conventional colony count procedures on membrane filters. A linear relation was observed between MPNGU and the number of GUs, at levels up to 30,000 GUs, for pure cultures of bacteria and for samples of natural waters. The HGMF has great potential for reducing the labor required in quantitative microbiology, since it allows, with one filter, enumeration of microorganisms over a very large concentration range and therefore reduces the need to make dilutions.     

Quantification of Listeria monocytogenes in fermented sausages by MPN-PCR method

AIMS: To combine the principles of most-probable-number (MPN) statistics and the conventional PCR technique to enumerate Listeria monocytogenes in fermented sausages. METHODS AND RESULTS: A simple method to enumerate L. monocytogenes in fermented sausages was developed and compared with direct plating in Palcam agar. Species-specific MPN-PCR, but not direct plating, made the enumeration of L. monocytogenes possible in all assayed samples. CONCLUSIONS: MPN-PCR proved to be a rapid and reliable method for enumerating L. monocytogenes in fermented sausages, including low contaminated samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This MPN-PCR technique may facilitate the enumeration of L. monocytogenes for routine analyses in fermented sausages without excessive work.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10¹ and 10⁵ cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Glucose and plant exudate enhanced enumeration of bacteria capable of degrading polycyclic aromatic hydrocarbons

Enumerating environmental microbial isolates capable of polycyclic aromatic hydrocarbon (PAH) degradation can provide insight into the microbe-plant interactions that facilitate PAH removal. We examined a known PAH degrader ( Pseudomonas putida G7), a nondegrader ( Agrobacterium tumefaciens LBA4404), and several microorganisms isolated from the environment by using a PAH cocktail in an enumeration medium?with or without 0.025% (m/v) glucose and (or) root exudates. Compared with the standard most probable number (MPN), the addition of glucose and root exudates in a modified MPN method resulted in a 3- to 11-fold enhancement of PAH degraders being enumerated among microorganisms found in PAH-contaminated soils. High-performance liquid chromatography analysis verified that PAH levels were reduced using this modified enumeration method. Low levels of glucose, perhaps in concert with other materials in exudates, may promote microbial metabolism, thereby enhancing PAH degradation.     

EVALUATION OF TWO NONRADIOACTIVE GENE PROBES FOR THE ENUMERATION OF VIBRIO PARAHAEMOLYTICUS IN CRABMEAT

This study evaluated two nonradioactive DNA probe procedures for the detection and enumeration of Vibrio parahaemolyticus in crabmeat by comparing counts obtained by direct plating and by most probable number (MPN) procedures. The nonradioactive probes evaluated were an alkaline phosphatase-labeled thermolabile direct hemolysin (AP-tdh) and a digoxigenin-labeled thermostable direct hemolysin (DG-tdh) for detection and enumeration of total and pathogenic V. parahaemolyticus, respectively. Inoculated samples (50 g each) of steamed crabmeat were analyzed by nine analysts in seven laboratories. Samples were inoculated with various Vibrio strains and combinations of strains at different levels of inoculation (0 to 92,000 cells/g). The results indicated that the AP-tlh probe was reliable for identification and enumeration of total V. parahaemolyticus and the DG-tdh probe was specific for the pathogenic strains. Both probes required less effort and expense than the biochemical testing and hemolysin assays that have previously been used. The MPN and direct plating procedures using the nonradioactive probes were both effective for enumeration of total and pathogenic V. parahaemolyticus in the absence of competing microflora, but direct plating was preferable to MPN for enumeration of V. parahaemolyticus, especially pathogenic strains, in the presence of competitors.     

Evaluation of recovery methods to detect faecal streptococci in polluted waters

AIMS: This paper compares the faecal streptococci count on 25 samples of polluted waters obtained with three techniques: most probable number (MPN), membrane filtration (MF) and pour plate (PP) methods. Although the PP method is a simple technique, familiar to water bacteriologists, it is not recommended in the international methods. METHODS AND RESULTS: For the MPN method, azide dextrose broth and ethyl violet azide broth were employed. For the MF technique, Millipore filters were placed onto azide maltose agar (KF agar), while for the PP method, 1 ml of a decimal water dilution was added to (Kennel Faecal) KF medium. Regression analysis and Friedman's ANOVA were performed to determine the relationship between faecal streptococci counts obtained with the three techniques. Statistical analysis of the results showed that the MPN, MF and PP techniques were equally valid with respect to faecal streptococci enumeration in polluted waters. CONCLUSION: Since the PP method was found to be as good as the other techniques, it may be preferred in polluted waters. It is more economical in terms of both time and materials than the MPN count, and it is as accurate as the MF count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that the PP method, although not recommended internationally, is a reliable alternative to MF and MPN.     

Microplate MPN-enumeration of monocyclic- and dicyclic-aromatic hydrocarbon degraders via substrate phase-partitioning

The high aqueous solubility of monoaromatic and some diaromatic oil components may hinder classical growth-based MPN enumeration of bacterial mono- and di-aromatics degraders because these aromatics are toxic in high concentrations. We developed a microplate MPN method for the enumeration of toluene-, xylene-, naphthalene-, biphenyl- and benzothiophene-degraders on the basis of phase-partitioning of substrate between a biologically inert organic phase and an aqueous mineral salt medium. This way, it was possible to maintain non-toxic, aqueous concentrations in the microplate wells. Depletion of aqueous aromatics by growth of the degraders was prevented by the continuous solubilization of aromatics from the silicone phase. The method was validated by MPN enumerating degrader cultures both with phase-partitioned aromatics and with tryptic soy broth as carbon sources. The applicability of the method was demonstrated by MPN-enumerating mono- and di-aromatic degraders in soils of varying hydrocarbon pre-exposure. An erratum to this article can be found at     

A statistical comparison of two culturing methods for enumerating sulfate-reducing bacteria

The addition of alkaline pyrogallol-soaked cotton wool plugs has been used by other workers to remove oxygen from the headspace gas in culture tubes for the growth of sulfate-reducing bacteria (SRB). This study compares the enumeration fo SRB using the most probable number (MPN) method in tubes with and without the pyrogallol plugs. In both cases, the liquid medium contained two iron finishing nails which help reduce the redox potential of the medium. For each of the 25 samples from oil fields, cooling water systems and natural environments, the time-consuming method using pyrogallol plugs in screw cap tubes yielded virtually the same MPN values as the same method without the plugs and using Kaput® closures on the tubes. However, the pyrogallol plug method, which requires approximately 10-fold more technician time, was superior for pure culture enumeration and in cases where SRB greatly outnumber heterotrophs.     

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.     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.