Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium

Based on results from eight experiments, no overall difference was found between roll tube and three- and five-tube most-probable-number (MPN) methods for estimating total numbers of ruminal bacteria. However, standard errors for the replicate means within an experiment were higher with the MPN procedures. Visual growth and pH were the criteria used for scoring the MPN tubes. Total numbers were significantly higher in MPN medium containing 40% ruminal fluid, as compared with a complete medium without ruminal fluid. By using a broth medium containing ball-milled cellulose and soluble carbohydrates as energy sources, it was possible to estimate both total and cellulolytic ruminal bacterial numbers in the same MPN series. Disappearance of cellulose and decrease in pH were used to determine growth. Values did not differ from those obtained in separate MPN assays. By using this method, diurnal changes in total and cellulolytic bacterial numbers were estimated in sheep fed forage or a concentrate-type diet.     

Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium.

Based on results from eight experiments, no overall difference was found between roll tube and three- and five-tube most-probable-number (MPN) methods for estimating total numbers of ruminal bacteria. However, standard errors for the replicate means within an experiment were higher with the MPN procedures. Visual growth and pH were the criteria used for scoring the MPN tubes. Total numbers were significantly higher in MPN medium containing 40% ruminal fluid, as compared with a complete medium without ruminal fluid. By using a broth medium containing ball-milled cellulose and soluble carbohydrates as energy sources, it was possible to estimate both total and cellulolytic ruminal bacterial numbers in the same MPN series. Disappearance of cellulose and decrease in pH were used to determine growth. Values did not differ from those obtained in separate MPN assays. By using this method, diurnal changes in total and cellulolytic bacterial numbers were estimated in sheep fed forage or a concentrate-type diet.     

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.     

Automated statistical analysis of microbial enumeration by dilution series

Equations are formulated for the standard error and confidence interval for the MPN estimate of microbial density from a general dilution series. A statistical test of homogeneity is presented. This tests whether a handling error in the dilution series may have occurred which would invalidate the density estimate. The analysis may be automated using a Basic computer program which contains a fast algorithm for the solution of the general MPN equation. This allows the calculation of the MPN, standard error, 95% confidence interval and test statistic for any dilution series, with any degree of replication at each dilution level, with variable sample volumes at each dilution level, with variable dilution ratio between levels, and with any number of levels.     

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.     

Concentration of Naegleria fowleri in natural waters used for recreational purposes in Sonora, Mexico (November 2007-October 2008)

A survey was designed to know the concentration of Naegleria fowleri in recreational areas in Hornos, Sonora, during a year. Samples were taken monthly at La Isleta and Las Palmas and the total amoeba counts were obtained by the most probable number method (MPN). The identification of N. fowleri was made by PCR. The maximum concentration of total thermophilic amoebae was 9175 MPN/L for La Isleta and 3477 MPN/L for Las Palmas. Thermophilic Naegleria were present mainly during summer and fall. October’s concentrations were up to 201 MPN/L, at both places. The maximum concentrations of N. fowleri were 201 MPN/L and 18 MPN/L for La Isleta and Las Palmas respectively, and were isolated from August to October. The presence of N. fowleri in these particular natural bodies of water reinforces the need for adaptation of preventive measures to avoid cases of primary amoebic meningoencephalitis.     

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.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

A simple method to evaluate the concentration of pentachlorophenol degraders in contaminated soils

A new most probable number (MPN) method for the determination of pentachlorophenol (PCP) degraders in soil using the change in pH due to PCP degradation is compared with a well documented MPN method using radiolabeled PCP. The results of all MPN counts were similar within a 95% confidence limit. The results obtained in MPN per gram of dry soil using pH measurements were 1.8 (+3.1, -1.03) x10 (4) compared to 0.64 (+1.34, -0.42) x 10(4) when using production of [(14)C]CO(2).     

Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement

Conductance measurement for quantitative estimations of Escherichia coli in live bivalve shellfish was evaluated as an alternative to the conventional most probable number (MPN) method used in France. The sensitivity of the two techniques was comparable. A single regression line ( r =-0·968, P < 10^-6) between log₁₀ MPN and detection time (DT) was used to estimate E. coli concentrations for all shellfish examined. Estimation accuracy was ± 0·92 log unit. Repeatability was better for DT than the log₁₀ of MPN estimations (average coefficients of variation 2·7 and 9·3%, respectively). The conductance signal was attributable to E. coli in 96% of cases, and only 0·7% of E. coli cultures failed to exhibit a signal within 20 h. The conductance method reduces analysis handling time and is much easier to use than the MPN method. Moreover, results can be obtained within 5–9 h compared to 3 d for the MPN method.     

Comparison of nine brands of membrane filter and the most-probable-number methods for total coliform enumeration in sewage-contaminated drinking water.

Nine different brands of membrane filter were compared in the membrane filtration (MF) method, and those with the highest yields were compared against the most-probable-number (MPN) multiple-tube method for total coliform enumeration in simulated sewage-contaminated tap water. The water was chlorinated for 30 min to subject the organisms to stresses similar to those encountered during treatment and distribution of drinking water. Significant differences were observed among membranes in four of the six experiments, with two- to four-times-higher recoveries between the membranes at each extreme of recovery. When results from the membranes with the highest total coliform recovery rate were compared with the MPN results, the MF results were found significantly higher in one experiment and equivalent to the MPN results in the other five experiments. A comparison was made of the species enumerated by these methods; in general the two methods enumerated a similar spectrum of organisms, with some indication that the MF method was subject to greater interference by Aeromonas.     

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.     

Determination of the abundance of viable tintinnid cysts in marine sediments in Hiroshima Bay, the Seto Inland Sea of Japan, using a modified MPN method

A modified MPN method was developed for enumerating viable tintinnidcysts in marine sediments Using this MPN method the abundanceof tintinnid cysts was estimated in Hiroshima Bay, the SetoInland Sea of Japan.     

Use of a commercial gene probe assay kit for rapid MPN enumeration of Escherichia coli in drinking water

A commercial gene probe assay kit for presence/absence determination of Escherichia coli in food samples has been used in the standard UK six tube format most probable number (MPN) method for enumerating E. coli in drinking water samples. Presence/absence analysis with the gene probe kit (requiring 3 h) of all MPN tubes after a 21–24 h incubation (minerals modified glutamate; 37°C) enumerated confirmed E. coli in 24–27 h which offered an improvement of up to 48 h over the standard UK MPN method. MPNs determined by the gene probe method and the standard UK method agreed in nine of the 16 water samples which were analysed and for which E. coli concentrations were within the detection limits of the six tube MPN format. This was consistent with the gene probe method detecting one E. coli in a tube. For the other seven water samples, the gene probe method registered positive only 20 of the 30 tubes which the standard UK method determined to be positive. The sensitivity of the gene probe method for drinking water samples, although encouraging, needs improvement perhaps through kit quality control procedures.     

Most-Probable-Number Rapid Viability PCR method to detect viable spores of Bacillus anthracis in swab samples

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1 × 10⁴, 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.     

Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate

We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.     

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.     

Estimation of Low Numbers of Escherichia coli Bacteriophage by Use of the Most Probable Number Method

The estimation of low numbers of the Escherichia coli bacteriophage was made possible by use of the most probable number (MPN) method. This method is similar to the technique used for counting coliform bacteria. The statistical results were computed by referring to tables. The method makes it possible to record values as low as two particles per 100 ml of sample. The direct plate count and MPN method were found to be in good correlation for T2 bacteriophage and bulk T bacteriophage in samples obtained from a sewage treatment plant and from contaminated seawater.     

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.     

A rapid luminescent-phage based MPN method for the enumeration of Salmonella typhimurium in environmental samples

Materials such as soils, waters, sewage sludges and foods can contain low numbers of salmonellas. A most-probable-number (MPN) method that utilized a bioluminescent-bacteriophage is described that allowed the specific determination of as few as one Salmonella typhimurium cell/100 ml of material within 24 h. The method was developed with soil, lake water and sewage sludge inoculated with Salm. typhimurium and had an efficiency of 100% when tested against a traditional MPN method. The protocol is rapid, sensitive, inexpensive, has a low operator time compared to the traditional MPN method, allows for the repair of injured cells and is amenable to automation.