Enchanced accuracy of coliform testing in seawater by a modification of the most-probable-number method.

A 1-year study of marine water sample from six beach locations showed that the most-probable-number method failed to recover significant numbers of coli-forms. Modifying this method by transferring, after 48 h, presumptive negatives (growth and no gas production) to confirmed and fecal coliform media significantly improved recovery. Tests which were presumptive negative but confirmed as fecal coliform positive were designated as false negatives. Most-probable-number method false negatives occurred throughout the year, with 143 of 270 samples collected producing false negatives. More than 50% of fecal coliform false-negative isolates were Escherichia coli. Inclusion of false-negative tubes into the coliform most-probable-number method data resulted in increased violation of the California ocean water contact sports standard at all sites. More than 20% of the samples collected were in violation of this standard. These data indicate that modification of the most-probable-number method increases detection of coliform numbers in the marine environment.     

Modification of the standard most-probable-number procedure for fecal coliform bacteria in seawater and shellfish.

To determine whether or not presumptive tubes which take 48 h to become positive need to be transferred to EC medium during the fecal coliform multitube most-probable-number procedure, 572 seawater and shellfish samples were considered. When the 24-hour positive tubes alone were transferred to EC, it appeared that incorrect dilution data would be rare.     

Rate of occurrence of false-positive results from total coliform most-probable-number analysis of shellfish and estuaries.

The incidence of confirmed test, false-positive coliform most-probable-number results was compared with environmental parameters and was found to be inversely related to water temperature. It is concluded that the completed coliform test must be done when water temperatures drop below 15 degrees C.     

Modified agar medium for detecting environmental salmonellae by the most-probable-number method.

Salmonellae in the environment remain a potential source of disease. Low numbers of salmonellae have been detected and enumerated from environmental samples by most-probable-number methods which require careful colony selection from a plated agar medium. A modified xylose lysine brilliant green medium was prepared to control the loss of selectivity caused by heating the brilliant green component. Added agar reduced colony spreading. The medium contained 47 g of xylose lysine agar base per liter; the agar content was adjusted to 2%, autoclaved, cooled to 50 degrees C, and then amended just before pouring to include H2S indicator and 7 ppm (7 ml of 1:1,000 brilliant green per liter) of unheated brilliant green dye. H2S-positive salmonellae were easily detected from sewage sludge compost to the exclusion of most other gram-negative bacteria. As a result, fewer non-salmonellae were picked for further most-probable-number analysis, greatly reducing the work load associated with the most-probable-number method. Direct plating was possible for enumerating salmonellae in laboratory composts containing ca. 10(3) or more salmonellae.     

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.     

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.     

Modified agar medium for detecting environmental salmonellae by the most-probable-number method

[This corrects the article on p. 1027 in vol. 48.].     

Modified agar medium for detecting environmental salmonellae by the most-probable-number method

Salmonellae in the environment remain a potential source of disease. Low numbers of salmonellae have been detected and enumerated from environmental samples by most-probable-number methods which require careful colony selection from a plated agar medium. A modified xylose lysine brilliant green medium was prepared to control the loss of selectivity caused by heating the brilliant green component. Added agar reduced colony spreading. The medium contained 47 g of xylose lysine agar base per liter; the agar content was adjusted to 2%, autoclaved, cooled to 50 degrees C, and then amended just before pouring to include H2S indicator and 7 ppm (7 ml of 1:1,000 brilliant green per liter) of unheated brilliant green dye. H2S-positive salmonellae were easily detected from sewage sludge compost to the exclusion of most other gram-negative bacteria. As a result, fewer non-salmonellae were picked for further most-probable-number analysis, greatly reducing the work load associated with the most-probable-number method. Direct plating was possible for enumerating salmonellae in laboratory composts containing ca. 10(3) or more salmonellae.     

Quantification of methicillin-resistant Staphylococcus aureus strains in marine and freshwater samples by the most-probable-number method

Recreational beach environments have been recently identified as a potential reservoir for methicillin-resistant Staphylococcus aureus (MRSA); however, accurate quantification methods are needed for the development of risk assessments. This novel most-probable-number approach for MRSA quantification offers improved sensitivity and specificity by combining broth enrichment with MRSA-specific chromogenic agar.     

Analysis of sample preparation procedures for enumerating fecal coliforms in coarse southwestern U.S. bottom sediments by the most-probable-number method

The determination of bacterial densities in aquatic sediments generally requires that a dilution-mixing treatment be used before enumeration of organisms by the most-probable-number fermentation tube method can be done. Differential sediment and organism settling rates may, however, influence the distribution of the microbial population after the dilution-mixing process, resulting in biased bacterial density estimates. For standardization of sample preparation procedures, the influence of settling by suspended sediments on the fecal coliform distribution in a mixing vessel was examined. This was accomplished with both inoculated (Escherichia coli) and raw, uninoculated freshwater sediments from Saguaro Lake, Ariz. Both test sediments were coarse (greater than 90% gravel and sand). Coarse sediments are typical of southwestern U.S. lakes. The distribution of fecal coliforms, as determined by the most-probable-number method, was not significantly influenced by sediment settling and remained homogenous over a 16-min postmix period. The technique developed for coarse sediments may be useful for standardizing sample preparation techniques for other sediment types.     

Analysis of sample preparation procedures for enumerating fecal coliforms in coarse southwestern U.S. bottom sediments by the most-probable-number method.

The determination of bacterial densities in aquatic sediments generally requires that a dilution-mixing treatment be used before enumeration of organisms by the most-probable-number fermentation tube method can be done. Differential sediment and organism settling rates may, however, influence the distribution of the microbial population after the dilution-mixing process, resulting in biased bacterial density estimates. For standardization of sample preparation procedures, the influence of settling by suspended sediments on the fecal coliform distribution in a mixing vessel was examined. This was accomplished with both inoculated (Escherichia coli) and raw, uninoculated freshwater sediments from Saguaro Lake, Ariz. Both test sediments were coarse (greater than 90% gravel and sand). Coarse sediments are typical of southwestern U.S. lakes. The distribution of fecal coliforms, as determined by the most-probable-number method, was not significantly influenced by sediment settling and remained homogenous over a 16-min postmix period. The technique developed for coarse sediments may be useful for standardizing sample preparation techniques for other sediment types.     

Modification of the 14C most-probable-number method for use with nonpolar and volatile substrates.

A method was developed to allow the use of volatile and nonpolar substrates in 14C most-probable-number tests. Naphthalene or hexadecane was sorbed to filter paper disks and submerged in minimal medium. The procedure reduced the volatilization of the substrates while allowing them to remain available for microbial degradation.     

Impact of verification media and resuscitation on accuracy of the membrane filter total coliform enumeration technique.

Verification of membrane filter total coliform colonies was compared in lauryl tryptose broth, and m-LAC broth primary media and brilliant green-lactose-bile broth and EC broth secondary media. Verification in m-LAC broth yielded the greatest number of aerogenic isolates for both untreated surface water and drinking water samples. Verification in brilliant green-lactose-bile broth increased the number of false-negative reactions. At least 90% of the isolates aerogenic in primary verification media and anaerogenic in brilliant green-lactose-bile broth were representative of the coliform genera. The addition of a resuscitation step in the membrane filter technique did not yield greater numbers of verified coliforms per sample. Verification of both typical and atypical colonies in m-LAC broth resulted in a 10-fold increase in coliform numbers from untreated surface water. With drinking water, verification of both colony types resulted in an increase from less than 1 coliform per 100 ml to greater than 1/100 ml. A single-step verification in m-LAC broth is proposed as a more rapid and sensitive coliform verification procedure than the standard technique.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

The significance of testing for Pseudomonas aeruginosa in recreational seawater beaches.

The occurrence of Pseudomonas aeruginosa and faecal coliforms in Mediterranean sea water from beaches was investigated. Water samples (1,598 in toto) were tested and P. aeruginosa was found in 222 samples (14%). In 31% of samples where P. aeruginosa was detected, faecal coliforms of less than ten bacteria per 100 ml were found. In a group of 98 samples which had > 500 faecal coliforms per 100 ml, 41% had no detectable P. aeruginosa. The inclusion of P. aeruginosa as an additional parameter for approving beaches for recreational activity is recommended.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus.

The effects of the addition of catalase (EC 1.11.1.6) or pyruvate on the enumeration of Staphylococcus aureus in Trypticase soy broth with 10% NaCl were examined using a most-probable-number technique. Addition of catalase or pyruvate to the broth increased enumeration of all heat-stressed S. aureus strains tested. Increases were also observed with nonstressed cells. Catalase and pyruvate were similarly effective when added to Trypticase soy broth-10% NaCl in enumerating staphylococci naturally present in low-temperature-rendered ground-beef samples.