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.     

Comparison of methods of enumerating coliforms after UV disinfection.

In view of the differences that have been found between the most-probable-number and membrane filtration methods for the recovery of coliforms from chlorinated samples, the survival of total and fecal coliforms in UV-irradiated effluent samples, as tested by the most-probable-number and standard single-step membrane filtration methods, was compared. There were no significant differences in the survival of total and fecal coliforms, as tested by the two methods. In a separate set of experiments comparing total and fecal coliform survival, as tested by the most-probable-number method, only a very small but statistically significant difference of 0.1 log survival units was found. For UV-disinfected wastewater effluents, standard one-step membrane filtration procedures are comparable to standard most-probable-number procedures.     

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.     

Membrane filter technique for the quantification of stressed fecal coliforms in the aquatic environment.

A two-layer membrane filtration (MF) medium (injury-mitigating MF [IM-MF]) and a procedure for the enumeration of injured fecal coliforms are described. These procedures included the addition of glycerol and acetate plus reducing agents to both layers of a two-layer medium and rinsing of the filter with a rich resuscitation medium. Some changes in incubation time and temperatures were used. This method was compared with the multiple-tube fermentation most-probable-number procedure and the one-step M-FC agar-membrane filter method (direct M-FC) in terms of fecal coliform recovery from various aquatic environments that cause bacterial injury. With chlorinated sewage effluents, results of the IM-MF technique were equal to or greater than the most probable number in 9 of 18 trials and were 1.3 to 19 times greater than the M-FC method. When sewage samples were chlorinated in the laboratory, fecal coliform counts with IM-MF equaled or exceeded the most probable number in 7 of 15 trials and always exceeded the M-FC. M-FC was exceeded by IM-MF in 30 of 33 trials with clean mountain stream water. Fecal coliform bacteria that were exposed to low levels of an iodophore in the laboratory produced IM-MF counts 3 to 10 times greater than those with M-FC. A biochemical rationale for the formation of the IM-MF medium is discussed.     

Membrane Filter Technique for the Quantification of Stressed Fecal Coliforms in the Aquatic Environment

A two-layer membrane filtration (MF) medium (injury-mitigating MF [IM-MF]) and a procedure for the enumeration of injured fecal coliforms are described. These procedures included the addition of glycerol and acetate plus reducing agents to both layers of a two-layer medium and rinsing of the filter with a rich resuscitation medium. Some changes in incubation time and temperatures were used. This method was compared with the multiple-tube fermentation most-probable-number procedure and the one-step M-FC agar-membrane filter method (direct M-FC) in terms of fecal coliform recovery from various aquatic environments that cause bacterial injury. With chlorinated sewage effluents, results of the IM-MF technique were equal to or greater than the most probable number in 9 of 18 trials and were 1.3 to 19 times greater than the M-FC method. When sewage samples were chlorinated in the laboratory, fecal coliform counts with IM-MF equaled or exceeded the most probable number in 7 of 15 trials and always exceeded the M-FC. M-FC was exceeded by IM-MF in 30 of 33 trials with clean mountain stream water. Fecal coliform bacteria that were exposed to low levels of an iodophore in the laboratory produced IM-MF counts 3 to 10 times greater than those with M-FC. A biochemical rationale for the formation of the IM-MF medium is discussed.     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Membrane filter method for recovery of fecal coliforms in chlorinated sewage effluents.

The standard one-step M-FC broth-membrane-filter procedure for recovery of fecal coliforms from chlorinated sewage effluents is much less effective than the multiple-tube (most-probable-number) technique. A two-step membrane-filter method, using a pre-enrichment technique with phenol red lactose broth and incubation at 35 degrees C for 4 h, followether 18+/-2 h, enhanced fecal coliform recovery from chlorinated effluents. The results of 126 comparisons using chlorinated effluents from five wastewater plants showed that fecal coliform recovery by using the two-step membrane-filter method is comparable to that using the multiple-tube procedure.     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water.

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Effect of noncoliforms on coliform detection in potable groundwater: improved recovery with an anaerobic membrane filter technique

A total of 529 well and distribution potable water samples were analyzed for total coliforms by the most-probable-number and membrane filter (MF) techniques. Standard plate count bacteria and MF noncoliform bacteria were also enumerated. Frequency of coliform detection, turbidity in most-probable-number tubes, and extensive overgrowth by noncoliforms on MF filters were directly proportional to standard plate counts. Recovery of coliforms was greatest by the MF method at low (less than 100 CFU/ml) standard plate count densities and better by the most-probable-number method (confirming gas and turbid tube) at high (greater than 500 CFU/ml) standard plate count densities. In the latter case, overgrowth by noncoliforms on MF filters suppressed sheen development and, in turn, masked coliform detection. Of 341 atypical (no sheen) MF colonies verified by parallel inoculation of lauryl sulfate broth and billiant green-bile broth, 156 were aerogenic in the latter medium. Of atypical isolates, 84% were identified as either Citrobacter or Enterobacter species. A 4.3-fold reduction in numbers of overgrown MF filters and an 2.2-fold increase in numbers of coliforms recovered from 127 water samples was accomplished by anaerobic incubation of MF cultures. This anaerobic modification of the standard MF technique significantly reduced overgrowth and enhanced recovery of coliforms from potable groundwater. This technique is simple, cost effective, and suitable for monitoring of untreated ground water common to some small water systems and private water supplies.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Effect of noncoliforms on coliform detection in potable groundwater: improved recovery with an anaerobic membrane filter technique.

A total of 529 well and distribution potable water samples were analyzed for total coliforms by the most-probable-number and membrane filter (MF) techniques. Standard plate count bacteria and MF noncoliform bacteria were also enumerated. Frequency of coliform detection, turbidity in most-probable-number tubes, and extensive overgrowth by noncoliforms on MF filters were directly proportional to standard plate counts. Recovery of coliforms was greatest by the MF method at low (less than 100 CFU/ml) standard plate count densities and better by the most-probable-number method (confirming gas and turbid tube) at high (greater than 500 CFU/ml) standard plate count densities. In the latter case, overgrowth by noncoliforms on MF filters suppressed sheen development and, in turn, masked coliform detection. Of 341 atypical (no sheen) MF colonies verified by parallel inoculation of lauryl sulfate broth and billiant green-bile broth, 156 were aerogenic in the latter medium. Of atypical isolates, 84% were identified as either Citrobacter or Enterobacter species. A 4.3-fold reduction in numbers of overgrown MF filters and an 2.2-fold increase in numbers of coliforms recovered from 127 water samples was accomplished by anaerobic incubation of MF cultures. This anaerobic modification of the standard MF technique significantly reduced overgrowth and enhanced recovery of coliforms from potable groundwater. This technique is simple, cost effective, and suitable for monitoring of untreated ground water common to some small water systems and private water supplies.     

Immunofluorescent evidence of Proteus mirabilis swarm cell formation on sterilized rat feces.

Swarming Proteus spp. were detected with the use of proteometry (a most-probable-number technique) in the fecal material of selected animal species and in raw sewage from a local sewage treatment plant. Proteus spp. were not detected in any of several soil and freshwater samples examined. Since rat feces harbored high numbers of Proteus mirabilis compared with other habitats examined, we chose to examine it for the possibility of supporting swarming. Immunofluorescent studies with a strain-specific conjugate revealed the morphogenesis of short forms into elongated swarm cells upon the surface of sterilized rat feces that had been inoculated with short forms of P. mirabilis. the same phenomenon was not observed consistently when nonsterile rat feces were inoculated and examined with immunofluorescence.