Modification of delayed-incubation procedure for detection of fecal coliforms in water.

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Modification of delayed-incubation procedure for detection of fecal coliforms in water

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Rapid Determination of the Presence of Enteric Bacteria in Water

A rapid and sensitive method is described for the detection of bacteria in water and various other natural substrates by the isolation of specific bacteriophage. By the addition of large numbers of the organism in question to the sample, the presence of virulent bacteriophage can be demonstrated in as little as 6 to 8 h. Fecal coliform, total coliform, and total coliphage counts were determined for over 150 water samples from several geographical areas over a period of 2 years. Computer analysis of the data shows a high degree of correlation between fecal coliforms and the coliphage present in the samples. With a high correlation coefficient between fecal coliform and coliphage counts, predictions of the fecal coliforms may be made by enumeration of the phage.     

Soil microbial populations, community composition, and activity as affected by repeated applications of hog and cattle manure in eastern Saskatchewan

A field site near Humboldt, Saskatchewan, was annually treated with hog or cattle manure and cropped to canola, spring wheat, barley, and canola from 1997 to 2000. During each growing season, soil was analyzed for microbial populations in terms of activity and community structure, and crops were assessed for root rot and foliar diseases. Microbial activity in soils treated with cattle manure was higher than in soils treated with hog manure or urea. Similarly, nitrous oxide emissions from soil increased with increasing rates of hog and cattle manure. Potential human pathogens, including Rahnella, Serratia, Proteus, Leclercia, and Salmonella species, were identified in soils that received cattle manure, whereas pseudomonads were the dominant species in the hog-manure-treated soil. Fecal coliforms were confirmed in soils that received hog or cattle manure. However, Enterobacteriaceae populations were 10-fold higher in soils receiving cattle manure than in soils receiving the other treatments. Increasing cattle manure rates increased fecal coliform population, but there was no indication that increased hog manure rates increased fecal coliform populations. Addition of urea, hog manure, or cattle manure to the soil did not increase foliar disease in wheat, barley, and canola and had variable effects on root rot incidence in cereals.     

Effect of dredge spoil deposition on fecal coliform counts in sediments at a disposal site.

The most-probable-number of fecal coliforms in sediments was monitored at the New London dump site in Long Island Sound during the deposition of dredge spoil from the Thames River. Although the geometric mean for fecal coliforms at five stations in the river was 14,000/100 ml before dredging commenced, the deposition of this material did not increase the incidence of fecal coliforms at 17 spoil stations and 13 control stations in the disposal and surrounding areas. Fecal coliforms appear to occur only in the surface sediment material and are diluted by the subsurface material during the dredging operation. Fecal coliform analyses of bottom waters during high and low tides indicated that the flow of water from the Thames River played a major role in determining the most-probable-number of fecal coliforms in the sediments at the disposal site.     

Effect of dredge spoil deposition on fecal coliform counts in sediments at a disposal site.

The most-probable-number of fecal coliforms in sediments was monitored at the New London dump site in Long Island Sound during the deposition of dredge spoil from the Thames River. Although the geometric mean for fecal coliforms at five stations in the river was 14,000/100 ml before dredging commenced, the deposition of this material did not increase the incidence of fecal coliforms at 17 spoil stations and 13 control stations in the disposal and surrounding areas. Fecal coliforms appear to occur only in the surface sediment material and are diluted by the subsurface material during the dredging operation. Fecal coliform analyses of bottom waters during high and low tides indicated that the flow of water from the Thames River played a major role in determining the most-probable-number of fecal coliforms in the sediments at the disposal site.     

Evaluation of Coli-Count Samplers for Possible Use in Standard Counting of Total and Fecal Coliforms in Recreational Waters

Millipore Coli-Count Samplers were used to enumerate colonies of laboratory cultures of coliform bacteria. The samplers gave significantly lower counts than standard membrane-filter procedures for both total and fecal coliforms. Although the samplers are useful for semiquantitative analysis as indicated by the manufacturer, they are not suitable for standard examinations of recreational waters.     

Persistence and distribution of pollution indicator bacteria on land used for disposal of piggery effluent.

Numbers of pollution indicator bacteria (fecal coliforms and fecal streptococci) were assessed on land to which effluent from intensively housed pigs had been applied. Topsoil (to a 30-mm depth) was found to provide a more favorable environment for fecal coliform persistence than was pasture or subsoil. Times required for a 90% reduction in number (T90) in topsoil (calculated by linear regression of log counts obtained in a 6-week period after effluent application) ranged from 7 to 20 days (mean T90, 11 days). T90 values for fecal coliforms fell within this range irrespective of the season of application and for a number of soil types and climatic conditions. The range in die-off times was encountered irrespective of the fecal coliform count in the applied effluent or the application regimen (125 to 1,000 kg of elemental nitrogen in the form of effluent per ha; return periods, 3 to 12 months). Autumn and winter conditions were conducive to the persistence of a survivor tail of these bacteria at 10(1) to 10(3) cells per g of topsoil. Fecal streptococci survived similarly on soil and pasture (T90, ca. 14 days) and appeared slightly more suited to survival in the environment than did fecal coliforms. Contamination of subsoils after effluent applications occurred at a rate well in excess of the infiltration capacity of the soil, presumably by percolation of the effluent through soil cracks. Contamination levels of subsoils in the experimental area generally remained low.     

Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.     

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

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 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.     

Release of Sediment-Bound Fecal Coliforms by Dredging

Fecal coliform concentrations increased significantly (F test) in the immediate vicinity of a maintenance dredging operation in the Mississippi River navigation channel. Increased counts were attributed to the disturbance and relocation of bottom sediments by dredging and a concomitant release of sediment-bound fecal coliforms.     

Occurrence of Microbial Indicators and Clostridium perfringens in Wastewater, Water Column Samples, Sediments, Drinking Water, and Weddell Seal Feces Collected at McMurdo Station, Antarctica

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.     

Isolation of Fecal Coliform Bacteria from the Diamondback Terrapin (Malaclemys terrapin centrata)

Total and fecal coliform bacteria were isolated from the cloaca and feces of the estuarine diamondback terrapin. The majority of samples contained fecal coliforms. Escherichia coli was the predominant fecal coliform species isolated, and members of the genus Salmonella were isolated from 2 of 39 terrapins. Fecal coliform numbers are used to regulate shellfish harvests, and diamondback terrapins inhabit the brackish-water habitats where oyster beds are found; therefore, these findings have implications for the efficacy of current regulatory parameters in shellfishing waters.     

Modification of M-FC medium by eliminating rosolic acid.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).     

Modification of M-FC medium by eliminating rosolic acid.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).     

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.