Recovery Of Fecal Coliforms Research Articles

Comparison of methods to determine the microbial quality of alternative irrigation waters

The availability of water for crop irrigation is decreasing due to droughts, population growth, and pollution. Implementation of the Food Safety and Modernization Act (FSMA) for irrigation water standards will discourage growers to use poor microbial quality water for produce crop irrigation. We evaluated the applicability of a novel concentrator method for assessment of microbiological quality of alternative waters including secondary-treated wastewater (STWW), roof-harvest rainwater (RHW), and creek water (CW) in comparison to the standard membrane filtration method. Water samples of 100 ml were filtered through a 0.45 μm membrane filter using a vacuum manifold or concentrated to ∼250 μl using the innovative concentrator. Then they were directly enumerated on specific agars, or enriched to monitor the populations of fecal bacterial indicators (Escherichia coli, enterococci, total and fecal coliforms) and bacterial pathogens (Salmonella, Listeria monocytogenes, and E. coli O157:H7). Presumptive pathogens were confirmed by real-time quantitative PCR. In total, 25 samples of alternative water were analyzed including 7 STWW, 9 RHW, and 9 CW. No significant differences between both detection methods were observed when enumerating indicator bacterial populations and detecting the presence of pathogens in RHW and CW samples. Recovery of fecal coliforms in STWW samples by concentrator analysis was significantly lower than the membrane filtration technique. Results suggest that performance of the concentrator method is equivalent to membrane filtration method in determining the microbiological quality of CW and RHW waters; the type of the water sources may influence the accuracy and sensitivity of the concentrator analysis.

Evaluation of Colilert-18® for Detection and Enumeration of Fecal Coliform Bacteria in Wastewater Using the U.S. Environmental Protection Agency Alternative Test Procedure Protocol

This study compared recovery of fecal coliform bacteria from sewage by Colilert-18 and Standard Methods 9222D (membrane-Fecal Coliform medium) in accordance with the U.S. Environmental Protection Agency (EPA) Alternative Test Protocol (ATP). Samples were collected from 10 different wastewater treatment plants in the northeastern United States and tested in a single laboratory. Twenty replicates of each sample were analyzed by each method, and 200 positive and 200 negative responses were confirmed for each method. Recovery of fecal coliforms by Colilert-18 was significantly higher than (8 of 10 sites) or statistically equivalent to (1 of 10 sites) recovery by the reference method (Standard Methods 9222D) for samples from all but one site. Both methods had low false-positive rates (< 2%); however, the false-negative rate observed with Standard Methods 9222D (21.5%) was substantially higher than that observed with Colilert-18 (7%). The accuracy rates of the two methods were calculated as 96.5 and 88.9% for Colilert-18 and Standard Methods 9222D, respectively. The results of this study demonstrate that Colilert-18 meets the acceptance criteria for alternative methods specified in the EPA ATP.

Comparison of Escherichia coli, Total Coliform, and Fecal Coliform Populations as Indicators of Wastewater Treatment Efficiency

Escherichia coli, total coliform, and fecal coliform population data were collected from two wastewater treatment facilities, a subsurface flow artificial wetlands, and a receiving stream. Results are presented from individual wastewater treatment process streams, final effluent, and river sites upstream and downstream of the treatment facilities. The QuantiTray technique with 4‐methylumbelliferyl‐β‐glucuronide‐based Colilert media was an effective method for quantifying E. coli and total coliform populations in these waters. Thermotolerant Klebsiella pneumoniae present in the effluent from one treatment facility interfered with recovery of fecal coliforms on m‐FC media using the delayed‐incubation membrane filtration technique; Klebsiella interference was not observed in the enumeration of E. coli by the QuantiTray technique. Both stream standards and discharge permits can be revised to apply E. coli as the indicator of fecal contamination. The results support development of E. coli‐based effluent and stream standards to protect public health.

Comparison of a Rapid Plate Count and MPN Methods for Enumeration of Fecal Coliforms and Escherichia coli in Soft-Shell Clams

A direct elevated temperature plate count method utilizing modified fecal coliform agar with rosolic acid (ETPC/mFC) was compared to 5-tube and 3-tube most probable number (MPN) procedures for its accuracy in enumerating fecal coliforms and Escherichia coli in naturally and artificially contaminated soft-shell clams (Mya arenaria). The results indicated that the extent of overall recovery of fecal coliforms was similar in the two methods tested. Therefore, the ETPC/mFC method may be considered as a rapid procedure for fecal coliform screening during depuration of soft-shell clams.

Improved membrane filtration method incorporating catalase and sodium pyruvate for detection of chlorine-stressed coliform bacteria

In vitro pure culture studies were conducted on three different strains of Escherichia coli (K-12, EPA 00244, and SWEI) to determine the effect of chlorination on catalase activity. In each case, stationary-phase cells exhibited significant (P less than 0.001) reductions in enzyme activity following exposure to chlorine. Mean differences in activity between control and chlorine-stressed cells ranged from 8.8 to 20.3 U/mg of protein for E. coli SWEI and EPA 00244, respectively. Following initial enzyme studies, resuscitation experiments utilizing the membrane filtration technique were conducted on chlorinated sewage effluent. Five different amendments, including catalase (1,000 U per plate), heat-inactivated catalase (1,000-U per plate), sodium pyruvate (0.05%), a catalase-sodium pyruvate combination (1,500 U/0.01%), and acetic acid (0.05%), were tested for the ability to enhance detection of chlorine-stressed cells on M-fecal coliform (M-FC), mT7, M-Endo, and tryptone-glucose-yeast extract (TGY) media. Significant (P less than 0.001) increases in recovery of fecal coliforms on M-FC, total coliforms on mT7 and M-Endo, and total heterotrophs on TGY were obtained on plates containing catalase, pyruvate, or the combination of these compounds. Supplementation with heat-inactivated catalase and acetic acid did not improve recovery of chlorine-stressed cells compared with recovery on nonamended media. Subsequent analysis of colonies from plates containing compounds which enhanced recovery indicated coliform verification percentages of greater than 80% on M-FC, greater than 90% on mT7, and greater than 94% on M-Endo media. These data suggest that the addition of peroxide-degrading compounds to various standard recovery media may improve detection of both coliform and heterotrophic bacteria in chlorinated waters.

Improved recovery of fecal coliforms from the Ottawa River by membrane filters in the presence of food debris.

In the absence of food debris, Sartorius and Millipore HA filters recovered substantially fewer fecal coliforms from Ottawa River water than did Millipore HC. On addition of a small quantity of sterile blended carrot to water samples, recovery by the poorer filters equalled that on Millipore HC. Scanning electron microscopy revealed bacteria sheltered in crevices formed by carrot fibres and thus protected from the normal stresses of exposure. Addition of carrot debris (e.g., 0.03 g carrot to 100 mL of sample) thus provides a convenient and inexpensive means of reducing variations in fecal coliform recovery between brands of membrane filters.

Anaerobic incubation of membrane filter cultures for improved detection of fecal coliforms from recreational waters.

Anaerobic incubation of membrane filter cultures significantly enhanced detection of fecal coliforms in surface-water samples from recreational beaches. In contrast to standard aerobic incubation, anaerobic incubation suppressed overgrowth of masking, noncoliform bacteria but did not increase the frequency of fecal coliform recovery.

Recovery of Fecal Coliforms and of Escherichia coli at 44.5, 45.0 and 45.5°C

Recovery rates of fecal coliforms and of Escherichia coli were determined at 44.5, 45.0 and 45.5°C in raw milk, ground meat and raw sewage. MPN values based on gas production (Standard MPNs) and on gas production and/or growth only (Total MPNs) were calculated for both fecal coliforms and for E. coli. The expected trend towards lower MPN values with increasing incubation temperature was more pronounced for the Standard MPNs than for the Total MPNs. The temperature effect was also strongly product - specific in that the Total and Standard MPNs for the fecal coliforms and the Standard MPNs for E. coli for sewage only differed significantly from one another within each determination at the three different incubation temperatures. The effect of length of incubation time on the ratios of E. coli to fecal coliforms was most pronounced at 45.5°C. Product specificity was again observed. The greatest increase in the recovery rate of aerogenic E. coli between 24 and 48 h of incubation time occurred in sewage (66%). For meat, the increase was 57% and for milk 46%. In terms of combined (aerogenic and anaerogenic) E. coli (expressed as Total MPNs), the increases were considerably less, but highest for the meat (33%), followed by sewage (29%) and by milk (21%). A breakdown of the E. coli isolates recovered from both gas-positive and gas-negative primary (fecal coliform) EC broth tubes showed that for the three products combined there were eight times as many false-positives at 44.5°C as at the other two incubation temperatures. In contrast, there were 12% false-negatives at 45.5°C compared to 3% at 45.0°C and 2% at 44.5°C. Since the high incidence of false-negatives (loss of E. coil) at 45.5°C is not counter-balanced by an enhanced specificity (fewer false-positives) over 45.0°C, the latter temperature is to be preferred. Meat yielded the lowest rate for false-positives at any of the three incubation temperatures. In contrast, at 45.5°C, it gave 21% false-negatives compared to only 9% for sewage and 10% for milk. On the other hand, milk contributed the most false-positives at 44.5°C (20%), compared to only 1% for meat and 3% for sewage. A potential loss of 21% of E. coli - containing EC broth tubes is hardly tolerable, reinforcing the contention that gas formation at evaluated temperatures is not a valid criterion of fecal origin.

Comparison of four membrane filter methods for fecal coliform enumeration.

Four membrane filter methods fecal coliform enumeration were evaluated and compared in six laboratories based on determination of accuracy, specificity, upper counting limit, and recovery comparability. Recovery accuracy with pure cultures ranged from 89 to 100% for m-FC, mTEC (a procedure developed for thermotolerant Escherichia coli), and m-FC2 methods (the latter incorporating a 2-h, 35 degrees C resuscitation period), but was less than 60% for the MacConkey membrane broth method. These figures dropped by approximately 40 to 55% when the cultures were subjected to temperature (10 degrees C) stress. Close to 800 colonies were verified to determine specificity. False-positive colonies occurred most frequently with the m-FC2 method (18%), whereas false-negative colonies were most common on MacConkey membrane broth (26%). In counting range experiments using a variety of samples, the highest upper counting limit was 130 colonies per filter with the mTEC procedure. Recovery comparisons were based on over 130 samples including raw surface waters, raw sewage, and chlorinated and unchlorinated sewage effluents. In general, recoveries were significantly higher with the m-FC2 and mTEC methods; however, on m-FC2, growth of nontarget background organisms was also higher in most cases. Highest recoveries from chlorinated sewage effluents were obtained by the mTEC method, and the addition of a similar resuscitation period to the m-FC procedure (m-FC2) improved fecal coliform recovery from such samples. The best overall performance for fecal coliform enumeration was obtained with the mTEC method with high recovery and low levels of background colonies, good specificity and accuracy, and a high upper counting limit. This procedure also offers the advantage of enumerating E. coli within 24 h.

Interlaboratory Evaluation of the AOAC Method and the A-l Procedure for Recovery of Fecal Coliforms from Foods

An interlaboratory evaluation was made of the 96 h AOAC method and the 24 h A-1 procedure for the enumeration of fecal coliforms in samples of yellow corn meal, rye flour, mung beans, raw ground beef, and raw oyster homogenate. Results indicated that the efficiency of the A-1 procedure, measured in terms of recovery of fecal coliforms, and the reproducibility of that recovery were dependent on the particular food being analyzed. Accordingly, until its efficiency can be more fully demonstrated, the A-1 procedure is recommended only as a screening procedure for fecal coliforms in foods.

Comparison of Modified A-1 Method with Standard EC Test for Recovery of Fecal Coliform Bacteria for Shellfish

This study is one of a series in which variations of the A-1 method for the detection and enumeration of fecal coliforms and Escherichia coli in seawater and foods were evaluated. The tests were conducted jointly by the Food and Drug Administration and state and provincial laboratories that support shellfish control programs in the United States and Canada as part of the National Shellfish Sanitation Program's Microbiology Task Force activities. Results showed significantly higher recovery of fecal coliforms from naturally contaminated shellfish by the AOAC official A-1-M method than by the American Public Health Association standard method. There was no significant difference in recovery of E. coli by the 2 methods.

Microbiological Analysis of Alligator (Alligator mississippiensis) Meat

The two tail muscles, ilio caudalis and ischio caudalis, along with hide surface areas from four alligators, Alligator mississippiensis, were evaluated for microbial numbers and types. Microbial analyses of hide surfaces yielded a mean aerobic plate count (APC, 35 C) of approximately 4.45 logs/cm2 at 35 C, and total coliform counts were low. Salmonella was not recovered from hide or tissue samples. APCs of fresh meat samples were low (2.88 – 3.02 logs/g) regardless of muscle type, with no recovery of fecal coliforms or Escherichia coli. Microbial development in meat stored at 1.7 C for up to 15 days was comparable to that reported for fresh beef. Typical microorganisms recovered included Corynebacterium, Staphylococcus, Micrococcus, Flavobacterium, Pseudomonas, Acinetobacter, Arthrobacter and yeasts.

Comparison of the surface structure, metal binding, and fecal coliform recoveries of nine membrane filters

A comparative study was made of nine commonly used membrane filters from five manufacturers, all recommended for enumeration of coliform bacteria. Bacterial recoveries and flow rates were examined from three types of water and were found to correlate with the surface pore structure determined by scanning electron microscopy. The sorption of metals was also determined. The results of these studies indicate that the five best membranes for fecal coliform recovery could be placed in two groups: Millipore HC and Gelman, followed by Johns-Manville SG and AG and Sartorius 13806.

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.

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.

Evaluation of Recovery Methods to Detect Coliforms in Water

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.

Influence of Coliform Source on Evaluation of Membrane Filters

Four brands of membrane filters were examined for total and fecal coliform recovery performance by two experimental approaches. Using diluted EC broth cultures of water samples, Johns-Manville filters were superior to Sartorius filters for fecal coliform but equivalent for total coliform recovery. Using river water samples, Johns-Manville filters were superior to Sartorius filters for total coliform but equivalent for fecal coliform recovery. No differences were observed between Johns-Manville and Millipore or Millipore and Sartorius filters for total or fecal coliform recoveries using either approach, nor was any difference observed between Millipore and Gelman filters for fecal coliform recovery from river water samples. These results indicate that the source of the coliform bacteria has an important influence on the conclusions of membrane filter evaluation studies.

Improved Membrane Filter Method for Fecal Coliform Analysis

A two-layer agar method has been developed which consistently yields higher recovery of fecal coliforms on membrane filters when compared to the existing membrane fecal coliform procedure. This method has been evaluated by three laboratories using samples of raw and chlorinated waste water, and reservoir, river, and marine waters. Verification of 1,013 fecal coliform colonies isolated from 61 water samples averaged 92% on this proposed procedure. Comparison with the Standard Methods membrane fecal coliform procedure revealed the two-layer agar method had an overall increased sensitivity to fecal coliform detection in these waters. It is therefore proposed that this procedure be evaluated as an alternative to the Standard Methods fecal coliform membrane Filter test in the examination of chlorinated secondary effluents, marine waters, and any natural waters that may contain pollutants with heavy metal ions.