Enumeration Of Fecal Coliforms Research Articles

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.

Evaluation of Media Used in Rapid Methods for the MPN Enumeration of Fecal Coliforms in the Sydney Rock Oyster (Crassostrea commercialis)

MacConkey and A-1 broths were compared for the MPN enumeration of fecal coliforms in the Sydney Rock Oyster (Crassostrea commercialis), using an incubation regime of 2 h at 37 ± 1 C followed by up to 46 h at 44.5±0.5 C. A higher recovery was obtained with MacConkey broth, with ratios of the geometric mean MPN values of 111 samples tested in A-1 and MacConkey broth of 0.5460 and 0.5654 after 24 h and 48 h incubation, respectively. Incubation for 48 h produced significantly higher counts than 24-h incubation for both broths.

Coprostanol in Severn Estuary sediments.

The detection of sewage pollution in the environment is of considerable importance for health, aesthetic and ecological reasons. Sewage may contain various pathogenic organisms representative of such conditions as polio, typhoid, dysentery, tetanus and tuberculosis. Although the contamination of potable water supplies with dangerous micro-organisms is an obvious hazard, bathing in polluted waters presents an appreciable, if lesser, risk. Pathogenic bacteria and viruses usually occur in small numbers in sewage and are extremely difficult to isolate and enumerate (AGG et al. 1978). Thus detection of faecal pollution is generally achieved by the use of an indicator substance, either microbiological or chemical, which is present in sewage in larger amounts. These indicators are not necessarily harmful substances in themselves. Among chemical indicators that have been proposed are the determination of phosphate (MURPHY and RILEY 1962), of ammoniacal nitrogen (DUEDALL et al. 1975), and more recently of siloxanes (PELLENBARG 1979). However, most chemical methods have been shown to be unsatisfactory as they are either non-specific or insufficiently sensitive. Non-chemical indicators such as the concentration of tomato seeds (SHELTON 1971) have also been studied as a possible method for the detection of faecal pollution. However, the method of choice has, in general, been microbiological, involving the enumeration of faecal coliforms in samples (ANON 1969). Whilst this method is simple and sensitive it has been sho~m that coliform counts can be highly variable. Even at a single sampl~ng point they may range over an order of magnitude in the course of a few minutes (AGG et al. 1978). There are also considerable problems in establishing firm associations between faecal coliforms and pathogenic bacteria. For instance, coliforms may be destroyed under conditions such as variation of temperature, sunlight or salinity in which pathogenic bacteria survive. There is thus considerable scope for the development of a satisfactory chemical indicator of faecal pollution to be used in conjunction with, or alternative to, the present microbiological methods.

Evaluation of membrane filter methods for enumeration of faecal coliforms from marine water

A modified membrane filter (MF) technique for recovery of faecal coliform bacteria, employing a pre-incubation temperature of 35°C for five hours followed by 18 ± 1 h at 44·5°C was evaluated against the standard MF technique and the multiple-tube (or (most-probable-number (MPN)) procedure in estuarine and marine environments. It was demonstrated that while both MF and MPN procedures supplied substantially the same information, only 75% of the samples analysed with the standard MF procedure fell within the 95% confidence limits of the MPN. When the modified MF was used, 90% of the counts fell within the confidence limits. On average, the modified MF yielded 50% higher counts than did the standard MF method.

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.

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.

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).

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.

Identification and Quantification of Fecal Coliforms using Violet Red Bile Agar at Elevated Temperature

A procedure was developed for identification and quantification of fecal coliforms by colony size obtained in Violet Red Bile agar incubated at 44.5±0.5 C for 24 h in an air incubator. Five strains of Escherichia coli, 5 strains of Enterobacter aerogenes, 37 coliform isolates from sewage, and 76 coliform isolates from water were tested by this procedure. The practical usefulness of this procedure was studied by comparative quantification of 11 water samples using this procedure, the Millipore method, and MPN method. Statistical analysis showed that this procedure is as reliable as the other procedures for enumeration of fecal coliforms. The advantages of this method over the commonly used methods of enumeration of fecal coliforms are ease of operation, simplicity, and speed.

A Comparison of Seitz and Millipore Membrane Filters for the Enumeration of Fecal Coliforms

In a comparison of two commonly used membrane filters for enumerating fecal coliform bacteria it was demonstrated that Seitz type M filters recovered statistically more colonies of bacteria than did Millipore HAWG 047S1 filters from pure cultures of Escherichia coli incubated at 44 °C. The membranes were grown on 0.4 % Teepol agar. On incubation at 37°C no significant discrepancy was found. As a reference method was used pour plating in plate count agar (Difco). It was demonstrated that incubation at 44°C did not per se inhibit propagation of fecal coliforms. Both types of filters examined were sterilized by the manufacturers with ethylene oxide. The discrepancy found can therefore not be due to sterilization procedures.

Rapid Method for Detection and Enumeration of Fecal Coliforms in Fresh Chicken

A rapid method for enumerating fecal coliforms in foods was developed employing an agar pour-plate medium. After 7 h of incubation at 41.5 +/- 0.05 C, this medium effectively allows the growth of fecal coliforms only. This rapid method was compared with the Association of Official Analytical Chemists multiple-tube dilution method for Escherichia coli, by using 21 samples of fresh, cut-up chicken and a surface rinsing procedure for sample preparation. Verification of picked colonies was carried out in EC broth using parallel incubation temperatures of 45.5 and 44.5 +/- 0.05 C. Verifications for these temperatures averaged 79 and 98%, respectively. All positively verified isolates were E. coli types I and II, as were the negatives. Geometric means for the verified 7-h plate count were within 12% of the standard means for both EC broth incubation temperatures.