drinking water supplies under the regulations of the Safe Drinking Water Act (33). The use of total coliforms as indicator bacteria has itself been the subject of ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1981, p. 130-138 0099-2240/81/010130-09$02.00/0
Vol. 41, No. 1
Failure of the Most-Probable-Number Technique to Detect Coliforms in Drinking Water and Raw Water Suppliest T. M. EVANS, C. E. WAARVICK, RAMON J. SEIDLER,* AND M. W. LECHEVALLIER
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331
A procedure was developed to detect false-negative reactions (interference) in the standard most-probable-number (S-MPN) technique for coliform enumeration of untreated surface water and potable water supplies. This modified MPN (M-MPN) procedure allowed a quantitative assessment of the interference with coliform detection in untreated surface water and potable water supplies. Coliform interference was found to occur in the presumptive, confirmed, and completed tests of the S-MPN technique. When coliforms were present, interference with their detection occurred in over 80% of the samples. The inferior nature of the SMPN was revealed by the 100% increase in the incidence of completed coliformpositive drinking water samples obtained with the M-MPN technique. The MMPN procedure was also superior to the standard membrane filter technique. Eight different species of coliforms were recovered from false-negative tests, including Citrobacter, Enterobacter, Klebsiella, and Escherichia coli (in decreasing order of occurrence). The use of standard MPN techniques for monitoring potable water supplies may lead to a false security that the drinking water supply is potable, i.e., free from indicator bacteria.
when the standard plate count bacteria exceeded 500 cells/ml. The authors suggested that the standard plate count bacteria were interfering with the detection of gas production by the coliform bacteria. This conclusion is consistent with the earlier studies of Chambers (7), who demonstrated that a slight reduction in coliform numbers could result in the failure to detect gas production. Interference by bacteria which are antagonistic to coliforms (18, 27, 34) as well as the inhibitory nature of the media used in the fermentation tube technique have been suggested as factors contributing to the masking of coliform detection (7, 29). In this study, we present a modified MPN (MMPN) procedure which has allowed the first quantitative assessment of the magnitude of coliform interference in raw surface water supplies and in potable drinking water. MATERLALS AND METHODS Sampling area Samples were collected from the
Enumeration of the total coliform bacterial population by the fermentation tube procedure has been used by microbiologists for some 60 years as an indicator of water quality (12). This technique is still used for monitoring the quality of potable drinking water supplies throughout the world. In this country, the most-probablenumber (MPN) fermentation tube procedure is one of two techniques permitted for monitoring drinking water supplies under the regulations of the Safe Drinking Water Act (33). The use of total coliforms as indicator bacteria has itself been the subject of debate (9). However, the object of this paper is not to debate this indicator concept, but rather to illustrate the shortcomings of the fermentation tube procedure as currently practiced in the recovery of coliform bacteria from potable drinking water and from raw surface water supplies. Several studies have suggested that the recovery of coliforms by the fermentation tube technique is potentially subject to various kinds of interferences, especially at the presumptive stage (13, 18). In potable drinking water supplies, pathogens have been isolated with few if any detectable coliforms present (1, 27, 31). Geldreich et al. presented results from a community water supply survey involving over 2,400 samples (14). The data revealed a decrease in the percentage of coliform-contaminated samples
finished drinking water supply of an Oregon coastal community serving 14,000 residents and from the two coast range streams supplying the raw water to the city. The intake points of the raw water supplies are located behind small concrete retention dams about 1.2 to 1.5 m high and constructed across the streams. At one intake, water flows by gravity into a 30.5-cm main line and, after about 45 min of flow time, receives gaseous chlorine injection by a flow proportional device resulting in about 1.5-mg/liter total initial chlo-
t Technical paper no. 5669, Oregon Agricultural Experiment Station.
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rine concentration. At the other intake, water is pumped from the diversion dam into a "settling reservoir." The reservoir water effluent receives a similar dose of gaseous chlorine before entering the distribution system. The chlorine contact time is approximately 30 min before reaching the first service connection. There is no physical or chemical treatment of the raw water except for chlorination. Although the watershed does not receive industrial or domestic wastes, logging operations have left some of the upstream slopes reduced in ground cover, making the hillsides subject to erosion. The watersheds also harbor populations of elk, deer, and some beavers. Due to the extensive winter and spring precipitation periods in the study area and other physical properties of the watershed, rains occasionally carried particulate material into the streams, leaving the intake and finished waters turbid. Collection and microbiological techniques. Raw surface water and finished drinking water samples were collected in 4-liter sterile polypropylene containers. Sodium thiosulfate was added to neutralize any free chlorine residual in the drinking water samples. Free and combined chlorine residuals were determined with a Hach field test kit (model CN-70), using the N,N-diethyl-p-phenylenediamine colorimetric technique (2). Temperature of all water samples was determined upon collection with a YSI-Telethermometer 400 series. Samples were placed on ice and transported back to the laboratory within 3 h after collection, and analyses were completed within 7 h. Unless otherwise noted, all bacteriological techniques and the time and temperature of incubation conformed to "Standard Methods" (2) and to the procedures of the Microbiological Methods for Monitoring the Environment (4). In the standard multiple-tube fermentation technique, volumes of 10, 1.0, and 0.1 ml were inoculated into two sets of 9 or 15 tubes. One set contained lactose broth (LB; lot no. 652242 and 662331; Difco Laboratories), and the other set contained lauryl tryptose broth (LTB; Difco lot no. 663637). Both media were supplemented with phenol red (Sigma Chemical Co.) indicator at 18 mg/liter. Parallel MPN analyses using presumptive media with and without added phenol red have indicated that phenol red has no effect on coliform recovery. The confirming medium was brilliant green lactose bile broth (BGLB; Difco lot no. 666632). The completed test agars were eosin methylene blue (EMB; Difco lot no. 610498) and m-Endo agar LES (m-LES; Difco lot no. 663068). Typical and atypical colonies were picked from both EMB and mLES agar media and streaked onto slants of tryptic soy yeast extract agar (TSYA) containing tryptic soy broth (Difco lot no. 663068) supplemented with 1.5% agar (Difco) and 0.3% yeast extract (Difco lot no. 656810). After a 24-h incubation at 35°C, growth from the slant was removed for Gram staining and transferred into secondary broth tubes of LB and LTB. In the M-MPN procedure, the same media were used as in the standard MPN (S-MPN) method, except that EC broth (Difco lot no. 641057) was added as an additional confirmatory broth and incubated at 350C. The M-MPN scheme consisted of the S-MPN technique plus additional manipulations designed to
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recover coliforms from any tests which were negative (Fig. 1). In the M-MPN scheme, coliform masking or interference was defined as the recovery of a completed coliform in pure culture from a tube or plate which failed to yield a coliform by the S-MPN procedure. Masking could have occurred at any of the three stages in the S-MPN scheme. An increase in the coliform MPN index would occur whenever a completed coliform was obtained from the M-MPN and not from the corresponding S-MPN scheme. In the M-MPN procedure, acid and/or turbid (gasnegative) presumptive tubes were subcultured onto m-LES and EMB, and 1 ml was transferred to sterile tubes of BGLB and EC broths (Fig. 1). If gas was produced in either BGLB or EC, the "Standard methods" completed test was performed, using both m-LES and EMB agar media. When either presumptive or confirmatory broths were subcultured onto m-LES or EMB plates, two procedures were used to determine whether growth on the plates contained coliforms. One procedure, designated isolated colony (IC), conformed to the "Standard Methods" procedure and consisted of transferring one isolated colony from the plate to secondary tubes of LB and LTB broth. If gram-negative gas-producing (LB or LTB) bacteria were recovered, masking in the presumptive tube was demonstrated. In the second procedure, designated multiple inoculation (MI), growth from the heavy portion of the streak or three to five isolated typical coliform colonies were transferred into LB and LTB broth tubes. Tubes with gas formation were again streaked back onto sterile plates of m-LES and processed by the IC procedure (Fig. 1, presumptive test). Isolated, typical coliform colonies were picked and inoculated into LB, LTB, and TSYA for Gram staining to fulfill the concept of Koch's postulates, i.e., gas formation from lactose by a pure culture of a gramnegative rod. Masking in the original presumptive broth could thus be demonstrated by the MI technique as well. Masking could also be demonstrated in the confirmed (BGLB) medium. Positive presumptive tubes were subcultured into BGLB and EC media. If the BGLB tube was negative, the positive presumptive tube was processed as described previously through step I of the M-MPN scheme (Fig. 1). Coliform masking was positive if the S-MPN confirmatory step was negative and completed coliforms were recovered either by step I of the M-MPN scheme or from the EC confirmatory broth. Masking in the completed step was demonstrable in samples which contained presumptive and confirmed positive tubes but failed to yield gram-negative gasproducing rods when single, isolated "typical" colonies were picked into secondary LB or LTB tubes. When typical completed coliform isolates were recovered from the positive presumptive or positive confirmed tubes and not from the completion agar media, masking was demonstrated in the S-MPN completed test stage. The M-MPN index was calculated on the basis of a compilation of positive completed tests from both the S-MPN and M-MPN techniques. The magnitude of masking in the standard technique was calculated from the difference in the MPN indices (total coli-
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PRESUMPTIVE TEST
COMPLETED TEST
GASU
"EGATIVE PSTIVE
NEGATIVE POSITIVE
O-
EfOMG RO
INON-SPOREFOINMINGR
FIG. 1. Flow scheme for M-MPN procedure. Abbreviations: a, lactose broth (LB) and lauryl tryptose broth (LTB); b, tubes examined after 24 and 48 h at 35°C; c, m-Endo agar LES and eosin methylene blue (EMB) agar; d, incubated for 24 h at 35°C; e, brilliant green lactose bile (BGLB) broth and EC broth; f, multiple inoculation (MI) technique; g, inoculation of an isolated colony (IC); h, tryptic soy yeast extract agar (TSYA). See Materials and Methods for further explanations.
forms/100 ml) derived from the S-MPN and M-MPN tube profiles. Membrane filter (MF) enumeration of total coliforms was conducted according to standard procedures (2). Gelman GN-6 membranes (pore size, 0.45,um) and m-LES were used. Duplicate 250- and 100-ml volumes of drinking water and 10- and 1-ml volumes of untreated surface water were routinely analyzed. Typical colonies were submitted to LTB verification (2). At least 50% of the typical colonies were randomly selected for verification. In samples where the number of typical colonies was less than 20 per plate, colonies were picked from replicate plates. In this case, when possible, a total of 10 colonies were submitted to the verification scheme. Identification of total coliform bacteria. Coliforms from the greatest sample dilutions were selected for identification. Three coliforms were identified from the S-MPN technique where LTB was the presumptive medium, and three were identified where LB was the presumptive medium. Coliforms isolated from each masked test were also identified. Coliforms were identified by use of triple sugar iron agar slants (Difco), the IMViC (indole, methyl red, Voges-Proskauer, citrate) tests, lysine and ornithine decarboxylase broths, arginine dihydrolase broth, and mucate and malonate fermentation. Cultures from the American Type Culture Collection were used to ensure that proper reactions were obtained in the media and to aid in the identification of the coliform isolates. In
addition, the API 20E system (Analytab Products, Inc., Plainview, N.Y.) was used to confirm the identities of 10% of the isolates. A quality assurance program as outlined elsewhere (4) was used throughout. Quality control consisted of monitoring incubator and autoclave temperature on a per-use basis. Each lot of medium was checked for performance. The sterility of media and materials used for each sampling event was verified. Statistical comparisons were made on the basis of the paired t-test on logarithmically transformed data
(32). RESULTS Inadequacies of the S-MPN technique. Coliforn interference occurred at all tests in the S-MPN technique (Table 1). With both types of water samples, the presumptive test was the most susceptible to interference. LB presumptive tubes exhibited higher percentages of masking than did LTB. The percentage of unmasked columns summarizes the relative incidence of failure in each test of the S-MPN as revealed by the special procedures used in the M-MPN technique. For the drinking water specimens with LB as the presumptive medium, coliforms were masked at one or more tests in 34 of 37 detectably contaminated samples. For these 34 sam-
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TABLE 1. Number offalse-negative results (masking) in the presumptive, confirmed, or completed tests in the S-MPN technique based on analysis of 100 drinking water and 15 untreated surface water samples LTB
LB
Sample source
No. of samples
with false-negative results at each test
Drinking water Presumptive Confirmed Completed Untreated surface water Presumptive
% Unmasked at each test
85a 41 12
29 14 4
No. of samples with false-negative results at each test
% Unmasked at each test
18
58b
18 4
58 13
11 lOOc 12 4 4 33 4 3 25 Completed medium. aA total of 34 samples exhibited masking when LB was the presumptive b A total of 31 samples exhibited masking when LTB was the presumptive medium. total of 12 samples exhibited masking when LB was the presumptive medium. dA total of 13 samples exhibited masking when LTB was the presumptive medium.
Confirmed
85d 31 31
CA
ples, 29 (85%) contained one or more false-negative (no gas) presumptive tubes from which a completed coliform was isolated by the M-MPN technique. When LTB was used in parallel analysis on the same set, coliforms were masked at one or more tests in 33 samples. Eighteen of these samples (58%) contained one or more falsenegative presumptive tubes. The confirmatory step of the S-MPN technique exhibited higher percentages of masking for drinking water than for untreated surface water samples. However, the completed step was masked at higher percentages for untreated water (25 to 31%) than for drinking water samples (12 to 13%). The magnitude of coliform masking (the difference between the M-MPN and S-MPN coliform indices) was not found to be correlated with the temperature of the water sample or precipitation events in the watershed. The coliform species most commonly recovered from false-negative presumptive and confirmed tests of untreated surface water were Citrobacterfreundii (8, 10, 11) and Enterobacter agglomerans, which comprised 60% of the total isolates identified. Escherichia coli, Klebsiella pneumoniae, Yersinia enterocolitica, and Hafnia alvei each comprised 10% of the isolates also recovered from false-negative S-MPN tests. The most commonly recovered coliform species from false-negative presumptive and confirmed tests of drinking water was C. freundii, which comprised about 70% of the total isolates identified. Escherichia coli, K. pneumoniae, Enterobacter aerogenes, E. agglomerans, E. cloacae, H. alvei, and C. diversus each comprised approximately 3% of the isolates also recovered from false-negative S-MPN tests. Comparison of the S-MPN, M-MPN, and MF techniques for the enumeration of col-
iforms. The M-MPN technique detected greater numbers of coliforms than did the SMPN technique in 80% (12/15) of the untreated raw water samples when LB was used and in 85% (13/15) of the samples when LTB was used (Fig. 2). Analysis of all the data points indicated that in 7 of the 15 samples, the M-MPN was greater than the upper limit of the 95% confidence interval (jagged line) of the S-MPN values.
The underestimation of coliforms by the SMPN technique was even greater in drinking water samples than in raw water samples. In 92% (34/37) of the occasions when coliforms were detected, the M-MPN index was greater than the S-MPN index (Fig. 3). In addition, at least 81% (30/37) of the M-MPN indices were greater than the upper limit of the 95% confidence interval of the S-MPN values (Fig. 3). A comparison of the geometric mean number of coliforms detected in raw water (Table 2) indicated that the M-MPN recovered 4.9-fold (4.4-fold, LB presumptive broth; 5.6-fold, LTB presumptive broth) more coliforms than the SMPN technique (P < 0.005) and 2-fold (comparable values for both presumptive media) more coliforms than the MF technique (P = 0.07). There was no significant difference in coliform recovery by LTB or LB (P > 0.5) in either MPN technique in the examination of raw or potable water. In potable drinking water, when coliforms were detected, the geometric mean of the MMPN revealed 4.6-fold more coliforms (4.3-fold, LB presumptive broth, P < 0.005; 5-fold, LTB presumptive broth, P < 0.005) than the S-MPN and 5-fold (4.5-fold, LB; 5.5-fold, LTB) more coliforms than the MF technique (P < 0.005). The advantage of the M-MPN technique rel-
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