Vol. 41, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1981, p. 130-1380099-2240/81/010130-09$02.00/0

Failure of the Most-Probable-Number Technique to DetectColiforms in Drinking Water and Raw Water SuppliestT. M. EVANS, C. E. WAARVICK, RAMON J. SEIDLER,* AND M. W. LECHEVALLIER

Department ofMicrobiology, Oregon State University, Corvallis, Oregon 97331

A procedure was developed to detect false-negative reactions (interference) inthe standard most-probable-number (S-MPN) technique for coliform enumera-

tion of untreated surface water and potable water supplies. This modified MPN(M-MPN) procedure allowed a quantitative assessment of the interference withcoliform detection in untreated surface water and potable water supplies. Coliforminterference was found to occur in the presumptive, confirmed, and completedtests of the S-MPN technique. When coliforms were present, interference withtheir detection occurred in over 80% of the samples. The inferior nature of the S-MPN was revealed by the 100% increase in the incidence of completed coliform-positive drinking water samples obtained with the M-MPN technique. The M-MPN 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 decreas-ing order of occurrence). The use of standard MPN techniques for monitoringpotable water supplies may lead to a false security that the drinking water supplyis potable, i.e., free from indicator bacteria.

Enumeration of the total coliform bacterialpopulation by the fermentation tube procedurehas been used by microbiologists for some 60years as an indicator of water quality (12). Thistechnique is still used for monitoring the qualityof potable drinking water supplies throughoutthe world. In this country, the most-probable-number (MPN) fermentation tube procedure isone of two techniques permitted for monitoringdrinking water supplies under the regulations ofthe Safe Drinking Water Act (33).The use of total coliforms as indicator bacteria

has itself been the subject of debate (9). How-ever, the object of this paper is not to debatethis indicator concept, but rather to illustratethe shortcomings of the fermentation tube pro-cedure as currently practiced in the recovery ofcoliform bacteria from potable drinking waterand from raw surface water supplies.

Several studies have suggested that the recov-ery of coliforms by the fermentation tube tech-nique is potentially subject to various kinds ofinterferences, especially at the presumptivestage (13, 18). In potable drinking water supplies,pathogens have been isolated with few if anydetectable coliforms present (1, 27, 31). Geld-reich et al. presented results from a communitywater supply survey involving over 2,400 sam-ples (14). The data revealed a decrease in thepercentage of coliform-contaminated samples

t Technical paper no. 5669, Oregon Agricultural Experi-ment Station.

when the standard plate count bacteria exceeded500 cells/ml. The authors suggested that thestandard plate count bacteria were interferingwith the detection of gas production by thecoliform bacteria. This conclusion is consistentwith the earlier studies of Chambers (7), whodemonstrated that a slight reduction in coliformnumbers could result in the failure to detect gasproduction.

Interference by bacteria which are antagonis-tic to coliforms (18, 27, 34) as well as the inhib-itory nature of the media used in the fermenta-tion tube technique have been suggested as fac-tors contributing to the masking of coliformdetection (7, 29).

In this study, we present a modified MPN (M-MPN) procedure which has allowed the firstquantitative assessment of the magnitude of col-iform interference in raw surface water suppliesand in potable drinking water.

MATERLALS AND METHODSSampling area Samples were collected from the

finished drinking water supply of an Oregon coastalcommunity serving 14,000 residents and from the twocoast range streams supplying the raw water to thecity.The intake points of the raw water supplies are

located behind small concrete retention dams about1.2 to 1.5 m high and constructed across the streams.At one intake, water flows by gravity into a 30.5-cmmain line and, after about 45 min of flow time, receivesgaseous chlorine injection by a flow proportional de-vice resulting in about 1.5-mg/liter total initial chlo-

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FAILURE OF MPN TECHNIQUE TO DETECT COLIFORMS 131

rine concentration. At the other intake, water ispumped from the diversion dam into a "settling res-ervoir." The reservoir water effluent receives a similardose of gaseous chlorine before entering the distribu-tion system. The chlorine contact time is approxi-mately 30 min before reaching the first service con-nection. There is no physical or chemical treatment ofthe raw water except for chlorination.

Although the watershed does not receive industrialor domestic wastes, logging operations have left someof the upstream slopes reduced in ground cover, mak-ing the hillsides subject to erosion. The watershedsalso harbor populations of elk, deer, and some beavers.Due to the extensive winter and spring precipitationperiods in the study area and other physical propertiesofthe watershed, rains occasionally carried particulatematerial into the streams, leaving the intake and fin-ished waters turbid.

Collection and microbiological techniques.Raw surface water and finished drinking water sam-ples were collected in 4-liter sterile polypropylenecontainers. Sodium thiosulfate was added to neutralizeany free chlorine residual in the drinking water sam-ples. Free and combined chlorine residuals were de-termined with a Hach field test kit (model CN-70),using the N,N-diethyl-p-phenylenediamine colorimet-ric technique (2). Temperature of all water sampleswas determined upon collection with a YSI-Telether-mometer 400 series. Samples were placed on ice andtransported back to the laboratory within 3 h aftercollection, and analyses were completed within 7 h.

Unless otherwise noted, all bacteriological tech-niques and the time and temperature of incubationconformed to "Standard Methods" (2) and to theprocedures of the Microbiological Methods for Mon-itoring the Environment (4).

In the standard multiple-tube fermentation tech-nique, volumes of 10, 1.0, and 0.1 ml were inoculatedinto two sets of9 or 15 tubes. One set contained lactosebroth (LB; lot no. 652242 and 662331; Difco Labora-tories), and the other set contained lauryl tryptosebroth (LTB; Difco lot no. 663637). Both media weresupplemented with phenol red (Sigma Chemical Co.)indicator at 18 mg/liter. Parallel MPN analyses usingpresumptive media with and without added phenolred have indicated that phenol red has no effect oncoliform recovery. The confirming medium was bril-liant green lactose bile broth (BGLB; Difco lot no.666632). The completed test agars were eosin meth-ylene blue (EMB; Difco lot no. 610498) and m-Endoagar LES (m-LES; Difco lot no. 663068). Typical andatypical colonies were picked from both EMB and m-LES agar media and streaked onto slants of trypticsoy yeast extract agar (TSYA) containing tryptic soybroth (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 fromthe slant was removed for Gram staining and trans-ferred into secondary broth tubes of LB and LTB.

In the M-MPN procedure, the same media wereused as in the standard MPN (S-MPN) method, ex-cept that EC broth (Difco lot no. 641057) was addedas an additional confirmatory broth and incubated at350C. The M-MPN scheme consisted of the S-MPNtechnique plus additional manipulations designed to

recover coliforms from any tests which were negative(Fig. 1). In the M-MPN scheme, coliform masking orinterference was defined as the recovery of a com-pleted coliform in pure culture from a tube or platewhich failed to yield a coliform by the S-MPN proce-dure. Masking could have occurred at any of the threestages in the S-MPN scheme. An increase in thecoliform MPN index would occur whenever a com-pleted coliform was obtained from the M-MPN andnot from the corresponding S-MPN scheme.

In the M-MPN procedure, acid and/or turbid (gas-negative) presumptive tubes were subcultured ontom-LES and EMB, and 1 ml was transferred to steriletubes of BGLB and EC broths (Fig. 1). If gas wasproduced in either BGLB or EC, the "Standard meth-ods" completed test was performed, using both m-LESand EMB agar media. When either presumptive orconfirmatory broths were subcultured onto m-LES orEMB plates, two procedures were used to determinewhether growth on the plates contained coliforms.One procedure, designated isolated colony (IC), con-formed to the "Standard Methods" procedure andconsisted of transferring one isolated colony from theplate to secondary tubes of LB and LTB broth. Ifgram-negative gas-producing (LB or LTB) bacteriawere recovered, masking in the presumptive tube wasdemonstrated. In the second procedure, designatedmultiple inoculation (MI), growth from the heavyportion of the streak or three to five isolated typicalcoliform colonies were transferred into LB and LTBbroth tubes. Tubes with gas formation were againstreaked back onto sterile plates of m-LES and proc-essed by the IC procedure (Fig. 1, presumptive test).Isolated, typical coliform colonies were picked andinoculated into LB, LTB, and TSYA for Gram stainingto fulfill the concept of Koch's postulates, i.e., gasformation from lactose by a pure culture of a gram-negative rod. Masking in the original presumptivebroth could thus be demonstrated by the MI techniqueas well.

Masking could also be demonstrated in the con-firmed (BGLB) medium. Positive presumptive tubeswere subcultured into BGLB and EC media. If theBGLB tube was negative, the positive presumptivetube was processed as described previously throughstep I of the M-MPN scheme (Fig. 1). Coliform mask-ing was positive if the S-MPN confirmatory step wasnegative and completed coliforms were recoveredeither by step I of the M-MPN scheme or from theEC confirmatory broth.

Masking in the completed step was demonstrable insamples which contained presumptive and confirmedpositive tubes but failed to yield gram-negative gas-producing rods when single, isolated "typical" colonieswere picked into secondary LB or LTB tubes. Whentypical completed coliform isolates were recoveredfrom the positive presumptive or positive confirmedtubes and not from the completion agar media, mask-ing was demonstrated in the S-MPN completed teststage.The M-MPN index was calculated on the basis of

a compilation of positive completed tests from boththe S-MPN and M-MPN techniques. The magnitudeof masking in the standard technique was calculatedfrom the difference in the MPN indices (total coli-

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132 EVANS ET AL.

PRESUMPTIVE TEST CONFIRMED TEST

APPL. ENVIRON. MICROBIOL.

COMPLETED TEST

GASU

"EGATIVE PSTIVE O- EfOMG RO

NEGATIVE POSITIVE INON-SPOREFOINMINGR

FIG. 1. Flow scheme forM-MPNprocedure. 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, multipleinoculation (MI) technique; g, inoculation ofan 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-MPNtube profiles.Membrane filter (MF) enumeration of total coli-

forms was conducted according to standard procedures(2). Gelman GN-6 membranes (pore size, 0.45,um) andm-LES were used. Duplicate 250- and 100-ml volumesof drinking water and 10- and 1-ml volumes of un-treated surface water were routinely analyzed. Typicalcolonies were submitted to LTB verification (2). Atleast 50% of the typical colonies were randomly se-lected for verification. In samples where the numberof typical colonies was less than 20 per plate, colonieswere picked from replicate plates. In this case, whenpossible, a total of 10 colonies were submitted to theverification scheme.

Identification of total coliform bacteria. Coli-forms from the greatest sample dilutions were selectedfor identification. Three coliforms were identified fromthe S-MPN technique where LTB was the presump-tive medium, and three were identified where LB wasthe presumptive medium. Coliforms isolated fromeach masked test were also identified.

Coliforms were identified by use of triple sugar ironagar slants (Difco), the IMViC (indole, methyl red,Voges-Proskauer, citrate) tests, lysine and ornithinedecarboxylase broths, arginine dihydrolase broth, andmucate and malonate fermentation. Cultures from theAmerican Type Culture Collection were used to ensurethat proper reactions were obtained in the media andto 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 identi-ties of 10% of the isolates.A quality assurance program as outlined elsewhere

(4) was used throughout. Quality control consisted ofmonitoring incubator and autoclave temperature on aper-use basis. Each lot of medium was checked forperformance. The sterility ofmedia and materials usedfor each sampling event was verified.

Statistical comparisons were made on the basis ofthe paired t-test on logarithmically transformed data(32).

RESULTSInadequacies of the S-MPN technique.

Coliforn interference occurred at all tests in theS-MPN technique (Table 1). With both types ofwater samples, the presumptive test was themost susceptible to interference. LB presump-tive tubes exhibited higher percentages ofmask-ing than did LTB. The percentage of unmaskedcolumns summarizes the relative incidence offailure in each test of the S-MPN as revealed bythe special procedures used in the M-MPN tech-nique. For the drinking water specimens withLB as the presumptive medium, coliforms weremasked at one or more tests in 34 of 37 detect-ably contaminated samples. For these 34 sam-

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FAILURE OF MPN TECHNIQUE TO DETECT COLIFORMS 133

TABLE 1. Number offalse-negative results (masking) in the presumptive, confirmed, or completed tests inthe S-MPN technique based on analysis of 100 drinking water and 15 untreated surface water samples

LB LTB

Sample source No. of samples No. of sampleswith false-nega- % Unmasked at with false-nega- % Unmasked attive results at each test tive results at each test

each test each test

Drinking waterPresumptive 29 85a 18 58bConfirmed 14 41 18 58Completed 4 12 4 13

Untreated surface waterPresumptive 12 lOOc 11 85dConfirmed 4 33 4 31Completed 3 25 4 31

aA total of 34 samples exhibited masking when LB was the presumptive medium.b A total of 31 samples exhibited masking when LTB was the presumptive medium.CA 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.

ples, 29 (85%) contained one or more false-neg-ative (no gas) presumptive tubes from which acompleted coliform was isolated by the M-MPNtechnique. When LTB was used in parallel anal-ysis on the same set, coliforms were masked atone or more tests in 33 samples. Eighteen ofthese samples (58%) contained one or more false-negative presumptive tubes. The confirmatorystep of the S-MPN technique exhibited higherpercentages of masking for drinking water thanfor untreated surface water samples. However,the completed step was masked at higher per-centages for untreated water (25 to 31%) thanfor drinking water samples (12 to 13%).The magnitude of coliform masking (the dif-

ference between the M-MPN and S-MPN coli-form indices) was not found to be correlatedwith the temperature of the water sample orprecipitation events in the watershed.The coliform species most commonly re-

covered from false-negative presumptive andconfirmed tests of untreated surface water wereCitrobacterfreundii (8, 10, 11) and Enterobacteragglomerans, which comprised 60% of the totalisolates identified. Escherichia coli, Klebsiellapneumoniae, Yersinia enterocolitica, and Haf-nia alvei each comprised 10% of the isolates alsorecovered from false-negative S-MPN tests.The most commonly recovered coliform spe-

cies from false-negative presumptive and con-firmed tests of drinking water was C. freundii,which comprised about 70% of the total isolatesidentified. Escherichia coli, K.pneumoniae, En-terobacter aerogenes, E. agglomerans, E. cloa-cae, H. alvei, and C. diversus each comprisedapproximately 3% of the isolates also recoveredfrom 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 detectedgreater numbers of coliforms than did the S-MPN technique in 80% (12/15) of the untreatedraw water samples when LB was used and in85% (13/15) of the samples when LTB was used(Fig. 2). Analysis of all the data points indicatedthat in 7 of the 15 samples, the M-MPN wasgreater than the upper limit of the 95% confi-dence interval (jagged line) of the S-MPN val-ues.The underestimation of coliforms by the S-

MPN technique was even greater in drinkingwater samples than in raw water samples. In92% (34/37) of the occasions when coliformswere detected, the M-MPN index was greaterthan the S-MPN index (Fig. 3). In addition, atleast 81% (30/37) of the M-MPN indices weregreater than the upper limit of the 95% confi-dence 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, LTBpresumptive broth) more coliforms than the S-MPN technique (P < 0.005) and 2-fold (compa-rable values for both presumptive media) morecoliforms than the MF technique (P = 0.07).There was no significant difference in coliformrecovery by LTB or LB (P> 0.5) in either MPNtechnique in the examination of raw or potablewater.

In potable drinking water, when coliformswere detected, the geometric mean of the M-MPN revealed 4.6-fold more coliforms (4.3-fold,LB presumptive broth, P < 0.005; 5-fold, LTBpresumptive broth, P < 0.005) than the S-MPNand 5-fold (4.5-fold, LB; 5.5-fold, LTB) morecoliforms than the MF technique (P < 0.005).The advantage of the M-MPN technique rel-

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APPL. ENVIRON. MICROBIOL.

x

z

z

a.

a 1-IL8o 10

00a

0 -

0

0

0

Ie o

o

10 100STANDARD COMPLETED MPN INDEX

,rB2

-jK)00

FIG. 2. Relationship between the S-MPN and M-MPN techniques when used to detect coliforms inuntreated surface water. MPN analyses were con-ducted in parallel, using LB (-) and LTB (U) as thepresumptive media. Numbers after data points indi-cate number of occurrences. The straight line repre-sents the theoretical line of equality, and the jaggedline represents the upper 95% confidence limit of theS-MPN (nine-tube) value.

ative to the S-MPN and MF techniques in de-tecting coliforms in drinking water sampleswhich appeared coliform-free is documented inTables 3 and 4 and Fig. 4. The M-MPN tech-nique increased the incidence of coliform-posi-tive drinking water samples over the S-MPN by100% regardless of the presumptive broth used(Table 3). The MF technique recovered verifiedcoliforms in 23 drinking water samples, the S-MPN did so in 22 samples, and the M-MPNdetected completed coliforms in 41 samples (Fig.4). The M-MPN, when both presumptive mediawere used, never failed to detect coliforms whenMF-verified coliforms were detected (Table 4).However, there were 18 samples which con-tained completed coliforms by the M-MPN tech-nique, and no MF-verified coliforms were de-tected. The latter results are surprising since theMF sample size was 100 ml whereas the M-MPNused 33.3 ml. Consistent with these results arethe data from raw water, where the M-MPNprovided higher counts than MF in 14 of 15samples.

Coliform masking in the S-MPN techniquewas found to affect compliance with the SafeDrinking Water Act regulations (Table 5). Datashow that for 6 of 8 months, the M-MPN tech-

nique indicated that the drinking water supplycontained coliforms in excess of the allowablelimit (33). However, both standard techniques(MPN and MF) indicated noncompliance foronly 3 months. Noncompliance in 1 month (2/79) was the result of coliforms detected by MFin a single sample. The M-MPN technique de-tected coliforms in this sample (2/79) in all threeLB presumptive tubes inoculated. If five tubesper dilution had been used, the M-MPN tech-nique would probably have indicated noncom-pliance for this month as well.

Coliforms from drinking water were recoveredby all methods most frequently in samples takenat dead-end distribution line sites. The resultsof coliform analyses of drinking water (Fig. 3;Tables 3-5) do not represent a random samplingof the water supply. It is recognized that thephysical, chemical, and bacteriological condi-tions in dead-end lines do not represent all con-ditions within the distribution system. However,since coliforms were also detected in drinkingwater samples with free residual chlorine (mainline samples), the M-MPN technique is clearlyapplicable to the isolation of coliforms from allwaters within a distribution system and rawwaters as well.

DISCUSSIONInterference with the detection of coliforms

(masking) was found to occur at all steps in the,^^An\IUUUJ

x

z 100 -z

a.0 LB2

aw cO- 10 *nLTB3w

-J

0.

LTB3

0W LB 10a: il LTB8a LTB2 NO2 '

<3_I ,,1

S

ol LTB 2

S

0

* *LTB2

LTB3LTO3.

<3 "03 12STANDARD COMPLETED MPN INDEX

24

FIG. 3. Relationship between the S-MPN and M-MPN techniques when used to detect coliforms inpotable drinking water samples. Symbols and linesare as in Fig. 2.

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FAILURE OF MPN TECHNIQUE TO DETECT COLIFORMS 135

TABLE 2. Geometric mean number of total coliforms enumerated by various techniques from untreatedsurface water and drinking water samples

Geometric mean total coliforms/100 ml

S-MPNa M-MPNaSample source (presumptive media used in (presumptive media used in par-

parallel analysis) allel analysis) MF

LB LTB LB LTB

Drinking water' 2.1 2.2 9.0 11.0 2.0

Untreated surface water 28.8 23.0 126.9 128.5 64.0a Defined in the text.'Means calculated only for samples when coliforms were detected by any technique.

TABLE 3. Comparison of the S-MPN and M-MPN techniques for the detection of coliforms in 15 untreatedsurface and 100 drinking water samplesa

Presumptive me- No. ofSample source dium used in paral- Comparison samples

lel analysis

Drinking water LTB S-MPN coliform (+) 16M-MPN coliform (+) 33M-MPN index greater than S-MPN index 31M-MPN coliform (+), S-MPN coliform (-) 17

LB S-MPN coliform (+) 17M-MPN coliform (+) 37M-MPN index greater than S-MPN index 34M-MPN coliform (+), S-MPN coliform (-) 20

LTB or LB M-MPN coliforn (+), S-MPN coliform (-) 19M-MPN index greater than S-MPN index 38

Untreated surface water LTB M-MPN index greater than S-MPN index 13LB M-MPN index greater than S-MPN index 12LTB or LB M-MPN index greater than S-MPN index 14

a Both techniques were completed through EMB and/or m-LES agar media followed by LTB and/or LB.

S-MPN technique and will be a problem in anyroutine MPN coliform analysis of freshwaterand contaminated drinking water. Masking inthe contaminated untreated surface water wasrecorded in one or more tests in 93% (14/15) ofthe samples. Naturally, the chlorinated drinkingwater samples had a lower incidence of contam-ination. However, the M-MPN technique dou-bled the incidence of coliform detection com-pared with that detected by the S-MPN tech-nique.The largest incidence ofmasking was recorded

in the presumptive test. Coliform masking inpresumptive media has been suggested by otherstudies (7, 14) and was demonstrated quantita-tively in the presumptive step ofMPN analysesof seawater samples (23). However, parameterswhich influence the detection of coliforms fromseawater (6, 24) are probably not relevant tofreshwater supplies (salinity, divalent cations).Indirect evidence for coliform interference wasreported in a study of the bacteriological qualityof 969 community drinking water supplies (14).In that study, the presence of noncoliform bac-teria in excess of 500 organisms/ml correlated

with a reduced frequency of coliform isolationfrom potable water samples. Geldreich et al. (14)speculated that competition by noncoliform bac-teria for lactose and the presence of bacteriaantagonistic to coliforms may result in coliforminterference. The results from several studiessupport this. Hutchison et al. found that whenantagonistic bacteria were inoculated simulta-neously with E. coli into LB tubes, a 28 to 97%reduction in the MPN index resulted (18). Com-petition between noncoliforms and coliforms forlactose has also been reported by others (27).The detection of coliforms in the presumptive

test is dependent on the fermentation of lactosewith the production ofgas in sufficient quantitiesso as to be visible in the Durham tube. Culturalconditions which influence either the activity orquantity of the enzymes responsible for gas pro-duction will ultimately influence the outcome ofthe presumptive tests. In this study, coliformswere isolated from negative presumptive tubeswhen growth in the medium produced acid (4.5)or slightly alkaline (7.2) pH conditions. ThesepH values have been reported as inhibitory toeither the activity or inducibility of the formic

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TABLE 4. Comparison of the standard MF to the S-MPN and M-MPN techniques for the detection of

coliforms in 100 drinking water suppliesaPresump-tive me-dium . No. ofused C a samplesparallelanalysis

LTB S-MPNcolifor(-),MF 11coliform (+)

S-MPN colform(+),MF 3coliforn (-)

M-MPN colfo (-),MF 2coliform (+)

M-MPN coliform(+),MF 12coliform (-)

LB S-MPN colifo (-),MF 10coliform (+)

S-MPN colfor (+),MF 6colifo (-)

M-MPN coform(-),MF 1coliform (+)

M-MPN coliform(+),MF 15coliform (-)

LTB or LB M-MPN colifor (-), MF 0coliform (+)

See Table 3.

hydrogen lyase enzyme system (15, 17, 20, 25).Another cause of coliform masking in pre-

sumptive media may result from the injury ofcoliforms in the aquatic environment. The phe-nomenon of coliform injury has been shown tooccur in surface waters (3) and chlorinated wa-ters (5). One of the physiological effects of injuryor stress was shown to be an increased lag time,compared with nonstressed cells, when inocu-lated into complex media (3). Since many bac-teria are not as susceptible to stress as coliforms(22), noncoliform bacteria may have a reducedlag time when inoculated into lactose-containingmedia. Therefore, noncoliform bacteria may beable to overgrow coliforms and produce condi-tions unsuitable for gas production.

Coliform maskirng in confirmatory MPN testswas also found to be a significant cause for theunderestimation of coliforms in drinking anduntreated surface water by the S-MPN proce-dure. The failure of coliforms to grow in BGLBwhen subcultured from lactose-containing mediahas been well documented (7, 26, 29, 30) and wasextremely prevalent when chlorinated potablewater samples were examined (30). Even thoughthe suitability of BGLB as a confirmatory me-dium has been demonstrated (19, 21, 28), BGLBmay not be the best medium to use in theconfirmatory test when chlorinated drinking wa-ter or untreated surface water supplies are ex-amined.

False-negative completed tests in the S-MPNtechnique occurred at a lower frequency than

the false-negative presumptive or confirmedtests. This was evident in both types of watersupplies examined. Coliform masking in thecompleted step may be a function of the inhibi-tory nature ofEMB agar or overgrowth by non-coliform bacteria on the agar medium obscuringtypical colonies. The use of the multiple inocula-tion procedure and the use of both LB and LTB,where an isolate was checked for the ability toferment lactose, recovered significantly morelactose fermenting isolates than did the tech-nique recommended by "Standard Methods"(2).Masking of the various coliform genera in the

S-MPN procedure did not appear to be a simplefunction of their relative numbers in the watersample. In potable drinking water samples, C.freundii was the most frequently recovered spe-cies from both the S-MPN and M-MPNschemes. Other species of coliforms did not fol-low this pattern in the untreated surface watersamples. For example, E. coli and K. pneumo-niae were the predominant coliforms recoveredin the S-MPN technique from surface water,whereas C. freundii was recovered most fre-quently from masked MPN tests. Different sus-ceptibilities of specific coliform bacteria to theinhibitory nature of the media used in the MPNtechnique, to competition for lactose by noncol-iform bacteria, and to antagonistic products pro-duced by noncoliform bacteria may result incertain coliforms being more prone to maskingthan others. Therefore, the probability of re-covering a specific coliform genus from a maskedMPN test is a function of both the number

MODFED MEMBRANE STANDARDMOST PIOB8LE FLTRATION MOST PROBABLENUMBER NUMBER

TECHNIQUE

FIG. 4. Number ofpotable drinking water samplesin which coliforms were recovered by theMF, S-MPN,and M-MPN techniques. The MPN analyses are ex-pressed as the results obtained when LTB (A) or LB(C) was used in parallel analyses and as the totalnumber of samples (B) in which coliforms could berecovered by either presumptive medium.

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FAILURE OF MPN TECHNIQUE TO DETECT COLIFORMS 137

TABLE 5. Impact of using the MF, S-MPN, and M-MPN techniques on the compliance with the SafeDrinking Water Act regulations

Coliform detection by given methodDate MF,cohfvanalyzed S-MPN, LB M-MPN, LB S-MPN, LTB M-MPN, LTB

2/79 78/1,600b, c /48d 3/48d 2/48d 2/48d3/79 3/1,600 1/48 1/48 2/48 4/484/79 7/1,600 1/48 5/48C 0/48 2/485/79 0/1,400 1/42 5/42C 4/42 7/42c6/79 9/1,900 2/57 14/57C 2/57 12/57c7/79e 36/2,100c 7/65c 26/65C 6/65 19/65c8/79 13/400c 3/20C 12/20c 1/20 15/20c9/79 116/700c 5/35c 11/35c 3/35 13/35C

Presumptive medium used in parallel analysis.b Volumes of 100 ml were sampled, and typical colonies were submitted for verification; thus the 16 samples

examined yielded 78 verified coliforms.c Indicates occurrences in excess of allowable limit.d Number of positive tubes/total number of tubes inoculated.'Twenty or more samples analyzed this month.

relative to other coliform species and the abilityto grow and produce gas in presumptive andconfinnatory media.The use of the M-MPN procedure showed

that both the S-MPN and MF techniques failedto detect as well as underestimated the numberof coliforms in potable drinking water. There-fore, the use of standard techniques in theirpresent form may lead to situations where thedrinking water supply is considered potable, yeta health hazard or potable water treatment de-ficiency may exist. In addition, failure of thestandard techniques to recover coliforms whenindeed they are present, as illustrated here andelsewhere (1, 13, 31), may be one of the impor-tant factors leading some to criticize the indica-tor organism concept (9). The M-MPN is not apractical technique to increase the efficiency ofcoliform detection. Alterations of the techniqueare currently being tested in field trials, where itis hoped that increased recovery of coliforms canbe achieved with less time and expense.

ACKNOWLEDGMENT1SThis research was supported by cooperative agreement

CR806287 from the U.S. Environmental Protection Agency,Drinking Water Research Division.

The suggestions and contributions of Harry D. Nash, U.S.Environmental Protection Agency, Office of Drinking WaterResearch, and David Tison, Department of Microbiology,Oregon State University, are greatly appreciated.

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