evaluation of standard and modified m-fc, macconkey, and

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1981, p. 192-199 Vol. 42, No. 20099-2240/81/080192-08$02.00/0

Evaluation of Standard and Modified M-FC, MacConkey, andTeepol Media for Membrane Filtration Counting of Fecal

Coliforms in WaterW. 0. K. GRABOW,* C. A. HILNER, AND P. COUBROUGH

National Institute for Water Research, Council for Scientific and Industrial Research, Pretoria 0001, SouthAfrica

Received 29 January 1981/Accepted 5 May 1981

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FCagar with a resuscitation top layer, Teepol agar, and pads saturated with Teepolbroth, were evaluated as growth media for membrane filtration counting of fecalcoliform bacteria in water. In comparative tests on 312 samples of water from awide variety of sources, including chlorinated effluents, M-FC agar without rosolicacid proved the medium of choice because it generally yielded the highest counts,was readily obtainable, easy to prepare and handle, and yielded clearly recogniz-able fecal coliform colonies. Identification of 1,139 fecal coliform isolates showedthat fecal coliform tests cannot be used to enumerate Escherichia coli becausethe incidence of E. coli among fecal coliforms varied from an average of 51% forriver water to 93% for an activated sludge effluent after chlorination. Theincidence of Klebsiellapneumoniae among fecal coliforms varied from an averageof 4% for the activated sludge effluent after chlorination to 32% for the riverwater. The advantages of a standard membrane filtration procedure for routinecounting of fecal coliforms in water using M-FC agar without rosolic acid asgrowth medium, in the absence of preincubation or resuscitation steps, areoutlined.

Fecal coliforms are selected members of thecoliform group of bacteria which are able toferment lactose at 44.50C, are fairly specific forthe feces ofwarm-blooded animals, and are com-monly used as indicators of fecal pollution inwaters such as wastewater effluents, rivers, ma-rine environments, recreational waters, and rawsources of drinking water supplies (1, 5, 8, 11,22). The total coliform group is less specific forfecal pollution, includes various species able tomultiply in a variety ofwater environments, andis generally used as a sanitary parameter forevaluating the quality of drinking water andswimming pool water (1, 11, 13, 20). Coliformsin water are enumerated by means of relativelysimple, rapid, and cheap techniques. Unfortu-nately, however, a wide variety of methods,which differ in accuracy and reliability, is beingused. This implies that results from differentlaboratories can hardly be compared, the imple-mentation of water quality standards is of lim-ited value, and unreliable tests may even havefar-reaching health implications (4-6).

In this study, counts obtained by means ofdifferent growth media recommended for mem-brane filtration (MF) counting of fecal coliformswere compared, and isolates were identified to

establish the composition of fecal coliforms invarious water environments. The media were M-FC agar used in countries such as the UnitedStates (2) and Canada (16) and recommendedby the World Health Organization (30), M-FCagar modified by the elimination of rosolic acid(24, 27) or the addition of a resuscitation toplayer (26), Teepol media used in Britain (7), andMacConkey agar specified in South Africa (28)and Canada (16). Most-probable-number esti-mates of fecal coliforms were disregarded be-cause these methods are inferior to MF for gen-eral purposes (10, 13, 17, 26). The results con-tribute to information urgently needed for thestandardization of coliform techniques (4, 5).

MATERIALS AND METHODSMF techniques. Laboratory procedures complied

with the specifications of the American Public HealthAssociation (2), Department of Health and Social Se-curity, England (7), and the South African Bureau ofStandards (28). Resuscitation of organisms counted onMacConkey agar by preincubation on enrichment me-dium (28), and on Teepol medium by preincubation at25°C (7), was omitted. Sartorius filter holders, GelmanGN-6 membranes (pore size, 0.45 ,m), and disposableplastic petri dishes (diameter, 65 mm) with loose-fit-ting lids were used. All counts are expressed as the

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COUNTING FECAL COLIFORMS IN WATER 193

average of tests done in triplicate. Incubation was ina water bath at 44.5 ± 0.20C for 22 to 24 h withcylindrical brass containers for immersion of mem-brane cultures (7).

Media. Dehydrated M-FC broth base (code 0883-01; Difco Laboratories, Detroit, Mich.) was obtainedcommercially and solidified by the addition of 1.5%agar (2). This standard M-FC agar was also modifiedby the elimination of rosolic acid (24) or the additionof a resuscitation top layer. The latter consisted of 13g of lactose broth (Difco), 15 g of agar, and 1,000 ml ofdistilled water. A 2.6-ml amount was poured on top of6.5 ml of solidified M-FC agar per petri dish less than1 h before use as specified by Rose, Geldreich, andLitsky (26). Membrane cultures on this two-layer me-dium were first incubated at 350C for 2 h and then at44.5 ± 0.20C for 22 to 24 h (26). Membrane-enrichedTeepol broth (0.4 ET) was prepared according to theformula of the Department of Health and Social Se-curity (7) and contained 40 g of peptone (Difco), 6 g ofyeast extract (Difco), 30 g of lactose (Merck & Co.,Inc., Rahway, N.J.), 50 ml of a 0.4% aqueous solutionofphenol red, 4 ml ofTeepol 610 (British Drug Houses,Poole), and 1,000 ml of distilled water. Membraneswere either incubated on pads saturated with thisTeepol broth or on the medium solidified by theaddition of 1.5% agar. MacConkey agar was preparedby the following formula (13): 15 g of agar (Difco), 20g of peptone (Difco), 10 g of lactose (Merck), 5 g ofbile salts (no. 3; Difco), 5 g of sodium chloride, 0.12 gof bromocresol purple, and 1,000 ml of distilled water.

Analysis of water samples. Samples of settledsewage, biofilter effluent, humus tank effluent (settledbiofilter effluent), activated sludge effluent before andafter chlorination to a total chlorine content of 1 to 4mg/liter, and a mixture of biofilter and activatedsludge effluents before and after tertiary treatment(chlorination to a total chlorine content of about 4mg/liter followed by sand filtration), were collected atthe Daspoort wastewater purification works in Preto-ria (13). The Apies River was sampled just upstreamof the inflow of treated sewage from the Daspoortpurification works (13). The raw water intake (acti-vated sludge effluent) and active carbon-treated waterfrom an experimental 4,500 m3/day multiple-barrierwastewater reclamation plant (Stander plant) in Pre-toria were also sampled (12). Bottles used for collectingsamples of chlorinated water contained sodium thio-sulfate for dechlorination (2, 13). Concentrations ofchlorine were measured by the DPD (N,N-diethyl-p-phenylenediamine) ferrous titrimetric method (2).Samples were collected during the period January toDecember 1980. They were homogenized in a Sanyomixer for 60 s at a speed selector setting of 4 (12) andprocessed within 2 h after collection.Identification of fecal coliform isolates. Mem-

branes with well-spaced colonies were selected. Allcolonies which conformed to the definition of fecalcoliforms (2, 7, 16, 28) were picked from these mem-branes and purified on the same medium for identifi-cation by means of the commercial API 20E system(13, 15, 25). The indole, methyl red, Voges-Proakauer,citrate (2, 13), and cytochrome oxidase (13) tests weredone additionally on all isolates. Terminology of bac-teria is that of the 8th edition of Bergey's Manual of

Determinative Bacteriology or otherwise that used bythe manufacturers of the API test system (AnalytabProducts Inc., Plainview, N.Y.).

RESULTSComparison of counts on different

growth media. For technical reasons it was notpossible to compare all media simultaneously.Comparative evaluations therefore consisted ofseries of tests on 312 samples of water in whichselected combinations of media were used (Ta-bles 1 to 6). Counts on MacConkey agar weregenerally about 50% lower than on the othermedia (Tables 1, 5, and 6). Apart from one seriesof tests on river water (Table 3), M-FC agar withtop layer yielded higher average counts andmore often a higher count than did standard M-FC agar (Tables 1, 3, 5, and 6). Average countson M-FC agar without rosolic acid were higherthan on standard M-FC agar (Tables 3 to 6)except for some experiments with limited num-bers of tests where differences were negligible(Tables 4 and 5). Apart from one series of testson the activated sludge effluent after chlorina-tion (Table 5) and a series of tests on the mixtureof biofilter and activated sludge effluents (Table6), M-FC agar without rosolic acid yieldedhigher average counts and more often a highercount than did M-FC agar with top layer (Tables3, 5, and 6). In one series of tests on the mixtureof biofilter and activated sludge effluents afterchlorination and sand filtration, M-FC agar withtop layer had the higher average count, whereasM-FC agar without rosolic acid more often hadhigher individual counts. In total, M-FC agarwithout rosolic acid yielded the highest count in

TABLE 1. MF fecal coliform counts for differentwaters with three growth media

Fecal coliform count (per 100ml) on:

Water No. ofsamples Mac- Standard M-FC

Conkey M-FC agar +agar agar top layer

Settled sewage 11 46b 178b 189b3_153b 5-60Wb 6-8.0b

(0) (2) (7)Biofilter effluent 11 108C 231' 264C

19-390' 35-790c 34-860c(0) (3) (8)

Humus tank 11 48' 81' 114'effluent 9-185c 13-290' 15-460'

(0) (3) (8)Activated sludge 35 25' 47' 61'

effluent 3-120c 6-181' 10-212'(0) (7) (24)

a Average count followed by the range of counts and, inparentheses, the number of samples for which the mediumyielded the highest count.

b Values are x 105.' Values are x 103.

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TABLE 2. MF fecal coliform counts for different waters with four growth mediaFecal coliform counta (per 100 ml) on:

Water No. of sam-ples Standard M-FC agar Teepol TeepolM-FC agar + top layer broth agar

Activated sludge effluent 5 25b 44b 33b l9b14-37b 22-91b21"47b 9-37b

(0) (3) (2) (0)Carbon-filtered waterc 5 3 3 7 1

0-10 0-6 0-17 0-4(0) (1) (3) (0)

a Average count followed by the range of counts and, in parentheses, the number of samples for which themedium yielded the highest count."Values are x 103. I

'Treatment stage in the Stander multiple-barrier wastewater reclamation plant.

TABLE 3. MF fecal coliforn counts for different waters with five growth mediaFecal coliform counts' (per 100 ml) on:

Water No. of sam-ples Standard M-FC agar M-FC agar Teepol Teepol

M-FC agar - rosolic acid + top layer broth agar

Apies River 33 53b 58b 50b 46b 54b3_400b 3_380b 3-260b 2-290b 3-390b

(5) (13) (5) (2) (7)Tertiary effluentc 34 90 103 92 77 89

0-360 0-380 0-430 0-410 0-420(6) (10) (6) (5) (6)

aAverage count followed by the range of counts and, in parentheses, the number of samples for which themedium yielded the highest count.bValues are x 103.'Mixture of biofilter and activated sludge effluents after chlorination and sand filtration.

TABLE 4. MF fecal coliform counts for differentwaters with two growth media

Fecal coliform counta(per 100 ml) on:

Water No. of MFsamples Standard M-FCM-FC agaragar - rosolic

Apies River 8 160" 260b1-660b 0-720b

(2) (5)Activated sludge 8 25b 22b

effluent 9-97b 0_100b(2) (4)

Tertiary effluentc 8 12 120-54 0-51

(3) (3)a Average count followed by the range of counts and,

in parentheses, the number of samples for which themedium yielded the highest count.bValues are x 103.c Mixture of biofilter and activated sludge effluents

after chlorination and sand filtration.

98 comparative tests, and M-FC agar with toplayer yielded the highest count in 61 (Tables 3,5, and 6). In an experiment with a limited num-ber of tests, counts were higher on pads satu-

rated with Teepol broth than on Teepol agar(Table 2). In a more comprehensive study ondifferent waters, however, the opposite was ob-served (Table 3). Counts on Teepol media weregenerally comparable to those on standard M-FC agar (Tables 2, 3, and 5). The results oftriplicate tests with individual media on eachsample showed no difference in the reproduci-bility of counts recorded on each medium. Theconsiderable difference in the range of countsfor some sampling points was due to differencesin individual samples as a result of variation inpollution load, treatment processes, and climaticconditions, and not to lack of homogeneity insamples or variability inherent in the methodsor media.At the sampling point the average chlorine

content of the activated sludge effluent afterchlorination was 0.4 mg of chlorine per liter totaland 0.1 mg of free residual chlorine per liter.The retention period was about 3 min. Thistreatment resulted in an average reduction infecal coliform counts of 92%, as evaluated on M-FC agar without rosolic acid (Tables 5 and 6).The tertiary effluent (mixture of activatedsludge and biofilter effluents after chlorinationand sand filtration) had an average of 0.5 mg of

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chlorine per liter total and 0.1 mg of free residualchlorine per liter at the sampling point. Thechlorination and sand filtration treatment had aretention period of about 10 min and resulted inan average reduction in fecal coliform counts of97%, evaluated on M-FC agar without rosolic

acid (Table 6).Identification offecal coliform isolates. A

total of 1,139 isolates that conforned to thedefinition of fecal coliforms on the growth me-dium from which they were recovered were iden-tified (Tables 7 to 13). The incidence of Esche-richia coli among isolates from individual sam-pling points varied from 40 to 66% for the ApiesRiver water (Table 8) to 83 to 100% for the

activated sludge effluent after chlorination (Ta-ble 13). After E. coli, Klebsiella pneumoniaewas the most common isolate, with an incidenceranging from 21 to 42% for the river water (Ta-bles 7 and 8) to 0 to 12% for the activated sludgeeffluent after chlorination (Table 13). In oneseries of tests a relatively high incidence of En-terobacter cloacae (28 to 33%) was recorded forthe tertiary effluent (Table 11).The specificity of different media for species

of bacteria did not differ meaningfully (Tables 7to 13). However, the percentage of E. coli iso-lates tended to be relatively low on M-FC agarwithout rosolic acid (Tables 8 to 11) and rela-tively high on MacConkey agar (Tables 8 to 10)

TABLE 5. MF fecal coliform counts for different waters with five growth mediaFecal coliform counta (per 100 ml) on:

Water No. ofsamples MacConkey Standard M-FC agar M-FC agar Teepol

agar M-FC agar - rosolic acid + top layer agar

ASEb before chlorination 9 33c 65c 79C 70C 53C1-92c 8-160c 11-203c 9-170c 8-120c

(0) (2) (4) (3) (0)ASEb after chlorination 6 31" 57d 57d 61d 56d

3-139d 15-163d 7-169d 21-199d 9-182d(0) (2) (2) (2) (0)

a Average count followed by the range of counts and, in parentheses, the number of samples for which themedium yielded the highest count.

b Activated sludge effluent.'Values are x 103.dValues are x 102.

TABLE 6. MFfecal coliform counts for different waters with four growth mediaFecal coliform count' (per 100 ml) on:

Water No. of sam-ples MacConkey Standard M-FC agar M-FC agar

agar M-FC agar - rosolic acid + top layerApies River 23 26b 63b 83b 72b

1-133b 4-243b 5_370b 5-320b(0) (1) (17) (5)

ASEC before chlorination 27 30b 63b 70b 65b1-258b 4_507b 4_487b 4_503b

(0) (2) (16) (9)ASE' after chlorination 27 47d 122d 132d 128d

9-506d 19-667d 15-717" 17-720"(°) (4) (12) (11)

Secondary effluent' 25 17b 36b 36b 39b0.01_90b 0.04-170b 0.06-170b 0.05_190b

(0) (6) (9) (10)Tertiary effluentf 26 456 927 1,120 1,122

3-3, 200 6-6, 700 4-7, 900 4-8, 100(0) (1) (15) (10)

'Average count followed by the range of counts and, in parentheses, the number of samples for which themedium yielded the highest count.

b Values are x 103.c Activated sludge effluent.dValues are x 102.'Mixture of biofilter and activated sludge effluents.f Mixture described in footnote e after chlorination and sand filtration.

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196 GRABOW ET AL.

TABLE 7. Identification ofpositive isolates in fecalcoliform tests on 14 samples ofApies River water, by

MF with three growth mediaNo. (and %) of positive isolates on:

Isolate M-FC M-FCagar Teepolagar -ro- + top brothsolic acid layer

Escherichia coli 23 (45) 22 (45) 29 (55)Citrobacter freundii 2 (4) 3 (6) 2 (4)Enterobacter 8 (16) 6 (12) 2 (4)

cloacaeEnterobacter 1 (2)agglomeransa

Enterobacter 1 (2)aerogenes

Klebsiella 16 (31) 10 (21) 20 (37)pneumoniae

Aeromonas 2 (4)hydrophila

Serratia 3 (6)liquefaciens

Serratia rubidaea 1 (2)Salmonella 2 (4)

arizonae

Total 51 49 53a Erwinia herbicola.

TABLE 8. Identification ofpositive isolates in fecalcoliform tests on 10 samples ofApies River water by

MF with four growth mediaNo. (and %) of positive isolates on:

Stan- M-FC M-FCIsolate Mac- agarConkey dard - ro- agaragar agar solic +atoPacid layer

E. coli 33 (66) 24 (48) 20 (40) 29 (58)C. freundii 1 (2) 3 (6) 2 (4)E. cloacae 1 (2) 5 (10) 6 (12) 1 (2)E. agglomeransa 2 (4) 2 (4) 2 (4)K. pneumoniae 14 (28) 18 (36) 21 (42) 15 (30)S. dysenteriae 1 (2)

Total 50 50 50 50a Erwinia herbicola.

and Teepol broth (Tables 7 and 11). M-FC agarwith top layer occasionally had relatively highincidences of isolates which did not belongto the Escherichia-Citrobacter-Enterobacter-Klebsiella group of bacteria (Tables 7, 8, and11).Tests for indole reaction at 36°C yielded pos-

itive results for 733 of 735 (99.7%) E. coli, 6 of 7(85.7%) Aeromonas hydrophila, 5 of 250 (2.0%)Klebsiella, 2 of 99 (2.0%) Enterobacter, and 3 of40 (7.5%) Citrobacter isolates.General features of the media. Colonies

which conformed to the definition of fecal coli-

forms were generally easy to recognize on all themedia. Colony differentiation was particularlyclear on M-FC agar without rosolic acid (fecalcoliform colonies bright blue), whereas the darkblue colonies of fecal coliforms were not asclearly defined on M-FC agar with top layer ason standard M-FC agar. Distinction of yellowfecal coliform colonies from pink and light redcolonies on Teepol media was sometimes uncer-tain, particularly on crowded membranes. Nocorrelation was detectable between colonialmorphology and identity of fecal coliform iso-lates on any of the media, apart from Klebsiellacolonies, which tended to be mucoid. Using padssaturated with Teepol broth proved time-con-suming, tedious, and inconvenient, and the padscannot be prepared in advance and stored forimmediate use like the agar-based media. Inaddition, Teepol became increasingly difficult toobtain. Using M-FC agar with top layer alsoproved inconvenient since the bottom and toplayers have to be prepared separately, their vol-

TABLE 9. Identification ofpositive isolates in fecalcoliform testsa by MF with four growth media

No. (and %) of positive isolates on:

Stan- M-FC M-FCIsolate Mac- dard agar agar

Conkey M-FC - ro- + topagar agar solic

acid

E. coli 45 (90) 34 (68) 34 (68) 36 (72)C. freundii 1 (2) 1 (2) 3 (6) 2 (4)E. agglomeransb 1 (2) 1 (2)E. aerogenes 2 (4)K. pneumoniae 4 (8) 15 (30) 11 (22) 9 (18)S. liquefaciens 1 (2)

Total 50 50 50 50

a Tests were on 10 samples of a mixture of activated sludgeand biofilter effluents before chlorination and sand filtration.

b Erwinia herbicola.

TABLE 10. Identification ofpositive isolates in fecalcoliform testsa by MF with four growth media


Stan- M-FC M-FCIsolate Mac- dard agaragadConkey M-FC - ro- +agarpagar solicagsr acid

E. coli 40 (80) 38 (76) 32 (64) 37 (74)C. freundii 1 (2) 2 (4) 1 (2)E. cloacae 1 (2)E. agglomeransb 2 (4)K. pneumoniae 9 (18) 9 (18) 17 (34) 11 (22)

Total 50 50 50 50

a Tests were on 10 samples of a mixture of activated sludgeand biofilter effluents after chlorination and sand filtration.bErwinia herbicola.

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umes in each petri dish have to be accuratelymeasured, the top layer can only be addedshortly before use, and the preincubation step iscumbersome, particularly with regard to testswhich cannot be completed within normal work-ing hours. A useful advantage of M-FC agar isthat the medium does not require sterilization(2), and the elimination of rosolic acid (a freshstock solution has to be prepared separately)obviously reduces the cost, materials, labor, and

TABLE 11. Identification ofpositive isolates in fecalcoliform testsa by MF with three growth media


Isolate M-FC agar M-FC agar Teepol- rosolic + top layer brothacid

E. coli 13 (27) 16 (32) 22 (41)C. freundii 6 (13) 1 (2) 4 (8)E. cloacae 16 (33) 15 (31) 15 (28)E. agglomeransb 3 (6) 1 (2) 2 (4)K. pneumoniae 8 (17) 13 (27) 10 (19)A. hydrophila 2 (4) 3 (6)

Total 48 49 53a Tests were on 15 samples of a mixture of activated

sludge and biofilter effluents after chlorination andsand filtering.

b Erwinia herbicola.

time required to prepare the medium. Mac-Conkey agar has to be autoclaved for 15 min(28), and Teepol broth has to be steamed for 30min on 3 successive days (16). Although Mac-Conkey and Teepol media can easily be preparedfrom basic ingredients in the laboratory, thehigher cost of commercial dehydrated M-FCmedium is justified by higher counts, savings intime and labor, convenience, and the advantageof using a medium of relatively homogenouscomposition.

DISCUSSIONIn comparative tests on samples of water from

a wide variety of sources, counts on MacConkeyagar were consistently so much lower than on

the other media (Tables 1, 5, and 6) that themedium failed to justify consideration for fecalcoliform testing. The higher counts on M-FCagar with top layer and preincubation at 35°Ccompared with standard M-FC agar withoutpreincubation (Tables 1 to 3, 5, and 6) are inagreement with results of Rose et al. (26). How-ever, Tables 3, 5, and 6 show that M-FC agarwithout rosolic acid, top layer, and preincuba-tion generally yielded even higher counts. M-FCagar without rosolic acid generally had the high-est count of the six media for all the waterstested, which included river water and secondary

TABLE 12. Identification ofpositive isolates in fecal coliform testsa by MF with five growth media


Isolate MacConkey Standard M-FC agar M-FC agar Teepol


E. coli 23 (85) 22 (79) 24 (83) 17 (81) 17 (94)C. freundii 1 (3) 1 (6)E. aerogenes 1 (3)E. agglomerans' 1 (3)K. ozaenae 1 (5)K. pneumoniae 4 (15) 6 (21) 2 (8) 3 (14)

Total 27 28 29 21 18a Tests were on eight samples of activated sludge effluent before chlorination.bErwinia herbicola.

TABLE 13. Identification ofpositive isolates in fecal coliforn testsa by MF with five growth mediaNo. (and %) of positive isolates on:

Isolate MacConkey Standard M-FC agar M-FC agar Teepol


E. coli 23 (96) 24 (96) 22 (88) 21 (100) 15 (83)C. freundii 1 (4) 2 (11)E. hafniaeb 1 (6)K. pneumoniae 1 (4) 3 (12)

Total 24 25 25 21 18a Tests were on eight samples of activated sludge effluent after chlorination.b Hafnia alvei.

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treated wastewater before and after chlorinationunder different conditions, resulting in differentlevels of reduction in fecal coliform counts. Sincecounts on M-FC agar with top layer may be inclose agreement with most-probable-number es-timates (26), counts on M-FC agar without ro-

solic acid are probably higher than most-proba-ble-number estimates, even for chlorinated ef-fluents (Tables 3, 5, and 6). The higher countson M-FC agar without rosolic acid comparedwith standard M-FC agar (Tables 3 to 6) are inagreement with findings that pads saturatedwith M-FC broth yield higher counts in theabsence of rosolic acid (24, 27). The difference isprobably due to the inhibitory effect of rosolicacid, particularly on injured bacteria (24). LikeM-FC agar without rosolic acid (Table 3), aTriton X-100 agar medium also yielded highercounts than Teepol broth (23). The relative dif-ferences in counts indicate that the Triton me-dium may yield results comparable to those ofM-FC agar without rosolic acid. The results forTeepol broth and Teepol agar in Tables 2 and 3support earlier evidence that agar-based mediaare preferable to pads saturated with liquid me-dia because they generally give higher countsand their preparation and handling are easierand less time-consuming and cumbersome (13,16, 21).The identity of randomly picked colonies

which conform to the definition of fecal coli-forms (Tables 7 to 13) shows that fecal coliformtests cannot be used to enumerate E. coli be-cause the incidence of E. coli among fecal coli-forms isolated from different waters varied froman average of 51% for the Apies River water(Tables 7 and 8) to an average of 93% for theactivated sludge effluent after chlorination (Ta-ble 13). The finding that 733 of 735 identified E.coli isolates (99.7%) had positive indole reac-tions, whereas only 16 of the remaining 407identified isolates (3.9%) conforming to the def-inition of fecal coliforms were positive indicatesthat the addition of an indole specification tofecal coliform tests may result in reasonablyreliable E. coli evaluations. However, even thenit should be kept in mind that some waters maycontain high incidences of indole-positive K.pneumoniae (3) or indole-negative E. coli (19).The high incidence of K. pneumoniae among

fecal coliform isolates, which varied from an

average of 4% for activated sludge effluent aftercholorination (Table 13) to 32% for the ApiesRiver (Tables 7 and 8), is in agreement withfindings of Bagley and Seidler (3).The relatively low incidence of E. coli among

fecal coliforms on M-FC agar without rosolicacid recorded in some series of tests (Tables 8 to

11) may be statistically insignificant because insome series of tests the incidence compared fa-vorably with that on other media (Tables 7, 12,and 13). It would therefore not seem that rosolicacid increases the incidence of E. coli on M-FCagar, which is supported by findings that theelimination of rosolic acid from M-FC brothincreased the percentage of organisms amongfecal coliform isolates that produce gas in ECbroth at 44.5°C (24), gas in lactose-peptone wa-ter, and indole in tryptone water at 44.5°C (27).

Evaluation of the advantages and disadvan-tages of the media considered in this study showsthat M-FC agar without rosolic acid and prein-cubation or resuscitation procedures is thegrowth medium of choice for general routineMFcounting of fecal coliforms in water. This me-dium generally yielded the highest counts for awide variety of waters, including chlorinatedeffluents, is easy to obtain, simple and quick toprepare and handle, and colonies are clearlyrecognizable. The elimination of rosolic acidfrom M-FC agar is in agreement with a state-ment in Standard Methods (2) which specifiesthat this may be done if equivalent results areobtained. It is important, of course, to use goodquality membranes, irrespective of the growthmedium (18, 29).

In view of the key role fecal coliform countsplay in the evaluation of water quality (1, 2, 6-9,11, 16, 28, 30), it is imperative to consider tech-niques which are reliable, simple, and cheap forpurposes of standardization to permit partici-pation of the many laboratories all over theworld which are limited with regard to trainedstaff, facilities, and funds, particularly in under-developed and developing countries. Althoughcoliform counts may have certain limitations(14, 31), their value as a practical indicator offecal pollution, sanitary quality, and health riskhas not yet been disproved (1, 6, 11, 14). Shouldthe need arise for increasing the sensitivity ofcoliform tests for routine water quality evalua-tion, for which there is potential since currenttechnology seems to only cover the "tip of theiceberg" (9), practical approaches, such as re-ducing acceptable limits of counts or increasingthe volume of water to be tested (1), should bepreferred to complicated, expensive, and time-consuming procedures which are limited to thecapabilities of laboratories with sophisticatedfacilities and highly trained staff.

ACKNOWLEDGMENTSThanks are due to 0. W. Prozesky and L. S. Smith for their

advice, Martella du Preez and Loes Alexander for technicalassistance, and N. P. Nicolle, Chief Chemist of the PretoriaMunicipality, for permission to sample the Daspoort purifi-cation works and the Apies River. This paper is published

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with the approval of the Director of the National Institute forWater Research.

LITERATURE CITED

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4. Bordner, R. H. 1977. The membrane filter dilemma, p.8-11. In R. H. Bordner, C. F. Frith, and J. A. Winter(ed.), Proceedings of the Symposium on the Recoveryof Indicator Organisms Employing Membrane Filters.U.S. Environmental Protection Agency, Cincinnati,Ohio.

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11. Geldreich, E. E. 1978. Bacterial populations and indicatorconcepts in feces, sewage, stormwater and solid wastes,p. 51-97. In G. Berg (ed.), Indicators of viruses in waterand food. Ann Arbor Science, Ann Arbor, Mich.

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14. Haley, C. E., R. A. Gunn, J. M. Hughes, E. C. Lippy,and G. F. Craun. 1980. Outbreaks of waterborne dis-ease in the United States, 1978. J. Infect. Dis. 141:794-797.

15. Hayek, L. J., and G. W. Willis. 1976. A comparison oftwo commercial methods for the identification of theEnterobacteriaceae-API 20E and the enterotube-with conventional methods. J. Clin. Pathol. 29:158-161.

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17. Janardan, K. G., M. C. Long, and D. J. Schaeffer.1977. Recovery of coliforms by the MPN and MF tech-niques using a 2'-factorial experimental design. 11EQDocument 77/07, Illinois Institute for EnvironmentalQuality, Chicago.

18. Lin, S. D. 1977. Comparison of membranes for fecalcoliform recovery in chlorinated effluents. J. WaterPollut. Control Fed. 49:2255-2264.

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21. McCarthy, J. A., J. E. Delaney, and R. J. Grasso.1961. Measuring coliforms in water. Water & SewageWorks 108:238-243.

22. Mood, E. W. 1977. Bacterial indicators of water quality inswimming pools and their role, p. 239-246. In A. W.Hoadley and B. J. Dutka (ed.), Bacterial indicators/health hazards associated with water. American Societyfor Testing and Materials, Philadelphia.

23. Opara, A. A., D. D. Mara, and D. W. F. Wheater. 1977.Teepol and Triton media for the enumeration of Esch-erichia coli by membrane filtration. Water Res. 11:949-954.

24. Presawood, W. G., and D. K. Strong. 1978. Modifica-tion of M-FC medium by eliminating rosolic acid. Appl.Environ. Microbiol. 36:90-94.

25. Robertson, E. A., and J. D. MacLowry. 1974. Mathe-matical analysis of the API enteric 20 profile registerusing a computer diagnostic model. Appl. Microbiol.28:691-695.

26. Rose, R. E., E. E. Geldreich, and W. Litsky. 1975.Improved membrane filter method for fecal coliformanalysis. Appl. Microbiol. 29:532-536.

27. Sartory, D. P. 1980. Membrane filtration faecal coliformdeterminations with unmodified and modified M-FCmedium. Water S.A. 6:113-115.

28. South African Bureau of Standards. 1971. Specifica-tion for water for domestic supplies. South AfricanBureau of Standards, Pretoria.

29. Tobin, R. S., and B. J. Dutka. 1977. Comparison of thesurface structure, metal binding, and fecal coliformrecoveries of nine membrane filters. Appl. Environ.Microbiol. 34:69-79.

30. World Health Organization. 1976. Surveillance ofdrinking-water quality. W.H.O. Monograph Series no.63. World Health Organization, Geneva.

31. World Health Organization Scientific Group. 1979.Human viruses in water, wastewater and soil. W.H.O.Technical Report Series no. 639. World Health Orga-nization, Geneva.

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