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HALLS, SHEILA & AYRES, P. A. (1974). J . appl. Bad. 37, 105-109. A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater SHEILA HALLS AND P. A. AYRES Ministry of Agriculture, Fisheries and Food, Fisheries Laboratory, Burnham-on-Grouch, Essex, England (Received 28 June 1973 and accepted 12 November 1973) SUMMARY. Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published ev
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  HALLS, SHEILA AYRES, P. A. (1974). J. appl Bad. 37 105-109. A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater SHEILA ALLS ND P. A. AYRES Ministry of Agriculture, Fisheries and Food, Fisheries Laboratory, Burnham-on-Grouch, Essex, England (Received 28 June 1973 and accepted 12 November 1973) SUMMARY. Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fcrrnenting colonies from seawater, developing at 44 on a 4% enriched Teepol medium, are E. eoli type I. A total of 1352 colonies from seawater was bsted for production of intlole and for gas from lactoso at 44 . In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonios tested ranged from lO-lOO , depending on the number of colonies on the membrane. Many of tho colonies (81.9 ) to which IMVEC tests were applied were E coli type I; a further 10.90/, were Irregular type I. The practical implications of these findings are discussed. MEMBRANE ILTRATION has become an accepted method for concentrating bacteria in liquid samples which, because they contain few cells/unit volume, are not easily enumerated by other techniques. The method can be adapted to estimate specific groups of bacteria by the use of seleotive media and choice of incubation temperature. We have used the technique to enumerate Escherichia coli in seawater samples to monitor the extent and degree of sewage pollution in coastal and estuarine areas. In this work we have assumed that all lactose-fermenting colonies appearing on mem- brane filters after overnight incubation at 44 re E. coli type I. This assumption is based on the results of t,he examinat>ion of molluscan shellfish by Clegg Sherwood (1947), who found that 96.9% of red colonies appearing on MacConkey agar incubated at 44 were either E coli type I or Irregular type I. However, little published evidence exists for judging the specificity of the technique when used to examine fresh or saline waters. The aim of much of the published work on membrane filtration has been to develop a technique which would enumerate, in 18 h, numbers of E coli equal to or greater than those obtained by a most probable number (MPN) method employing incubation for 48 h at 37 followed by confirmation at 44 . The work described in the present paper was designed to test what proportion of the lactose-fermenting colonies developing during an 18 h test at 44 on a 4% enriched Teepol medium were E. wli type I. Materials and Methods Samples Seawater samples were collected in sterile 8 oz medical 'flats', using a simple weighted sampler. The majority were taken at various points in the estuaries of the Rivers D51  106 Sheila Halls and P. A. Ayres Roach and Crouch, Essex, where salinities ranged from 26-36%, and the remainder were taken off the coast at Whitstable, Kent. Thirty samples were stored overnight at 8 prior to examination, but most were examined within 8 h of collection, being kept in a cool place and out of direct sunlight until testing. Examination of samples All samples were filtered through 5 cni diam., 0.45 p pore size, membranes (Oxoid), using a stainless steel filter head (Gallenkamp). Volumes (100 and 10 ml) of each sample were filtered through separate membranes and placed on Whatman absorbent pads previously soaked uith the cnriclied Tccpol broth. The filters and pads were incubated in purpose-made plastic trays each holding 8 filters placed in a heavy gauge polythene bag. A small pad of moistcncd cotton wool was included in each bag to prevent drying of the membranes, and some lead shot was added to sink it in the water bath. Incubation was carried out in a thermostatically-controlled water bath fitted with a time switch so as to provide resuscitation at 30 for 4 h (Taylor, 1968), followed by incubatlion for at least 14 h at 44 0.2 . Media Membranes were incubated on an enriched Teepol broth (Burman, pers. cornm.) of the following composition: peptone, 40 g; yeast extract, 6 g; lactose, 30 g Teepol 610 BDH), 40 ml; 0.4% aqueous phenol red, 50 ml; distilled water, 1000 ml, autoclaved at 121 for 15 niin (final pH value, 7.4). The media used for characterizing colonies were tryptone water, brilliant green-bilc broth, Koser's citrate and MRVP medium (all Oxoid). Colony characterizatioTL After incubation, yellow lactose- ermenting colonies on each membrane were counted and, depending on the numbers present, either all or a proportion of them picked off singly into tryptone water and incubated at 37 for 24 h, when further sub-cultures were made into brilliant green-bile broth, Koser's citrate and MRVP media. The tryptone water cultures were incubated for a further 24 h and then tested for indole production by extraction with xylene and the addition of a few drops of Ehrlich's reagent. Growth and the product'ion of gas in brilliant green-bile broth, after 18 h at 44 (Eijkman test), were recorded. Koser's citrate medium was incubated at 37 for 3-5 days. Cultures in MRVP medium were incubated at 30 for 5 days and the contents of each tube divided into two; one half was tested with 0.04% mcthyl red solution and thc othcr by Barritt's (1936) method for the Voges-Proskauer reaction. These 5 tests formed the IMVEC series (Mushin Ashburner, 1964). The IMVEC reactions of the coli-aerogenes solates are given in Table 1 (taken from Mushin Ashburner, 1964). Since it was not possible to distinguish between E coli type I1 and Irregular type I11 using this scheme, colonies giving the reactions of these groups were placed together as E. coli type 11.  Enumeration of E. coli in seawater 107 Results Numbers and percentages of E. coli type I amongst colonies tested, separated on the ba.sis of our tests, are shown in Table 2. The percentage recovery of E. coli type I obtained by applying the full IMVEC series of tests to 622 colonies from 30 samples was Sl.9yo. TABLE Characterization of colonies from seawater samples by IM VE C tests IMVEC* reactions Number Colonies testod E oli type I1 and Irregular 111 I , coli type I - - + - erobacter aerogenes I E. coli Irregular I --- I1 - - - I VII --- C - D - J -- - Unidentified 622 510 4 68 18 7 1 2 2 4 4 1 - 100 81.9 0.3 0.6 10 9 2.8 1.1 0.2 0.3 0.3 0.6 0.6 * I, Indole; M, Methyl rod; V, Voges Proskauer; E, Eijkman; C, Citratc. A further 730 colonies from 58 samples were tested only for production of indole and of gas from lactose at 44 (i.e. IE tests). Of these, 78.4% gave positive results with both tests and were regarded as E oli type I (Report 71, 1969). If either or both tests were negative, a culture was not characterized further. TABLE Isolation of E. coli type I from seawater samples Tests applied IMVEC* IE No. of samples examined 3 58 No. of colonies testod 622 730 yo of E. coli type I 81.9 78.4 No. of E coli typo I 510 573 * Abbreviations as in Table 1 Table 1 shows the number and percentage of colonies other than E coli type I from the results of IMVEC tests. The largest single group was E. coli Irregular type I occasional isolates of Irregular types 11, VT, VII, C, and J were found, as well as Aerobacter aerogenes type I and E. coli type 11. Four colonies out of the 622 tested could not be placed in any group on the basis of the tests used.  108 Sheila Halls and P. A. Ayres Discussion The percentage recovery of colonies identified as E. coli type I using the IWEC and IE tests was very similar. It is not clear why the more rigorous IMVEC tests should identify more colonies as type I than the IE tests. However, the close agree- ment between the 2 methods suggests that identification on the basis of IE tests only is a valid one, with the limitation that the remaining organisms can be described only as 'other than E coli type 1 . Of the colonies subjected to the full IMVEC test, 16.2% were classified as irregular coli-aerogenes, but they could not be distinguished from E. coli type I by their appear- ance on membrane filters incubated at 44 . The public health significance of these organisms in estuarine water is uncertain, but they are known to occur in the intestinal tracts of man and animals (Holden, 1970 . It would seem, therefore, that there may be some merit in assuming that all lactose fermenters growing under these conditions are of faecal srcin. Aerobacter aerogenes constituted only 0.6y0 of the lactose fer- menters, and the errors of including non-faecal organisms would, therefore, appear to be low. Although there are no British bacteriological standards for shellfish-growing uaters, and the relationship between the level of pollution in shellfish and in the surrounding water is complex (Wood, 1964), water sampling can be valuable in identifying the source, extent and degree of sewage pollution in estuarine waters. Since a survey of this type depends on comparisons of the E. coli content of samples taken at the same time from different stations, or at different times from the same station, absolute numbers are not required. Provided that each sample is heated similarly, it does not seem unreasonable to assume that the proportions of colonies which are not E. coli type I are of the same order. However, when comparisons are made with other techniques (MPN, roll tube or plate count), or where an absolute estimate of the number of E. coli in seawater is required, the fact that < 20% of colonies assumed to be E. coli are not type I may be of some relevance. There is little published information on the characterization of colonies isolated from samples of freshwater by similar techniques. Mr H. G. Coles (pers. comm.) tested 1059 lactose-fermenting colonies from membranes incubated on 0.4 enriched Teepol broth at 44 ; 86% of these were confirmed as E. coli by the Eijkman test in brilliant green-bile broth and the production of indole at 44 . This is of a similar order to our figure of Sl*9%, lthough the medium used in the work described here contained 10 times the concentration of Teepol used by Coles. Preliminary comparisons in this laboratory have shown consistently that colony counts on the 0.4 y enriched Teepol broth were higher than those on 4% Teepol broth, but insufficient data are available to indicate whether the same proportion of colonies was of E. coli type I. Thus, although the IE test method lacks precision, most of the lactose-fermenting colonies are E. coli type I, and where a large number of samples has to be examined, the method has much to commend it. In our hands, when working in the field, 3040 samples can be handled in 1 h by 1 person and, in our view, this outweighs the disadvantages of relative lack of precision.
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