A chemically defined fluid medium—improved formate lactose glutamate—is described of which the cost is less than a quarter that of standard MacConkey broth and which is at least equally suitable for routine bacteriological examination of water. It has actual advantages in that the medium after fermentation is clearer and that, therefore, acid and gas production is more easily seen; moreover more isolations of both coliform bacteria and ofEsch. coliare obtained with fewer false positive reactions. Trials with a solid version of the medium suggest that the relative freedom from false positive reactions arises from the failure of the medium to growCl. welchii—one of the commonest causes—and enterococci, another suspected participant. In fact, without any inhibitory agent the medium appears to be virtually specific for the coliform group and particularly favoursEsch. coli.It remains to be seen whether certain aerobic spore-bearers found from time to time in surface waters may, to some slight extent, interfere with the almost complete specificity so far discovered.The medium is recommended in place of MacConkey broth and other peptonecontaining media, not only for the prospect of better results, in that suppression of the coliform group does not occur, but also for cheapness and for freedom from the inevitable variability associated with media incorporating peptone and, particularly, bile salt. It is hoped that this medium will shortly become available commercially in dehydrated form at a price still considerably less than that required to make up MacConkey broth from its constituents.Some labour-saving curtailments in the routine of water examination are also suggested.It is hoped not only that the fluid medium will supplant MacConkey broth for the dilution method of water analysis but that the solid medium, without material alteration, will also prove suitable for water examination by membrane filtration.I am deeply grateful to my laboratory staff, particularly Mr J. H. Evans, F.I.M.L.T., and Mr G. H. Lowe, F.I.M.L.T., for undertaking so much of the work when routine work was particularly heavy, and for many valuable suggestions. My thanks in addition are due to Mrs Peggy Johnson and Mrs Marilyn Childs, who made up the medium, and also to the directors of the participating P.H.L.S. laboratories not only for having the work carried out but for helpful comments.
During a period of 13 months, 1273 consecutive samples of water received at this laboratory were submitted to a modified presumptive coliform test which included single tubes of four different media. These were: (1) MacConkey broth, (2) formate lactose glutamate medium (pH 7·5), (3) lactose glutamate medium (pH 7·5), and (4) Folpmers's glucose glutamic acid (pH 6·0). 955 samples gave exactly the same results in all four media, but the remaining 318 samples produced differences which enabled comparisons to be made between the media.As compared with MacConkey broth, the three glutamic-acid media produced between 24–30% more isolations ofEsch. coli, and the formate and the glucose media produced at least the same total number of true coliform organisms (includingEsch. coli) with appreciably fewer false positive reactions. The lactose glutamate medium gave no false positive reactions but, through the suppression of coliform organisms other thanEsch. coli, reduced by 16% the total coliform yield. MacConkey broth gave the largest early (18 and 24 hr.) yield of positive reactions, but the results at the end of 24 hr. showed the formate medium to be not far behind MacConkey broth and appreciably ahead of the glucose medium.Further experiments with the formate lactose glutamate medium adjusted to different pH's, ranging from 6·0–7·5, indicated that a medium of pH 6·7 provided optimal conditions for the early development of both acid and gas. It was therefore decided to test this observation more fully with routine samples. During a period of 4 months, 279 unselected consecutive samples of water (of which 78 gave positive results) and 57 consecutive unchlorinated water samples (56 of which gave positive results) were examined by a multiple-tube technique in both MacConkey broth and formate lactose glutamate medium (pH 6·7). In the former series the MacConkey broth contained the inhibitory bile salt previously used; in the latter series this was replaced by a relatively non-inhibitory bile salt. In both series the formate medium yielded an appreciably increased total of coliform organisms (12 and 17%) including an increased total ofEsch. coli(27% in the former and 13% in the latter series), and in both series also the results at 18 hr. were abreast with, and at 24 hr. ahead of, those obtained in MacConkey broth.With certain reservations, it is considered that formate lactose glutamate medium (pH 6·7) can be offered as a suitable alternative to MacConkey broth for the presumptive coliform test of water. Wider trials of this medium would be required before this claim could be fully substantiated.My thanks are gratefully accorded to Drs W. H. H. Jebb and A. H. Tomlinson of the Oxford Public Health Laboratory for invaluable advice during the investigation and to Dr Ian Sutherland of the M.R.C. Statistical Research Unit for statistic appraisal of the results. I am also greatly indebted to my technicians Mr J. H. Evans, F.I.M.L.T., and Mr G. H. Lowe, F.I.M.L.T., for their unstinting help.
SUMMARYAs part of the bacteriological quality control programme of the Public Health Laboratory Service, we were asked to investigate the possibility of providing simulated water samples for distribution to the laboratories. For this purpose it was necessary to find some means whereby suspensions of coliform organisms and Escherichia coli could be kept relatively stable in number at room temperature for a period of 7-10 days. This, it was finally found, was best achieved by adding selected strains of the organisms to improved formate lactose glutamate medium (Gray, 1964) without the lactose but with added boric acid to a final concentration of 1.8 %.The procedures adopted in the successful quality control programme are described. INTROD'UCTIONIn the preparation of simulated samples of polluted water for the purpose of the quality control programme, the main problem was to find some means of maintaining the stability of suspensions of coliform organisms and Escherichia coli at ambient temperature for periods up to 10 days.It is well known that coliform organisms have a limited survival time in water. Variations occur even after 6 hr. storage, with significant decreases in numbers occurring more frequently than increases (Reports, 1952(Reports, , 1953 Fisher (personal communication) found that some cultures of E8ch. coli after freeze-drying had lost their ability to ferment lactose at 440 C. when reconstituted in the manner described.Two methods employing chemical agents for the preservation of bacteria have been described. Hoather (1952, 1957) suggested that the addition of sodium thiosulphate to water samples was effective in the short term in stabilizing the 4 HYG 76
A quantity of 0.1 ml. of a 3% solution of crystalline sodium thiosulphate in a 6 oz. bottle is sufficient to neutralize up to 5 p.p.m. of residual chlorine; the use of large quantities of thiosulphate is unnecessary, except possibly for some swimming-bath samples. The investigation has shown that this amount of thiosulphate has no significant effect on the coliform or faecal coli content of non-chlorinated water samples during 6 hr. storage at refrigerator temperature. There therefore appears to be no disadvantage in adding this amount of thiosulphate to all water sampling bottles.
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