It has been observed that during the treatment of sewage by biological filtration the numbers of coliform bacteria are considerably reduced (Allen, Tomlinson & Norton, 1944; Allen, Brooks & Williams, 1949). As it was thought that this reduction might be due to the action of bacteriophage known to be present in sewage, a short investigation to obtain evidence on this point was made at Stevenage sewage works.At this works sewage from the residential and industrial areas of the town is mixed and screened, one-third of the flow being then treated in the old section of the works and two-thirds in a modem extension. In each section the sewage is settled in continuous flow tanks, treated biologically in percolating filters, and settled again in humus tanks before it flows to a sump, common to the two sections, from which it is pumped intermittently. METHODSEnumeration and isolation of coliform organisms Counts of coliform bacteria were made from sewage taken at different stages of treatment by spreading 041 ml. of a suitable dilution in saline over the surface of Bacto eosin-methylene-blue agar and counting typical coliform colonies after incubation for 24 hr. at 370 C. This medium was almost completely specific for coliform organisms.Over a period of several weeks pure cultures of forty-nine strains of coliform organism were obtained from the influent crude sewage and a further fifty from the effluent in the final sump by plating on double strength Bacto eosin-methyleneblue agar and subculturing typical Bacterium coli colonies twice on single strength eosin-methylene-blue medium; the resulting cultures were maintained on nutrient agar. All the organisms were subsequently confirmed biochemically to be either Bact. coli, Bact. aerogenes, or intermediates.Isolation and enrichment of specific bacteriophage Ten ml. of each of the sewage samples, of which a portion had been used for inoculation on to eosin-methylene-blue agar, was immediately treated with 3 drops of toluene at 37°C. for 2 hr. This procedure killed the majority of the bacteria in the sewage without affecting the bacteriophage count. The purified bacteriophage suspensions were stored in a refrigerator until the pure culture of the coliform isolated from the same sample was available. Fifteen ml. of nutrient broth was then mixed with 5 ml. of toluene-treated suspension, drops of free toluene being
SUMMARY: A strain of Bacterium coli capable of growth in broth and in an ammonium +glucose + salts medium at 37' and in broth at M', died in the defined medium at 4 4 ' . The addition of glutamic acid to this medium allowed the growth temperature to be raised slightly ; the further addition of nicotinamide allowed a further increase in growth temperature.One of the criteria on which Public Health bacteriologists rely to identify strains of Bacterium coli as being of faecal origin is the ability to ferment lactose with the evolution of gas in a bile salt medium a t 44". The experiments recorded in this paper show that ability to multiply a t 44" depends not only on the strain of Bact. coli but also on the nutritive materials supplied. METHODSThe defined medium used throughout the work was Bigger's (1950) modification of Gale's (1947) medium which contains only organic salts and glucose, the sole nitrogen source being ammonia. Broth, where used, contained 1 yo Lab-Lemco and 1 yo peptone. Experiments were carried out with 5 ml.volumes of medium in small glass bottles covered with loosely fitting aluminium caps. When other substances were required in the medium, additions were made either in bulk or by adding to the individual bottles a sufficient number of drops (Dreyer pipette) of a suitable dilution of the substance. Serial dilutions, to halve the concentration of a substance, were made by transferring 5 ml. amounts from bottle to bottle. Bact. coli strain no. 18 was used as test organism, except in certain experiments designed to determine whether the phenomenon described occurred with other strains of Bact. coli. The cultural characteristics of Bact. coli strain no. 18, which was isolated from faeces, are given in Table 4. Inoculation was, in all cases, carried out by adding to 5 ml. of the medium, one drop (Dreyer pipette) of a 1/10,000 dilution in buffered saline of the test organism grown in defined medium at 3 7 ' . Cultures were examined every 24 hr. during incubation. One 5 mm. loopful of each culture which had remained clear was spread over the surface of an agar slope, which was examined after 24 hr. incubation, to determine the colonies resulting from the viable organisms in the original culture. RESULTSGrowth curves were obtained from viable counts (2 hr. intervals) on Bmt. coli growing in defined medium or in broth at 37 and 44". It was found that whereas the organism multiplied freely in both media a t 37" and in broth a t 44", it died when exposed to 44" in defined medium. The effect of adding certain
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