An agar diffusion method for the assay of colicins A, B, D, E 2 , E 3 , and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about ± 10%.

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An agar diffusion method for the assay of colicins A, B, D, E2, E3, and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about-410%.

IT is often assumed that the serum of a healthy uninoculated rabbit contains only insignificant amounts of antibacterial agglutinins. This view is largely supported by the findings of Gibson (1930), but Boyd (1939-40) and Messer (1943) have drawn attention to the occurrence of agglutinins in considerable concentration in some " normal " rabbit sera. Indeed, with a agglutinin, the natural antibody titres may be as high as those found in sera from immunised animals (Stamp and Stone, 1943-44 ; Francis and Buckland, 1945).I n the present work, certain paracolon bacilli isolated from desoxycholate-citrate-agar plate cultures during the examination of fieces from cases of enteritis or their contacts were found to have biochemical reactions superficially resembling those of certain specific pathogens and to be agglutinated to titre by a considerable number of batches of Oxford standard agglutinating sera prepared against different organisms. I n preliminary absorption experiments with antisera to Shigella sonnei and Shigella jlexneri, types Newcastle and Boyd 103, the paracolon bacilli failed to remove the specific agglutinins for the dysentery bacilli, and the homologous dysentery bacilli had no effect on the ability of the sera to agglutinate the paracolon strains. Subsequently the paracolon strains were found to be agglutinated to high titres by the sera of two healthy uninoculated rabbits. These rabbit sera also agglutinated lactose-fermenting and non-lactosefermenting coliforms from human fieces or contaminated war wounds. The agglutinins in these sera were studied in absorption experiments and the results form the subject of this paper. MethodsStrains. Four strains of paracolon bacilli (PR, PW and PM from faxes, P 6 from a contaminated war wound) and 4 strains of Bacterium coli of Wilson et al. (1935), type I (CA and CB from fseces, C 5 and C 7 from wounds) were used for the main absorption experiments. Part of the work was repeated with three known tc strains, including the classical 1721, kindly provided by Dr Doris Stone.Bacterial suspensions. The organisms grown on Lemco-agar slopes for 18 hours at 37' C. were washed off in saline (0.85 per cent. sodium chloride)

PURIFIED colicines have usually proved to be potent antigens (Goebel, Barry and Amano, 1957; Hutton and Goebel, 1962; Barry, Everhart and Graham, 1963; Barry et al., 1965; Hinsdill and Goebel, 1966), and colicines K and V appear to be indistinguishable from the 0 (somatic) antigens of the organisms producing them (Amano, Goebel and Miller Smidth, 1958;Hutton and Goebel). Five strains of Escherichia coli known to produce colicines of the B group have been investigated serologically to determine any possible relationships between their 0-antigens and B-colicines.