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When used to determine immunopurified antibacterial globulins in saline-phosphate buffer, the detection sensitivities of direct competitive and indirect immunoradiometric assays were respectively 25 and 2 ng. Normal human and rabbit sera interfered in both types of assay, markedly decreasing sensitivity and precision. Various pre-treatments of sera and modifications of reaction conditions substantially decreased interference in the competitive assay without affecting the activity of added antibody, but they had only marginal effects in the indirect assay. When serum samples taken from rabbits after vaccination with the Live Vaccine strain of Francisella tularensis were tested with the two assays and agglutination tests, newly synthesised circulating antibody was usually detected earlier by the competitive assay than by the other methods.

SUMMARY: The survival characteristics of washed stationary phase Aerobacter aerogenes organisms suspended in buffered sodium chloride solution and stored at room temperature, or at 37° with aeration, depended on the medium used for growing the bacteria. Populations of bacteria harvested from tryptic meat broth or tryptone glucose medium remained almost completely viable for longer periods than bacteria from a simple ammonium salt + mannitol medium in which carbon was limiting. Analyses of washed freeze-dried preparations of freshly harvested bacteria showed that the amounts of protein, carbohydrate and ribonucleic acid present varied according to which of the above media was used for growth. During the initial stages of storage at 37°, when the viability of the population remained apparently unchanged, a progressive loss in bacterial dry weight occurred, due to degradation of these cell constituents. Endogenous glycogen was degraded and oxidized; bacteria which contained glycogen survived well. However, the addition of glucose to suspensions stored under aerobic or anaerobic conditions did not favour survival. Utilization of substances made available by degradation of various endogenous macromolecular constituents may be an important factor concerned with the survival of bacteria in unfavourable environments.

SUMMARY: Small numbers of several bacterial species were rapidly and specifically detected with the 125I-labelled antibody method described previously. Multibacterial species detection was achieved in one operation with 125I-labelled mixtures of type-specific anti-bacterial globulins but the level and variability of the blank value increased, and sensitivity decreased, with the number of globulin components in the mixture. An indirect radio-assay that involved successive treatments of bacteria with unlabelled rabbit anti-bacterial serum and 125I-labelled immunopurified goat anti-rabbit globulin was less sensitive and reproducible than the direct radio-assay. A modified assay, developed to detect and determine bacteria filtered on to a membrane filter, allowed the detection of small numbers of bacteria in large volumes of aqueous sample. The feasibility of rapidly and specifically detecting small numbers of virus particles with 125I-labelled anti-viral globulin was investigated with bacteriophage T7 as a model particle. Methods for the rapid separation of phage-125I-labelled globulin complex were developed and a minimum of about 5 × 105 total phage particles was detected.

‘Substrate-accelerated death’ (Postgate & Hunter, 1963a, 1964) was observed with carbon-limited but not ammonium-, phosphate- or sulphate-limited Aerobacter aerogenes grown at 37° in defined medium and starved at 37° in aerated saline buffers containing the growth-limiting substrate. Carbon sources besides the one limiting growth increased the death-rate of starved mannitol-, glycerol-, galactose- and ribose-limited bacteria. Glycerol-accelerated death depended on the rate of oxidation of glycerol and the bacterial concentration; with bacteria fully adapted to glycerol, populations of less than 1-2 x 109 organisms/ml. died at a faster rate the denser the population and above this concentration the death-rate decreased with increasing bacterial concentration. Death was delayed when aerated bacterial suspensions containing glycerol were dialysed at 37° against saline buffer containing the substrate. Bacteria-free filtrates, from populations dying in the presence of glycerol, accelerated the death of fresh bacteria to a greater extent than did glycerol alone. In contrast, bacteria-free filtrates from dense populations surviving in the presence of glycerol partially protected fresh bacteria exposed to glycerol. Mg2+ abolished glycerol-accelerated death but not the lethal effect of filtrates from dying populations. Compared with its influence on glycerol-accelerated death, population density had much less influence on the death-rate of glycerol- or mannitol-limited organisms starved in the presence of glucose or mannitol. Irrespective of bacterial concentration, α-ketoglutarate had no effect and pyruvate, citrate, malate, succinate and oxalacetate had less effect than glycerol on the death-rate of starved glycerol-limited bacteria.

SUMMARY: Aqueous extracts of disintegrated spores of Bacillus cereus and non-virulent B. anthracis contained an enzyme which produced visible lysis of the isolated cell walls of vegetative B. cereus. Optimum activity occurred at pH 7-8 in the presence of cobalt or manganese ions (10 p.p.m.) at 58°. Activity was destroyed during heating at 100° for 15 min. The lytic preparation released non-dialysable components containing αe-diaminopimelic acid (DAP), glutamic acid, alanine, amino sugars and glucose. Although lysis was less obvious, the enzyme preparation released similar material from cell walls of other Bacillus species, spore coats of B. megaterium and coats of autoclaved B. cereus spores. Extracts of freshly harvested B. cereus spores were more active than those from spores which had been stored for several weeks at 2°. Extracts from disintegrated spores of B. megaterium had no enzymic activity; the enzyme system was associated with the insoluble spore coat fraction. The action of the enzyme differed from that of lysozyme or glucosaminidase; the reaction products did not give a significant reaction for N-acetylhexosamine and visible lysis proceeded more rapidly with cell walls of B. cereus than with B. megaterium. Possible functions of the enzyme may be to release ‘spore peptide’ from the spore coat during germination and to lyse the sporangium and free the spore during sporulation.

SUMMARY: When washed sporulating cells of Bacillus cereus were incubated in buffer at 37°G in the presence of toluene, a partial autolysis occurred resulting in the freeing of mature and immature spores. The autolysate contained lytie enzymes which attacked vegetative cells and cell-wall preparations, releasing hexosaminecontaining peptides of characteristic constitution. The most active enzyme preparations were obtained from sporulating cells incubated for 1-2 hr. in buffer at pH 5-0-6-0. Two water-soluble lytic systems, enzyme V and enzyme S with pH optima near 4-5 and 8-0 respectively, were separated from the autolysate. Enzyme S is probably identical with the lytic system present in spores of B. cereus and other Bacillus species and further observations on this system are described. When non-sporulating cells of B. cereus were incubated under similar conditions no obvious lysis or sporulation occurred and no cell-wall lytic activity could be demonstrated. In growing cultures of Bacillus cereus, considerable amounts of hexosamine-containing peptides were released into the medium during the period between the appearance of intracellular spores and free spores. It is suggested that enzyme V may be mainly concerned with the release of free spores from sporangia and enzyme S with the lytic processes which accompany spore germination.

SUMMARY: An assay for rapidly detecting and determining sparse populations of vegetative bacteria or bacterial spores (minimum number about 500; 2 to 4 x 10-10g. equivalent bacterial dry weight) is described. A sample is treated with 125I-labelled purified homologous antibody, filtered and washed on a Millipore membrane filter, and the radioactivity of the separated labelled immune complex is measured. The assay is specific, accurate and completed within 8 to 10 min. Sensitivity and accuracy decrease if the assay is applied to samples that contain particulate matter that non-specifically attaches antibody and is retained by a membrane filter. This type of interference is decreased by pretreating samples with clarified normal rabbit serum for a few minutes before assay.