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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.

SUMMARY: The death-rate of washed exponential phase Aerobacter aerogenes chilled in saline phosphate buffer (pH 6.5) at 0° was increased by ribonuclease (RNase) but not by deoxyribonuclease, trypsin, pepsin or lysozyme; none of these enzymes had any immediate effect on the viability of similar bacterial suspensions at 20°. Leakage products from chilled A. aerogenes, Mg2+ and, to a smaller extent, 0.3M-sucrose, antagonized the lethal effect of RNase on chilled organisms. RNA degradation occurred when bacteria were chilled and then incubated in fresh diluent at 37°; organisms exposed to RNase during chilling degraded RNA at 20-25° when the rate of auto-degradation of RNA was low. As the salt content of the environment was decreased, the amount of RNase adsorbed by the bacteria and its lethal effect increased at both 0° and 20°; in distilled water RNase was more lethal at 20° than at 0°. Anilino-naphthalene-8-sulphonate penetrated into bacteria chilled in buffer containing this dye. Acid or alkali accelerated the death rate of bacteria to greater extents at 0° than at 20°. RNase increased the lethal effect of freezing and thawing on a population from a continuous culture and augmented subsequent degradation of RNA.

AFTER capsulated B. anthracis had grown in a modified synthetic medium of Brewer et al.1, a mixture of growth products was isolated from the glass-filtered medium which gave a precipitate with copper sulphate. Crude polyglutamyl peptide was obtained from this precipitate2. An attempt was made to separate the components of the original mixture of growth products by paper electrophoresis using a tank as described by Flynn and Mayo3, and it was found that the peptide could not be detected using naphthalene black, bromo-phenol blue, ninhydrin or ultra-violet light4. We found it could be detected by taking advantage of the acidic nature of the peptide, using the following technique : (1) paper strips with the peptide applied are run in a suitable buffer, for example, veronal/veronal sodium at pH. 8.6 or acetic acid/sodium acetate at pH 4.5, and dried at 100°; (2) the buffer is washed out with two changes of 80 per cent ethanol; (3) the strips are treated with N/50 hydrochloric acid in 90 per cent ethanol; (4) the acid is thoroughly removed with ethanol.; (5) after drying, the strips are dipped into N/300 alcoholic sodium hydroxide containing 0.04 per cent bromo-cresol purple, which is repeated if necessary until colour differentiation is obtained, and the strips are blotted.

SUMMARY: The lethal effect of cold shock on Aerobacter aerogenes suspensions depended on the time of exposure to low temperature, the growth phase, the concentration of bacteria, the diluent. No death occurred when weak suspensions of susceptible bacteria (about 108/ml.) in buffered saline (pH 6.5) were rapidly cooled to 0° and immediately warmed to 20°, but loss of viability was progressive during 1 hr. at 0°. Bacteria harvested from defined medium at intervals during the exponential growth phase varied in sensitivity to chilling but were more susceptible than stationary phase organisms. While growing in partially synchronized culture the sensitivity of bacteria did not increase significantly during the division lag phase. The viability of dense suspensions (about 1010 bacteria/ml.) in buffered saline was little affected by chilling for 1 hr. at 0°, irrespective of the growth phase. A bacteria-free filtrate from a chilled concentrated suspension of exponential-phase organisms substantially protected a dilute suspension from the lethal effect of chilling. Substances found in protective filtrates were amino acids, adenosine triphosphate and nucleic acid constituents. When added to the diluent in which susceptible bacteria were chilled, a mixture of amino acids afforded some protection; small amounts of adenosine triphosphate had no effect. Other substances found to protect susceptible bacteria were sucrose (0.3 M), magnesium or calcium ions (5 x 10−3M) and, to a much smaller extent, spermine (10−5M). The present results support the suggestion that the lethal effect of chilling is at least partly due to interference with the functioning of a bacterial permeability control mechanism.

SUMMARY: The viability of microbial populations recovered from aerosols was determined by a slide culture technique and/or the radioactively labelled antibody method and the results were compared with those obtained by the more widely used Bacillus subtilis var. niger spore tracer technique. With Escherichia coli MRE 162, results with the three methods were in good agreement. Slide culture gave inaccurate results with Pasteurella tularensis, live vaccine strain (LVS) and E. coli MRE 160. Results with the spore tracer and radio-antibody methods in conjunction with determinations of viable numbers were in fair agreement when P. tularensis, E. coli MRE 160 and coliphage T 7 were tested.

WHEN certain bacteria are suspended in sucrose solution of suitable concentration and incubated with lysozyme, the rigid cell-walls are dissolved away, leaving relatively stable spherical protoplasts1. Lytic enzymes which dissolve the isolated cell-walls of vegetative Bacillus cereus have been found in extracts of mechanically disintegrated resting spores2 and partial autolysates of sporulating cells3 of this organism. When heat-treated, intact vegetative cells were treated with preparations of these enzymes, the walls were dissolved, leaving the coagulated cell-contents apparently unchanged. It has now been found that when viable organisms are suspended in sucrose solution and treated with these enzymes, relatively stable protoplasts are obtained in good yield.

SUMMARY: “Substrate-accelerated death” (Postgate & Hunter, 1963a; 1964) occurred when a nitrogen-limited variant of Aerobacter aerogenes NCTC 418 (Postgate & Hunter, 1962) was starved at growth temperature (37° or 40°) in aerated saline buffers containing ammonium ion; it was not observed when the parent strain of A. aerogenes or Escherichia coli (MRE 162) was grown and starved under similar conditions. Sulphate ion increased the lethal effect of ammonium ion on the variant and magnesium did not abolish either the effect of ammonium or ammonium + sulphate ions. The A. aerogenes variant differed from the parent strain in morphology, colonial appearance on nutrient agar, biochemical and immunological reactions and ability to synthesize polysaccharide. In ammonium-limited medium at 37° or 40° at a dilution rate near 0·25 hr−1 the variant contained 3-5% and the parent strain 12-16% of dry weight as polysacharide; in spent medium or phosphate buffer at 37° with added glycerol the rate of polysaccharide synthesis by the variant was about 25% that of the parent strain. When grown in nitrogen-deficient medium with excess glycerol in batch culture, populations of the variant containing 25% polysaccharide were obtained; the survival of the polysaccharide-rich variant was not affected by ammonium ion.

SUMMARY: The death-rate of washed Aerobacter aerogenes in aqueous suspension at 47° depended on the nature of the growth medium, the composition of the liquid used to wash and resuspend the bacteria, the bacterial growth phase, the bacterial concentration in heated suspensions, the pH value, the oxygen tension and the composition of the diluent in which bacteria were heated. The relative resistance of bacteria in different growth phases differed according to the growth medium and the washing fluid; stationary phase bacteria were not more resistant than exponential phase organisms under all conditions. Starvation increased the thermal resistance of exponential and stationary phase bacteria. High bacterial concentration favoured survival at 47° under most conditions; cell-free filtrate from a heated dense suspension (1010 bacteria/ml.) protected a sparser population of fresh bacteria (107-109/ml.) heated in it. Protective material in filtrate was heat-stable (100°/15 min.) and diffused through cellophan. The optimum pH value for survival at 47° was near pH 6·5. Aerobic conditions favoured survival in distilled water but not in salt solutions or phosphate saline (pH 6·5). The effects of various concentrations of NaCl and KCl on the survival of bacteria at 47° under aerobic conditions were different, K+ concentrations above 0·1 m being more lethal than equivalent concentrations of Na+; the lethal effect of heating in mixtures of these salts (total m > 0·1) increased with K+ concentration. Growth medium, Mg2+ (0·01–5 mm) and, to a lesser extent, Mn2+ (0·5 mm) or Co2+ (5 mm) decreased the death-rate, whereas ethylenediamine tetraacetic acid (mm), or various sugars, increased it. Mg2+ but not Mn2+ reversed the lethal effect of sugars. Generally, conditions which accelerated the death-rate of Aerobacter aerogenes at 47° also increased the rate of degradation of endogenous RNA. This was accompanied by an increase in the ultraviolet absorption of cold acid-extracts of bacteria and of the suspending fluid. Bacterial protein was degraded to a smaller extent. Depletion of RNA is probably not the primary cause of death at 47° but the effect on bacterial metabolism of a rapid increase in endogenous pool constituents resulting from RNA degradation may contribute to the lethal effect.

Bacillus anthracis contains a polysaccharide which appears to be part of the cell wall1. Ivanovics2 first isolated the polysaccharide which contained galactose 34 per cent, glucosamine 34 per cent, nitrogen 4.2 per cent and α-carboxyl amino-nitrogen 0.8 per cent. Strange and Belton3 obtained from culture filtrates of B. anthracis a polysaccharide which contained galactose 39–43 per cent, glucosamine 34 per cent, nitrogen 3.9 per cent and α-carboxyl amino-nitrogen 1.26 per cent. The differences between the total nitrogen and glucosamine nitrogen values together with the figures for α-carboxyl amino-nitrogen in these preparations indicated the presence of 5–10 per cent of amino-acid residues. Smith and Zwartouw4 obtained a polysaccharide from B. anthracisgrown in vivo. It contained galactose 38–43 per cent, glucosamine 38–44 per cent, nitrogen 4.0 per cent and α-carboxyl amino-nitrogen 0.3 per cent. Since this preparation satisfied a number of rigorous tests for homogeneity it was concluded that a small peptide moiety (2–3 per cent) was firmly attached to the polysaccharide. We are concerned here with the constitution of this peptide moiety.

SUMMARY: The magnesium and RNA contents of Aerobacter aerogenes, growth-limited by Mg2+, K+, NH4+ or carbon source, in defined media at 35° increased with growth rate. The results support the view that the amounts of these constituents are functions of the growth rate and are inter-dependent. Up to 26% of the total Mg2+ of bacteria freshly harvested from cultures containing excess magnesium was loosely bound to the bacterial surface; this adsorbed Mg2+ was removed by washing with 0·85% (w/v) NaCl but was unaffected by distilled water. Mg2+-limited bacteria had no surface-adsorbed magnesium. Surface-adsorbed Mg2+ stimulated polysaccharide synthesis, and affected the response of bacteria in saline buffer to stresses including starvation, heat-accelerated and substrate-accelerated death, and cold shock.

THE lethal effect of chilling on certain exponential phase Gram-negative bacteria (‘cold shock’)1 may be due to interference with bacterial permeability control mechanisms2. Evidence supporting this view is that, on chilling, susceptible bacteria release normal endocellular constituents into the environment3 and are more permeable than similar unchilled bacteria to various substances including ribonuclease4. Cold shock also affects the stability of endogenous RNA. Thus, if susceptible bacteria are chilled and then incubated in fresh diluent at 37°, the rate of RNA-degradation increases with the duration of chilling4. In view of these symptoms of cold shock, the protection afforded to chilled bacteria by magnesium ions3 may be due to the metal's stabilizing effect on bacterial membranes concerned with osmotic control and/or endogenous RNA. In this connexion it is well known that magnesium stabilizes isolated protoplast membranes5, spheroplasts6 and ribosomes7. This note reports the use of the ‘optical effect’8 to determine the influence of magnesium on permeability control in exponential phase Aerobacter aerogenes during chilling.

SUMMARY: Aerosolization decreased the accumulation of methyl-[α-D-gluco]pyranoside (αMG) in two strains of Escherichia coli. The inactivation was apparently not due to damaged permeases but to detachment from the bacteria of components involved in the transport of substrates through bacterial membranes. Transport activity was partially restored when aerosolized bacteria were incubated with leaked components. Aerosolization also decreased accumulation of isopropyl-thio-β-D-galactopyranoside (IPTG) by E. coli but increased permeability to O-nitrophenyl-β-D-galactopyranoside (ONPG). Loss in viability of airborne bacterial populations correlated with the decrease in αMG accumulation by bacteria recovered from aerosols one second after generation of the cloud.

THE endogenous metabolism of micro-organisms in aqueous suspension in the absence of added nutrients may involve the utilization of endogenous protein and RNA1, as well as amino-acid pool constituents and reserve substances such as glycogen and polyhydroxybutyric acid (see reviews by Dawes and Ribbons2). Investigations with Aerobacter aerogenes1, Escherichia coli and Pseudomonas fluorescens have shown that starvation in aerated saline buffer at 37° results in a considerable loss of protein during a period when viability of the populations remains at 95–100 per cent. Since analyses of ultracentrifugal fractions separated from A. aerogenes after various periods of starvation showed decreases in the protein content of the insoluble, ribosomal and soluble fractions3, the loss apparently represents a depletion of various cell proteins. It is interesting, therefore, that induced β-galactosidase in Escherichia coli was found to be relatively stable while other proteins were being rapidly degraded during turnover4. The relative stability of induced β-galactosidase in a wild-type strain of E. coli during starvation has been investigated and this communication records the relevant data.

IN certain conditions, bacteria accumulate relatively large amounts of polyglucose compounds with properties similar to those of animal glycogen1. An interpretation of bacterial “glycogen” production is that it provides a food and/or energy reserve for the organisms in unfavourable environments; in other words, bacteria rich in glycogen should survive longer than bacteria without such reserves. Experimental results apparently supporting this teleological interpretation were obtained with Aerobacter aerogenes2 and Escherichia coli3 but not with Sarcina lutea4; glycogen-rich S. lutea died at a faster rate than cells without glycogen during starvation in aerated phosphate buffer at 37° C. A feature of glycogen reserves in bacteria is their rapid depletion during starvation which suggests that any contribution glycogen makes towards maintenance and survival is of short duration5. It is possible that growth conditions which stimulate the accumulation of glycogen give rise to bacteria better able to resist stress for reasons not concerned with their glycogen content. The effect of glycogen reserves on bacterial survival was examined with E. coli; cells containing different amounts of glycogen were grown in a chemostat and their survival properties were determined in aerated saline phosphate buffer with and without magnesium at 37° and 48° C.

AN enzyme system catalysing the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate or glucose-6-phosphate has been demonstrated in Neurospora crassa 1,3, rat liver2,3 and Escherichia coli 3. Ghosh et al. 3, who purified the enzyme from these various sources and studied its properties, have called it L-glutamine-D-fructose-6-phosphate transamidase. We have determined transamidase activity in fresh, cell-free extracts of Bacillus cereus, Micrococcus lysodeikticus, Aerobacter aerogenes and Escherichia coli. Results indicate that, for a given weight of cells, activity is higher in Gram-positive than in Gram-negative bacteria, and for a given organism, activity increases markedly during the lag and early exponential phases of growth, but decreases during the stationary phase. The high transamidase activity associated with dividing cells is probably related to the synthesis of cell-wall basal structure4, which contains amino-sugars. During these investigations, using a system with standard conditions of pH, substrate concentrations and reaction time at 37°, it was found that the amount of glucos-amine-6-phosphate synthesized rarely exceeded 25 per cent theoretical, and frequently was not proportional to enzyme concentration when different volumes of the same cell extract were tested in parallel. An attempt was made to explain these findings.

CEBTAIN bacteria in the logarithmic growth phase become non-viable when chilled in aqueous suspension. The phenomenon has been observed with strains of Escherichia coli1, Pseudomonas pyocyanea2 and Aerobacter aerogenes3. Loss of viability of logarithmic phase A. aerogenes suspended in chilled phosphate-saline is accompanied by the leakage of endogenous solutes, and the leakage products, in sufficient concentration, protect the bacteria against the lethal effect3. Thus, loss of viability is greater the sparser the population and is insignificant in chilled suspensions containing more than 5 × 109 organisms/ml. Correlation between loss of viability and leakage of endogenous solutes has now been obtained by observing the effects of chilling on three Gram-negative bacteria.