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Direct probe mass spectrometry of single colonies of different bacteria produced spectra which could be distinguished by calculations based on the intensities of 25 selected ions. Direct probe mass spectrometry has enormous potential as a rapid technique for characterizing micro-organisms.

The discrimination of a set of 53 strains, taken from four closely related Bacillus species (Bacillus subtilis, B. pumilus, B. licheniformis and B. amyloliquefaciens), was examined using pyrolysis mass spectrometry. Strains were grown on six different media to examine the effect of media variation, especially batch-to-batch variation of a single medium, on the pyrolysis mass spectra and strain discrimination achieved. Long-term reproducibility over a period of 14 months was also examined. It was shown that batch-to-batch media variation is insufficient to affect spectra and strain discrimination significantly, but different media types do affect this. It was shown that species groups could still be recovered from the data, however, with an appropriate data-handling system. It was not possible to directly compare spectra produced 14 months apart, but the strain and species discrimination achieved using each data-set were highly comparable.

Nine strains of Bacillus mycoides, 10 of Bacillus cereus and 18 of Bacillus thuringiensis were analysed using pyrolysis gas—liquid chromatography. Application of canonical variates analysis to the resultant pyrograms showed that these organisms formed three distinct groups. Stepwise discriminant analysis with jacknifing showed that of the original 27 peak heights used to define the canonical variates axes, only two were needed to obtain reasonably stable discriminant functions. These discriminant functions (Dixon, 1975) are potentially valuable in identification. Jacknifing also indicated that, had it been an unknown, one strain of B. mycoides would have been incorrectly identified as B. cereus.

Eight strains each of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, and Bacillus amyloliquefaciens were analyzed by using pyrolysis gas-liquid chromatography. Statistical analysis with canonical variates gave four well-separated groups, which represented the four species. Further analysis of the same strains by deoxyribonucleic acid-deoxyribonucleic acid hybridization and API identification systems confirmed the discrimination obtained with pyrolysis gasliquid chromatography. However, analysis by biochemical tests performed in the classical way gave only three groups since it was not possible to achieve separation of the strains representing B. subtilis from those of B. amyloliquefaciens when these tests were used.

SUMMARY: The existence of subgroups within Bacillus megaterium has been reported previously on the basis of DNA-DNA hybridization and DNA base composition studies. In this study the strains used to define these subgroups have been reanalysed by pyrolysis gas-liquid chromatography. The resultant two-group classification of the test strains was directly comparable with that obtained from the previous nucleic acid analyses at the between-group level. However, comparisons of the test strains at the within-group level proved less successful.

Summary Twentyfive separate isolates of serotype 4B of the aerobic sporeforming insect pathogen, Bacillus thuringiensis, have been obtained over the period 1961–68. Most of the isolates of this particular serotype were obtained from storage products insects infesting such places as grain stores, flour mills or ships carrying grain materials. In one instance the organism was isolated from insects infesting Guinea corn growing in the field. In comparison with the other serotypes of this bacillus the association with stored products environments is striking and, on several occasions, it has been possible to trace the distribution of the bacteria by movements of infested materials from one part of the world to another.

SUMMARY Spore, vegetative O and vegetative H agglutinogens were demonstrated as separate entities in Bacillus species. Spore precipitinogens were distinct from those of the vegetative stage, the latter being associated with the H antigen. Injection of viable spores into rabbits resulted in the formation of antibodies for the vegetative phase as well as spore antibodies, while injection of autoclaved spores gave rise to spore antibodies only. Spore agglutinogens varied in their strain specificity, B. cereus and B. megaterium especially showing much subspecies specificity, but the spore precipitinogens showed a high degree of species specificity and could be of considerable taxonomic value. The multiplicity of spore precipitinogens in certain species was demonstrated and inter-species cross reactions were studied.

Summary. Thin sections of methane-utilizing bacteria have been examined in the electron microscope and the fine structure of the cells is described for the first time. The outstanding feature of all the isolates studied is the existence of intracellular membrane structures resembling those seen in nitrifying and photosynthetic bacteria. The possible significance of these membranes is discussed.

Summary The results of an electron microscope survey of flagellation in the genus Azotobacter are presented and the implications of the findings are discussed.

Summary. Forty-six isolates of protein crystal-forming bacteria pathogenic for insects were studied in an attempt to correlate classifications based on biochemical reactions, flagellar antigens, and esterase patterns obtained when cell extracts were analysed by electrophoresis in starch gel. Nine serotypes were represented, one being subdivided into two subtypes on the basis of secondary H antigens. There was a close correlation between the serological classification and that based on esterase analysis. The relationships between the three types of classification are described and the significance of esterase analysis in this context is discussed.

SUMMARY: A lytic principle associated with cultures of Bacillus cereus is described and some of its properties discussed. The principle is relatively non-specific, acting on several species of aerobic spore-formers. It has properties in common with similar agents reported recently and comparisons with these are made. Lysis results in the loss of cell contents whilst at least part of the cell-wall structure remains intact.

Summary Starch gel electrophoresis showed that Bacillus cereus esterase and catalase are not single enzymes but multimolecular species and that they undergo changes during sporulation. Heat resistant spore catalase was detected in vegetative cells before mature spores were present.

Calcium was essential for the growth of A. vinelandii, A. chroococcum, A. beijerinckii and A. insigne in the presence and absence of combined nitrogen. A. agile and A. macrocytogenes were able to grow in the absence of calcium but growth was stimulated by the cation. Calcium could be replaced by strontium at roughly the same molar concentration. The authors present evidence suggesting that the calcium sparing effect of ammonium and sodium acetates reported in the literature may be dependent on the size of inoculum used.

Crystalline inclusion bodies resembling those seen in Clostridium cochlearium were detected in cultures of Bacillus thuringiensis infected with bacteriophage.

SUMMARY: The ‘negative contrast method’ was used to study by electron microscope four previously described Bacillus cereus bacteriophages. The main features of the structure of these phages are described and the observations corrected with similar work on phages associated with other genera.

SUMMARY: The use of pyrolysis mass spectrometry in the characterization and identification of Bacillus species was studied. Fifty-three strains of four closely related groups, Bacillus subtilis, B. pumilus, B. licheniformis and “B. amyloliquefaciens”, were used in a study of both sporulated and non-sporulated cultures. Pyrolysis was carried out using a Pyromass 8-80, a novel pyrolysis mass spectrometer specifically designed for fingerprinting complex samples. The pyrolysis data obtained were analysed using multivariate statistical techniques. All four groups could be differentiated using data from non-sporulated cultures but the data from sporulated cultures did not separate B. subtilis from “B. amyloliquefaciens” or B. pumilus. In contrast, B. licheniformis was more clearly differentiated from the other three species using these data. Culture maturity affected the mass spectra obtained from non-sporulated cultures.

Low resolution pyrolysis gas-liquid chromatography could differentiate the following groups of Clostridium botulinum and related organisms: (1) Cl. botulinum type A. proteolytic types B and F and Cl. sporogenes; (2) Cl. botulinum types C and D. and (3) Cl. botulinum type E and non-proteolytic types B and F. Toxin types A and B could be distinguished from type E and from type F.

Baillie, Ann (University of Glasgow, Glasgow, Scotland), and John R. Norris . Antigen changes during spore formation in Bacillus cereus . J. Bacteriol. 87: 1221–1226. 1964.—Ultrasonic extracts of Bacillus cereus cells harvested at different stages in spore formation were analyzed by immunoelectrophoresis with antisera prepared by injecting similar extracts into rabbits. Seven antigens resistant to heating at 80 C for 10 min were detected in extracts of young vegetative cells, and there were at least eight additional thermolabile antigens present at this stage. During spore formation, many of the thermolabile antigens disappeared from the cells and new antigens could be detected. Mature spores contained four of the heat-resistant antigens present in young vegetative cells and an additional five heat-resistant antigens. Only three thermolabile antigens were detected in spore extracts.

SUMMARY Four phages isolated from bacteria of the Bacillus cereus group are described. They show cross-reactions between crystal-forming and non-crystal-forming strains which are deficient in ability to produce a lecithinase C. The implications of this finding are discussed.

Summary. The morphologies of 4 new bacteriophages active against various strains of Bacillus thuringiensis have been examined by electron microscopy. The sensitivity patterns to these bacteriophages of B. thuringiensis strains and of other Bacillus spp. are described.

SUMMARY: Agglutinogens and precipitinogens have been detected in the spores of many species of aerobic and anaerobic spore-formers and these antigens are distinct from those of the vegetative growth phases of the corresponding organisms. Spore antigens are heat resistant and antibody response is elicited by the injection of autoclaved spores into rabbits. Injection of living spores leads to the development of antibodies reacting with vegetative cell antigens in addition to spore antibodies. Agglutinogens tend to show subspecies distribution in some aerobic species but spore precipitinogens appear to be mainly species specific in aerobes. Less information is available about anaerobes where a similar pattern of specificity is suggested. Data about the antigenic structure of the spore and the antigenic changes accompanying sporulation and germination are scant; it is in these aspects of the subject that the most significant progress is to be anticipated.

Summary A soil perfusion apparatus is described which features an enclosed system of small size. It has been successfully used to isolate micro-organisms capable of utilizing various aromatic compounds as sole sources of carbon and energy.

PREVIOUS attempts at studying the antigens of Azotobacter have met with only limited success. Azo and Yoshida in a brief report1 described complement-fixation by Azotobacter cells in the presence of specific antisera but gave few experimental details. Jensen and Petersen2 obtained an agglutinating antiserum for Azotobacter chroococcum. Recently, Petersen3 studied the agglutinogens of Az. chroococcum, Az. vinelandii, and Az. agilis. Az. chroococcum and Az. vinelandii had antigens in common and showed serological affinities to Beijerinckia sp. Az. agilis appeared to be antigenically distinct. Eisenstark et al., on the other hand, using apparently adequate technical methods, failed to produce antisera to Az. agilis or “numerous other azotobacter species”. Heidelberger (quoted in Lawson and Stacey5) showed Az. chroococcum polysaccharide to be antigenic.