- Volume 15, Issue 2, 1956

Summary: Washed suspensions of a strain of Rhodospirillum rubrum S1 grown anaerobically in the light and incubated in the light under argon metabolized many of the 15 amino acids tested and produced ammonia and carbon dioxide as the principal extracellular products. Hydrogen was also produced in the presence of glutamic acid. Of the glutamic acid metabolized 80 % of the carbon and 55–70% of the nitrogen were converted into intracellular products. The magnitude of the catabolism of the other amino acids, as measured by ammonia and carbon dioxide production, appears to be an inverse function of the closeness to which the elemental composition of the amino acid approximates to that of cell material.

When the organism was grown aerobically in the dark, studies of the metabolism of amino acids by washed suspensions, under aerobic conditions, were complicated by the relatively high rate of endogenous respiration. In the presence of certain amino acids this was decreased by 9 % (with alanine) to 45 % (with aspartic acid), and glutamic acid, aspartic acid and alanine, at least, were oxidized to completion by the organism.

Summary: When Bacillus cereus was grown on a medium solidified with agar but of otherwise known composition (salts, glucose, urea) impression preparations made after 2½ hr. of growth at 30° showed simple round nuclei by the osmic acid-hydrochloric acid-Giemsa technique. Similarly treated preparations made after 24 hr. of incubation showed well-marked unstained refractile inclusions. These inclusions, whose lipid nature was indicated by staining with Sudan Black, were closely associated with nuclear material, often appearing to be surrounded by an unbroken ring, a granular band, or a horse-shoe shape of nuclear material. These observations support the suggestion of Delaporte (1950) that lipid inclusions may distort nuclear shapes. These distortions might be misinterpreted as mitotic figures.

Filtrates from suitable cultures of Bacillus anthracis contain a phospholipase which slowly hydrolyses the phospholipid in egg-yolk broth but has no action on free egg-lecithin. This enzyme does not appear to be the anthrax toxin.

SUMMARY:Stocks of staphylococcal phage 47C (serological group A) contained some group B phage. This was found to have originated in a lysogenic staphylococcus previously used to propagate phage 47 C and which had subsequently lost its lysogenicity. The lysogenic phage was thus perpetuated in the phage stocks as a lytic phage. The characters of the two phages are described. When they lysogenized five different strains of staphylococci changes in typing pattern were produced. There was evidence which indicated that, in the prophage state, the two phages occupy different sites and that there is no cross-immunity between them. The propagation of the phages in different hosts resulted in changes in their host range. A virulent mutant of the B prophage was induced by the application of a variety of phages to the strain carrying it.

SUMMARY:Paramecium aurelia, var. 4, stock 47.8 (sensitive) grown in an axenic medium required the following amino acids: dl-tyrosine, l-phenylalanine, l-tryptophan, dl-methionine, dl-threonine, l-leucine, dl-isoleucine, l-lysine, l-histidine, l-arginine, and dl-serine. dl-valine, l-proline and glycine were synthesized at a slow rate. Tyrosine, methionine and serine were required by stock 51.7 (sensitive). Other essential metabolites required by stock 47.8 (s) were thiamine, riboflavin, folic acid, nicotinic acid, pantothenic acid, and possibly pyridoxal. 2,6-diaminopurine inhibited growth at concentrations as low as 1–4 μg./ml. The growth rates (expressed as final population density/ml./transfer) of various stocks in identical media are different. An apparent correlation was found to exist between the growth rates and the chromosome number of these stocks, those with the largest chromosome number attaining the highest population density.

SUMMARY: The enhanced pathogenicity of mouse hepatitis virus (MHV 1) in mice infected with Eperythrozöon coccoides is shown to result from the increased probability of virus particles actively infecting cells in the presence of the blood parasite. The ratio (α) of the probability that a virus particle will infect a cell in the absence of E. coccoides to the probability of its doing so in the presence of E. coccoides has been estimated by two independent methods. One method depends upon the increased infectivity of dilute suspensions of MHV 1 for mice pretreated with E. coccoides and gives values for α varying between 0·34 and 0·77, with a mean of 0·5. The other method, in which relatively large doses of MHV 1 are used, depends upon estimating the slope of the regression line of survival time on time of infection with E. coccoides, and yields a value for α of 0·49, with 0·95 probability limits of 0·37 and 0·61. The enhancing effect of E. coccoides on the pathogenicity of MHV 1 is attributed to the parasite increasing the ratio of active to latent infections of cells by the virus.

SUMMARY: Bacterial counts made, over a period of 27 months, on skin, gut and gill samples of freshly caught skate and lemon sole, using sea water-based and tap water-based media in parallel, revealed a seasonal variation in the size of the bacterial populations on the fish throughout the year. Evidence is presented for the view that the occurrence of maximum bacterial populations on fish is correlated with plankton outbursts. Sea water-based medium was generally superior to a tap water-based medium for isolating bacteria from fish; the importance of this as evidence of a specific marine bacterial flora is discussed.

SUMMARY: The effects of phenol and 2-phenoxyethanol on the oxygen uptake of washed suspensions and a disrupted cell preparation of Escherichia coli, with different substrates, has been further investigated. Stimulation of oxygen uptake with mannitol or glucose as substrate was not due to an increase in the viable population or associated with an uncoupling effect. The results obtained with a disrupted cell preparation capable of oxidizing glucose and lactic acid to pyruvic acid, on comparison with results with intact organisms, suggest that enzyme location may account for the differences in inhibitory activity obtained with different substrates.

SUMMARY: Four strains of Streptococcus pyogenes were found to have lost their group and type serological reactions after acquiring resistance to tetracycline antibiotics in vitro. These degraded variants were non-haemolytic and weakly fibrinolytic; they failed to grow in fresh human blood. Some of the degraded strains were found to have acquired the property of fermenting raffinose. Antisera prepared against three degraded strains revealed some antigenic relationship between degraded forms derived from different parent strains, and between degraded and parent organisms

Thiobacillus thiooxidans oxidizes elemental sulphur more rapidly in a shaken than in static liquid medium. Various wetting agents either had little favourable effect or inhibited growth in static media at 100 p.p.m. In shaken media, some wetting agents were toxic, but ‘Tergitol 08’ and ‘Tween 8’ increased sulphur oxidation. ‘Carbowaxes’ and propylene glycol had relatively little effect on sulphur oxidation.

Summary: Investigations were continued on the nature of the growth factor requirements of Corynebacterium diphtheriae gravis, strain Dundee. Hypoxanthine, or adenine + guanine, or adenine + xanthine, were found capable of replacing concentrates of the growth factor from liver and yeast.

Nucleoside and nucleotide fractions, prepared from electrophoretically-purified ribonucleic acid, were unable to replace the free purine bases. A purine extract was obtained from an active fish-liver concentrate by copper precipitation. In this extract, a large amount of hypoxanthine and a smaller amount of xanthine were found by chromatography to be the major purine constituents; traces of adenine and guanine were also detected. Chromatographic fractionation and spectro-photometric examination of the fractions confirmed the presence of hypoxanthine and xanthine, whose concentrations were estimated.

The purine extract supported less growth than the original fish-liver concentrates. Growth on the purine extract could be reproduced by a mixture of hypoxanthine and xanthine at an equivalent concentration.

The taxonomic value of the different ‘groups’ into which the Enterobacteriaceae are divided is assessed, and consideration is given to the classification of different groups within the family. No scheme fits the requirements of all workers and two different approaches are made, one for workers in applied bacteriology, and a more logical one for taxonomic purists. For the former scheme it is suggested that common (vulgar) names should be used to describe the groups and their subdivisions. Linnaean binomials are essential for the more systematic scheme, in which the Arizona group is merged with Salmonella; Ballerup-Bethesda is combined with Citrobacter freundii; Sonne’s bacillus and Bacillus alcalescens are included in Escherichia; Cloaca and Hafnia (32011) are included as species of Klebsiella, and Providence (29911) as a species of Proteus. Few of these suggestions are new, but the scheme is a classification of a family and not a collection of ‘groups’.

Further experience of semi-solid agar media has fully confirmed the earlier opinion (Knox, 1955a) of their great value for culture of Mycobacterium tuberculosis. The media mainly used were those of Kirchner, Fisher and Dubos, made semi-solid with 0.125% agar and containing serum or bovine albumin. Large inocula of M. tuberculosis H 37 Rv strain in Kirchner semi-solid agar gave easily visible growth in 2–3 days when the simple method of viewing described was used; small inocula gave countable colonies in 10–14 days, or in 7–10 days when a hand-lens was used. Maximum or nearly maximum viable counts were reached in about 2 weeks, and the final number of viable organisms recovered was as high as or higher than in any other media used. The medium also gave rapid growth of other strains of M. tuberculosis including drug-resistant variants of H 37 Rv, strains of M. tuberculosis var. bovis, and of B.C.G. and strains isolated from patients’ sputa. Semi-solid media are easy to prepare and safe to handle. Contamination rates are low, evaporation does not occur when rubber stoppers are used, and large numbers of cultures can be inoculated and incubated in a small space. Repeated readings can be made without opening the culture tubes. Viable counts and drug sensitivity tests are easy to read and record. For drug sensitivity tests semi-solid media, besides giving, when serial drug dilutions are used, an initial end-point which is as easy to read as with liquid media, also give information not given by liquid media as to the proportion of resistant organisms present in a given culture. Semi-solid agar media are of great value for any work involving speed in culture, drug sensitivity tests or viable counts of M. tuberculosis.

Summary:Direct observation techniques were used to determine the antibiotic effects of eight actinomycete species on Helminthosporium sativum in soil and in vitro. In actinomycete-inoculated sterilized soils, the inhibition of germination of fungal spores corresponded with the degree of inhibition of the fungus produced by the same actinomycetes in Petri plate culture. The effects of the actinomycetes on the vegetative growth of H. sativum in soybean-supplemented soils varied with the individual actinomycetes and, with one exception, were identical with the effects caused by the actinomycete antibiotics in vitro. These hyphal changes in soil included: suspension of further mycelial development; lysis; characteristic morphological effects such as stunting, distortion, excessive branching and the formation of hyphal protuberances. Lysis of the fungal hyphae only occurred in the presence of soil and was shown to be due to the combined effect of the antibiotic and some unidentified soil factor. Evidence was also obtained which demonstrated the ability of the actinomycetes to produce antibiotics in unsupplemented soils. Additional proof of antibiotic activity in soil was obtained by using the antibiotic actinomycin and strains of Streptomyces antibioticus, the organism responsible for its production. In Petri plate culture, both crystalline actinomycin and the actinomycetes produced a characteristic swelling, distortion and stunting of the vegetative growth of Helminthosporium sativum. When introduced into sterile soils, identical morphological changes were evidenced by the fungus in the presence of either the pure antibiotic or the actinomycetes.

Summary:The bactericidal effects of ozone solutions were tested against Escherichia coli suspensions at 1°, and the lethal concentration was found to be that quantity of ozone necessary to produce a detectable residue in the suspension; under the conditions of our experiments this was 0·4–0·5 mg./l. A comparison of the bactericidal activity of chlorine under similar conditions emphasized the different modes of action of the two agents.

Summary:The factors which influence the initiation of growth from small inocula of Clostridium parabotulinum type A in the defined medium previously described were re-examined. CO2 was found essential for rapid and regular growth from small inocula; it could be partially replaced under conditions of strict CO2 depletion by an unidentified factor present in enzymic hydrolysate of casein or yeast extract.

SUMMARY: Factors which govern toxin production by Clostridium parabotulinum type A were studied in growing cultures and in non-proliferating cell suspensions. With growing cultures high toxin titres, comparable to those obtained in complex media, were obtained in a defined medium containing the following essential constituents: arginine, cysteine, histidine, isoleucine, leucine, methionine, phenylalanine, serine (or glycine), tyrosine, tryptophan, valine, glucose, vitamins, phosphate and magnesium. Most of the toxin was formed only after cessation of growth.

Resting suspensions of organisms produced toxin at a rapid rate; a three- to fivefold increase being obtained after 4 hr. incubation. It appears that the toxin is synthesized within the organisms and is liberated into the medium by autolysis. Release of toxin on artificial lysis by sonic oscillation, provided decisive evidence for this conclusion.

The effect of different metabolic inhibitors on toxin synthesis by resting organism suspensions was examined. Ethylenediamine tetracetic acid in amounts which did not inhibit growth suppressed completely toxin production. Some broad spectrum antibiotics (streptomycin, chloramphenicol, chlorotetracycline at 100 μg./ml.) inhibited toxin formation to about 50%. Penicillin did not affect toxin synthesis even at concentrations 1000-fold higher than those required to arrest growth of the organism.

SUMMARY: A growth requirement for haematin by a streptomycin-resistant strain of Staphylococcus aureus was abolished by adding pyruvate or acetate. Growth in the absence of haematin was improved by addition of purines and uracil. Under anaerobic conditions growth occurred in the presence of nitrate when haematin was added. Suspensions of organisms grown in the absence of haematin reduced nitrate to nitrite when incubated in buffered glucose with haematin, and the amount of nitrite formed was proportional to the concentration of haematin over a range from 0·03 to 0·25 μm-mole/ml. This method was used to assay iron protoporphyrin formed by suspensions of Rhodopseudomonas spheroides. This organism synthesizes iron protoporphyrin as well as free porphyrins when incubated anaerobically in the light with δ-aminolaevulic acid, iron salts and an oxidizable substrate; cobalt ions inhibit the formation of iron protoporphyrin.

SUMMARY: One hundred and fifty-eight strains of vibrio, predominantly Vibrio cholerae, were tested for their nutritional requirements. Half of the strains were able to grow on a simple inorganic medium with ammonium ions as the sole source of nitrogen; the other strains required purines in addition. The simplest purine which produced adequate growth was hypoxanthine. The purine-requiring strains were able to grow on inorganic media containing human, rabbit or goat serum, but not on the serum from mouse, rat, guinea pig or horse.