- Volume 46, Issue 2, 1967

A simple chemically defined medium for examining the motility of Escherichia coli k12 was designed. The essential components were: (1) a chelating agent to protect the motility against inhibition by traces of heavy metal ions; (2) a buffer to keep the pH value at the optimum between pH 6·0 and 7·5; (3) an energy source to stimulate the motility above that allowed by an endogenous energy source. Oxygen was required unless an energy source was provided which yielded energy anaerobically. A temperature optimum was determined.

A chemically defined growth medium capable of producing motile bacteria was devised. It was found that the presence of glucose or growth above 37° prevented synthesis of flagella.

This paper deals with variability within certain species of the genus Mallomonas as revealed under the electron microscope. New varieties and two new species are described (M. annulata; M. pillula), one of these having previously been considered a variety, partly through confusion. An attempt is made to consider different kinds of variability and to make a decision on what range can be tolerated within the limits of a species. The importance of examining the whole organism under the electron microscope as well as a few scales is now realized. It is desirable also to examine the flagellum since several species which have been described as belonging to the genus Mallomonas must be separated from it because they prove to have two flagella.

The influence of dissolved oxygen concentration on the metabolism and respiration of growing Klebsiella aerogenes NCTC 8017 was studied by means of a continuous-flow culture technique. Different dissolved oxygen tensions (equivalent partial pressures) were obtained by varying the partial pressure of oxygen in the gas phase. The respiration rate (oxygen uptake rate per unit mass organism) was independent of dissolved oxygen tensions above 10–15 mm. Hg when the culture was said to be in the ‘excess oxygen state’. With dissolved oxygen tensions below about 10 mm. Hg, the ‘limited oxygen state’ developed. In the transition to this state, complex oscillations in the dissolved oxygen tension occurred. The oscillations reflected alternate stimulation and inhibition of the respiration rate. A decrease in dissolved oxygen tension to below 5 mm. Hg immediately increased the respiration rate; an increase in dissolved oxygen tension from 5 to 10 mm. Hg immediately decreased the respiration rate. These effects formed part of the mechanism of the oscillations which could be sustained indefinitely. The oscillations in respiration rate occurred irrespective of change from pH 6·0 to 7·4 or whether growth was limited by glucose or ammonium supply. The pattern of oscillations in respiration rate varied with growth rate over the range 0·2–0·5 hr–1. It is suggested that decreasing the oxygen tension to about 5 mm. Hg acted like an uncoupler of oxidative phosphorylation.

In the excess oxygen state with excess glucose and growth limited by ammonium supply, the glucose carbon was largely accounted for as CO2, pyruvate and organisms, irrespective of the pH value. Pyruvate was not accumulated when growth was glucose-limited. In the limited oxygen state, the glucose carbon was largely accounted for as: organisms, CO2, 2:3-butanediol, ethanol, acetic acid, formic acid, lactic acid. The proportions of these fermentation products varied with oxygen supply, pH value and whether growth was ammonium-limited or glucose-limited.

The aerobic growth of Klebsiella aerogenes was studied in chemically defined media consisting of mineral salts (ammonium as N source) and various benzenoid compounds singly as sole source of carbon+energy. A strain adapted to a defined glucose mineral salts (ammonium as N source) medium grew readily in concentrations of benzaldehyde, benzoate, p-hydroxybenzoate or phenylacetate of the order 10–3 m to 10–2 m. The initial growth rate in benzoate, p-hydroxybenzoate or phenylacetate increased and then decreased with increasing concentration of benzenoid compound, according to a relationship which is interpreted by analogy with enzyme inhibition by excess substrate. The kinetics of growth inhibition by the above compounds in glucose salts medium were similar to those in the simplest case of inhibition of an enzyme or heterogeneous catalyst. During repeated subculture in benzoate, p-hydroxybenzoate or phenylacetate as sole carbon source, the bacteria showed various adaptive responses which include an immediate decrease in the lag and an increase in the growth rate. As the concentration of benzoate to which the bacteria had become adapted to utilize as sole carbon+energy source was increased, the molar growth yield and the growth rate changed in a related manner and the inhibitory action of benzoate in glucose salts medium decreased.

Actinomyces naeslundii fermented glucose primarily by the Embden–Meyerhof pathway, as based on 14C-glucose fermentation data and enzyme studies. Enzymes of the oxidative pentose phosphate cycle were also present, but functioned only to a minor extent. Growth on glucose was increased 2-to 4-fold in the presence of substrate amounts of CO2 or O2. This increase was attributed to the additional energy (ATP) made available from the breakdown of pyruvate to acetyl coenzyme A. In the absence of CO2 or O2, pyruvate was reduced to lactate. The weight of organism produced/mole ATP (Y ATP) was 15–18 g. units under anaerobic conditions with CO2, dependent on the growth medium, and 20 under aerobic growth conditions.

The morphological effects on bacterial walls of the early stages of inhibition of mucopolymer synthesis have been investigated in Escherichia coli. Inhibition was achieved in strain b by penicillin treatment and in auxotrophic strains m203 and m173–25 by deprivation of diaminopimelic acid (DAP). Only growing organisms were affected; the walls of organisms in stationary phase remained unchanged. The first effects of treatment with penicillin appear in 20 min., whereas changes in DAP-deprived auxotrophs become visible only after 40–60 min. The soft layers of the wall formed bag-like protrusions and, at the same time, wide gaps or holes developed in the proteinaceous portion of the rigid layer. Widening of these gaps was paralleled by a weakening of the rigid layer, resulting eventually in bursting of the cell, or in protective media of high osmotic strength, in the formation of spheroplasts. The initial appearance and the subsequent widening of gaps in the rigid layer, observed almost exclusively in organisms multiplying logarithmically, are taken to indicate the presence of a mechanically weak mucopolymer formed under conditions that prevent crosslinking. The discontinuities, interpreted as sites of mucopolymer synthesis, were randomly distributed over the bacterium. Their appearance at the poles of the bacteria seemed frequently to be suppressed by media of high osmotic pressure, in which plasmolysis was first seen in the polar regions. The observations suggest that morphogenesis of the protein layer is dependent on the secondary structure of the mucopolymer in newly synthesized wall areas.

Salmonella enteritidis, S. pullorum and S. gallinarum were found to be sensitive to complement-mediated killing by fresh normal human serum but far less so to sera of the mouse, rat or rabbit. S. pullorum but not S. gallinarum was sensitive to the microbicidal activity of adult fowl serum. S. enteritidis was slightly sensitive to the latter serum. Day-old-chick serum was unable to kill either S. gallinarum or S. pullorum even when undiluted serum was used. When human serum was absorbed at 4° by dense suspensions of dead organisms it was found that all antimicrobial activity against the three organisms was absorbed only when the homologous organism was used as the absorbing strain. This suggests that minor somatic antigenic differences occur between the three related organisms. S. pullorum was extremely sensitive to the action of monospecific rabbit antisera. However, serum concentrations which were highly effective against S. pullorum were less effective for S. enteritidis. Even highly microbicidal serum concentrations were unable to kill 100% of the S. enteritidis within the time period of the test. The relevance of these findings to the known differences in the virulence of these three salmonellas for the mouse and the chicken is discussed.

The nutritional requirements of the Gram-negative Bacterium ncib 8250 (formerly referred to as ‘Vibrio 01’) have been determined. Growth was tested on almost 450 compounds, of which over 100 served as carbon+energy sources and almost 50 as nitrogen sources. The effects of various factors such as pH value, temperature and aeration on the growth rate and cell yield of organism have also been measured.

Adansonian analysis of 31 Acinetobacter lwoffii and 8 A. anitratus strains is described. The mean S-value was 81·9 and 87·3% for groups A. lwoffii and A. anitratus, respectively. The intergroup value was 71·9%. The computer distinguished well the Alcaligenes faecalis strains from the former two species. According to the computer analysis, Bordetella bronchiseptica is closely related to A. faecalis. Pseudomonas aeruginosa and Aeromonas liquefaciens link with the former groups at a much lower level.

Griseofulvin induced various morphological abnormalities of the hyphae of Verticillium albo-atrum and V. dahliae. The growth-response curves obtained were unusual, and were due to the behaviour of the antibiotic. Repeated sub-cultivation of mycelium on Dox medium agar containing constant concentrations of griseofulvin produced trained mycelium which was however unstable. True adaptation of V. alboatrum was obtained when faster growing sectors of mycelium were produced at the periphery of inhibited colonies. This adapted mycelium showed decreased morphological abnormality but still showed marked inhibition of growth. Chitin production, estimated as glucosamine, following growth in the presence of griseofulvin appeared to be stimulated in the walls of non-adapted mycelium, but remained at a relatively constant value in the walls of the adapted mycelium.

The activation of spores of Clostridium bifermentans by various treatments was investigated. Mercaptoacetate did not activate the spores but inhibited the spontaneous activation which occurred in buffer alone. Sodium borohydride effected activation of spores by increasing the pH value above pH 10·0 rather than by its reducing action. The pH value of a suspension had a marked effect on activation; at 37° spores held at less than pH 3·0 or at pH 10·0 or more were activated within 1 hr. Continued incubation at an alkaline pH value led to a decrease in the specific requirements for germination, an effect not produced by acid pH values. Heat-activated spore suspensions became partially de-activated on storage unless they were continuously aerated. Spores activated at 37° and pH 2·0 or pH 7·4 readily became de-activated on storage, whereas spores activated at pH 10·5 remained activated. A specific requirement of heat-activated spores for sodium ions for germination was also found.

This study reports the investigation of tissue specificity of interferon induced with Chikungunya virus in lung and kidney cells obtained from 17-day chick embryos. Lung cells produced significantly more interferon than kidney cells under essentially the same conditions. Both lung and kidney interferon preparations repressed plaque formation on homologous and heterologous cells and lung cells were more sensitive to the protective effects of all the interferon preparations tested. No evidence for tissue specificity was obtained.

The minimal inhibitory concentrations of various antibiotics for a strain of Streptococcus pyogenes and a stable l-form of it were determined. These results were evaluated with those of other investigators similarly performed with S. faecalis and derived protoplasts. Differences in reaction to puromycin, bacitracin and ristocetin were apparent only between the wall-less derivatives. The polyene antibiotics nystatin and filipin and two antimicrobial agents, isatin 3-thiosemicarbazone and 1-methyl-isatin 3-thiosemicarbazone, were ineffective or only slightly inhibitory on growth of S. pyogenes and its l-form. Labelled penicillin studies with this group A streptococcus and its l-form indicated that the binding sites for penicillin were probably distinct from those concerned with streptococcal wall formation.

The two flagella of the dinoflagellates examined possess the normal axoneme structure with 9+2 filaments, the nine outer doublets forming triplets in the basal region. Unlike other flagella which possess one basal disc, the dinoflagellates have two basal discs and two diaphragms in the transition region. The two flagella differ from each other in their external morphology and extra-axonemal structures. The longitudinal flagellum contains much packing material in its proximal two-thirds, whereas the distal third contains only the axoneme. This flagellum bears short fine hairs. The transverse flagellum differs from any other type which has been described. It has a helical ribbon-like form due to the presence of a striated strand, which is shorter than the axoneme, and an expanded sheath. It also bears long fine hairs in a unilateral array. The distal end of this flagellum is attached to the cell in the region of the flagellar bases. The structure of the two flagella appears to be compatible with their respective forms of movement.

Phase-bright bacterial spores are deposited singly and rapidly by means of a glass microloop at pre-marked sites on the surface of a cast block of agar gel. The block is then dissected and portions, each carrying a single spore, are transferred to fluid for heat-resistance or other tests. Under optimal conditions of culture 96–100% of these single spores germinated and gave visible growth.