- Volume 24, Issue 2, 1961

SUMMARY: Methods are described for the purification of Murray Valley encephalitis virus from baby mouse brain extract. The method finally chosen involved treatment with protamine, ultracentrifugation, adsorption to and elution from hydroxyl apatite and a second ultracentrifugation. The degrees of recovery of virus infectivity and haemagglutinin were about 15 % and 50 %, respectively. Measurement of the absorption in the ultraviolet region and examination in the electron microscope suggested that a substantial degree of purification had been achieved. The stability of the infectivity of purified virus was less than that of crude virus. Purified virus yielded infective ‘ribonucleic acid’ on treatment with phenol. The yield was consistent with the possibility that the potential infective ‘ribonucleic acid’ in crude virus was contained in infective virus particles.

SUMMARY: An organism capable of growing at pH values up to 11·0 appeared, presumably as a contaminant, in the course of ‘training’ experiments designed to produce alkali-resistant strains of Bacillus cereus Frankland & Frankland; this organism has now been characterized as B. circulans Jordan. Of 26 other strains of B. circulans studied, none grew at pH 10·7. The alkali-resistant B. circulans strain showed little loss of resistance after many transfers on neutral medium. When it grew in alkaline media it lowered the pH value of these media.

SUMMARY: When Escherichia coli organisms were suspended in distilled water and freeze-dried the maximum loss of viability did not occur during the drying process proper, but during the time of contact of the dried organisms with air between the primary and the secondary drying periods. By substituting other gases for air at this stage, it was proven that oxygen was the active agent involved. The dried organisms which were exposed to different pressures of air and oxygen at different temperatures proved to be extremely sensitive to traces of oxygen, even at very low temperatures. The implications of this oxygen effect in connexion with existing freeze-drying procedures, as well as some preliminary kinetic experiments concerning the shape of the survival curve, are discussed.

SUMMARY: Inter-species conversion has been observed in thiobacilli, starting from isolates of single bacilli.

SUMMARY: A bacterial species which degrades the pyrimidines, uracil, thymine and cytosine by induced enzymes has been characterized as Nocardia corallina (strain S). All other strains of N. corallina investigated oxidized thymine, but varied in their abilities to oxidize uracil and cystosine.

Organisms adapted to pyrimidines converted uracil to barbituric acid and thymine to 5-methylbarbituric acid. Oxidation of uracil by thymine-grown organisms was almost entirely by a pathway in which barbituric acid was an intermediate. Oxidation of thymine by uracil-grown organisms was similarly almost entirely via 5-methylbarbituric acid.

Oxidation of uracil by uracil-grown organisms and of thymine by thymine-grown organisms occurred, at least in part, through the respective barbituric acids. Discrepancies between the theoretical and observed values for O2 uptake suggested however that other pathways may also occur in these cases.

Pyrimidine-grown organisms oxidized 2-thiouracil to 2-thiobarbituric acid and 2-thiothymine to a compound which was probably 5-methyl-2-thiobarbituric acid. These products were not further degraded by the organism.

Barbituric acid was oxidized by uracil-grown organisms to CO2 , NH3ss and urea with concurrent oxidative assimilation. The oxidation of barbituric acid was inhibited by isobarbituric acid and sodium azide although barbiturase activity in cell-free extracts was not affected by these substances. Barbiturase preparations converted barbituric acid anaerobically to malonic acid, CO2 and NH3, but barbituric acid was not degraded by whole organisms under anaerobic conditions. Whole organisms, grown on uracil, degraded urea but did not oxidize malonic acid. Acetic and propionic but not malonic or barbituric acids were activated by cell-free extracts as judged by hydroxamate formation. From the evidence presented, it is unlikely that free malonic acid is an intermediate in the breakdown of barbituric acid.

SUMMARY: The development and mechanism of formation of Mycoplasma and bacterial L-form colonies were studied. The micro-organisms penetrate into the agar within a few hours after inoculation. It is suggested that penetration is caused by capillary forces which draw the minute plastic organisms into the dried agar gel, together with the water surrounding them. Penetration does not take place when the agar surface is very moist. The organisms appear to develop in the interstices of the fibrillar network of the agar gel and form a firm and elastic ball-like colony growing in all directions. When reaching the agar surface the growth spreads into the thin free water film which covers the agar, forming the peripheral zone typical of mature colonies. Factors which decrease the thickness of the free water film, like drying the medium or increasing the agar concentration, cause a decrease of the peripheral zone dimensions. Factors which retard growth, such as high concentrations of inorganic salts or hydrogen ions, inhibit the formation of the peripheral zone by limiting the initial ball-like growth inside the agar, rendering it incapable of reaching the agar surface. Typical colonies composed of both central and peripheral zones appear also on other fibrillar media such as the cellulose pellicle of Acetobaeter xylinum and gelatin.

SUMMARY: The relationship of the pH value of the suspension medium to inorganic phosphate accumulation by Tetrahymena pyriformis, strain W, in the presence and absence of 2:4-dinitrophenol was studied. Investigations were conducted to ascertain whether variations of the conditions of culture influenced phosphate accumulation. The pH value of the suspension medium had an influence on the amount of radiophosphate accumulation. The pH optimum, under the conditions used, was 6·5. The pH value of the culture medium, in the range tested, had little or no influence on the pH optimum observed in the suspensions, although differences in total phosphate accumulation were noted. Approximately 70% of the orthophosphate accumulated by the organisms was in the form of inorganic phosphate, which leads to the postulate of an active transport mechanism for phosphate accumulation in Tetrahymena. 2:4-Dinitrophenol was most effective in lowering radiophosphate accumulation in an acidic medium, and became less effective with increasing alkalinity. The results can be explained on the basis of the penetration of only the undissociated molecule into the organisms. It is suggested that 2:4-dinitrophenol interferes with the transport mechanism involved in phosphate entry.

SUMMARY: A disease of finnock (immature Salmo trutta) is described. The causal organism was found to be a Gram-negative, motile, curved rod which was also pathogenic to eels, perch, plaice and saithe. On comparison with other pathogenic vibrios from fish it was apparent that the organism was a variant of Vibrio anguillarum ; the name V. anguillarum type C is suggested for it.

SUMMARY: The sugar composition of cell walls of various streptococci and pneumococci was investigated by paper chromatography. Rhamnose was found in the walls of most streptococci bearing Lancefield group haptens but was not identified in the walls of pneumococci or most ‘viridans’ streptococci. In the group as a whole galactose and glucose were more regularly present than rhamnose. A significant correlation was found between the distribution of rhamnose and haemagglutination pattern, as determined by the presence of a widely distributed red cell sensitizing antigen and the production of an agent capable, like the receptor-destroying enzyme of the influenza virus, of modifying the antigenic properties of the red-cell surface.

SUMMARY: Morphological and biochemical features of 42 strains of ‘atypical’ mycobacteria and one strain of Mycobacterium tuberculosis were studied. Of the 42 atypical mycobacteria, 16 were originally classified in Runyon’s group I, 4 in group II, 19 in group III, and 3 in group IV. The characteristics studied were bacillary morphology and staining properties on Kirschner and Löwenstein-Jensen media; colonial morphology on 7H-10 agar medium; pigmentation in the dark and after exposure to light; rate of growth and temperature requirements, with different methods of inoculation; growth on blood and nutrient agar plates, and in gelatin stabs; catalase activity on drug-free and on isoniazid-containing media; nicotinic acid (niacin) production. The sensitivity of the majority of the strains to 6 chemotherapeutic drugs was tested. The niacin test proved to be the most useful method for distinguishing the atypical mycobacteria from M. tuberculosis. In identifying strains of group I, their ability to produce yellow pigment after exposure to light was of most value, and their colonial morphology and their periodic acid-Schiff staining were also helpful. Strains of group II were identified by their ability to form yellow pigment in the dark, by their periodic acid-Schiff staining and by their colonial morphology. Strains of group III were identified by their rate of growth and buff pigmentation. Exceptionally a yellow pigment was formed, and such strains were identified principally by their colonial morphology and periodic acid-Schiff staining. Among the 19 strains classified originally as group III, 3 were reclassified into group IV. Strains of group IV were identified by their ability to grow on blood and nutrient agar plates within 3 days and in gelatin stab cultures within 2 weeks.

SUMMARY: Successful isolation of azotobacter phages from soil samples was accomplished by using a modified Burk’s nitrogen-free medium with sucrose as the carbon source. The natural azotobacter flora in the soil samples served as the enrichment strains and the medium was not further enriched by the addition of laboratory cultures of the bacteria. Phage titres as high as 5·5 × 109 plaque-forming units/ml. were obtained when the indicator strain for phage assays was Azotobacter vinelandii O. On the basis of plaque morphology, nine phage isolates were obtained and purified by standard techniques. The plaques formed by the phages consisted of a central clear area surrounded by a halo and ranged from 1 to 7 mm. in diameter. Antiphage sera were produced in rabbits against a previously isolated phage and the new isolates; on the basis of cross-neutralization experiments with homologous and heterologous antisera, the 10 phages were placed into four major serological groups. Groups I and II contained four phages each, and groups III and IV contained one phage each. The degree of serological relatedness among the phages within groups I and II was investigated. A survey of 48 azotobacter strains showed that 11 out of 12 A. vinelandii strains and 14 out of 25 A. chroococcum strains showed plaque formation by one or more of the phages. Strains of A. agilis, A. macrocytogenes, A. insigne and A. indicus were not lysed by the phages. The value of the present series of phages in the classification of the genus Azotobacter was discussed.

SUMMARY: Enrichment cultures of mesophilic sulphate-reducing bacteria were readily obtained from soils, mud and corrosion products which had been heated to 90°, yet pure cultures of Desulfovibrio desulfuricans were killed by heating to 60° even when absorbed on dried sterilized soil. Examination of the populations from heated samples revealed exclusively organisms resembling Desulfovibrio orientis; unheated samples contained either D. orientis alone or both D. orientis and D. desulfuricans. The belief that D. desulfuricans sporulates is questioned.

SUMMARY: The walls and cell contents of a number of lactobacilli were prepared by mechanical disruption of the organisms followed by differential centrifugation. The nature of the teichoic acid present in the separated fractions was determined by extracting it with dilute trichloroacetic acid, precipitating with ethanol and identifying the precipitated polymer by hydrolysis to ribitol or glycerol phosphates and other recognizable degradation products. All the organisms contained a glycerol teichoic acid within the cell. This and similar observations with other bacteria indicates that glycerol teichoic acids play an important part in cellular metabolism. Both glycerol and ribitol teichoic acids occur in walls. The presence and type of teichoic acid in the walls correlate with the serological behaviour of lactobacilli and provide a useful means of classification. It is suggested that teichoic acids themselves may possess antigenic properties.

SUMMARY: A tryptophan auxotroph of Escherichia coli produced an altered tryptophan synthetase which could not convert indole to tryptophan but converted indole-3-glycerol phosphate to indole. As distinct from the normal tryptophan synthetase, which also catalysed this reaction, both pyridoxal phosphate and serine stimulated the activity of the mutant enzyme system. Fractionation and chromatography of the mutant tryptophan synthetase separated it into its two protein components, A and B. Examinations of the separated components showed that the A protein was normal, while the B protein was altered. Studies of the effect of serine on the pH-activity response of mutant preparations in the indole-3-glycerol phosphate → indole reaction demonstrated that different pH-activity responses were obtained, respectively, in the presence and absence of serine. The curves obtained were characteristic of the serine-requiring and serine-non-requiring reactions, respectively, of normal tryptophan synthetase. The saturation curves of the mutant component B by normal component A, with and without serine added, suggest that one role of serine and pyridoxal phosphate in the stimulation of the indole-3-glycerol phosphate → indole reaction is to bind together the A and B proteins in a catalytically effective complex.