- Volume 16, Issue 2, 1957

SUMMARY: The active growth of Gelasinospora tetrasperma in a chemically defined liquid medium was completed in 5 days. Cytochrome was detected at all ages (spectroscopically and by manometric assay) and a cytochrome-cytochrome oxidase system was mainly responsible for Oa uptake. Phenol oxidase was not detected until the cultures had been growing 48 hr., and even then the proportion of the Oa uptake of the mycelium due to this enzyme was small, since light reversed carbon monoxide inhibition of the Oa uptake almost completely.

SUMMARY: The requirements for growth factors, amino acids and inorganic constituents were determined for three strains of Clostridium perfringens (Veillon & Zuber) Holland. A number of substances were tested as energy sources for this organism, and the influence of pH value and temperature on growth was determined. The minimal medium evolved contained: alanine, arginine, aspartic acid, cystine, glutamic acid, histidine, isoleucine, leucine (methionine), phenylalanine, threonine, tryptophan, tyrosine and valine; ammonium chloride, magnesium chloride, ferrous chloride, sodium-potassium phosphate buffer; glucose; adenine, biotin, calcium pantothenate and pyridoxine.

For maximal growth the presence of lysine, glycine and serine was necessary. Maximal growth was affected by the balance of amino acids in the medium; the balance of sodium and potassium ions was also important. Certain strain differences were noticed with respect to amino acid and vitamin requirements. Methionine was needed by one strain only; none of the strains required serine (reported essential for strain BP6K of Boyd, Logan & Tytell, 1948b), whereas aspartic acid was essential for all strains tested but not for BP 6 K. Riboflavine, an essential growth factor in BP6K, had no effect on growth of the strains tested. One of the strains showed a need for added nicotinamide when transferred to a nicotinamide-free medium after several transfers in peptone water.

SUMMARY: The utilization of several inorganic and organic sulphur compounds by three strains of Clostridium perfringens and the production of hydrogen sulphide from these compounds was investigated. Sulphate, sulphite, thiosulphate and sulphide cannot supply sulphur to the organism, neither do they have any effect, when added in non-toxic concentrations, on the growth in media containing utilizable organic sulphur. Cystine or cysteine, which can be replaced by glutathione, were required by the three strains tested; no additional sulphur source was needed by two of the strains, the third requiring also methionine, which can be replaced by homocyst(e)ine. Hydrogen sulphide is produced by growing cultures from sulphite, thiosulphate, cystine, cysteine and glutathione, but not from methionine. Different enzyme systems are concerned with hydrogen sulphide production from cystine and sulphite, respectively. The sulphite-reducing capacity of some cultures was often decreased after several passages in laboratory media, the presence of nicotinamide being then required for this reduction. Sulphite was reduced to sulphide by suspensions of resting organisms. The presence of a hydrogenase system is suggested by preliminary experiments, the organisms being able to activate molecular hydrogen for reduction of methylene blue and sulphite. Other hydrogen donors are utilized by the organisms, in particular glucose. The addition of sulphites does not induce growth on substrates which do not support growth in sulphite-free media.

SUMMARY: Glutamine synthesis by Staphylococcus aureus is inhibited by the basic triphenylmethane dyes crystal violet, methyl green, fuchsin, pararosaniline, brilliant green and malachite green. In the pH range 6·5–8·5, the inhibitory action of crystal violet is independent of pH, whilst with fuchsin, brilliant green and malachite green, the degree of inhibition is increased by increasing the hydrogenion concentration of the medium. At pH 7·5, inhibitory activity increases in the order: brilliant green, fuchsin, malachite green, methyl green, crystal violet. The effectiveness of a basic triphenylmethane dye as an inhibitor of glutamine synthesis can be correlated with the basic dissociation constant of the dye.

SUMMARY: An immunological study was made of Paramecium aurelia variety 2. The antigens which produce immobilization antibodies were studied and a survey of the antigenic types, or serotypes, manifested by variety 2 stocks carried out. The eight types found were titrated against several sera. Serotype B, when found in different stocks, was distinguished from stock to stock on the basis of reactions to homologous and heterologous sera. Stock-specific C serotypes were also found and the conditions for maximum stability of the C serotype occurring in different stocks determined. It has been shown by absorption experiments that: (a) antiserum against a single serotype has at least two antibodies directed against the immobilization antigen(s); (b) if there are two or more determinant groups in a serotype, these groups are on a single molecule. The possible serological bases for cross-reactions of serotypes controlled by the same genetic locus are discussed.

SUMMARY: Series of experiments were carried out both in vitro and in vivo on single conidial isolates of Venturia inaequalis and V. pirina. Relationships between colony type and sporulation in vitro were apparent in both species. In addition, isolates of V. inaequalis showed relationships between degree of resistance of host source, width of host range and stability of cultural characters and pathogenicity during storage in culture. Isolates of V. pirina showed no such relationships, cultural characters being invariably stable while pathogenicity rapidly declined in the storage conditions. Studies of nutritional requirements suggested no deficiencies in the synthetic abilities of either pathogen, but limiting nutritional factors were important. Isolates of both species which differed widely in pathogenicity and other characteristics, reacted similarly to various culture media.

SUMMARY: A simple glass slide technique has been devised for the continuous microscopical observation of growth and infection of root hairs of clover seedlings. The method involves an aseptic cultivation of seedlings on microscope slides which are partly immersed in a mineral salts medium. The roots are protected by a cover-slip.

By this procedure, the root hairs of white clover inoculated with nodule bacteria were studied. The earliest infection was observed to take place within 48 hr. of inoculation, on 4-day-old seedlings. In branched hairs the growth of the thread from a lateral branch towards the hair tip is tentatively explained as an effect of the position of the hair nucleus relative to the site of infection.

SUMMARY: Two strains of bacteria which produced flagella when grown at 36° but not at 44° were examined; one was a strain of Salmonella typhimurium and the other of Proteus vulgaris. These organisms were grown on membranes for electron microscopy, being incubated at 36° so that the parent bacteria of each microcolony possessed a normal quota of flagella, and then transferred to 44° so that no more flagella were produced. In the microcolonies, after several divisions, it appeared that all these flagella were retained by the original parents, in accordance with the theory that bacteria of this type divide by budding from a growing-point at one pole.

SUMMARY: From the caeca of 252 adult bovines 131 yeast strains were isolated, distributed as shown (in brackets) among the following species: Saccharomyces cerevisiae (12), S. chevalieri (3), S. drosophilarum (3), S.fragilis (1), Pichia bovis (1), P. membranaefaciens (3), P. fermentans (3), Cryptococcus diffluens (1), Torulopsis glabrata (4), Torulopsis sp. (2), Candida tropicalis (45), C. parapsilosis (3), C. krusei (33), C. macedoniensis (1), C. utilis (1), C. bovina (1), Trichosporon cutaneum (5). Candida albicans was not isolated. The apparent absence of C. albicans from the bovine intestinal tract may explain the rareness of oral and intestinal moniliasis in bovines. Infection in certain forms of yeast mastitis (caused neither by C. albicans nor by Cryptococcus neoformans) may be derived from the intestinal tract.

Descriptions and Latin diagnoses of Pichia bovis n.sp. and Candida bovina n.sp. are given.

SUMMARY: Mice were challenged by mouth with a suspension containing equal numbers of streptomycin-sensitive (Str−) and streptomycin-resistant (Str+) variants of Salmonella typhimurium. These variants were of equal virulence but the Str+ variant grew more slowly in vivo than the Str− variant. The LD50 dose contained c. 5 × 105bacteria. Heart blood obtained from mice dying from many LD50 doses nearly always contained a great excess of the Str− variant, but blood from mice dying from less than one LD50 dose contained either Str−, Str+, or a mixture of Str− and Str+ variants. The appearance of the Str+ variant alone in the latter mice strongly suggests that these fatal infections were initiated by a very small number of organisms or possibly by a single organism. It is therefore concluded that these organisms were acting independently. In this system, it is likely that any bacterium which enters the tissues from the gut can initiate a fatal infection and that the probability of effecting such an entrance almost entirely determines the probability of an inoculated bacterium causing a fatal infection.

SUMMARY: A number of proteins can be separated from purified preparations of tobacco mosaic virus; they differ from each other antigenically and all differ from the intact virus by not possessing all antigenic determinant groups possessed by the virus. Some of these proteins are easily detached from the virus by such mild treatments as placing it in a protein solution or in agar gel. These are antigenically identical with the ‘ X-protein ’ which remains in the supernatant fluid when the virus is sedimented by ultracentrifugation from sap of infected plants. More of these proteins are detached from the virus by placing it in borate buffer at c. pH 8·7. When a more drastic treatment is applied, such as incubation at c. pH 10, which disintegrates a proportion of the virus, still more of these proteins are released, but then some proteins antigenically different from those of ‘ X-protein ’ are also released.

SUMMARY: Refractive index measurements were made on the spores and vegetative cells of strains of Bacillus cereus, B. cereus var. mycoides and B. megaterium by phase contrast and interference microscopy with protein immersion. The refractive indices of the spores were found to be very high and comparable with that of dehydrated protein, suggesting that they contained very little water. Those of the vegetative cells were much lower, and indicated a solid content of about 30 %, w/v.

SUMMARY: A study has been made of the uridine pyrophosphoglycosyl compounds present in a non-capsulated pneumococcus (Streptococcus pneumoniae, strain R19, derived from a type II organism) and a capsulated pneumococcus (type III; Streptococcus pneumoniae, strain A66), and also of certain enzymes involved in the metabolism of these compounds. It has been found that both the pneumococcal strains contained considerable amounts of uridine pyrophosphoglucuronic acid (UPPGA) and uridine pyrophosphoacetylglucosamine (UPPAG), with lesser amounts of uridine pyrophosphoglucose (UPPG), uridine-5′-monophosphate (UMP), uridine pyrophosphate (UPP) and uridine triphosphate (UTP). The patterns shown by these two strains with respect to uridine nucleotide content were very similar.

Cell-free extracts of strain R19, derived from a type II organism, were obtained; these extracts contained the following enzymes: glucose-6-phosphate dehydrogenase, uridyl transferase, inorganic pyrophosphatase, nucleoside diphosphokinase, hexo- kinase and phosphoglucomutase. Examination of the strain A 66 capsulated (type III) organism showed the presence of uridyl transferase, nucleoside diphosphokinase and inorganic pyrophosphatase.

SUMMARY: Four mutant strains of Neurospora crassa, due to repeated mutation within the same short chromosome region, required arginine as a nutrient and failed to respond to citrulline. These strains accumulated argininosuccinic acid in the mycelium when grown on a medium supplemented with arginine. When the medium also contained citrulline this accumulation of argininosuccinic acid was enhanced, and citrulline also accumulated in the mycelium. Citrulline depressed the growth of the mutants at a concentration at which it had no effect on growth of the wild type.

Cell-free extracts of the wild type contained an argininosuccinase which catalysed the reversible splitting of argininosuccinic acid to arginine and fumaric acid. A procedure is described for the approximately fivefold purification of the enzyme. The pH optimum for the synthesis of argininosuccinic acid from arginine and fumaric acid was close to pH 7·0.

Neither cell-free extracts nor partially purified preparations from the four mutants showed any trace of argininosuccinase activity; 1–2 %, and 0–2 %, of normal wild-type activity should have been detectable in crude cell-free extracts and purified preparations, respectively. Neither the argininosuccinase activity of crude wild-type extracts, nor that of the partially purified preparations was appreciably altered by the addition of corresponding preparations from one of the mutants. A mixture of wild-type and mutant mycelium extracted together yielded the amount of activity to be expected on the assumption that the mutant mycelium was neither contributing to, nor decreasing, the yield.

Arginase and fumarase activities were similar in the wild type and one of the mutants.

SUMMARY: Nine mutants of Neurospora crassa which require arginine as a nutrient but cannot use citrulline were obtained from various sources. These fall into two classes, according to location in linkage group I or VII. (Enzymic tests, reported elsewhere, indicate that the two classes of mutants affect the enzymes which control the two reactions between citrulline and arginine.) Heterokaryon tests between mutants of the same group were negative, and crosses between mutants of the same group were semi-sterile, most of the ascospores being non-viable. Crosses between the five group I mutants produced no arg+ progeny, and separate mapping tests on four of them indicate that they are either allelic or closely linked. All crosses between the group VII mutants gave many arg+ progeny. For the one pair of mutants which was studied in detail, origin of the arg+ by means other than crossing-over (or gene conversion) has been virtually eliminated. However, mapping studies place the two mutants only 0–6 units apart. It is concluded that the high arg+ frequency is due to selection, and that the mutants might be pseudoalleles.

SUMMARY: Fluorescent antisera to a number of isolates of rumen bacteria have been prepared and used to demonstrate the presence of these organisms in situ in rumen contents. The serological tests for a specific organism in rumen contents of a number of calves agree with the isolations of this organism from the same sources. A mieroseope suitable for this work, the preparation and purification of antisera and the preparation of specimens is described.

SUMMARY: Certain bacteria of the genus Pseudomonas attack the tartaric acids by means of inducible stereospecific dehydrases. Each dehydrase converts its specific isomeric substrate to oxaloacetic acid; in crude cell-free extracts the oxaloacetic acid is in turn converted to pyruvic acid, which accumulates. By treatment of the crude extracts with ethylenediaminetetraacetic acid (EDTA), substantial accumulations of oxaloacetic acid can be obtained from the meso- and d-tartaric acids, and assays for these two dehydrases, based on the accumulation of oxaloacetic acid in the presence of EDTA, have been developed. This procedure cannot be used to assay the l-tartaric acid dehydrase, which is itself very sensitive to EDTA. The patterns of inhibition of the three dehydrases by compounds sterically related to their substrates have been explored, and the findings are interpreted in terms of the minimal steric requirements for enzyme-substrate combination.

SUMMARY: Pseudomonas strains, capable of utilizing as carbon sources one or more isomers of tartaric acid, were isolated from soil by specific elective culture methods. Most strains were markedly stereospecific, utilizing only the isomer or isomers upon which they had developed during the elective cultivation. Enzymic study showed that the commonest mechanism for the dissimilation of the tartaric acids by this group of strains is dehydration to oxaloacetic acid, through the action of stereospecific dehydrases. The attack on the tartaric acids is strictly inducible, and in many strains the induced state is rapidly lost in the absence of the substrate- inducer. Enzymic assay showed that such loss of induction cannot be accounted for by loss of the specific dehydrase, which is often present in large quantities in organisms which have become incapable of attacking the substrate. This indicates that the metabolism of the tartaric acids requires specific inducible transport systems for bringing the tartaric acids into the cell, in addition to the specific, inducible dehydrases. The existence of such transport systems is further indicated by the fact that the inhibitory interactions between the isomers of tartaric acid, demonstrable with the cell-free dehydrase preparation, do not exist in vivo.

SUMMARY: Interference was demonstrated with pantropic Rift Valley fever virus in appropriately ultraviolet-irradiated mouse serum in which a residuum of active virus remained. The degree of interference varied with the amount of irradiation to which the virus had been exposed. With the smallest doses of ultraviolet radiation, which left a fair amount of active virus, the only manifestation of interference was a prolongation of the incubation period. With virus which had received optimal irradiation, concentrated inocula elicited no symptoms but the same material in higher dilution produces the typical fatal illness. With doses of ultraviolet radiation between the minimum and the optimum, virus suspensions were obtained which showed interference, but many of the mice developed signs of neurological involvement after a prolonged incubation period. Although the symptoms elicited were neurological in origin, the virus recovered from the brain was unaltered pantropic variety. The distribution of this virus in the affected mice was determined. It is suggested that the residual live virus contained in irradiated material undergoes a growth cycle in a limited number of liver and other susceptible cells not protected by the interference effect of inactivated virus. The late neurological manifestations are attributed to this newly formed virus and the lesser affinity of the brain cells for the interfering inactivated virus. Many of the mice which survived inoculations of concentrated irradiated virus were shown to be immune to subsequent infection with 100 MLD of Rift Valley fever virus. This immunity is considered to be due to a latent infection initiated by residual live virus present in the irradiated material. Virus subjected to prolonged irradiation probably loses its immunizing power because of complete inactivation rather than destruction of the antigenicity of individual virus particles.