- Volume 31, Issue 1, 1963

Microbiological, physical and chemical changes were followed in worm casts ageing in the field. Filamentous fungi and yeasts increased in number rapidly after the cast was produced, but not bacteria or actinomycetes which were initially numerous. Measurements of hyphal length confirmed the increased growth of fungi. Ageing casts showed a declining respiratory activity, possibly because the bacteria formed resting stages. Aggregate stability increased rapidly as casts age, probably due to increasing amounts of fungal hyphae. Polysaccharide content of casts was much greater than that of soil, but did not vary with changes in stability. Total and mineral nitrogen levels of casts were greater than those of soil; the major part of the inorganic nitrogen occurred as ammonia which was rapidly converted to nitrate.

A mono-quaternary amine, cetyltrimethylammonium bromide (Cetrimide), and bisquaternary diamine, triclobisonium chloride (Triburon), were examined for their action on Aspergillus niger. Cetrimide, at 30 μg./ ml. inhibited growth, but the organism was not inhibited by as much as 1–28 mg. Triburon/ml. Cetrimide at 20 μg./ml. inhibited citric acid accumulation. Triburon, at concentrations up to 160 μg./ml., increased the citric acid concentration, the maximum yield being at 40 μg. Triburon/ml.; at higher concentrations of Triburon there was an inhibition of citric acid production. The effect of Triburon was still perceptible in the presence of ferric ions. Cetrimide and Triburon brought about an initial increase in sterol concentration, followed by a decrease at higher concentrations of these compounds. The effects of these compounds on citric acid production by preformed mycelia were similar to the effect on cultures growing from spores.

This paper describes an investigation into the degree of homogeneity of the substance termed collocalia mucoid obtained from edible bird-nest. This material is a potent inhibitor of influenza virus haemagglutination and additional information is given here regarding its ability to neutralize infectivity. The crude material obtained by simple extraction was found to contain three components differing in molecular size and biological activity. Some separation of these was obtained by means of preparative ultracentrifugation. The slowest moving component differed from the others not only physically, but also in having much less inhibitor activity.

The isolation genetic analysis of a new class of mutants of the mould Aspergillus nidulans is described. The mutants were detected by their inability to utilize specific carbohydrates as sole carbon source for growth. All of the mutants are recessive analysis of 27 mutants has resulted in the description of 10 new loci concerned with the utilization of carbohydrates in the organism; the loci have been allocated to linkage groups and 6 were mapped meiotically. Two loci which control lactose utilization are not linked and neither are 5 loci which control the utilization of galactose. A series of sorbitol mutants is unusual in that they are all non-complementary when combined in heterokaryons, although certain pairs of mutants complement in the corresponding heterozygous diploids.

An examination has been made of serologically reactive products obtained from four strains of Lactobacillus casei (serological groups B and C) and two strains of Lactobacillus casei var. rhamnosus (serological group C). Cell wall preparations were hydrolysed with a preparation of Streptomyces muralytic enzyme and the soluble indiffusible products isolated. Rhamnose is the major component of both the cell wall and soluble extracts from group B organisms, whereas glucose is the major component of preparations from L. casei group C. Two products differing considerably in composition have been isolated from each strain of L. casei var. rhamnosus.

The serological properties of the preparations have also been studied the results confirm that strains of Lactobacillus casei can be divided into two serological groups. It is concluded that group B specificity is primarily dependent on rhamnose group C specificity on glucose, which is probably joined by a 1–6 β linkage to the adjacent sugar. The two fractions isolated from L. casei var. rhamnosus differ in their serological properties; antibodies formed on injection of this organism are predominantly directed against one component (in fraction I) whereas extracts react with sera against strains of L. casei group C because of the presence of a second component (in fraction II).

A comparison has been made between the serological properties of 35 strains of Lactobacillus casei and the carbohydrate components of corresponding cell-wall preparations. All strains of L. casei var. rhamnosus belonged to serological group C, whereas strains of L. casei var. casei belonged to group B or C. Rhamnose was the major cell-wall component of group B strains, galactose glucose being minor variable components. Of the group C strains only those classified as L. casei var. rhamnosus contained significant amounts of rhamnose in the cell wall. All strains in group C, however, contained galactose and glucose as major cell-wall components.

A low-protein medium is described for the production of reasonable yields of Clostridium welchii (perfringens) e-prototoxin. The e-antigen in young cultures on this medium was virtually non-toxic, 99.8% being in the form of prototoxin. Its purity, estimated in terms of the protein content of crystalline e-prototoxin, was 50%. Fractionation of concentrates of the antigen on columns of diethylaminoethyl- and carboxy-methyl-celluloses suggested that a number of proteins intermediate between e-prototoxin and e-toxin were present which differed from the latter in their isoelectric points in their activation ratios. The concentration and number of these intermediates increased as the incubation of cultures was prolonged. Since they could be fully converted to e-toxin by trypsin it is probable that they were formed from e-prototoxin by proteolytic or spontaneous degradation. In toxicity tests, death caused by unactivated antigen may be delayed. This evidence suggested that the intermediates were intrinsically non-toxic but could become activated in the animal body. The activation ratios might then be regarded as a measure of the ease with which the intermediates could be converted to toxin in vivo. Physical data and the amino acid composition of crystalline e-prototoxin are presented.

Granular cultures of Trichophyton mentagrophytes changed their growth habit when grown on a medium containing, as the source of nitrogen, the 19 L-amino acids found in hydrolysed casein, a response not seen when methionine was omitted. In contrast, pleomorphic cultures of T. mentagrophytes grown on amino acid medium were not modified. The methionine effect has been used successfully to differentiate between normal granular cultures mutant pleomorphic cultures. Also, isolates characterized as atypical pleomorphs could be classified as metabolically granular or pleomorphic.

A group of epidemiologically related strains of Staphylococcus aureus, which were untypable by standard methods, became sensitive to typing phage 187 after the inocula had been held at 56° for 2 min. The strains were found to be lysogenic. Evidence is presented to show that the phage carried, phage 5504′, was exerting interference against phage 187 which could be partially overcome by heat treatment. Phage 187 grown in heated cocci was found to have undergone a host-induced modification which rendered it capable of lysing unheated lysogenic organisms. Active multiplication of the carried phage, phage 5504′, appeared to be necessary for the multiplication of phage 187. Ultraviolet irradiation also increased the sensitivity of the strain to phage 187, but to a lesser degree than heat treatment. It is suggested that the effect of heating is to induce active multiplication of phage 5504′ in the lysogenic culture, possibly by destroying a cytoplasmic substance responsible for maintaining the carried phage in its prophage state.

The metabolism of glycyl-L-leucine in a leucine auxotroph of Escherichia coli strain K-12 was investigated with respect to the bacteriostatic effect of the peptide on growth, its hydrolysis by bacterial preparations, and its uptake by resting organisms. Growth inhibition by the peptide is relatively small if the organisms used as the inocula for the growth tests are taken from actively multiplying cultures (young organisms), but is marked if the inocula consist of organisms from cultures aged in an acid-producing, poorly buffered medium; ageing in a well-buffered or neutral medium does not produce peptide-sensitive organisms. Aged organisms that are very sensitive to the peptide readily take up the compound from the external medium, both in the absence and presence of chloramphenicol. Whole-cell preparations of these aged organisms do not effect the rapid hydrolysis of glycylleucine, but cell-free extracts have as much enzymic activity as cell-free extracts of young organisms. Whole-cell preparations of young organisms also have high enzymic activity. The available data suggest that the uptake of glycylleucine by young organisms is followed immediately by its hydrolysis, whereas the peptide taken up by aged (peptide-sensitive) organisms is hydrolysed only very slowly. The growth of inocula composed of organisms taken from yeast extract + peptone + agar slopes is markedly inhibited by glycylleucine. The hydrolytic activity of whole organisms taken from slopes is significantly less than that of young organisms, but disruption of slope-grown organisms to produce cell-free extracts does not lead to any significant increase in enzymic activity.

Transformable streptococci produce an exocellular factor provoking competence under certain conditions. Non-competent cultures become competent upon addition of this factor. The kinetics of conversion is concomitant with an enzymic reaction; the process is time and temperature dependent and the factor itself is heat sensitive. The action of the hypothetical enzyme on cells of a non-transformable streptococcus results in provocation of competence.

In a strain of Bacillus megaterium the Gram reaction was investigated by comparing its effect on intact bacilli with its effects on bacilli treated with lysozyme in several different ways. The lysozyme-treated bacteria varied from bacilli showing only polar separation of the cell wall from the protoplasm to protoplasts free from cell wall. The uptake of the primary dye (crystal violet) by intact bacteria was higher than that of all lysozyme-depolymerized cell forms. Iodine uptake depended on the previous uptake of crystal violet. Therefore it was lower for the lysozyme-depolymerized forms. Cell wall took up crystal violet and iodine in small quantity. The same dye iodine complex was formed in all cell structures (cell wall and protoplasm). Gram differentiation was obtained with 95% (v/v) ethanol in water, this was found to extract much less of the dye iodine complex from intact bacteria than from the lysozyme-depolymerized forms. The latter lost about 80–90% of their net dye and iodine content, whereas intact bacilli lost only 20–45%. This was due to the cell wall which formed a barrier to the ethanol extraction of the dye iodine complex, for organisms whose cell walls had been depolymerized or even dissolved by lysozyme during or after the iodine step of the Gram reaction lost much more dye and iodine than intact bacilli. Chemical integrity of the cell wall is a prerequisite for Gram positivity.

The alimentary canal of adult bees is the main source of ‘Bacterium eurydice’ which spreads between bees and to larvae, pollen and honey, from the mouths of infected adults. Most natural spread happens when foraging activity is greatest, probably because infected adults then collect contaminate pollen which is soon eaten by the young brood-rearing bees.

The effect of biotin deficiency on the osmotic behaviour of Saccharomyces cerevisiae was studied by following the changes in the extinction of dilute suspensions of the yeast in phosphate buffer containing different concentrations of NaCl. The extinction of suspensions of biotin-optimal yeast increased as the NaCl concentration was increased to 1.5M, but with yeast grown in unsupplemented biotin-deficient medium the readings decreased with increasing NaCl concentration. Yeast grown in biotin-deficient media supplemented with aspartate, or with oleate + aspartate, showed the same type of osmotic behaviour as yeast grown in unsupplemented biotin-deficient medium. But with yeast grown in biotin-deficient medium supplemented with oleate alone, the extinction changes in buffer containing up to 0.7M-NaCl resembled those of suspensions of biotin-optimal yeast. These differences in osmotic behaviour were not due to breakage of the osmotic barrier by the osmotic pressure differences across the cytoplasmic membrane, but to an increased permeability of the membrane to Cl-. This was accompanied by an increased permeability to H2PO- 4 to higher molecular weight solutes, including bovine plasma albumin. The increase in permeability of biotin-deficient yeast was not as great as that of biotin-optimal yeast in which the osmotic barrier had been broken with 5% (v/v) n-butanol.

In HeLa cells infected with a single infectious unit of lymphogranuloma venereum virus and stained with acridine orange, one particle (initial body) of ribonucleic acid (RNA) about 2 μ diam. was seen by fluorescence microscopy in the cell cytoplasm after incubation for 6–8 hr. at 37°. After 11–12 hr. of incubation, an average of 2 particles/infected cell was found. Thereafter the number increased exponentially with a mean generation time of 2–2 1/4 hr. The particles then remained discrete and in a circumscribed area in the cytoplasm until 18–21 hr., when a vacuole was formed around the initial bodies. By 21–24 hr. smaller particles ranging in diameter from 1 μ to about 0.25μ, and in colour from the orange fluorescence characteristic of RNA to yellowish green, were detected amongst the initial bodies. At 33 hr., the initial bodies were almost entirely replaced by smaller particles or elementary bodies, most of which stained yellowish green and had the green fluorescence of deoxyribonucleic acid (DNA) only after treatment with ribonuclease. The elementary body of lymphogranuloma venereumum thus consists of a DNA particle surrounded by a layer containing a detectable amount of RNA. After 33 hr. the number of elementary bodies decreased; presumably because infective virus had been released, since at 40–44 hr. initial bodies appeared in previously uninfected cells. HeLa cells infected with more than one infectious unit often contained more than one focus of infection, indicating that replication is not confined to a single site in the cytoplasm.

Crystals which occurred within colonies of Staphylococcus aureus growing on nutrient agar were identified as struvite (magnesium ammonium phosphate hexahydrate). Other organisms, all of which increased the pH value of the medium during growth, formed similar crystals.