- Volume 3, Issue 2, 1949

SUMMARY: By the use of anti-bacteriophage sera prepared in rabbits, thirty-nine staphylococcal phages were divided into six serological groups. The first group (A) comprised phages lysing coagulase-positive staphylococci of human origin. They were stable at 20° but inactivated at 49°. They multiplied in broth cultures containing sufficient tryptophan but rarely produced clearing of such cultures. The second group (B) lysed both bovine and human coagulase-positive staphylococci. They were markedly sensitive to heat and required growth factors present in the vitamin B complex. Group C comprised phages of ovine origin which were antigenically related to group B phages and also resembled them in their growth requirements. Group D comprised phage K, which lysed both coagulase-positive and negative staphylococci and was antigenically related only to phage W. Phage W belonged to group E and lysed only some coagulase-negative staphylococci. Group F was related in its general characters with the phages of group A.

A staphylococcus was found carrying two serologically distinct phages, one of which was detected during the process of adaptation of a phage filtrate to a new propagating strain.

Since many strains of staphylococci are lysogenic, lytic filtrates may contain contaminating phages which manifest themselves during adaptation. Adequate serological characterization of the phages used for typing and for investigations of phage-bacterium relationships and of apparent mutation is therefore necessary.

SUMMARY: Using a filtrate from an emulsion of skin nodules, lymph node and a milk-duct nodule from a calf dead of bovine Lumpy Skin Disease as starting material, a filtrable agent was isolated by serial passage in chick embryos. The agent affects mainly the skin structures of the chick embryo and results in a characteristic shrunken featherless embryo tightly wrapped in its amnion, and with almost complete disappearance of amniotic fluid.

The agent appears to be a filtrable virus approximately 10-25 mμ in diameter. It was not pathogenic for day-old chicks and adult fowls.

The virus was neutralized by the serum of fowls which had received repeated intramuscular injections of the virus, suggesting that the virus is etiologically related to Lumpy Skin Disease in cattle. Further work is needed before such a relationship can be regarded as established.

The virus withstood heating to 60° for 20 min., survived in 50% glycerol for at least 1 week, and at a temperature of +4° for at least a month. It was destroyed by boiling for 2 min. and by incubation at 37° overnight. Desiccation destroyed a large part of the virus in a suspension, but that part which withstood the drying process survived for at least a month.

SUMMARY: Streptomycin may be estimated in body fluids by allowing the fluid to diffuse through nutrient agar seeded with a culture of Staphylococcus aureus contained in small-diameter (3 mm. internal) glass tubes. The zone of inhibition produced is affected neither by the anaerobic conditions within the agar nor by the volume of the test fluid. Acidity of the test fluid invalidates the results, since the depth of the zone of inhibition is decreased. Increase in the size of the inoculum of the test organism also decreases the depth of the zone of inhibition. Small errors arise from variations in timing during the setting-up of the test. The zones produced in the presence of different human sera differ slightly and to a degree similar to that found in the presence of various urines.

The available mathematical expressions suggest that the square of the depth of the zone of inhibition is linearly related to the log of the concentration of an antibiotic in the test fluid. Experimentally this relationship does not hold for low concentrations of streptomycin, probably due to the assumption of boundary conditions which cannot be defined with certainty; but it is a better approximation than the assumption of a linear relationship between the depth of the zone of inhibition (unsquared) and the log of the streptomycin concentration. A method of statistical analysis is given in which a weighted regression line is fitted to the squared values of the zones of inhibition in a manner analogous to probit analysis. The routine method may be refined without undue labour to the point where assays are accurate to within + 5 %.

SUMMARY: The total numbers (active + cystic) and the numbers of active amoebae in plots treated with complete minerals + ammonium sulphate and with farmyard manure were much higher than in the untreated plots. The complete minerals plot of Barnfield had a just significantly lower total count of amoebae than the farmyard manured plot, although no significant difference existed between the counts of active amoebae. The difference in the numbers of both the total and the active amoebae between the complete minerals and farmyard manure-treated plots on Broadbalk was not significant. No correlation was found between the percentage of organic carbon in the soils and the number of amoebae.

SUMMARY: Using the corresponding amino-acid decarboxylases, the six amino-acids arginine, glutamic acid, histidine, lysine, ornithine and tyrosine were found to be free inside the cells of yeast. They are present when growth takes place in the absence of amino-acids, but their concentration may be increased by growing the organisms in media rich in amino-acids. Uptake of glutamic acid from the external medium is dependent on a source of energy which can be provided by the simultaneous fermentation of glucose. The presence of an ammonium salt in the medium decreases both the rate at which glutamic acid enters the cells and the amount of glutamic acid which can be taken up. Certain other amino-acids exert a similar sparing action on the assimilation of glutamic acid, which can be related to their efficiency as nitrogen sources for growth. The free glutamic acid content of cells remains practically constant when the cells are suspended in salt solutions without amino-acids, but if glucose is also present then the concentration of free glutamic acid inside the cells decreases steadily. When ammonia is also present the concentration of glutamic acid remains constant, suggesting that glutamic acid is synthesized by the cells under these conditions, or alternatively that the ammonia is assimilated and utilized preferentially.

Summary: Strains of Sporocytophaga myxococcoides were cultivated in a mineral nutrient medium with glucose. In media containing 0·1% glucose sterilized by Seitzfiltration growth readily occurred, whereas in corresponding media with 0·5 and 1·0% glucose there was a pronounced lag in development that sometimes lasted for several weeks.

The duration of the lag period appeared to depend on the number of cells in the inoculum, and could be diminished by incubation at 25 ° instead of 30°, although the latter temperature is nearer to the optimum temperature for growth. Apparently adaptation to the high glucose concentration takes place during the lag period.

Cells adapted to high glucose concentrations grow readily in media with the same concentration of mannose, whereas non-adapted cultures do not grow in these media, or only after a long lag period. None of the strains investigated showed any development in media in which either fructose or xylose was the sole source of carbon; nevertheless, these sugars did not inhibit, or only slightly, inhibited cellulose decomposition.

Stanier’s observation, that the development of Sp. myxococcoides is inhibited when the glucose is autoclaved in the nutrient medium, was confirmed. If, however, due care be taken to avoid an alkaline reaction, glucose autoclaved either separately or in the medium sustains growth in the same way as does glucose sterilized by filtration.

Summary: The physical condition of soil is improved by adding readily decomposable organic material. Microbial cells and metabolic products affect soil structure by binding loose soil particles into water-stable aggregates.

Experimentally, the relative aggregating power of pure cultures of micro-organisms was as follows: fungi > actinomycetes and a few gum-forming bacteria > many gum-producing bacteria > yeasts, proactinomycetes, and many bacteria; the last three groups did not improve aggregation. Fungal hyphae entangled soil particles into stable aggregates; weaker crumbs were formed by the frailer threads of actinomycetes. A few bacterial strains produced gums capable of glueing soil into water-stable aggregates, but the majority of bacterial slimes were almost useless because they remained water-soluble after drying. The cementing properties of these gums was not improved by treatment with H or Ca ions. Bacterial gums stabilized the aggregates produced from completely dispersed soils and kaolin, but not those formed with bentonite or ferric hydroxide. The pH value of the soil played a very minor part in influencing the aggregation produced by pure cultures of micro-organisms or even by soil inoculum.

Mixed cultures of fungi or of actinomycetes gave slightly better aggregation than pure cultures, but neither capsulated nor non-capsulated bacteria in mixtures gave better results than single strains. More complex mixtures containing fungi, actinomycetes and bacteria gave good aggregation when all micro-organisms wrere compatible, but poor results when antagonistic bacteria inhibited the growth of either fungi or actinomycetes. The fair aggregation obtained with soil inoculum was reproduced in the laboratory by inoculating sterilized soil with complex mixtures of micro-organisms.

A study was made of the relative merits of glucose, starch, blood, yeast, fungal mycelium, straw, clover and farmyard manure for encouraging aggregation by mixtures of fungi, actinomycetes and bacteria.

Aggregates bound by mycelia did not last long because the hyphae were decomposed by bacteria. The temporary improvement of soil structure after the addition of organic materials can be partly explained by the action of microbes, but the permanent crumb structure of many soils must be due mainly to other causes.

Summary: Bacillus cereus and B. mycoides produce lecithinases which split lecithin into phosphorylcholine and a diglyceride in the same way as the lecithinase (α-toxin) of Clostridium welchii. These enzymes also possess most of the biological activities associated with Cl. welchii α-toxin, e.g. produce the Nagler reaction and the egg-yolk reaction, lyse red blood cells, and are dermonecrotizing and lethal.

The enzymes are activated by Ca and Mg ions, but inhibited by Na, K, NH4 ferric and Al ions. Optimal enzyme activity requires the presence of Ca ions within a narrow range of concentration of 1–4 × 10−3 m. It is interesting that at this concentration of Ca ion lecithin flocculates most readily from its emulsion. The enzyme thus seems to have a maximal affinity for lecithin when the latter adsorbs an optimal amount of Ca ion in reaching its isoelectric point.

Like the Cl. welchii lecithinase, the B. cereus lecithinase is fairly resistant to heat. The lysis of red blood cells and the hydrolysis of free lecithin by B. cerBus lecithinase was strongly inhibited by normal sera of all the animals tested. But when lecithin was bound in egg-yolk lipoproteins, its hydrolysis by the enzyme was unaffected by normal serum. Specific serum, on the other hand, was capable of inhibiting the hydrolysis of both free lecithin and protein-bound lecithin. The B. cereus and B. mycoides lecithinases are immunologically related, but they are not so related to Cl. welchii lecithinase.

Summary: Penicillinase-producing strains of Staphylococcus pyogenes isolated from human infections and kept under various conditions were plated out and fifty colonies from each plate tested for penicillin-sensitivity to determine the permanence of their resistance to penicillin.

Of 32 strains kept in Lemco broth and tested once, 5–12 months after isolation, two yielded only penicillin-sensitive colonies and fifteen others yielded a proportion of such.

Of six strains preserved by the gelatin-ascorbic acid drying process and kept for over a year, only one yielded penicillin-sensitive colonies.

Of six strains kept in Lemco broth with and without regular subculture and tested at intervals for 9 months, all gave rise to at least one penicillin-sensitive variant colony and more than half the colonies of one strain became penicillin-sensitive.

The natural tendency of these strains to yield penicillin-sensitive variant colonies was not appreciably accelerated by treatment with X-rays or by growing them with other organisms.

SUMMARY: In the classification of yeasts it is customary to use an infusion of bakers’ yeast as the basal medium for fermentation tests. This extract frequently contains variable amounts of trehalose. A number of yeasts were observed to ferment yeast extract and trehalose. The fermentation of yeast extract is serious from a taxonomic point of view, since it gives the impression of positive fermentation of a sugar which actually may not be fermentable. Dilute yeast autolysate should be used as the basal fermentation medium since during autolysis trehalose is destroyed. The fermentation of yeast extract (without added sugar) is easily observed in Durham tubes by the collection of gas in the inserts, but when Einhorn fermentation tubes are used gas production is seldom apparent.

One culture (N-18) isolated from spoiled apricots, and identified as Candida tropicalis showyed adaptive trehalose fermentation.

The ability of various yeasts to ferment trehalose was investigated, using 133 cultures, representing twenty genera and seventy-three species. Sixteen species representing seven genera fermented yeast extract and trehalose.

The fermentation of trehalose is worthy of consideration as a character for use in differentiating certain species of Candida, and perhaps other yeasts.

Summary: We have examined by a uniform technique the sensitivity of twentyfive viruses to ethyl ether. Evidence of the sensitivity of ten other viruses is available from the literature. Viruses seem to be either very sensitive or highly resistant. Of the viruses pathogenic to animals, most of the resistant ones are either in the pox group or amongst the very small viruses. The results may prove of use to workers who at times need to separate one virus from mixture with others or with bacteria. The findings may also be of value in any attempt at virus classification.

Summary: Influenza and related viruses were studied by a new method in which virus is adsorbed on the membranes of laked fowl red cells for examination in the electron microscope. The numbers of virus particles adsorbed per unit area of red-cell membrane were estimated from direct counts in micrographs of palladium-shadowed and unshadowed specimens. There was a definite saturation level of adsorption for each strain of virus, the value varying also according to the particular batch of cells and their age after storage at 0°. For subsaturation conditions the number of particles adsorbed was proportional to the concentration of virus and the concentration of cells, and a function both of time of contact and temperature. The relationships were complicated by the fact that elution began before adsorption was complete.

Summary: A description is given of apparatus and methods which have been used to produce large numbers of satisfactory and reproducible chromatograms of bacterial culture filtrates. Some of the factors which influence the preparation and development of chromatograms are discussed.

Summary: Some 300 strains of bacteria representing twenty genera were grown on an acid-hydrolysed casein medium. Using the paper chromatographic technique of Woiwod (1949) a preliminary survey was made of the amino-acid and polypeptide composition of the bacterial culture filtrates. It was proved that: (a) changes may occur in the filtrate chromatogram which have group or species significance; (b) bacteria with simple nutrient requirements, i.e. which utilize ammonia, do not affect the chromatogram in the initial growth stage; (c) whenever the chromatogram is affected serine is the first amino-acid to be metabolized; (d) Gram-positive bacteria eliminate the aspartic acid spot but leave the basic amino-acid group unaffected, and the reverse occurs with Gram-negative bacteria; (e) many bacteria synthesize ninhydrin-positive material, presumably polypeptide, the synthesis of a given kind of polypep-tide being sometimes associated with a particular group or species of bacteria. By a disintegrator technique it was demonstrated that bacteria store many free amino-acids inside the cell.