- Volume 13, Issue 1, 1955

SUMMARY: The Staphylococcus aureus strain known as Staph. Oxford (NCTC 6571) produces, in any one mutational step towards penicillin resistance, a number of mutants, differing by various characteristics. One of these characteristics is the degree of penicillin resistance. The most resistant first-step mutant is about five times more resistant than the least resistant one. As such mutants are picked up from different penicillin concentrations, according to their higher or lower resistance, the resultant cultures may appear graded by the amount of penicillin present in the medium from which they were obtained. Mutants recognizable as members of one clone, however, show the same resistance from whatever concentration of penicillin they are isolated, within the range on which their growth is possible.

Another characteristic is the distribution of penicillin sensitivity in the population; it differs between mutant strains but, contrary to expressed views, the curves of distribution do not become flatter in mutants of increased resistance, provided one uses a logarithmic scale for the drug concentrations.

Prolonged culture on agar with sub-inhibitory concentrations of penicillin does not lead to increased penicillin resistance. In fluid medium with low penicillin concentrations, on the other hand, stable mutants, resistant to inhibitory concentrations, soon arise.

The growth rate of most penicillin-resistant strains is slower than that of the original strain, and is slowed down still further by the presence of penicillin, even by less than one-hundredth of the inhibitory concentration.

Variants and mutants of slightly increased penicillin-resistance are often pigmented.

Penicillin in sub-inhibitory concentrations enhances autolytic processes in the original organism and in all the mutants, and curious pictures of colony morphology are encountered which result from the interplay of autolysis and secondary growth.

SUMMARY: To see whether the initial damage caused by penicillin involved the osmotic barrier, certain characters of Staphylococcus aureus were examined at intervals during the first 2 hr. after addition of penicillin to growing cultures. In the 30 min. following addition of penicillin the cell appeared to expose nearly all its reserve penicillin-binding component (PBC), the penicillin uptake being double that which occurred without growth and the reserve PBC disappearing. The amount of PBC in the cell and its rate of exposure to penicillin in excess of normal synthesis (rate of turnover?) were both very small. Between 30 and 60 min. the uptake of Na, Mg, K, P and increase of total dry matter ceased abruptly. Continued increase in dry wt., while P, Na, Mg and K decreased slightly, resulted in the penicillintreated cells becoming relatively deficient in these elements. Synthesis of lipid phosphorus and some, but not all, large molecule phosphates still continued, and Fe and Co uptake were not affected. After 60-80 min. water entered and solutes began to leave the cell, and synthesis of large molecule P ceased. The primary site of action of penicillin is probably not concerned with gross assimilation of Na, K, Mg, P, Fe, Co, or any substance contributing more than 10% of the dry wt., or with gross synthesis of lipid P. It may, however, involve a reaction turning over 3000 times while the cell mass is doubled.

SUMMARY: Concentrations of fumagillin which allowed host growth prevented multiplication of phage 209 on Staphylococcus aureus 209. Fumagillin had no action on free phage and little on the adsorption of phage to its host, but when added 15 min. or less after the start of the latent period it prevented phage multiplication. Fumagillin present during adsorption was diluted out during the latent period; the longer fumagillin was present the smaller was the phage yield. Prolonged exposure of the host + phage mixture in Ringer′s solution to a concentration of fumagillin too low to prevent phage multiplication lengthened the latent period and decreased the yield of phage, whether fumagillin was present during the burst or not. Under these conditions, when sufficient fumagillin was added to produce a phage-inhibitory concentration and later diluted out, the yield of phage was constant when the dilution was made in the period 0–20 min.

Summary: A dialysed cell-free extract of Saccharomyces fragilis Jörgensen was found to catalyse the transfer of the amino group from alanine, aspartic acid, isoleucine, leucine, norleucine, methionine, ornithine, phenylalanine, tryptophan, tyrosine or valine to α-ketoglutaric acid with the production of glutamic acid and the corresponding keto acid. The transaminase reactions leading to the formation of alanine, aspartic acid, phenylalanine or tryptophan from glutamic acid and the corresponding keto acid were also catalysed by the yeast extract. The addition of pyridoxal phosphate to the incubation mixture increased the rate of transamination between α-ketoglutaric acid and any one of the following amino donors: aspartic acid, leucine, methionine, phenylalanine, tryptophan or tyrosine.

The rate of formation of glutamic acid from α-ketoglutaric acid and leucine was most rapid at pH 8, but the rate of amino transfer from aspartic acid did not vary with pH over the range 6·0–9·0. The equilibrium constant for the aspartic-glutamic transamination reaction was found to be 2·2 at pH 7·8 and 37 °.

The transfer of the amino group from aspartic acid or leucine to α-ketoglutaric acid was not inhibited by any one of five antibiotics tested, but was partially inhibited by 10−3 m-semicarbazide.

Summary: Sporulation of Bacillus sphaericus, NCTC 7582, in a complex medium was strongly stimulated by growth in mixed culture with a sporing strain of B. cereus and with sporogenous and asporogenous strains of B. subtilis, NCTC 85. There was a similar, but less pronounced, effect with a variety of Gram-positive and Gram-negative organisms. The degree of sporulation of B. sphaericus in these mixed cultures depended on (a) an external stimulus from the medium and (b) the previous cultural history of the B. sphaericus inoculum upon which depended its ability to respond to the external stimulus. Sporulation of B. sphaericus in pure culture was strongly stimulated by bicarbonate and ketoglutarate, suggesting that increased carbon dioxide production was the stimulating factor in mixed cultures.

Summary: Spore suspensions of Bacillus megaterium germinated spontaneously after heat treatment. The extent of germination depended on the degree of close packing of the suspension, the temperature and duration of heating, and on the presence of water. Spontaneous germination was not inhibited by heating with cyanide, azide, 2:4-dinitrophenol or sodium iodoacetate, but was significantly inhibited by sodium fluoride.

Freshly harvested spores of a laboratory strain of Bacillus cereus required either inosine or a mixture of alanine + tyrosine + adenosine for optimal germination. After prolonged storage or a short heat treatment, adenosine alone stimulated rapid and complete germination. Heat activation did not occur in the absence of water. It was inhibited to some extent by sodium fluoride. Similar results were obtained with B. cereus, NCTC 8035.

After preheating, spores of a virulent and an avirulent strain of Bacillus anthracis germinated more rapidly and completely in a mixture of adenosine + tyrosine + alanine, but not in adenosine alone. Inosine stimulated rapid and complete germination of heated suspensions, and was consistently more effective than adenosine. Adenosine deaminase activity has been found in extracts from resting spores of the laboratory B. cereus and the avirulent strain of B. anthracis but not in B. megaterium.

Summary: Physiologically definable groups within the genus Lactobacillus are morphologically distinct. There are marked differences between homofermentative and heterofermentative strains and between physiological groups within the former.

Summary: K12, K26 and K34 are three genetically distinct histidine-requiring mutants of Neurospora crassa. K12 and K26 are 0·69 units apart in the left arm of linkage group I, and K34 is in linkage group IV 32·6 units from 5531 (pan).

Response to histidine of these three mutants is markedly affected by the genetic background.

K26 accumulates two imidazole compounds, one of which may be histidinol and the other possibly compound II of Ames, Mitchell & Mitchell (1953) . Like other histidine-requiring mutants K12, K26 and K34 are inhibited by certain other amino acids in combination with either arginine or lysine. The degree of inhibition is affected by the genetic background. The effect of the inhibitors is to prevent uptake of histidine from the culture medium. Histidine is accumulated from the culture medium against a concentration gradient and stored in the mycelium, from which it gradually disappears with growth. Inhibitors applied after accumulation have no effect on utilization of this stored histidine. Histidine is similarly accumulated by wild type and uptake is also prevented by a combination of the inhibitors arginine and methionine. The histidine requirement therefore serves to accentuate an effect which already exists in the wild type.

Summary: Electron microscope observations were made on submerged cultures in defined medium of two species of the genus Streptomyces. The evidence obtained confirms the supposition of Klieneberger-Nobel (1947) , with surface cultures of four other species, that at an early stage in the life history of the organism concerned, fusion occurs between portions of the same or different hyphae, culminating in the formation of ‘initial cells’. In S. griseus initial cells were observed at 26 hr. and as late as 6 days. Certain unidentified bodies were observed. These may be related to initial cell formation.

Summary: The numbers of local lesions (x) produced by different leaves when inoculated with the same virus preparation deviate greatly from a normal distribution, and the standard errors of x’s depend on their magnitude. Before customary statistical analyses are applicable to results of infectivity assays, x’s must be suitably transformed. When mean values of x are greater than about 10, the transformation z = log10 (x + c) (where c is a constant), is satisfactory but inapplicable with smaller numbers. In some work the use of poorly infective inocula is unavoidable, and to allow statistical analysis of results in such work a new transformation z = log10½ [x + c + √(x 2 + 2cx)] is derived. This operates satisfactorily when mean numbers of x are greater than about 1·5. The results of the two transformations converge as x increases. Values of z are tabulated to make the use of the new transformations as quick as that of the other transformation.

Summary: Cultures of sixteen different soil actinomycetes were tested for their ability to inhibit the growth of eight pathogenic strains of Fusarium oxysporum Fr. on agar media. Three of the actinomycetes did not inhibit growth, and nine inhibited growth of all strains equally, but the other four actinomycetes consistently inhibited the growth of individual strains to different extents. The differences provide an in vitro test which distinguishes between certain pathogenic strains of Fusarium that are otherwise indistinguishable in culture.

Isolated cell walls of strains of Bacillus, Micrococcus and Caryophanon species obtained by mechanical disintegration, and in some instances by the action of trypsin, gave a specific cell-wall reaction, visible in the phase-contrast microscope on the addition of homologous antibody. The cell-wall structure, as revealed in the specific cell-wall reaction, is very similar to that observed in stained preparations. A method is described for staining the bacterial cell wall with Alcian blue without preliminary treatment with a mordant.

Summary: A named strain of each of the species Photobacterium phosphoreum, P. fischeri, P. albensis, P. pierantonii, P. splendidum, P. sepia and P. harveyi, together with a recently isolated strain of each of the first two named species, were studied in an attempt to elucidate their correct generic classification. On the basis of morphology, sensitivity to certain antibiotics and a vibriostatic substance, and method of dissimilation of carbohydrates, it was concluded that P. sepia and P. harveyi should be classified in the genus Aeromonas, and the remainder of the species in the genus Vibrio.

Summary: Progressive stages in the germination at 60 of the highly refractile spores of Micromonospora vulgaris were observed by means of phase-contrast microscopy. The development of individual spores was followed in slide cultures. Mass cultures were also prepared in liquid and solid media from spore suspensions in phosphate buffer which had been heated at 100 for periods of 1–20 min. and 30 min.-4 hr. The liquid cultures derived from heated and unheated spores were sampled at frequent intervals and examined in wet preparations containing dilute toluidine blue. Throughout, loss of refractility preceded germination, which occurred in 1–3 hr. Germinating spores appeared as dense opaque bodies by phase contrast and were then capable of absorbing the dyestuff to which non-germinating spores were impermeable.

Heat activation at 100 for 1 min. enhanced the initial germination rate within the first 3 hr. period. No differences were observed between heated and unheated spores during the processes of germination. Indications of an eccentric nucleus were occasionally found in phase-contrast examination of very lightly stained fixed spores from material heated for short periods, or in electron micrographs of unheated growths. Heating at 100 for the shorter periods (1–10 min.) resulted in a higher proportion of the spores becoming darkened than was the case with the longer exposures. Artificial darkening of the spores in the manner successfully used for eubacterial spores was not accomplished. Sectored coloniesthe sectors showing loss of aerial mycelium and impaired viabilityfrequently developed from spores which had been subjected for more than 1 hr. to a temperature of 100.

Summary: The pathogenic partially acid-fast lipophilic organism Nocardia sebivorans and a closely allied species were found capable of withstanding exposure to 90° for 10 min. when dispersed in phosphate buffer suspensions in small sealed ampoules. Denser suspensions withstood 3 min. at 100°. The nature of the medium on which the cells were grown did not seem to influence their thermoduric properties. Of the two morphotypes of N. sebivorans that were originally isolated, one (G) consistently showed a higher degree of heat-resistance. The morphotypes were further distinguished by slight differences in the ‘feel’ of the cells and in their response to micromanipulative procedures. Different single cell isolates of the two morphotypes varied as regards thermotolerance, but the more resistant were G isolates. The lag period following heat treatment was generally accompanied by a compressed and stunted mode of growth, featuring prolonged angular division. Subsequent growth was normal.

Two strains of the pathogenic Streptomyces madurae, as well as two saprophytic soil species of Nocardia, were killed by exposures of 1–2 min. at 65°. An organism received as a blood culture and belonging to the Streptomyces albus group, was able to withstand 90° for 1 min. Subjection to heat treatment increased the inherent autolytic tendency of the culture and affected the type of sporophores produced during the first generation.

Summary: Several isolates of monosporous lines of Streptomyces coelicolor, both normally pigmented and colourless variants, were obtained by microdissection of young vegetative mycelia. In each case their properties largely resembled those of the parents. This material was studied together with three strains of Streptomyces scabies of varying degrees of virulence, which had been isolated from potato scab lesions. Modifications of growth were noted with respect to gross colonial morphology on agars containing detergents, structure of aerial mycelium arising from various types of surfaces, and development of germinating spores in liquids and in contact with non-nutritive plane surfaces. In both species-groups those organisms which produced least aerial mycelium on stock nutrient agar were most susceptible to the inhibitory influence of 0·1% Gemex 29 on the development of aerial mycelium in simpler media. Distorted aerial growths on detergent agar resembled irregular swollen sporogenous filaments produced in pectin + ammonium salt liquid media. These were also reminiscent of cases of sporogenesis in submerged cultures described by other workers. Apart from surface tensions operating at the air/medium interface, the relative degree of humidity in the culture vessel was found to have some effect upon variation of spore size. Germinating spores and young filaments growing in contact with plane surfaces were more prone to elongation than those growing in liquids. Branching was also influenced to a considerable extent by the nature of the physical environment.

Summary: The growth on solid medium of organisms of the pleuropneumonia group as seen in electron micrographs was compared with the growth in liquid media described in a previous paper ( Klieneberger-Nobel & Cuckow, 1955 ). There is no evidence of a rigid cell wall in these organisms, a finding consistent with those electron micrographs of growth in liquid media and in light microscopical preparations. The newly formed minimal reproductive units, hitherto never demonstrated in the light microscope except when they have grown to a larger size, are here demonstrated in large numbers as a uniform phase of small, delicate elements, the diameter of which measures c. 100 mμ. in the electron micrographs.

Summary: Biochemical reactions of 153 Providence (29911 type) and 63 Proteus cultures were examined. Providence cultures are included in the genus Proteus, as their cultural characters are similar; this relationship is strengthened by the production of l-amino acid oxidase and glutamic acid decarboxylase by both. Proteus inconstans Ornstein nov.comb. is the name proposed for Providence cultures.