- Volume 3, Issue 1, 1949

SUMMARY: Working details are given for keeping as desiccates a collection of some 1500 strains of bacteria. The efficacy of drying, the various methods of freezing, and the effect of storage were tested by viable counts, using a spinning bottle modification of the roll-tube method. The survival rate of bacteria suspended in broth or other protective colloids, and subjected to freezing at − 78°, varied with the species from about 100 % with the resistant Staphylococcus aureus to about 10 % with the sensitive Neisseria gonorrhoeae, and from about 100 to 1·5 % or less, respectively, when the organisms were suspended in saline. The percentage of organisms which survive the freeze-drying process was found to vary with the species, from 100 % to less than 1·0 % when the organisms were suspended in broth and from 100 to no survivors when the organisms were suspended in saline. The storage loss of dried cultures was found to be a function of the storage temperature; however, suitably dried cultures could be kept at room temperature for very long periods. By far the most important factor influencing loss on storage was the presence of traces of moisture, and to ensure optimal survival the cultures must be as dry as possible. It appears that even with adequately dried cultures the presence of oxygen is deleterious.

The survival rate of bacteria in a sample of dried culture after heating to 80° for 1 hr., determined by plating a suspension of the heated sample in broth, provides a simple measure of the capacity of the particular batch of the dried culture to remain viable on storage at ordinary temperatures.

SUMMARY: The production of penicillin by Penicillium notatum in surface culture on a number of chemically defined media is described. Good results were obtained with media containing lactose, glucose, acetic, citric and phenylacetic acids, ammonium sulphate and inorganic salts. The yield of penicillin was raised by further addition of starch and an aliphatic base such as ethylamine. In the latter instance the penicillin yield was about the same as that obtained with media containing corn-steep liquor.

A suitable addition of mineral salts should include magnesium, potassium, iron, zinc, copper, manganese, cobalt, phosphate and fluoride.

The addition of starch and ethylamine increased the growth of the mould and produced crinkled felts similar to those obtained with corn-steep liquor. These media have a composition which is in many ways comparable with that of media containing corn-steep liquor, and it seems likely that corn-steep liquor owes its effectiveness to the fortuitous presence in it of several types of substances rather than to one substance.

SUMMARY: The factors affecting the growth of bacteria in fresh water stored in different containers, usually glass, were studied in order to reconcile the different results which have been obtained by previous workers. Growth occurred in two sitesin—the body of the water and at the surface of the container—and was affected by the constituents of the container.

Bacteria invariably grew on the sides of the container and wrere presumably dependent for their multiplication on having a site of attachment; thus the increase in the count per unit volume which occurred when bottles were vigorously shaken was greater in small bottles than in large bottles, and was due to removal of some of the cells attached to the walls. Bacterial growth was stimulated by soluble chemical substances in the wralls of containers; Bohemian glass and the glass of measuring cylinders were stimulatory; Pyrex glass and fused silica containers were inactive. It is probable that under the conditions of their experiments some workers have been observing bacteria which were dependent on the glass surface for their existence and which were unable to multiply in the body of the water sampled.

SUMMARY: Some strains of Streptococcus cremoris are slightly more heat resistant than the majority. After growth in milk at 37° for 5 hr. some of them show under the microscope involution forms which are sufficiently characteristic to serve as a useful means of identification.

Summary: There are many strains and perhaps several species of sulphate-reducing bacteria. They may be isolated by using a variety of media over a wide range of temperature. Crude cultures are readily obtained, but isolation of absolutely pure cultures is usually difficult. Considerable simplification in procedure may sometimes be effected by including 3% Na2SO3.7H2O in the media; the sulphite eliminates most of the contaminating organisms in crude cultures and facilitates subsequent purification. A method of maintaining stock cultures on sterile clay is preferred to the use of artificial media.

No growth takes place in media rendered ‘biologically free’ of iron, but the traces normally present in media constituents as impurities are sufficient for good development. Rapid and abundant growth free from ferrous sulphide has been obtained in a mineral salts + lactate + yeast extract medium containing ferrous ions below the saturation concentration for ferrous sulphide.

One thermophilic and four mesophilie strains have been shown to be facultative autotrophs, using the oxidation of hydrogen as energy source, with sulphate, sulphite, thiosulphate and elementary sulphur as hydrogen acceptors. No autotrophic growth takes place in the absence of hydrogen. Hydrogen can be provided in vitro by immersing metallic iron in the medium.

Summary: The production of trichothecin by strains of Trichothecium roseum was greatly increased wnen nutrients such as yeast extract, malt extract or corn-steep liquor were added to Czapek-Dox medium containing 5 % (w/v) glucose. Ammonium salts, especially ammonium tartrate, were better than nitrate as source of nitrogen, and high trichothecin titres were also obtained with asparagine or glycine as source of nitrogen. Maximum yields of the order of 100 mg./l. were obtained on a modified Czapek-Dox medium containing ammonium tartrate instead of sodium nitrate and including 5 % (w/v) glucose and 1 % (v/v) of corn-steep liquor, in about 30 days, with 4 cm. depth of medium.

Trichothecin had no antibacterial activity against Staphylococcus aureus, Bacillus subtilis or Bacterium coli. It was active against each of the twenty-seven species of fungi examined. The growth of Penicillium digitatum, the most sensitive species investigated, was completely inhibited at 0·64 mg./l., and 50 yo germination of its spores took place at trichothecin concentrations between 0·30 and 0.75 mg./l., according to the age of the spores and other factors.

Summary: The National Collection of Type Cultures, in its list of species maintained, uses unorthodox classifications of the genera Bacterium and Staphylococcus. For convenience in cataloguing Bacterium is divided into groups representing coliforms, plant pathogens, non-fermenting species and paracolons; to these is added a miscellaneous group of organisms of uncertain taxonomic position. These subdivisions are not intended to constitute a valid classification.

Coagulase-positive staphylococci, irrespective of pigment, form the species pyogenes; coagulase-negative strains are subdivided according to the pigment produced.

Summary: The pattern of differential inhibition of Gram-negative bacilli of intestinal origin depends not only upon a synergy of bile salts and electrolytes of the Hofmeister series but also upon aerobiosis and the nature of the surface upon which they are grown (Brodie, 1948). Identical patterns of inhibition are obtained if shallow-layer fluid cultures are employed. The media under discussion possess lethal qualities which appear to be modified by a process akin to adsorption and the results obtained with solid media to depend in part upon the physical nature of the gel.

Electron microscope studies suggest that bile salts alter the permeability of the cell-wall, facilitating the entry of electrolytes. The resultant swelling or disruption of cells exposed to bile-salt + electrolyte mixtures and subsequently to distilled water is a measure of their electrolyte content. The ease with which organisms can be inhibited in the media seems to be directly related to their altered cell-wall permeability. The observations direct attention to physiological differences between rough and smooth variants of a single culture. They indicate that one factor upon which such differences depend is the greater permeability of the rough variant to electrolyte in the presence of bile salts.

SUMMARY: The concentration of the group-specific substance in hydrochloric acid extracts by precipitation with ethanol has facilitated the serological identification of Streptococcus bovis and certain other serologically refractory streptococci as members of group D. The notorious difficulty of preparing potent group sera for Str. bovis was overcome by immunizing rabbits with organisms shaken with an abrasive in a magnetic vibrating machine. The relationship of Str. bovis to certain other streptococci and the chemical nature of the specific substance of group D are discussed.

SUMMARY: Bacilli in young cultures of Corynebacteria and Mycobacteria are multicellular. The individual cells are almost spherical, and a single bacillus may contain from one to twelve or more units. Reproduction may take place by division of the constituent cells, followed by simple fission of the bacillus, or alternatively by fragmentation into single cells which subdivide without separating, and grow into multicellular bacilli once more. The latter phenomenon may account for previous descriptions of life cycles in these genera.

The nuclear units are small, spherical granules, resembling those of some species of cocci. The characteristic morphology of Corynebacteria and Mycobacteria is an artefact, resulting from drying and heat-fixation. These genera do not appear to possess any morphological characters in common with the true Actinomyces, with which they are at present classified.

SUMMARY: The antigens of eight strains of influenza virus were compared by a simple and economical complement-fixation technique in which drops on Perspex sheets replaced larger volumes of reagents in racks of tubes. By this means more extensive tests were made with a limited supply of material than by standard tube techniques.

The specific antisera were prepared in mice, thus avoiding the elaborate isolation precautions necessary for ferrets, and sera from large groups were pooled to minimize individual variations between animals. The antigens were standardized in terms of the amount of complement fixed in the presence of excess homologous antiserum. The comparison of the strains is presented in the form of index numbers, ranging from 0 to 1·0: 1·0 represents complete identity of two strains, while the smaller numbers are taken to represent the degree of antigenic relationship between strains. Of the six strains examined which were isolated in different years, all were serologically distinct though the two B strains were closely similar and two of the A strains were fairly closely related. On the other hand, the three strains of influenza A virus isolated in 1947, two in London and one in the U.S.A., were identical.

SUMMARY: Experiments by the Thunberg tube technique have shown that tetrathionate can oxidize reduced Nile blue in the presence of tetrathionate-adapted cells of a Gram-negative coliform organism but not in the presence of cells lacking a developed tetrathionase system, viz. unadapted cells of the same organism and cells of Shigella sonnei. Concentrations of tetrathionate of the order of m/3200 and slight tetrathionase activity can be detected by the Thunberg tube technique, which may be useful also in investigating the effect of certain physical and chemical agents on the tetrathionase system. It appears that tetrathionate can act as a hydrogen acceptor for organisms capable of reducing it.

Summary: The swarming of Proteus vulgaris is inhibited on a heated blood-agar medium containing 0·1 % (w/v) boric acid. This boric acid concentration does not inhibit the growth of many organisms having more exacting metabolic requirements. The medium is equally successful in controlling certain swarming strains of Pseudomonas pyocyanea. This inhibition of swarming is possibly due to the formation of a boron-polysaccharide complex, resinous in character, the formation of which is associated with flagellar disintegration.

SUMMARY: Details are given of the ‘auxanographic𠀙 method for identifying growthfactor requirements of micro-organisms, and particularly of mutant strains differing from a parent strain in having additional growth-factor requirements. Cells are distributed in a nutritionally deficient agar medium which is allowed to set in a Petri dish. Possible required nutrients are then spotted on the agar surface, thus enabling the corresponding exacting mutants to grow at those localities. The technique is only a labour-saving device which can be extremely efficient in shortening the preliminary steps in the work. It can also be applied to other uses, such as the identification of competitive interactions between nutrilites.

Summary: Penicillin blocks the assimilation of glutamic acid by Staphylococcus aureus; the effective concentration of penicillin is of the same order as that required to inhibit growth of the organism whether the culture is sensitive or resistant to penicillin. Serial subcultivation in increasing concentrations of penicillin results in the selection of resistant mutants; as resistance increases, the ability of the cells to assimilate glutamic acid decreases. The efficiency of the assimilation process in highly resistant cells is poor, but they can synthesize all their amino-acid requirements from ammonia and glucose.

The decrease in assimilatory efficiency as resistance to penicillin increases is correlated with an increase in the ability of the cells to synthesize amino-acids. Reverse mutations, having decreased ability to synthesize certain amino-acids, were obtained from highly resistant strains and had increased sensitivity to penicillin. Strains of Staph. aureus requiring several amino-acids as nutrients have been ‘trained’, by subcultivation in media progressively more deficient in amino-acids, to dispense with the addition to the medium of all amino-acids other than cystine and histidine; the increase in synthetic ability was accompanied by a marked increase in penicillin resistance.

It is suggested that penicillin interferes with the mechanism whereby certain amino-acids are taken into the cell, and that the sensitivity of the cell to penicillin is then determined by the degree to which its growth processes are dependent upon assimilation of preformed amino-acids rather than upon their synthesis.

Summary: Soil-enrichment cultures in a medium containing 0·1 % (v/v) pyridine as the sole organic compound gave plate counts of more than 1000 million organisms/ml. Organisms of the genus Proactinomyces were able to utilize pyridine, aniline, nicotinic acid, nitrobenzene, or phenol + ammonium ion as sole source of carbon, nitrogen and energy.

SUmmary: The isolation, from sheep, of strains of the Bosworth type of Clostridium welchii is recorded; it is proposed to designate this type as Cl. welchii type E.

The toxicity of culture filtrates of Cl. welchii type E after short growth periods is increased by treatment with crude trypsin preparations; no such increase can be demonstrated when the filtrates are from growths older than 5–7 hr. The toxin which is activated in these filtrates is the toxin iota. It appears that Cl. welchii type E strains elaborate prototoxin convertible by enzymes into iota toxin proper.