- Volume 1, Issue 2, 1947

SUMMARY: The effect of temperature on the growth of Bact. coli under accurately controlled conditions has been examined in an apparatus which permits food to be supplied at any desired rate by means of an automatic syringe mechanism. Six temperatures were used, from 15° to 40° at 5° intervals, with a single rate of food supply. Total and viable cell counts were frequently made and growth curves constructed. In some cases the changes in numbers were followed after stopping the food supply.

At all stages the total counts greatly exceeded the viable. There was always first an ‘initial’ phase in which the daily increments in total cells varied with time. Very early in this phase cell division lagged behind cell growth. At 15° this condition probably persisted throughout the whole experiment. The initial phase was longest when the temperature was lowest, but the time taken to reach 300 × 106 total cells/ml. did not vary greatly, except at 15° when the time was markedly increased. The yield of total cells at the end of the initial phase was highest when the temperature was lowest and depended on the amount of food added, i.e. on the duration of the phase, but relatively more cells were formed in a long than in a short phase. The viable cell count at the end of the initial phase was also enhanced by low temperature, except at 15° where the viable cells were fewer than expected in comparison with the other experiments. The ratio of the total to the viable count at the end of the initial phase was often close to 2·0.

The initial phase was followed by a ‘steady’ phase in which there were, on the whole, constant daily increments in both total and viable counts, although there were indications of stepwise increments. In the steady phase the calculated rates of increase in both total and viable cells were highest when the temperature was low, and bore a linear relation to the temperature. The rate of increase in viable cells was almost zero at 35°, which was apparently a critical temperature for viability. At 40° there was only a slow decline in viable cells, but nevertheless at both 35° and 40° growth of the cultures occurred since the total counts continually increased, but approximately half the cells formed were non-viable.

To express the difference between the total and viable counts a non-viability index was calculated. This was constant throughout the steady phase and was smallest when the temperature was lowest. The amount of food previously shown to be required for the formation of a new cell at 35° was found to be not inconsistent with the experimental data at any of the other temperatures, but if this amount were constant the food used per cell for maintenance and wastage must have declined with decreasing temperature.

After cessation of the food supply the total count declined at both 15° and 30°, being slightly faster at the lower temperature. The viable count also declined during starvation at 15°; the rate of decrease of the viable count was greater at 30°.

SUMMARY: Strain selection and the addition of yeast extract to the medium have enabled high yields of tyrothricin to be produced from the culture fluids of Bacillus brevis. Contrary to the accepted view, this organism does ferment carbohydrates with the production of acid, and a description of the characters of the organism is given. A method of assaying tyrothricin has been developed and used to investigate the stability of aqueous solutions of the material. Tyrothricin produced in aerated submerged culture appears to be more stable in solution than that produced by surface culture.

SUMMARY: Synthetic media containing ammonium succinate as the sole source of nitrogen permit Bacillus brevis to grow and form tyrothricin in aerated submerged culture. The yield of the antibiotic is almost doubled by adding a complex of B-vitamins to the medium, the effect being due specifically to the presence of biotin.

Urea can be used as an alternative but less satisfactory source of nitrogen, but ammonium succinate cannot be replaced by ammonium salts of a number of other organic acids. The process can be operated successfully on a large scale.

Normally, B. brevis will grow, but will not produce tyrothricin, in submerged culture in the presence of complex nitrogen sources. An exception to this has been observed, but metabolic investigations show distinct differences in the behaviour of the organism (in submerged culture) in synthetic and in peptone media.

SUMMARY: One hundred and sixty-six strains of Fungi Imperfecti have been examined primarily for the production of antifungal substances, though antibacterial substances have also been sought in most cases.

Antifungal substances were produced by several species of Aspergillus and Penicillium, Fusarium caeruleum, Metarrhizium glutinosum, Stachybotrys atra, Tri-choderma viride, Gliocladium catenulatum and Trichothecium roseum. Antibacterial substances were produced by species of Aspergillus, Fusarium, Penicillium, Stachybotrys and Trichoderma.

The chemical nature of the substances responsible for this activity is known or can be suggested in some cases, but others are worthy of further investigation.

SUMMARY: The organisms whose antibacterial powers are to be investigated are grown in parallel streaks on cellophan superposed on an agar plate. The cellophan is stripped off and the test organisms are streaked on the sterile agar surface thereby revealed, at right angles to the first streaks. After incubation, growth of the test organisms will be interrupted in those areas in which an inhibitor has diffused through the cellophan from the first organisms. There are some limitations in the application of the method.

SUMMARY: The use of mechanical air filtration combined with ultra-violet irradiation of the filtered air has aided markedly in avoiding contamination by undesirable micro-organisms during pilot-plant scale work on penicillin production. By maintaining master cultures of Penicillium strains in sterile soil it has been found possible to eliminate strain variation and to maintain the penicillin-producing capacity of the strains. This procedure, followed by transfer of the organism to a rye grain substrate, gives a convenient method of securing large numbers of spores for large-scale work.

It has been found to be desirable to check the purity of the strains, their penicillin-producing capacity and the viability of the spores at various stages of the production sequence; methods have been developed for each of these requirements. Improvements in the cup assay method of estimating penicillin have eliminated certain difficulties encountered in this assay and have enabled the overall error of the method to be reduced to 5%. A colorimetric method of assay has been developed which gives results agreeing with those of the cup assay. With this new method the penicillin content of a sample can be estimated in 5 hr.

This paper gives details of a 50 gal. fermentation vessel designed for investigating the formation of antibiotics (or other metabolic products) by microorganisms grown in submerged culture. This vessel has been used for investigating the submerged culture production of penicillin by Penicillium chrysogenum X 1612 and Q176, and certain results relating to the size of the inoculum and the yields obtainable from these strains in synthetic and other media have been obtained. Culture fluids containing from 400 to 500 Oxford units penicillin/ml. have been obtained with cultures of Q176 in a corn-steep liquor medium.

A method of extracting penicillin from the broth has been worked out, based on solvent transfer, the method being applicable on virtually any scale of operation and involving only relatively simple equipment. It has the advantage of reducing the time of contact of penicillin with acid to such a degree that extraction at room temperature is possible, although extraction at still lower temperatures improves the yield. Using this method of extraction we have obtained calcium penicillin with a potency of 940 Oxford units/mg., the overall recovery from the broth being of the order of 35–50%.

SUMMARY: A pink, butyric acid-producing Clostridium isolated by Ch. Weizmann from South African hibiscus stems is described. It ferments sucrose, glucose, lactose, maltose, galactose, xylose, starch, but not cellulose, inulin, mannitol, glycerol and sorbitol.

The main products of fermentation of maize and glucose are butyric and acetic acids; acetone, butanol, ethanol and isopropanol are produced in slight or moderate amounts, lactic acid, formic acid and acetylmethyl-carbinol only in slight or negligible amounts. In its partial diastatic action on maize mashes and in the composition of its resultant metabolic products the new organism resembles members of the so-called butyric group of clostridia; but it differs markedly in liquefying gelatin.

In certain morphological and physiological features the organism resembles two orange-coloured Clostridia, Cl. felsineum and Cl. roseum (both belonging to the so-called butylic group), but is readily distinguishable from them. The two butylic organisms have a complete diastatic action on maize and butanol is the chief product of fermentation. The new organism is also distinguishable by its inability to ferment inulin and to disintegrate potato slopes.

On the basis of this evidence the organism is considered to be a new species, for which the name Cl. aurantibutyricum is suggested.

SUMMARY: The production of a thermolabile, somatic antigenic modification by growth of Erysipelothrix in media containing serum has been demonstrated in vitro by agglutinin-absorption methods and by passive protection of mice against infection. Suspensions produced by growth in serum media have been termed OL-suspensions, to signify their possessing both the thermostable O-antigen, and a labile L-antigen.

OL-suspension stimulated the production of immune sera in rabbits which protected mice fully against infection with the homologous strain and partly against infection with the other strains tested. Such sera contained agglutinins against the thermostable O-antigens, agglutinins against the thermo-labile L-antigen and protective antibodies. The three types of antibodies apparently exist both as separate molecules and united in the same molecule, suggesting that the antigenic groups stimulating them exist united in the same antigenic molecules in the organism. The bearing of this suggested polyspecificity of the antigenic substance of Erysipelothrix upon previous work is discussed.

SUMMARY: Tools required for the more delicate processes of micromanipulation are not commercially available and existing equipment for their making is expensive. Some cheaper equipment is described, comprising a gas forge and an electric forge which together suffice to prepare the tools needed for isolating single cells, for injecting and dissecting living tissue and living cells, and for determining their electrical potential.

A simple hanging-drop micromanipulation chamber is described.

SUMMARY: From the existing methods for identifying common antibiotics it is possible to select tests for recognizing one or more of fourteen known antibiotics in crude culture medium. These tests, given in tabular form, are: (1) stability at pH 2·0 and 9·5; (2) ether-water partition at pH 2·0, 6·0–7·0 and 9·0; and (3) relative activity towards specified strains of Staphylococcus aureus and Bacterium coli. Further tests depend on destruction of penicillin by penicillinase, colour reactions, and volatility of aspergillic acid in steam. For final confirmation, isolation of the antibiotic may be necessary.

SUMMARY: The action of several bacteriostatic substances on strains of pleuro-pneumonia-like organisms has been tested. Suitable concentrations of thallium acetate and penicillin in the media were inhibitory for ordinary bacteria, but allowed the pleuropneumonia-like organisms to grow in pure culture and facilitated their primary isolation. These two bacteriostatic substances were also useful for the isolation of strains on plate cultures. The addition of thallium acetate alone to media used for maintaining strains in subculture diminished the number of contaminations.

The media gave variable results, depending on the batch of serum used for enrichment. The routine addition of fresh yeast extract yielded consistently good growths of all strains of pleuropneumonia-like organisms tested. The effective factor in the yeast was not the ‘V’ factor required by Pfeiffer’s influenza bacillus.

SUMMARY: Penicillinase preparations strong enough to destroy all the penicillin in 1 hr. or less at room temperature should be used in carrying out sterility tests on solid penicillin. Otherwise, penicillin-sensitive contaminants may be killed by the penicillin before it is destroyed and samples containing them will be sterile by the test. A convenient unit of penicillinase is the quantity which will destroy 100 units of penicillin in 1 ml. in 1 hr. at room temperature (24°).

SUMMARY: A quantitative study of the survival of vegetative bacteria on drying in various suspending media led to a simple method for preserving bacterial cultures. Bacterial cells are suspended in melted nutrient gelatin cont.aining ascorbic acid or sodium ascorbate in concentration of 0·25–0·5%. Small quantities are dried over P2O5 at pressures of 100–300 mm, of mercury and stored in vacuo over P2O5 at room temperature. A wide range of bacterial species of medical and verterinary importance was preserved by this method for 4 years. The slow decline in the number of viable organisms and the high percentage survival rate at 4 years indicate the likelihood of survival for a much longer period.

The virulence of a number of pathogenic species was successfully maintained in the dry preparation. The method might well be applied to the preservation of living vaccines.

The survival rates with Chromobacterium prodigiosum dried by this method were better than those in preparations subjected to rapid freeze-drying processes. They were, however, unsatisfactory with a few species such as Vibrio cholerae and Neisseria meningitidies.