- Volume 1, Issue 3, 1947

SUMMARY: A strain of Lactobacillus plantarum has been isolated from Sauerkraut which can produce acetylcholine during growth and in washed suspensions. The conditions necessary for the production of acetylcholine are: (i) the presence of choline and (ii) the simultaneous fermentation of carbohydrate. The acetylcholine is formed inside the cells and subsequently passes into the medium. The amount formed can be about 5 μg. acetylcholine/mg. dry wt. cells/hr. The acetylcholine was identified by the usual biological tests and was also isolated and identified as the salt of hexanitrodiphenylamine. The mechanism of the acetylation has not been discovered. Cells grown in the absence of added pantothenate acetylate extremely slowly. The rate is restored by the addition of pantothenate to the cell suspension.

SUMMARY: The level of glutamic acid concentration measured inside the streptococcal cell during assimilation represents a balance between the rate at which the amino-acid is withdrawn from the external medium and the rate at which it is metabolized within the cell.

Treatment of the cells, prior to or during assimilation, with dyes of the triphenylmethane series results in raising the level of free glutamic acid attained within the cell. Evidence is presented that the triphenylmethane dyes prevent the metabolism of glutamic acid within the cell. The activity of the triphenylmethane molecule as an antibacterial agent and in raising the level of glutamic acid assimilation is increased by alkyl substitution and can be correlated with the lipid solubility of the dye.

SUMMARY: The effect on glutamic acid assimilation of the addition of penicillin to growing cultures of Staphylococcus aureus is described. When Staph. aureus is grown in media containing glutamic acid this substance accumulates in steadily increasing concentration in the cells. The addition of penicillin to the medium is followed after an interval by rapidly decreasing concentration of glutamic acid within the cells.

The assimilation of glutamic acid by normal washed cells is not affected by penicillin in high concentration. The assimilation of glutamic acid by cells which have grown in the presence of penicillin is impaired and may be completely inhibited. Complete inhibition of assimilation is brought about by bactericidal concentrations of penicillin, low concentrations requiring a longer time to become completely effective than high ones. The loss of assimilatory power can be correlated with loss of viability.

Comparison of the general properties of normal and penicillin-inactivated cells show that the respiration, glucose oxidation, glucose fermentation and lysine assimilation of the latter are normal. The internal metabolism of glutamic acid is normal in penicillin-treated cells, but, since the passage of glutamic acid across the cell wall is blocked, is limited by the existing internal concentration.

SUMMARY: The level of free glutamic acid accumulating within cells of certain Gram-positive cocci is lower in growing cells than in ‘resting’ cells, other conditions being equal. Part of the glutamic acid assimilated by growing Staphylococcus aureus is condensed into peptides or proteins, thus accounting for this apparent drop in glutamic acid accumulation. Sulphathiazole interferes with this condensation of glutamic acid into peptide form.

SUMMARY: Working details are given of a method for producing streptomycin by the surface culture of Streptomyces griseus in pint milk bottles on a papain digest of beef+meat extract+glucose+mineral salt medium. Streptomycin titres in the crude culture filtrates of 250 µg./ml. or more were obtained after 10–14 days’ growth.

Evans’s peptone, papain digest of spent pancreas from insulin manufacture, papain digest of yeast, and a proprietary casein-meat hydrolysate were found to be possible alternative sources of organic nitrogen. Growth was good on media containing peptic digest of beef or peptic-tryptic casein digest, but the streptomycin there was low.

The utilization of glucose and the production of streptomycin depended on the relative amount of nitrogen present in the medium.

SUMMARY: A method is described for extracting streptomycin from culture filtrates by adsorption on charcoal at pH 6–8, elution with 1·2% (v/v) aqueous phosphoric acid, readsorption of the eluate on charcoal at pH 7, elution with acidified methanol, followed by evaporation at reduced pressure and precipitation of streptomycin by dilution of the concentrated methanol eluate with 5 volumes acetone or amyl acetate. An indication is given of the order of recovery, and the potency of the product obtained. The stability of streptomycin under the conditions of pH and temperature to which it may be subjected during the extraction is outlined. The process has been carried out in pilot-scale production equipment handling 1500 1. batches of culture filtrate.

SUMMARY: A dilution method for the assay of streptomycin is described using a digest nutrient broth as the test medium and a certain strain of Escherichia coli (Bacterium coli) as the test organism. The inhibition end-point is estimated turbidi-metrically by comparison with that in a standard solution of streptomycin base. The accuracy of the method when employed as described is of the order of ± 15%. It is necessary to control the dilution of the test medium, the temperature of incubation of the tests, the size of the inoculum and hydrogen-ion concentration in order to obtain consistent results. With dilution of the medium the sensitivity of the test is increased while its accuracy is decreased. The use of an inoculum of constant size is important, and an incubation temperature of 28° is found to be more satisfactory than 37°. Medium with a hydrogen-ion concentration of pH 7·6 affords greater sensitivity than that with a lower pH value.

SUMMARY: When spores of Dictyostelium mucoroides or D. giganteum were added to sterilized soil containing a pure culture of an edible bacterium, the resulting myx-amoebae actively destroyed the bacteria in the soil. When spores of D. mucoroides were added to the centre of a Petri dish of sterilized soil containing bacterial food no fruiting bodies were formed at a moisture content of 15% or less. At 19% moisture fruiting bodies were formed at the centre only. At 33% moisture the central area over which fruiting bodies appeared steadily increased in size until it covered the whole soil surface. Species of Dictyostelium can also pass through the life cycle in fresh unsterilized soil.

The nature of the bacterial food supply affects the growth of species of Dictyostelium in soil as measured by fruiting body formation. Normal fructification occurs on soil containing certain bacterial strains which induce abnormal fructification on agar.

SUMMARY: By means of a simple atomizer the surface of agar media may be sprayed with a suspension of micro-organisms, so that an even growth is obtained. The method is suitable for the seeding of plates for the assay of antimicrobial substances. It is particularly valuable for the detection of microbial antagonisms, for a test organism may be seeded on to established plate cultures of other organisms, without disturbing the bacterial colonies already present on the agar surface.

SUMMARY: Optimal yields of penicillinase were obtained by the continuous addition of penicillin to cultures of certain strains of Bacillus subtilis during their logarithmic growth phase.

Lower yields of enzyme were obtained if the penicillin was added when growth had ceased or to partly grown cultures kept at rest and subsequently shaken.

Partial lysis of the bacteria was accompanied by an increased yield of enzyme, but complete lysis appeared to result in partial destruction.

SUMMARY: The production by certain species of Clostridium of enzymes disintegrating hide powder was investigated by measuring the lytic action of broth cultures and toxic filtrates on finely divided hide powder suspended in an agar gel.

Cl. histolyticum was the most active producer of enzyme, Cl. welchii A was less active and Cl. sporogenes and Cl. bifermentans only moderately active. Cl. tetani, Cl. oedematiens and Cl. septicum produced no such enzyme. The lytic enzyme of Cl. histolyticum is not the lethal toxin.

Among strains of Cl. welchii type A, enzyme production, α-toxin production and ability to cause fatal infection in guinea-pigs are associated.

There was some evidence that the different enzymes affecting hide powder are antigenically related, but no definite conclusion is possible, since the antisera employed may have contained antibodies to the lytic enzymes of a number of different organisms.

SUMMARY :Clostridium bifermentans elaborates a lecithinase which induces turbidity and curding in crude lecithovitellin preparations, and hydrolyzes egg-yolk lecithin with the liberation of acid-soluble phosphate, both over a pH range of 50–63 with a maximum in the region of pH 6·0. It does not induce turbidity in human serum alone, but does so in the presence of agar gels, which activate the reaction over a pH range of 50·80. The activities are not dependent on the presence of Ca++ ions. The enzyme responsible appears to be a lecithinase C.

In the concentration available the lecithinase haemolysed rabbit and mouse, but not human, horse, sheep or guinea-pig red cells; and it was not lethal on intravenous injection into mice, but was slightly toxic in the skin of guinea-pigs.

The Cl. bifermentans lecithinase C is antigenically related to the lecithinase C (the α-toxin) of Cl. welchii, though differing from the α-toxin in its pH optima, haemolytic powers, independence of Ca++ ions and relative non-toxicity. It is neutralized by Cl. welchii α-antitoxin, and Cl. bifermentans antilecithinase behaves in a reciprocal manner, neutralizing the lecithinase, necrotizing and lethal action of Cl. welchii α-toxin. The antigenic relation of the two lecithinases appears to be remote.