- Volume 4, Issue 3, 1950

SUMMARY: Studies on the anaerobic growth of aerobically adapted purple bacteria show that the same growth factors suffice for both aerobic and anaerobic growth, thus supporting van Niel's formulations of photosynthesis and carbon assimilation in these forms, and his thesis that the previously observed need for peptone or yeast extract could be attributed to their content of essential growth factors. Combinations of glutamate and succinate (or fumarate) were especially good promoters of growth in malate media; it is possible that their effectiveness was referable to their allowing a by-pass of the CO2 requirement. The production of molecular hydrogen by purple bacteria is discussed in relation to the reducing intensities attained in cultures.

A bottle technique for anaerobic cultures, and a flask technique for aerobic cultures, are described in detail.

SUMMARY: A yellow-pigmented Streptococcus was isolated from certain dairy cows. The three strains examined were closely related to Strep. faecalis but differed from it in fermenting raffinose. The organism produced a tyrosine decarboxylase. Serologically the organism belonged to Lancefield group D, Sharpe type 10.

SUMMARY: Phage-resistant mutants were obtained from four strains of Rhizobium trifolii. Mutation to phage resistance tended to coincide with mutations in other features, such as morphology of colonies or effectiveness in nitrogen fixation. The accompanying mutations occurred independently, and their frequency varied widely from strain to strain.

Some mutants seemed stable in their newly acquired features; others continued to mutate at high rates.

Some of the mutants as regards nitrogen fixation were stable or could eventually be stabilized, but other mutants derived from one of the parent strains remained unstable even after several successive replatings or passages through nodules.

SUMMARY: Seventeen representative strains of pleuropneumonia-like organisms were investigated to determine whether they could be identified by cultural methods.

The L1 organism was readily identified by its morphology and by its cultural appearances on solid and in fluid and semi-solid media. Differences, depending on whether growth occurred anaerobically, whether growth was smooth or granular and whether a precipitate was formed in horse serum media, served to distinguish most of the other strains.

The requirements of serum and yeast extract for promoting growth and the effect of pH were studied with each strain.

Several strains fermented carbohydrates. Young cultures of some strains reduced methylene blue. Haemolysis around colonies in horse blood agar was noted with some strains; young broth cultures of these strains discoloured suspensions of horse erythrocytes. The factor discolouring erythrocytes was found in filtrates of old cultures of Asterococcus bovis.

Two strains were pathogenic for mice; when recovered after passage they showed no alteration in their nutritional requirements.

The organisms were resistant to penicillin; one strain grew in a medium containing 3000 units/ml.

SUMMARY: The rate of germination (defined as loss of heat-resistance accompanied by change in staining properties under specified conditions, with maintenance of viability) of thick suspensions of Bacillus subtilis spores in phosphate-buffered l-alanine solution increased with the time from harvesting. The maximum rate of germination was reached after about 20’ days storage in water at 20°. This effect could be retarded, but not reversed, by storage at low temperatures. The rate of germination may be temporarily accelerated by heat treatment. Germination was considerably retarded after treatment with mercuric chloride, and was completely inhibited by 8-hydroxyquinoline (oxine) and by 2:3-dimercaptopropanol (BAL) at 10 mm concentration. The latter effect was partially reversed by the addition of metals.

SUMMARY: For viable counting, 0·1 ml. volumes of bacterial suspension were incorporated in 1·5–2 ml. nutrient agar, which after mixing was allowed to solidify in an almost horizontal test-tube so as to produce a 3–4 in. agar strip. After incubation in the vertical position, the counts of the strips gave estimates of a bacterial population which did not differ significantly from that obtained from plates inoculated with 1 ml. quantities.

SUMMARY: Recently isolated virulent strains of Haemophilus pertussis grow well in a partially defined medium, but are more exacting in their growth requirements and conditions of growth than avirulent strains. The optimal pH for growth is 7·6–7·8. Mechanical agitation by stirring, spinning or aeration with either nitrogen or air, inhibits growth. Gaseous conditions, however, are critical in that there is an optimal relationship of surface area to volume of medium. Mechanical agitation and aeration stimulate the growth of avirulent strains.

Virulent strains require starch for growth, and there is a close relationship between virulence, specific agglutinability and starch requirement. Avirulent strains with low specific agglutinability grow readily in the absence of starch.

When starch in a standard medium is replaced by amylose, there is an increase in the final amount of growth. Amylopectin, glycogen and dextrans are about one-third as effective as starch; other carbohydrates, gums and inorganic adsorbents support little growth. Charcoal can replace starch, but has only c. 70% of its effect.

The amino-acid requirements of H. pertussis are satisfied by acid-hydrolysate of casein, the optimal concentration being 7%. All strains utilize aspartic and glutamic acids, serine, threonine, glycine, alanine and proline, but the rate of utilization is greater with virulent than with avirulent strains.

For good growth yeast extract is needed but can be replaced by nicotinamide, nicotinic acid or cozymase, provided sulphur-containing amino-acid is also present. Virulent strains of H. pertussis require a sulphur-containing acid for growth, which may be supplied by yeast extract. Cysteine, cystine or glutathione, but not methionine, act as sources of essential sulphur.

SUMMARY: Enzymes elaborated by Clostridium histolyticum and by Cl. welchii type A that disintegrated the organic component of human tooth dentine were compared with those attacking muscle collagen.

The potency of specific antisera to neutralize the enzymes disintegrating the organic component of dentine was determined by using as indicator finely divided decalcified dentine suspended in an agar gel.

By comparing the potency values of the sera with those obtained when muscle collagen preparations were used as indicators, it was evident that the organic component of dentine was attacked by the collagenases of Cl. welchii type A (κ antigen) and Cl. histolyticum (β antigen) and probably by the γ antigen of Cl. histolyticum. The λ antigen of Cl. welchii type B had no action on decalcified dentine.

SUMMARY: Three antigenic components (α, β, γ) are present in toxic culture-filtrates of Clostridium histolyticum. Of these α is the lethal and necrotizing toxin, β is a collagenase, and γ is a cysteine-activated proteinase which attacks altered collagen (e.g. hide-powder or azocoll) but not native collagen. The β and γ-enzymes both attack gelatin. By methods based on the properties of these antigens, Cl. histolyticum antisera can be tested for the corresponding antibodies.

SUMMARY: The intermediate type I coliform organism 1433 can use, as hydrogen donators during the adaptive formation of the tetrathionase enzyme system, most of the carbohydrates that it can ferment, although in some cases the cells must first be grown on a medium containing the carbohydrate concerned. Not only the reduction of tetrathionate, but the process of adaptive formation of tetrathionase as well, is inhibited by oxygen gas, but the capacity to adapt when favourable conditions are restored is relatively unaffected by oxygen. The amount and the rate of formation of new enzyme are greatly increased by providing a source of available nitrogen. The rate of formation is also increased by incubating the cells with mannitol and phosphate before tetrathionate is added, but the total amount of enzyme formed is not increased: it is probable that the increased rate is due to the accumulation of intermediates within the cells; these intermediates have not so far been identified. None of the mathematical expressions investigated fits the course of tetrathionase adaptation exactly; that put forward by Spiegelman (1945) for the production of yeast galactozymase approximates most closely.

Cells of a tetrathionate-reducing coliform organism growing in semi-anaerobic conditions gained no advantage from the presence of tetrathionate at 41°, but at lower temperatures (e.g. 34°) they grew much better with than without it. When freely supplied with oxygen, the cells grew about as well at 41° as at 34°. The adaptive formation of tetrathionase in washed suspension has already been shown to diminish with increase of temperature from 34° to 44°, whereas the activity of the enzyme when formed increases with temperature in this range; nitratase, on the other hand, is still actively formed at temperatures as high as 44°. It is clear that whatever factors may be necessary for adaptation, one of them is more sensitive to heat than either nitratase formation or the overall growth process.

SUMMARY: The fluorescence of tubercle bacilli stained with auramine and rhodamine does not require ultra-violet light. It can be caused by blue light up to 496 mμ. A copper sulphate ammonia liquid filter, suitably diluted, will transmit this waveband. A gelatin screen filter, absorbing all light below 510 mμ. is used in the eyepiece. Normal, high intensity projection filament lamps, combined with lamp condensers of large aperture, provide suitable light sources. The numerical aperture of the microscope condenser must be fully used by immersion in glycerol. The fluorescence is very bright with the usual biological, and certain binocular, microscopes. The simplicity of the equipment enables fluorescence microscopy of tubercle bacilli to be used at low cost in any laboratory.

SUMMARY: A method is described for measuring the purine-pyrimidine absorption of Micrococcus pyogenes var. aureus (strain Duncan) (M. pyogenes; Staphylococcus aureus) in intact cell suspensions with the Beckman Model DU spectrophotometer, from which values for the total ‘nucleic acid’ content of the cells may be obtained. The approximate nature of the method is discussed, but results are presented which show that the values obtained throughout a normal growth of a culture of M. pyogenes compare very favourably with the values given by the Schmidt & Thannhauser (1945) technique. Some observations on Escherichia coli (strain H) suggest that the method may be applicable to organisms other than Micrococcus pyogenes.

SUMMARY: The sampling tube from the fermentation vessel is connected to a tube passing loosely through the specially fitted cap of a standard screw-capped bottle. By means of a mouthpiece suction can be applied so that a sample of fluid is drawn over into the bottle, which is then replaced by a similar empty sterile bottle. By fitting an extension of the tube to pass to the bottom of the bottle, and by blowing instead of sucking, fluid in the bottle may be driven over into the fermentation vessel. The device may be used for certain other operations such as removing or transferring sterile solutions or suspensions.

SUMMARY: The microcysts or resting cells of Bacteriaceae have three typical forms, which are regular and recognizable, and by which the Family may be divided into three groups.

The first type of microcyst is small, oval and eccentrically nucleate; it is typical of Bacterium coli, and Proteus, Pseudomonas and Salmonella spp. with the exception of Salm. typhi which forms a very large, oval microcyst, with a central nucleus. The microcysts of Shigella spp. resemble those of Salmonella typhi. Bacterium aerogenes produces large, spherical or oblong microcysts with a small, central nucleus.

SUMMARY: Marcescin, a thermostable polypeptide isolated from a strain of Serratia marcescens inhibits the growth of Corynebacterium diphtheriae at 0·01 μg./ml. and Staphylococcus aureus at 0·1 μg./ml. It is produced in good yield in 18–36 hr. at 24° in an aerated ammonium citrate glycerol medium. It was very toxic to mice and in sublethal doses failed to protect mice against sensitive organisms. It is very strongly adsorbed by bacteria so it is not possible to say whether it is bacteriostatic or bactericidal in high concentration.

SUMMARY: The individual animals in a large batch of European tree-frogs imported into England from Italy were found to be suffering from what proved to be, with two exceptions, a uniformly fatal attack of typical red-leg. Bacterium alkaligenes was isolatedin profusion in pure culture from the heart blood and lymph fluid of all the frogs examined. The strain was highly susceptible to streptomycin, and the only animals to recover were two that were treated with 1000 units of the antibiotic, in divided doses over 9 days.

SUMMARY: The method used in maintaining cultures of Aerobacter aerogenes affects the relationship between duration of lag phase and age of inoculum in a glucose ammonium salt medium. Organisms kept in broth may show pronounced early lag whilst those from agar slopes do not. Previous subcultivation with aeration decreases the lag. Bacteria showing no lag may do so when they have been washed; glutamate, α-ketoglutarate and succinate partially remove the lag so produced, whilst malate, fumarate and aspartate do not.

The initial growth rate of light inocula showing no lag is increased by the addition of glutamate, aspartate, α-ketoglutarate, succinate and oxalacetate; but pyruvate, fumarate and malate have no action or are slightly inhibitory. The effect of these compounds over a concentration range of 0 to 1·6 × 10−3 g. mol./l. was studied quantitatively. Filtrates from growing cultures have a similar effect on initial growth rate; those taken during the late logarithmic phase are more effective than those taken earlier. The effects of both filtrates and added compounds are confined to initial growth; the rate of growth in the later phase, when turbidity becomes visible, remains unaltered. This behaviour is dependent on the amount of O2 available in the medium.

Early lag is a phase of greatly reduced rate of growth rather than complete absence of cell division. Glutamate and other effective compounds increase the rate to values approaching normal.

SUMMARY: A colony illuminator is described which incorporates side illumination by means of a small ring fluorescent tube. Inspection of the Petri dish is facilitated by the use of a detachable 5 in. diameter plastic industrial inspection lens.