- Volume 7, Issue 1-2, 1952

With a note by K. E. COOPER on the theory of inhibition zones

SUMMARY: The time taken for agar plates to reach incubator temperature under different conditions was determined by thermocouple measurements. From 1 to 5 hr. was taken to reach within 1 of the incubator temperature, according to their mass, position, stacking and original temperature. Delay in heating slowed the growth of organisms and produced larger inhibition zones due to diffusing antiseptics. This leads to errors in sensitivity and thus in assay work unless technique is standardized to overcome these differences. Deviations from the calculations resulting from the formula of Cooper & Woodman (1946) can be overcome by such standardization.

An appendix discusses the theory of inhibition zones and compares the formulae proposed by Cooper & Woodman (1946) , by Vesterdal (1947) and by Mitchison & Spicer (1949) .

SUMMARY: Observations on the growth phases of Escherichia coli (Bacterium coli), American type ‘B’, on aerated media are reported. On casein-casein-yeast (CCY) medium the culture passes through the following phases: (a) an initial stationary phase; (b) a logarithmic lag phase; (c) a phase of rapid cell division; (d) a phase of equal division and growth rates; (e) a phase of diminishing growth and division rates. The average cell weight increases regularly through phases (a) and (b) to a maximum. It falls during phase (c) to a lower value at phase (d) and decreases finally to its initial value when the growth rate decreases more slowly than the division rate during phase (e). Similar phases can be recognized during growth on 2 % peptone. The onset of the phase of rapid cell division appears to be stimulated by the concentration of cell material.

SUMMARY: Wide fluctuations of phosphorus concentration in the cells occur during the cultivation of Escherichia coli (Bacterium coli) on aerated media. The concentration of cell phosphorus increases rapidly during the stationary phase and early lag phase to a peak which coincides with the formation of half the first generation; it declines until the end of this generation and increases once again to a second peak, declining subsequently when growth and division rates fall off. Analyses of the cells during cultivation show that the different concentrations of ribonucleic acid largely account for these variations. The association of the initial phosphorus peak with the first generation has been observed during the cultivation of Esch. coli on several liquid media and on CCY medium at different temperatures. The increase in phosphorus concentration during the first generation can be associated quantitatively with the increase in average cell weight.

Summary: A microtechnique has been developed for the growth of many cultures in one Petri dish. The agar substrate is in the form of small plaques pipetted so that each plaque is distinct and separate from its neighbour in the Petri dish. The method is used for the assay of antifungal materials and has been adapted to the isolation and biochemical characterization of micro-organisms. Work is in progress to adapt the procedure to yeast and bacterial counts.

Summary: The effect of various conditions on the rate of death of Bacterium coli and of Streptococcus faecalis in dilute buffer solutions was followed by making colony counts at intervals during prolonged periods of incubation and then constructing mortality curves. The form of these curves was characteristic for each organism. Of the pH values investigated (approximately 5, 6, 7 and 8) both organisms were least viable in the range 6 to 7. Strep, faecalis was not affected by the degree of oxygenation of the water but Bact. coli died much more rapidly under anaerobic than under aerobic conditions.

Growth of Bact. coli was observed with as little as 0·28 part per million organic matter in solution. A much higher concentration of nutriment was required for growth of Strep. faecalis; with a concentration slightly below that required for growth the viable population remained virtually stable for a long period. The rate of death depended on the age of the cells at the time of immersion in water.

Summary: A small lag section has been noticed in the standard curves obtained in the estimation of threonine using Streptococcus faecalis R. An increase in the acetate and phosphate buffers in the basal medium has been found to be advantageous.

Summary: Two mouse virulent strains of Staphylococcus aureus and their avirulent variants were tested for their resistance to the bactericidal power of fresh human blood and for toxin production.

The variants were much more easily killed by fresh blood than the parent strains and had lost the ability to produce α-haemolysin, dermonecrotic toxin and leucocidin.

The Panton-Valentine leucocidin test was supplemented by two others that gave more reliable results, one depending on direct observation of leucocytes emigrated from a clot on to the surface of a slide, the other on the destruction of bactericidal power of the leucocytes.

Summary: The growth of several adenine-independent strains of yeasts in defined media containing suboptimal concentrations of biotin and relatively high concentrations of methionine is associated with the formation of a pink pigment in the cells and of an arylamine in the culture fluid. Under similar conditions, pigment and arylamine are not produced by an adenine-requiring strain of Schizosaccharomyces octosporus. Culture filtrates containing arylamine promote the growth of Sch. octosporus in adenine-free medium, but an adenine-requiring lactobacillus responds only after a considerable lag. The results are interpreted to mean that the arylamine is, or is derived from, a precursor of adenine, whose conversion to adenine is impaired by deficiency of biotin.

Summary: A quantitative method for the study of the specific agglutination of micro-organisms is described. The degree of agglutination is estimated from the sedimentation curves of suspensions to which varying amounts of serum are added, which are compared with that of a control suspension without serum. The time of centrifugation required for sedimentation of 50% of the organisms in the serumsuspension mixtures related to the corresponding time for the control suspension gives the relative 50 % sedimentation times which, within a certain region, show a linear relation with the amounts of serum used. The activity of sera is expressed by their agglutinin content and by their agglutinating capacity, given by the slope of the time-serum regression line.

Rickettsias of the typhus group and their corresponding sera are clearly distinguished by the method described. Proteus OX19 agglutinating sera of different origins (pregnant women, murine typhus and Proteus-immune rabbit), with the same agglutinating titre by the dilution method, are distinguished according to their origin by the quantitative method. The application of the method to the study of other groups of micro-organisms is suggested.

Summary: The adaptation of a genetically deficient strain of Escherichia coli (Bacterium coli) to the utilization of lactose was shown to involve overgrowth of the population by a small number of mutants. The rate of mutation to lactose utilization is about 2 × 10−7 per bacterium per generation. This rate is not influenced by lactose or a series of substances related to that sugar. The distribution of these mutants among different cultures is in agreement with that expected on the basis of random mutation. These results are discussed in connexion with the theories of Hinshelwood on bacterial adaptation, and in relation to the problem of specifically induced mutation.

Summary: Counts were made of the total number of bacteria in cultures by a method that comprised suspension of the cells in molten agar, and the casting of this agar into a thin film in a haemocytometer chamber, followed by removal of the film from the chamber and drying in air. Counting of the bacteria was carried out with the use of a phase-contrast microscope. Statistical analysis of the counts indicated that the principal sources of variation lay in the differences between the numbers of organisms counted in successive microscope fields, between the total numbers in different traverses of the film, and between the primary agar dilutions of the culture.

Summary: Bacillus subtilis was grown in surface culture on media containing about 0·6 mC./ml. of 32P as phosphate, and harvested after 2 days. The spore suspension was heated at 60° for 1½ hr. and washed by centrifuging and resuspending in water six times. The fresh suspension contained about 2 × 109 spores/ml., of which 20 % were viable. The radioactivity was about 0°05 mC./ml. of which more than 95 % was carried by the spores. The radioactivity was sufficient for 103 spores to be readily detected and assessed by conventional Geiger-Müller counting methods. These highly radioactive suspensions are particularly useful in studying the respiratory retention of bacterial aerosols.

Summary: When Proteus vulgaris is grown in a simple defined medium with limiting concentrations of nicotinic acid but all other constituents in excess, growth as measured by dry-weight determinations continues for several hours after the disappearance of all detectable nicotinic acid from the medium. A linear phase of protoplasmic synthesis and of oxygen uptake follows this disappearance and lasts for 2·5–3 hr., while the amount of recoverable nicotinic acid in the cells remains constant. It is suggested that, during the period of linear growth, the rate of protoplasmic synthesis is determined by the presence within the total cell population of a more or less constant amount of nicotinic acid or its active derivatives.

Summary: Grown at 22° Pasteurella pseudotuberculosis was motile and had flagella and H antigen; grown at 37° it was non-motile and had neither flagella nor H antigen. The change occurred over a narrow range of temperature about 30°.Cells grown at 37° when transferred to 22° developed flagella without cell division; cells grown at 22° when transferred to 37° became non-motile, due to an unexplained adverse effect indicated by a high death-rate. It appeared that different metabolic processes were used at the two temperatures; it was not necessary to postulate the selection of a naturally occurring mutant and no evidence was obtained for the existence of an extracellular enzyme which would destroy flagella at 37°.

In addition to the ‘short-term’ effect there was a ‘long-term’ effect of temperature shown by a difference in degree of motility and rate of growth (at 22°)of cultures grown for several months at 22 or 37°.This may have been an extension of the ‘short-term’ effect, a period of training being required for the optimal functioning of the metabolic processes concerned. The long-term effect could be reversed by prolonged cultivation of the 22° substrain at 37° and vice versa.

Preliminary experiments indicated that virulence was affected by the temperature of cultivation.

SUMMARY: The distribution of antibiotic (or other substance) in the agar around a container or around a hole in a punch-plate can be expressed theoretically by an equation involving: the initial quantity of antibiotic, the depth of the agar layer, the diffusion constant, the concentration at a given distance from the container, and the time of diffusion. The validity of the equation was confirmed by measurement of the diffusion constants of penicillin, streptomycin and aureomycin, and of the critical concentration of these substances required to inhibit test organisms, followed by the use of the values so obtained to predict the sizes of the inhibition zones produced experimentally by these antibiotics after varying periods of diffusion.

The theory predicts that the square of the inhibition zone diameter will be proportional to the logarithm of the antibiotic concentration. This relationship was found to hold, when accurate assays were made, for a number of antibiotics but not for penicillin when tested with Bacillus subtilis. The most important factor determining the slope of the dose-response curve under given conditions is the diffusion constant of the antibiotic. The slope can, however, be increased by prolonging the time allowed for diffiusion.

Particular factors which affected both the sharpness of the zone edge and the nature of the dose-response curve were production of small amounts of penicillinase by strains of B. subtilis used for penicillin assay, and uptake of streptomycin by organisms used for streptomycin assay. Measurements of adsorption of streptomycin by B. subtilis, B. pumilus and by Staphylococcus aureus were made, and were shown to fit equations of the Freundlich isotherm type.

SUMMARY: The invention of glass electrothermal heating tape provides a simple method for heating a hot box or incubator without the expense and trouble of a water jacket. An outfit is described for observing microscopically and photographing cultures at controlled temperatures over long periods.

SUMMARY: In a suitable buffer at 39 ° the life of all three species of holotrich ciliates of the sheep’s rumen can be extended for one or more days by addition of glucose, fructose, galactose, sucrose, cellobiose, raffinose, inulin, bacterial levan, salicin or melibiose (the least effective). Mannose, glucosamine or galactosamine are definitely toxic in that they greatly shorten protozoan life. This toxic effect is observed even in the presence of glucose. Isotricha prostoma and I.intestinalis, but not Dasytricha ruminantium, will ingest vegetable starch of small grain size, e.g. rice starch, thereby prolonging protozoan life.

The nitrogenous requirements of these protozoa are best met by whole grass juice, which extends the life of the culture for several weeks; and even the ethanolic precipitate from boiled and cleared grass juice is a better nitrogenous supplement than cleared rumen liquor. The ash from this alcoholic precipitate will definitely extend protozoan life in absence of nitrogenous supplement. The effect is probably due to a ‘trace’ metal, which is not Zn, Fe, Sn, Sr, Mn, Cu or Ni, but may be Ti, Mo, Cr, Co or V.

The holotrich ciliates here studied do not ingest lactobacilli, either when starved or when in an actively motile state after addition of glucose to the medium.

SUMMARY: Culture filtrates of Trichothecium roseum contain two heat-stable substances that inhibit infection with plant viruses. One is trichothecin, an antifungal substance with the molecular formula C19H24O5; it sometimes visibly damages bean leaves. The other was isolated as a polysaccharide, [α] D 19° = −33° it contains 1·1–1·4% nitrogen, yields 60–70% reducing sugars (as glucose) on acidic hydrolysis, and the predominant (45%) component sugar is d-galactose. The polysaccharide does not combine with tobacco mosaic virus in vitro.

The extent to which infection is inhibited depends on the species of the host plant but not on the identity of the virus. Trichothecin, like ribonuclease, is relatively more effective with beans than with Nicotiana glutinosa, whereas the polysaccharide and two derivatives of trichothecin (trichothecolone and acetyltrichothecolone) affect N. glutinosa more than beans. Trichothecin inhibits infection when sprayed over leaves a day after they have been inoculated with viruses, but is ineffective when applied 2 days before. The polysaccharide inhibits when sprayed over leaves before inoculation but not after. It is suggested that inhibitors act by temporarily altering the metabolism of leaf cells so that introduced virus particles cannot multiply and are inactivated.

SUMMARY: Bacterial suspensions of capsulated Haemophilus pertussis, prepared from cultures freshly isolated from infected mice, or from Phase I strains maintained on Bordet Gengou medium, produced in rabbits antisera which contained specific agglutinating and complement-fixing antibodies and which protected mice against experimental pertussis infection. Organisms which had been washed three times with distilled water and freed from capsular material also produced similar antibodies. More than one antigen may be involved in the production of these antibodies.

On the other hand, bacilli-free preparations, containing the capsular material in a concentrated form, produced antisera with only low levels of agglutinating, complement-fixing and protective antibodies.

The experiments indicated that the antigens of H. pertussis which produced these antibodies are somatic rather than capsular.