- Volume 19, Issue 2, 1958

SUMMARY: A continuous culture apparatus was used to study the effects of air supply and of growth rate on the respiratory activities and the cytochrome content of a strictly aerobic fluorescent pseudomonad. The oxidizing capacity, measured as Qo2(succinate), of organisms grown with air as the growth-limiting factor was lower than that of organisms grown with succinate as growth-limiting factor, and with air supply in excess. There was no significant correlation between oxidizing capacity and growth rate under either of the two conditions. Estimation of the cytochrome content of whole organisms grown under these different conditions showed that this tended to decrease with increase in growth rate. Organisms grown with air as the growth-limiting factor always had about double the cytochrome content of cells grown at the same rates but with succinate as the growth-limiting factor.

SUMMARY: Monolayer cultures of HeLa cells were exposed to a high multiplicity of herpes virus per cell. Samples of cells taken at successive time intervals after infection were sectioned and examined by electron microscopy for characteristic ringed particles. Replicate cultures of cells were assayed for infective virus. Despite an estimated input of 6 HeLa plaque-forming units (pfu)/cell, an infective centre count indicated that only 6% of the cells yielded virus. No increase in infective virus was found in the cultures at 9 hr. and no characteristic particles were found in sections of 28 cells. At 12 hr. new virus appeared in the cell fraction and particles were found in the nuclei of 2 of 25 cells. At 26 hr. after infection and later, there was a large increase in virus in both cell fraction and medium, and large numbers of particles were present in the nucleus and cytoplasm and on the cell surface.

SUMMARY: Less than a tenth of the pock-forming particles of the HFEM strain of herpes virus are able to initiate infection in HeLa cells, even after passage in these cells. The pock-forming virus attaches firmly to the cells but most of it remains susceptible to antiserum and presumably does not penetrate the cell. A small proportion of virus initiates infection in the HeLa cells, and, after exposure to more than one HeLa infectious particle/cell, new virus first appears in the cell fraction 12 hr. after infection, and 4 hr. later virus is released into the medium. Virus in the cell fraction can be detected either by disruption of the cells, or simply by removing the cells from the glass with ethylenediaminetetra-acetic acid (EDTA) or saline. This ‘EDTA fraction’ may contain virus released from the cell surface. An attempt was made to determine the number of virus-yielding cells, by inoculation of whole cells on to the chick chorioallantoic membrane. The proportion which yielded virus was lower than would be expected from the input of HeLa-infectious virus. After removal of superficially attached virus with antiserum, it was not possible to detect infective virus during the latent period in the majority of cells which ultimately released virus.

Herpes virus is well known as a cause of latent infection, and it is our interest in latency which has prompted further investigation of the growth of this virus. Previous work on herpes virus multiplication has been carried out in the chorioallantoic membrane cavity of fertile hens’ eggs (Scott, Coriell, Blank & Gray, 1953; Wildy, 1954; Yoshino & Taniguchi, 1956a, b) and in explants of rabbit cornea (Scott, Burgoon, Coriell & Blank, 1953). In recent years a more quantitative approach has been possible through the use of cell suspensions and monolayer cultures, and growth studies have been reported by Gostling & Bedson (1956) and Gostling (1956) in chick embryo cells, and more recently by Kaplan (1957) in rabbit kidney cells. We have studied the growth of herpes virus in HeLa cells because of the desirability of propagating cells indefinitely when studying latent infection. HeLa cells are also convenient because they are suitable for isolation of cell clones, and for study of virus release from isolated single cells.

This paper describes some aspects of virus growth in monolayer cultures of HeLa cells. Studies on plaque formation in HeLa cells (Farnham, 1958), cytological observations (Ross & Orlans, 1958), chemical changes (Newton & Stoker, 1958), electron microscope studies (Stoker, Smith & Ross, 1958) and single cell experiments (Wildy, P., Stoker, M. G. P. & Ross, R. W., unpublished) will be given in other communications.

SUMMARY: Ammonium alginate wool has proved to be a suitable collecting filter for estimating a small number of micro-organisms in a large volume of air.

SUMMARY: The literature of ‘volutin’ granules is reviewed in an attempt to clarify the meaning of the term in respeet to trypanosomes. Fifteen species of trypanosomes have been examined by phase-contrast microscopy and the morphology and natural history of their cytoplasmic inclusions described. It is suggested that the formation of inclusions in trypanosomes circulating in the blood of mammals represents a constant phenomenon which is closely related in time to the immune reaction of the host; other evidence suggests that this relationship is not fortuitous. In addition, the formation of inclusions in Trypanosoma rhodesiense and T. lewisi appears to be related to polymorphism, notwithstanding the wide differences in the manner and time in which polymorphism (and associated cytoplasmic inclusions) occur within their cycles in the vertebrate host.

SUMMARY: Strains of Pasteurella pestis under certain conditions form bacteriocin-like material, for which the name ‘pesticin’ is proposed; it inhibits the growth of P. pseudotuberculosis. The conditions necessary for induction of pesticin formation are similar to those which lead to induction of formation of some known bacteriocins. Of 24 strains of P. pestis tested, all but one produced pesticin. A mutant of P. pseudotuberculosis which was resistant to pesticin produced by one strain of P. pestis was found to be resistant to pesticin produced by all the other strains.

SUMMARY: Nutritional experiments were carried out dealing with: (1) the utilization of carbohydrates; (2) the nitrogen source; (3) the action of different mineral salts and their interaction. The results showed that the strains of Zygorhynchus used grew well at 25° at pH 8·0 when buffered with nutritionally inert tris buffer and incubated for 12–14 days. Statistical analysis of the results showed that under the given conditions optimal growth measured as mg. dry wt. was obtained in liquid media containing 4% (w/v) glucose; 0·1 m tris buffer; 0·00625 m-(NH4)2SO4; 2 p.p.m. Fe, Zn and Mn; with the following optimal conditions for the different strains specified: (i) for Z. moelleri CMI 0·002 m-KH2PO4; 0·012 m-MgSO4. 7H2O; (ii) for Z. moelleri Baarn 0·002 m-KH2PO4; 0·006 m-MgSO4. 7H2O; (iii) for Z. vuillemini CMI 0·002 m-KH2PO4; 0·003 m-MgSO4. 7H2O; (iv) for Z. vuillemini Baarn 0·004 m-MgSO4. 7H2O. There was a balance between the possible pairs of the salts from KH2PO4, MgSO4. 7H2O and (NH4)2SO4 (in one case only between KH2PO4 and MgSO4.7H2O). There was an optimal balance between all the salts for two strains. The best carbon sources were glucose, fructose, mannose, xylose and arabinose. The best sources of nitrogen were l-glutamine, l-asparagine, glycine, (NH4)2SO4.

Three selective media are described for the isolation of Pasteurella pestis, P. septica, P. pseudotuberculosis and P. haemolytica from material heavily contaminated with other bacteria.

SUMMARY: During the 6- to 7-week active life of soybean root nodules produced by a single strain of Rhizobium japonicum, the respiratory activity of the bacteria isolated from the nodules changed according to a definite pattern: a depression occurred 2 weeks after nodules first appeared, followed by a rise, and with a further sharp rise 1 week before nodule breakdown. The initial depression coincided with the onset of the bacteroid condition and the beginning of nitrogen fixation. The limits within which respiration varied depended on host-plant growth. The nitrogen-fixation rate remained constant from 2–3 weeks after nodule appearance until a few days before nodule decay began. The bacteroids isolated from the nodule did not oxidize substrates of the tricarboxylic acid cycle to completion, but the number of mole O2 consumed/mole substrate did not change during nodule life, and were similar to values obtained for cultures of the free living bacteria. Changes in bacteroid dry weight and nucleic acid content were observed; these were within the limits of dry weight and nucleic acid content of the same bacterial strain grown in liquid culture. The ratio acid soluble nucleotides: nucleic acid for bacteroids was between 0·12 and 0·13 during nitrogen fixation; these values are characteristic of bacteria of the same strain from the mid-logarithmic phase of growth in culture in spite of the fact that bacteroids are non-proliferating cells.

SUMMARY: Concentrated Brucella abortus strain 19 vaccine is shown to deteriorate in time, even at low temperatures (4–7°). The loss of viability of vaccine sealed in pure nitrogen can be expressed as a coefficient. The value of this coefficient may be derived from the formula of a straight line fitting the means of the logarithms of the viability counts made 1 week to 6 months after freeze-drying.

SUMMARY: Strains of Pseudomonas and Achromobacter spp. isolated from soil, utilized quinic acid as a sole source of carbon. Protocateehuic acid was produced as an intermediate in this oxidation. Protocateehuic acid was also produced in the course of oxidation of p-hydroxybenzoic acid by these organisms. Sequential induction experiments indicate that p-hydroxybenzoic acid is not an intermediate in quinic acid oxidation by the organisms used.

SUMMARY: Above a value of one, repeatedly doubling the multiplicity of infection of chick embryo cells by vesicular stomatitis virus progressively shortened the latent period by about 0·6 hr.; this phenomenon is referred to as ‘shortened latency’. Varying the multiplicity above unity with dilute-passage stocks did not interfere with rate of infective virus release, number of cells infected, or final yield, i.e. there was no ‘von Magnus’ effect or other obvious interference phenomena.

The doubling time for virus release was also about 0·6 hr. This suggested that virus may have been growing as a simple intracellular pool equally accessible to all adsorbing virus, and that 1 particle was released when the pool reached a certain size (perhaps 20–200 units) irrespective of inocula. However, other explanations are possible, and of those allowing experimental test, earlier initial adsorption of virus, multiplicity reactivation amongst a partly inactivated population, more rapid elution of attached virus or more rapid release of accumulated internal virus could not account for shortened latency.

SUMMARY: Placing live or inactivated vesicular stomatitis virus of one serotype on chick cells in tissue culture prevented most of the cells from releasing infective virus of the other serotype when super-infected with it (heterotypic exclusion). Inactivated virus did not prevent super-infection with the same serotype and had no effect on the latent period or rate of virus release (homotypic non-exclusion and non-interference). The ‘Indiana’ serotype was more effective as heterotypic excluding agent than was the ‘New Jersey’ serotype, and exclusion was noticeable when only 12 min. elapsed between interfering and challenge virus. Each cell liberated virus of only one serotype when infected with live virus of both serotypes, but the serotype released was often (20–40 %) not that of the particle first adsorbed. Heterotypic exclusion in fact behaved as if it were reversible and dependent on the multiplicities of infection, at least within the latent period. Many inactivated particles per cell were adsorbed before heterotypic exclusion was achieved.

SUMMARY: Escherichia coli B10, a mutant strain of E. coli B, which exhibited a requirement for histidine + uracil under certain conditions of growth, became pyrimidine-independent when grown in a simple medium supplemented with these two substances. It was demonstrated that this change from uracil-dependence to non-dependence was not due to the selection of a competent back-mutant, but to the formation of the enzyme dihydro-orotic acid dehydrogenase, which is lacking in Uracil-requiring organisms. A study of the enzyme content of mutant and wild type E. coli at various stages of growth demonstrated that a high enzyme-forming capacity is associated with young organisms harvested from cultures in the late lag or early logarithmic period.

SUMMARY: As found by earlier workers, Escherichia coli, growing in broth at 37° was rendered incapable of gross multiplication either on nutrient agar or in nutrient broth by sudden cooling in many diluents at 4°. Killing is due to the joint action of a suitable diluent and of sudden chilling, since survival was complete either after sudden chilling in 0·3 m-sucrose or after gradual cooling in a potentially lethal diluent, such as Ringer’s solution. Organisms in the stationary phase of growth were completely resistant. The susceptibility of growing organisms to sudden chilling changed rapidly during the exponential phase. Comparison with the survival after exposure to streptomycin, another bactericidal agent which has no effect on stationary phase cultures, showed that survival after chilling was not due to a fraction of the population being in the stationary phase.

Sudden cooling of Escherichia coli, strain B, infected with phage T2, had the same effect as ultrasonic disruption; namely, destruction of infective centres in the first half of the latent period followed, in the second half, by release of intracellular mature phage.

SUMMARY: A series of fungi with varying degrees of sensitivity to soil fungistasis was selected as the result of agar disk and buried-slide tests with 17 fungi and one soil. Six out of 7 different soils exhibited a spectrum of inhibition of the test series of fungi similar to that of the first soil tested. The exception was a very acid raw humus soil which only inhibited the acid-sensitive fungus Acrostalagmus cinna-barinus. Fertilizer treatments of two series of plots at Rothamsted were found to affect soil fungistasis only through their influence on soil pH. Inhibitory effect decreased with increasing soil acidity, being absent from the most acid plot tested. From the results of experiments in which three fungi were pre-incubated before exposure to the influence of soil, it is suggested that spores are most sensitive to soil fungistasis at an early stage in the process of germination.

SUMMARY: Biotin and methionine are essential growth factors for the aerobic thermophilic actinomycete Micromonospora vulgaris. Biotin can be partially replaced by Tween 80 (sorbitan mono-oleate). No aerial growth is obtained in the absence of methionine. A chemically defined medium containing mineral salts, phosphate buffer (pH 6·8), soluble starch, a mixture of 18 synthetic amino acids (including methionine) and biotin will support growth of the organism.

SUMMARY: Two hundred strains of Bacillus megaterium recently isolated from soil were tested for an antagonistic effect on a phage-resistant strain of the same species. Fifty-one strains gave a zone of inhibition on a lawn of the indicator strain. This effect was increased by a small dose of ultraviolet radiation which also caused 41 other strains to show an antagonistic effect. Sixteen strains which were completely lysed after u.v. irradiation also produced confluent lysis of the indicator strain. When grown in liquid media, these strains gave irregular growth curves owing to partial lysis during the exponential phase. Lysis was almost complete when such cultures were incubated after exposure to small doses of u.v. radiation. Lysis was always associated with accumulation of the antibacterial principal previously named ‘megacin’. Irregular growth in liquid media and production of megacin were strictly correlated. However, only 2 megacinogenic strains were lysogenic and liberated phage. Populations of megacinogenic strains were examined by replicaplating for the presence of non-megacinogenic mutants. Spore suspensions of 4 megacinogenic strains contained non-megacinogenic mutants in various proportions depending on the strain and the particular sample. Non-megacinogenic mutants could not be made to lyse by exposure to u.v. radiation; did not produce any antibacterial effects; and all showed normal patterns of growth in various liquid media. It is supposed that megacinogeny and aberrant growth, as well as inducibility by u.v. radiation, of some B. megaterium strains are governed by a single hereditary unit (gene). It cannot be decided at present whether or not this unit is a highly defective prophage.

SUMMARY: Cysteamine and certain closely related derivatives induced lysis in a majority of 28 strains of Bacillus; cultures of all other genera tested were resistant. Forty-three additional sulphur and/or nitrogen containing compounds were unable to induce lysis. Optimum conditions for cysteamine activity included exposure of metabolizing post-log. phase organisms contained in shallow shaken layers of nutrient broth to the compound for 4–8 hr. Upon the completion of lysis, no formed cellular elements were visible by microscopy except flagella. The induction by cysteamine of a lysogenic bacteriophaxge or of an unusually large amount of an autolytic enzyme was not demonstrated.