- Volume 50, Issue 3, 1968

Taxonomic studies on the thermophilic oligosporic actinomycete chiefly responsible for the respiratory disease Farmer’s Lung support the view that the organism was incorrectly identified as Thermopolyspora polyspora Hens. The organism belongs to the genus Micropolyspora Lechevalier et al. and is described as a new species Micropolyspora faeni.

A mutant (dm-1a) obtained from colonies of a diseased isolate (d-1a) of Helminthosporium victoriae carried the causal agent of disease but snowed almost no symptoms. Typical disease symptoms were produced in healthy H. victoriae after hyphal contact with the diseased mutant (dm-1a). Hence, the character responsible for suppression of severe disease symptoms in the mutant did not appear to be transmitted with the agent of disease. Healthy mutant cultures that did not carry the causal agent of disease were obtained by isolation of hyphal tips from the diseased mutant. Such cultures inoculated with a second diseased isolate (d-1 b) remained nearly symptomless but were capable of transmitting the disease to healthy cultures. The diseased mutant (dm-1 a) inoculated with diseased isolate d-1b usually transmitted only d-1 a to healthy cultures and the diseased mutant (dm-1 b) inoculated with d-1 a usually transmitted only d-1 b to healthy cultures. This indicates that the two agents tend to be mutually exclusive.

The survival characteristics of washed Streptococcus lactis ml 3 organisms, suspended in phosphate buffer, have been examined at the growth temperature. Mg2+ markedly prolonged survival. High bacterial concentrations extended survival times, probably because Mg2+ was excreted by the bacteria. Surviving organisms in some conditions showed prolonged division lags, especially in the absence of Mg2+. Salt concentration had little effect on survival within wide limits; the optimum pH value for survival was near 7·0. Survival times increased considerably at lower temperatures. Agitation and aeration tended to decrease survival. Death was not influenced by the phase of growth at which cells were harvested from a lactose-limiting medium. Casamino acids increased survival markedly in the presence of Mg2+; arginine was almost as effective as the complete mixture of amino acids while other individual amino acids tested gave only slight increases in survival times. Fermentable carbohydrates accelerated death of starving organisms irrespective of the growth phase from which they were harvested and of the limiting nutrient; the accelerated death was reduced by addition of Mg2+.

A nisin-producing strain of Streptococcus lactis was grown in batch culture and at intervals during growth secondary cultures were derived from the parent culture and the length of lag of growth was estimated. Two media were used: a highly buffered ‘medium 22’ and an unbuffered (LTB) medium both without pH control and with pH controlled at about 6·7. In ‘medium 22’ the dry weight doubling time was 95 min. and at the end of growth the pH was 5·2. Neutralization of the culture in this medium did not markedly affect nisin production or dry weight. The total and cell-bound nisin formed/unit dry wt of organism tended to correlate with the length of lag phase of growth. In LTB medium the dry wt doubling time was 45 min. and the terminal pH was 4·5. Of the total nisin formed > 80 % was in the culture fluid when the pH was allowed to decrease below 6·0; total nisin in cultures without pH control did not correlate with length of lag phase of growth. Neutralization of the culture increased the dry wt of organism about threefold. Neutralization also increased the cell-bound nisin/unit dry wt organism in stationary phase and the length of lag of growth of such organisms was longer than comparable stationary-phase organisms grown in unneutralized culture. Chloramphenicol (1 μg./ml. medium) decreased the nisin/unit dry wt organism at stationary phase and the length of lag of growth of such cultures was shorter than in comparable cultures without chloramphenicol. Frequently the start of growth was characterized by a decrease of bio-assayable nisin and a shortening of the lag phase; these changes could be mimicked by a culture which had reached the stationary phase through glucose depletion. The characteristics of early growth were reproduced after the addition of more glucose.

Chloramphenicol was found to inhibit the growth of Pythium ultimum, showing a maximum dose-response at a concentration of 100 μg./ml. Mycelium grown in the presence of chloramphenicol 100 μg./ml., was devoid of the mitochondrial cytochromes aa 3 and b but contained increased cytoplasmic cytochrome c. Mitochondria in thin sections of hyphae grown in the presence of chloramphenicol resembled mitochondria in normal hyphae. In negatively stained preparations of isolated normal mitochondria stalked particles were observed on the cristae. These particles could not be demonstrated in mitochondria from hyphae grown in the presence of chloramphenicol 100μg./ml.

The rates of utilization of energy-yielding substrates (glucose and oxygen) by Penicillium chrysogenum in glucose-limited chemostat cultures were resolved into requirements or ‘rations’ for growth and maintenance. The maintenance ration of glucose was almost all oxidized to carbon dioxide. Over the growth rate range 0·023–0·075 hr‒1 only vegetative growth occurred; although the filamentous growth form predominated, the occurrence of pellets and swollen organisms increased with growth rate. At growth rates of 0·014 hr‒1 and below, conidiation occurred and was maximal at a specific growth rate of 0·009 hr‒1 (average doubling time 78 hr). After growth in chemostat culture the organism could be maintained in a non-growing state by supplying only the maintenance ration of glucose (0·022 g. glucose g. mycelial dry wt/hr). When growth in the chemostat was suddenly stopped by stopping the glucose feed, the mould autolyzed; autolysis was prevented by supplying the maintenance ration of glucose. When the glucose feed rate in chemostat cultures was decreased to the maintenance ration, mycelial differentiation occurred. Differentiation involved increased hyphal vacuolation, a decreased degree of oxidation of glucose, breakdown and resynthesis of nucleic acids and conidiation. The rates at which these changes occurred were inversely related to the growth rate prior to stopping growth. For maximum conidia formation there was an optimal glucose feed rate about 0·038 g. glucose/g. mycelial dry wt/hr, that is 1·7 × maintenance ration. The maintenance ration of glucose was shown to play a role in preventing autolysis and facilitating conidiation. Electron microscope studies showed that there was no change in the observed ultrastructure of cells (except degree of vacuolation) with change in specific growth rate from near the maximum to zero. The septa of the hyphae were found to be plugged.

A description is given of the biological properties of two substances produced extracellularly by a strain of Escherichia coli 078 K80, grown in aerated liquid culture. One of the materials was of high molecular weight and possessed the properties of endotoxin extracted from whole organisms by chemical methods. In mice this material was toxic, induced non-specific resistance to infection with Salmonella typhi, necrotized Sacroma 180, and elicited the local and general Shwartzman reactions. In rabbits it was strongly pyrogenic, dermonecrotic, and induced formation of precipitating antibodies. Heat, chemicals and enzymes did not affect its toxicity, but acid hydrolysis rendered it non-toxic. We have called this substance ‘free endotoxin’. The other material was of lower molecular weight and apart from inducing nonspecific resistance to infection with S. typhiin mice, it had none of the properties of the endotoxin.

Cell walls of Prototheca zopfii kruger 236/65 were prepared by differential centrifugation after disruption of the organisms in a French press. The isolated walls were free from whole organisms and subcellular particles as shown by examination by light and electron microscopy. A quantitative analysis of the walls was made with respect to total nitrogen, phosphorus, ash and lipid. Paper chromatography of hydrolysates of walls showed glucose, mannose, glucosamine and twelve amino acids. A quantitative amino acid analysis was made by ion-exchange chromatography; trace amounts of several other amino acids were detected. The structure of the walls was studied before and after treatment with NaOH and a variety of hydrolytic enzymes. The results are compared with those obtained by other workers with various species of Chlorella, Aspergillus and yeasts. The results are discussed with reference to the taxonomic position of Prototheca zopfii.

A mesophilic strain of Pseudomonas aeruginosa, grown at 30° and suspended to a concentration of equiv. 0·02 mg. dry wt/ml. in 30 mm-NaCl, showed rapid loss of viability when chilled rapidly from 30° to 0° or –2°. The viability of a psychrophilic pseudomonad, grown at 30°, did not decrease to the same extent when a dilute suspension (equiv. 0·02 mg. dry wt/ml.) was rapidly chilled from 30° to 0° or –2°. Concentrated suspensions (equiv. 3·0 mg. dry wt/ml.) of both the mesophile and the psychrophile released about the same proportion of total endogenous ultraviolet-absorbing compounds when rapidly chilled from 30° to 5°, 0° or –2°. Loss of viability following rapid chilling of a dilute suspension of the mesophile or the psychrophile was partly or completely prevented by 5 mm-Mg2+ and by bacteria-free filtrates from chilled concentrated suspensions of either bacterium. The viability of the bacteria grown at 10° did not decrease when dilute suspensions were rapidly chilled from 10° to –2°. Bacteria grown at 10° contained a greater proportion of unsaturated fatty acids than bacteria grown at 30°. Fatty acid analyses showed that susceptibility of the mesophile and psychrophile to cold shock could be correlated with the contents of unsaturated fatty acids in their lipids.

The antibacterial action of the skin germicides tetrachlorosalicylanilide, tribromosalicylanilide, trichlorocarbanilide and monochlorophenoxysali-cylanilide against Staphylococcus aureus, depends on the reversible adsorption of the germicides on the cell membrane. Bacteriostasis results from the adsorption of, respectively, 0·75 × 105, 2·0 × 105, 5·1 × 105 and 7·2 × 105 molecules of germicide per bacterium. The resistance of Escherichia coli to these compounds results from a decreased adsorption, which is a property of the cell wall. It is proposed that the membrane-active antibacterial compounds, detergents, phenols, quaternary ammonium compounds, poly-peptide antibiotics and the germicides under study in this paper, share a common mechanism of action, in which the adsorption of the compound on the cell membrane is a critical step. Resistance to these compounds results from the inhibition of the penetration through the cell wall to the combining sites on the membrane. At the cellular bacteriostatic concentration, the effect of tetrachlorosalicylanilide on the biochemical activities of Staphylococcus aureus has been studied. The energy-dependent transport of phosphate and amino acids into the bacteria is inhibited, whereas the energy-independent entry of phosphate, amino acids and glucose is unaffected. The energy-dependent incorporation of lysine and glucose into cellular material is also inhibited. The release of amino acids from the cell pool into the medium results from the inhibition of the energy-dependent processes involved in the maintenance of the amino acid pool. This inhibition of energy metabolism by the germicide at its bacteriostatic concentration is sufficient to cause the inhibition of growth.

When peritrichously flagellated cells such as normal, curly, paralysed-curly and small-amplitude strains of Salmonella abortusequi are suspended in 0·5% methylcellulose solution, flagellar bundles can be clearly seen by electron microscopy. In a bundle, five or more of the component flagella are tightly united in parallel with each other, and the bundle formed into a helix with a shape characteristic for each strain. These figures reveal the structural detail of the flagellar bundle observed under a dark-field microscope. Ten minutes after the cells were suspended in methylcellulose solution, bundled flagella could be seen in approximately 70 % of the cells of normal, curly and paralysed-curly strains; the remaining 30 % were dispersed. At this time among the normal cells, some were single-bundled and others were multi-bundled. The fraction of single-bundled cells was larger in both motile and paralysed curly-flagellated cells than in normal cells. The fraction of normal cells having single bundles increased with time. Methylcellulose was therefore presumed to enhance aggregation of flagella and/or to inhibit the dispersion of the aggregated flagella.

In the small-amplitude strain, transformation of flagellar shape to curly has been previously observed. In methylcellulose solution, this transformation occurs in bundled flagella but not in dispersed flagella. It is inferred that the tight association of the component flagella in methylcellulose solution enhances the stress among the flagella, thus causing the transformation.

Factors concerned in demonstrating haemadsorption and haemagglutination and their occurrence among different mycoplasmas were investigated. Haemadsorption occurred best to colonies which had recently developed on agar at pH 6·5. Mycoplasmas isolated from various bird and animal sources, e.g. Mycoplasma gallisepticum, M. agalactiae, M. bovigenitalium and M. pulmonis, haemadsorbed with erythrocytes from a wide range of species. However, not all strains within a serotype haemadsorbed. Thus, the ‘Negroni’ strain of M. pulmonis did not. Haemadsorption could be inhibited by crowding of colonies on agar and by the addition of specific antiserum to the colonies. Generally, antiserum titres obtained by haemadsorption inhibition were low in comparison with those obtained by metabolic inhibition, and haemadsorption inhibition was not useful as a routine serological technique.

The development of the haemagglutinin of Mycoplasma gallisepticum in liquid medium was studied in detail; a change in the pH value of the medium could be used as an index of its development and it was intimately associated with the organism. The centrifuged deposits of other mycoplasmas, from birds, cattle, goats, man, rodents and pigs, and which were grown in liquid medium also haemagglutinated, but generally to low titre. Haemagglutination occurred best in 𝖴-shaped cups at 37° and at pH 6·5–7·0 and could be inhibited by specific antiserum. There was lack of correlation between haemadsorption and haemagglutination; both these phenomena were exhibited by some mycoplasmas, others haemadsorbed only, and still others haemagglutinated only.

Crude tetanus toxin paralyses the cholinergic innervation of the sphincter pupillae of the rabbit eye. To test whether this activity was due to tetano-spasmin or to a non-spasmogenic fraction with a peripheral effect, the action of crude tetanus toxin and of the non-spasmogenic fraction prepared from it were compared in rabbits. The paralytic action of the crude toxin was not due to its content of the non-spasmogenic fraction and was probably due to tetanospasmin itself. A convenient method for the preparation of a mixture of the four most abundant gangliosides is given. The preparation of a ganglioside cerebroside complex and its use for the removal of tetanospasmin from a solution of crude tetanus toxin are described.

Very numerous small transparent colonies appeared on plates of nitrogen-poor agar inoculated with soils from various places in Rhodesia. Eighty-three cultures of bacteria were isolated from these colonies, all but two of them small Gram-negative rods. Two cultures contained Gram-positive cocci. Thirty-nine of the isolates fixed nitrogen, ten of them fixing as much as or more than a Beijerinckia culture grown under the same conditions. The Gram-negative isolates are not all alike; their identity is uncertain, but some might be Pseudomonas species, some Achromobacter (or Acinetobacter), and one good fixer might be a Flavobacterium. There are also a few isolates which attack glucose fermentatively. Nitrogen-fixers of these types—aerobic bacteria with very small colonies—appear to be numerous and widely distributed in Rhodesian soils.