- Volume 16, Issue 1, 1957
Volume 16, Issue 1, 1957
- Article
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A Bacteriolytic Principle Associated with Cultures of Bacillus cereus
More LessSUMMARY: A lytic principle associated with cultures of Bacillus cereus is described and some of its properties discussed. The principle is relatively non-specific, acting on several species of aerobic spore-formers. It has properties in common with similar agents reported recently and comparisons with these are made. Lysis results in the loss of cell contents whilst at least part of the cell-wall structure remains intact.
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Detection of Gas Production from Glucose by Heterofermentative Lactic Acid Bacteria
More LessSUMMARY:Several modifications of the Durham tube method for detecting gas production have been tested with a variety of heterofermentative lactobacilli, some of which had been described in the literature as failing to produce gas from glucose by this method. Gas production in Durham tubes was detected consistently when the surface of a tomato juice broth medium containing 0·5 % or 2·5 % (w/v) glucose was sealed with liquid paraffin or with washed agar, but less consistently when the surface of the medium was unsealed. A minority of the strains examined grew weakly in a peptone + yeast extract medium containing glucose and produced very little titratable acidity or detectable gas. These produced large quantities of gas in the same medium when maltose replaced glucose.
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Haemolytic Material from Aerobic Sporing Bacilli
More LessSummary: Material from a strain of Bacillus subtilis which causes haemolysis was investigated. A fraction possessing the solubility properties of an organic acid appeared to be responsible for the haemolytic activity of the organism, but in the culture filtrate this material was associated with protein which modified its solubility properties considerably. Similar material was found in the culture filtrates of other haemolytic aerobic sporing bacilli.
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The Preferential Suppression of Hyaluronidase Formation in Cultures of Staphylococcus aureus
More LessSUMMARY: When Staphylococcus aureus strain 524/SC/55 is inoculated from an overnight culture into fresh broth only small amounts of hyaluronidase are formed at first, but while growth proceeds at a constant exponential rate an increasing proportion of the cell protein is secreted into the medium as hyaluronidase. This increase in proportion continues for eleven generations. Thereafter, it remains constant even when the organisms are transferred to fresh medium. The organisms of the inoculum taken from overnight cultures are deficient in thiamine and a partial deficiency of this and other essential growth factors suppresses the proportion of hyaluronidase formed. The accumulation of α-aminobutyric acid in organisms from overnight cultures was demonstrated; the addition of this substance to cultures decreases the proportion of cell protein which is turned into hyaluronidase.
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Some Hypotheses on the Aetiology of Fatal Infections in Partially Resistant Hosts and their Application to Mice Challenged with Salmonella paratyphi-B or Salmonella typhimurium by Intraperitoneal Injection
More LessSummary: Some hypotheses are considered which describe the aetiology of a fatal infection in a partially resistant host; i.e. a host which does not invariably die after inoculation with one bacterium. The hypothesis of independent action postulates that the mean probability per inoculated bacterium of multiplying to cause (or help to cause) a fatal infection is independent of the number of bacteria inoculated and, for a partially resistant host, is less than unity (1 > p> 0). It predicts: (1) that the slope, b, of the probit-mortality/log-dose curve will be 2·0 or less at the LD 50 point; (2) that, while hosts dying after inoculation with many LD50 die as a result of the multiplication of many of the inoculated bacteria, most of those dying from 1 LD 50 or less do so following the multiplication of only one of the inoculated bacteria, regardless of the total number of bacteria inoculated. When a mixture of several equally virulent, distinguishable variants of a given pathogen are inoculated, fatal infections caused by the growth of one bacterium should result in the predominance of only one variant at post mortem. The hypotheses of maximum and of partial synergism postulate that inoculated bacteria co-operate so that the value of p increases as the size of the dose increases. They predict: (1) that the slope of the dose-response curve may be more than 2·0 at the LD50 point; (2) that all observed fatal infections will be initiated by more than one bacterium and that consequently the inoculation of a mixed inoculum will lead to the predominance of several variants at post mortem.
Variants of Salmonella paratyphi-B which carried one of the three flagellar antigens, b, i, or e,h, were prepared by transduction. Variants of S. typhimurium which either fermented or did not ferment xylose were also prepared. In each case, the variants did not differ detectably in growth rate in vivo, or in virulence. The value of b was 1·8 for mice inoculated with S. paratyphi-B by intraperitoneal injection (LD50 = 7·7 × 106 organisms). As predicted by the hypothesis of independent action, the relative frequencies of the variants in samples of heart blood obtained post mortem varied greatly from mouse to mouse when the dose was 1 LD50 or less, and became progressively more uniform (and similar to the inoculum) as the size of the dose increased. The value of b was 0·66 for mice challenged by S. typhimurium by subcutaneous injection (LD50 = 2 × l03 organisms); and challenge with a mixed inoculum gave similar results.
Despite this general conformity with prediction, far fewer mice than expected yielded only one variant at post mortem. This discrepancy possibly resulted from a terminal breakdown in resistance, which was demonstrated by experiment. It is concluded that these results are most economically explained by the hypothesis of independent action and that this hypothesis is probably applicable to many other infective systems.
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The Oxygen Requirement of Growing Cultures of an Aerobacter Species Determined by means of the Continuous Culture Technique
More LessSummary: A formula for calculating the oxygen demand of a growing culture is derived. The fate of substrate-glucose in cultures of Aerobacter cloacae was found to depend on the amount of available oxygen and the oxygen demand of the organisms. Anaerobically, cell synthesis and CO2 production are at their minimum levels and most of the glucose-carbon is converted into ethanol, formic acid, 2:3-butanediol, acetoin and acetic acid. A small supply of oxygen suppresses the formation of ethanol and formic acid but still permits the production of butanediol and acetoin and increases the proportion of glucose-carbon converted to acetic acid, cells and CO2. A larger supply of oxygen suppresses the formation of butanediol and acetoin and still further increases the yields of cells and CO2. With an excess of oxygen available, provided the growth rate of the organism is not too near its maximum value, acetic acid production is suppressed and complete conversion of the glucose-carbon into cell and CO2 occurs. At growth rates very close to the maximum a part of the glucose is converted into acetic acid even with an excess of available oxygen. The metabolism in a batch culture with excess oxygen resembles the metabolism with excess oxygen in a continuous cultures in which the organisms are growing at a rate close to the maximum. Means of recognizing and preventing an oxygen deficiency are discussed.
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The Nuclear Cycle of Myxococcus fulvus
More LessSummary: The life cycle of Myxococcus fulvus consists of the following stages: germinating microcysts, young vegetative cells, intermediate cells, mature cells and resting microcysts. Germination takes place by simple elongation, or germ-tube formation. The nucleus of the germinating microcyst is a single oval or elongate body. The young vegetative cell should probably be referred to as multinucleate because the nucleus consists of a string of lobate bodies. In intermediate and mature cells the nucleus is either bi-lobed or oval, with the presence of entire edges. Details of the electron micrographs suggest a nuclear membrane. Microcysts normally have a single nucleus, and possibly possess nucleoli. Resting microcysts consist of a thin outer shell, a thick inner shell, cytoplasm and a nucleus.
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The Influence of Oxygen and Arǵinine on the Motility of a Strain of Pseudomonas sp
More LessSUMMARY: The motility of a strain of Pseudomonas sp. was activated by molecular oxygen or by arginine. In the presence of sufficient oxygen to support motility, arginine was not required for this purpose. In the absence of sufficient oxygen, arginine supported motility and was broken down to ornithine; there is not yet enough evidence to indicate whether this breakdown supplied the energy for such arginine-activated motility. In the presence or absence of arginine, the organism exhibited chemotaxis towards an optimum oxygen concentration which was less than that at the air/suspension interface.
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The Kinetics of the Mating Process in Escherichia coli
More LessSUMMARY: When broth cultures of donor (HfrH) and recipient (F-) strains of Escherichia coli K-12 are mixed, zygotes are formed by the transfer of part of the donor chromosome to the recipient cell. The donor parent thus becomes dispensable as soon as transfer is accomplished. The kinetics of zygote formation can therefore be studied by treating samples, removed at intervals from a parental mixture, with virulent bacteriophage to which only the donor parent is susceptible. Only zygotes already formed at the time of treatment can segregate a recombinant ceil. A lag of 8-10 min. precedes a linear rise in the number of zygotes when selection is made for inheritance of the donor nutritional markers T + L + only. The formation of zygotes inheriting the marker Lac+ as well as T + L + shows a lag of about 18 min. These lag periods represent the times required for the genes T + L + and Lac +, respectively, to enter the F- cells and confirm the finding of Wollman & Jacob (1955) that chromosome transfer is an oriented process and that the donor genes penetrate the F- cell in the same order as their arrangement on the chromosome. The process of zygote formation in the equivalent F + × F- cross has also been studied by the phage method. Although the yield of T + L + recombinants is c. 2 × 104 times less than in the Hfr × F- cross under the same conditions, the times of entry of the donor genes T + L + and Lac + are the same in both crosses. In the Hfr × F- cross, significant zygote formation does not occur in unsupplemented buffer but requires the presence of both glucose and sodium aspartate. Zygote formation is a temperature-dependent process which occurs in the absence of multiplication of either parent and is unaffected by the presence of deoxyribonuclease. The number of zygotes (and therefore of recombinants) formed in a given time is a function of two independently variable factors: (i) the frequency and intimacy of chance concacts; (ii) the speed of chromosome transfer which is related to energy production. Decrease of temperature from 37° to 32° about doubles the time required for any given Hfr gene to be transferred to an F- cell. Alteration of the parental population density, or the pH values of the medium ( Fisher, 1957b ), does not affect the times of entry of Hfr genes into the F- cells but does modify the rate of effective contact formation. Segregation of haploid recombinant cells from Hfr × F- zygotes, at 37°, takes place in nutrient broth at about 140 min., and on minimal agar at about 160 min., after mixing the parental suspensions. The phenotypic expression of resistance to sodium azide, inherited from the Hfr parent, commences shortly after the zygotes are formed and becomes complete just before segregation; resistance to phage T1, however, is not expressed at all until the time of segregation, and requires four generations of the recombinant segregants for completion.
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The Role of the Krebs Cycle in Conjugation in Escherichia coli K-12
More LessSummary: Zygote formation between strains of Escherichia coli K-12 is dependent on a supply of free energy made available by the oxidation of carbohydrate via the Krebs cycle reactions. The simultaneous addition of glucose and a dicarboxylic acid of the tricarboxylic acid cycle, or an immediate precursor, to mating cells markedly stimulates the number of zygotes formed. Reagents known to inhibit reactions of the Krebs cycle inhibit zygote formation. Evidence was obtained that synthesis of protein or either pentose or deoxypentose nucleic acid was not necessary for zygote formation.
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The Nature of the Endergonic Processes in Conjugation in Escherichia coli K-12
More LessSummary: The energy required during zygote formation was found to be needed solely by the donor parent. The free energy is utilized by the donor cell in the establishment of effective contacts and subsequently in the transfer of the genetic material to the F- cell. Energy is not required to hold the cells in contact throughout the mating process. The effect of pH value on the recombination rate is described.
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The Pathway of Breakdown of 2:4-Dichloro- and 4-Chloro-2-methyl-phenoxyacetic Acid by Bacteria
More LessSummary: The metabolism of 2:4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid by a strain of Flavobacterium peregrinum and an Achromobacter sp., respectively, has been studied. Bacteria from young cultures were more active than those from older ones in oxidizing these substrates. Evidence is presented that adapted organisms dissimilate 2:4-dichlorophenoxyacetic acid through 2:4-dichlorophenol and 4-chlorocatechol, and that 4-chloro-2-methylphenoxyacetic acid is dissimilated through 5-chloro-2-cresol. Bacteria grown on 2:4-dichlorophenoxyacetic acid do not oxidize any of the other five possible isomers, but can oxidize 2:4-dibromo-, 4-bromo-2-chloro-, 4-chloro-, and, to a small extent, 2-chlorophenoxyacetic acids.
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Physiological Factors in the Production of an Iodophilic Polysaccharide from Pentose by a Sheep Rumen Bacterium
More LessSummary: A Gram-negative, non-flagellated, asporogenous, anaerobic, iodophilic, curved rod (2·5 μ. × 0·5 μ.) was isolated from the rumen of a sheep and designated Bacteroides amylogenes n.sp. This organism transforms a number of carbohydrates including pentoses to intracellular iodophilic polysaccharide which has been isolated and shown to be a polymer of glucose. Resting suspensions of this organism utilize this iodophilic material as a reserve substance under a variety of conditions. Bacteroides amylogenes grows optimally in vitro when bicarbonate and rumen liquor are included in the medium. On physiological and morphological grounds it is considered to be an authentic rumen bacterium. The chief fermentation product from xylose is butyric acid.
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Adaptive Variation in the Level of Oxidative Activity in Saccharomyces cerevisiae
More LessSUMMARY: After growth in a medium containing 2 % (w/v) galactose, a strain of Saccharomyces cerevisiae showed greater oxidative activity, an essentially unchanged anaerobic fermentation rate and an increase in total growth, compared with yeast grown in the same medium but containing instead 2 % (w/v) glucose as major carbon source. The galactose-grown yeast possessed a generally higher level of oxidative metabolism, with enhanced Q air O 2 values (up to 500 %) with glucose and other oxidizable substrates, a higher cytochrome content and a greatly increased cytochrome oxidase activity. These differences were associated with an inhibitory effect of glucose on the formation of oxidative enzymes when this sugar was present in the growth medium at concentration greater than 0· 05 % (w/v). Changes in the composition of the basal growth medium greatly altered the magnitude of the differences between the glucose- grown and galactose-grown organisms and the differences were not apparent with certain growth media. Possible interpretations of these findings are discussed.
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Autolytic Release and Osmotic Properties of ‘Protoplasts’ from Staphylococcus aureus
More LessSUMMARY:The cell wall of exponential phase Staphylococcus aureus (strain Duncan) loses its tensile strength in c. 2 hr. when the organisms are incubated at 25° in 1·2 m-sucrose at pH 5·8 and ionic strength 0·3. The ‘protoplasts’ thus released from the mechanical protection of the cell wall are stable in 1·2 m-sucrose but lyse in media of lower osmotic pressure. The mean internal osmotic pressure of the ‘protoplasts ’ is c. 20 atmospheres; they are permeable to glycerol but not to sucrose or NaCl. The rate of ‘protoplast’ release varies with the rate of growth of the organisms at harvesting. After osmotic explosion of ‘protoplasts’ released from slowly growing organisms the plasma membranes may be recovered as spherical shells which disintegrate and condense into small particles on washing, and 75 % of the weight of the cell walls may be recovered as hemispherical shells.
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The Structure of Influenza Virus Filaments and Spheres
More LessSUMMARY: Influenza virus filaments treated with acid on an electron microscope film developed along their length rows of spheres which were completely digested by trypsin. Influenza virus spheres similarly treated revealed trypsin-resistant polygonal rings which by their behaviour with enzymes have been identified as ribonucleoprotein.
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The Occurrence and Distribution of Bacterial Types on Flatfish
More LessSUMMARY: An investigation of the bacterial flora of the representative flatfish, skate and lemon sole, was carried out by direct counts of special groups of bacteria and by the analysis of over 1700 strains of bacteria isolated from the fish. Luminous and agar-digesting bacteria occurred seasonally on fish. Luminous strains occurred mainly in the gut contents. A group of sea-water-loving Pseudomonas spp. which seem to require sea water for growth on initial isolation was present on the flatfish in variable numbers throughout the year. A more or less distinct intestinal flora was present in North Sea flatfish in which a homogeneous group of micro-organisms provisionally labelled Gut Group vibrios predominates; this group includes luminous bacteria. The bacterial populations of skin and gills were similar in both the fish studied and were composed principally of Gram-negative rods of the Pseudomonas and Achromobacter genera. The composition of the bacterial flora of the two flatfish, as calculated from an analysis of strains isolated from sea-water agar plates, was: Lemon sole: Pseudomonas 60%, Achromobacter 14%, Alcaligenes 8%, Flavobacterium 5%, corynebacteria 1%, cocci 1%, Gut Group vibrios 9%, miscellaneous 2%. Skate: Pseudomonas 53%, Achromobacter 13%, Alcaligenes 6%, Flavobacterium 9%, corynebacteria 2%, cocci 3%, Gut Group vibrios 12%, miscellaneous 2%.
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The Type A Phages of Salmonella typhimurium: Identification by a Standardized Cross-immunity Test
More LessSUMMARY: The identification of the type A phages of Salmonella typhimurium has hitherto been based on antigenic structure, host range, and plaque characters. The discovery of an apparently phage-free strain of S. typhimurium, equally sensitive to all these phages, has enabled us to prepare standardized lysogenic bacteria, alike in all respects except in the symbiotic phage (prophage) they carry. With these it has been possible to test cross-immunity with a high degree of accuracy. The immunity pattern of a collection of 598 type A phages has been examined, and subdivisions in the original types have been revealed. Twelve types are now identified. The test is described and the significance of the findings is discussed.
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Viable Counting of Mycobacterium tuberculosis in a Silica Gel Medium
More LessSUMMARY: A method is described in which deep counts of Mycobacterium tuberculosis are made in a defined medium solidified with silica gel. The method was accurate, simple and safe. The contamination rate was low. Deep counts were higher than surface counts on plates. The defined medium was superior to Kirschner medium.
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A Cell-wall Lytic Enzyme Associated with Spores of Bacillus Species
More LessSUMMARY: Aqueous extracts of disintegrated spores of Bacillus cereus and nonvirulent B. anthracis contained an enzyme which produced visible lysis of the isolated cell walls of vegetative B. cereus. Optimum activity occurred at pH 7–8 in the presence of cobalt or manganese ions (10 p.p.m.) at 58°. Activity was destroyed during heating at 100° for 15 min. The lytic preparation released non-dialysable components containing ae-diaminopimelic acid (DAP), glutamic acid, alanine, amino sugars and glucose. Although lysis was less obvious, the enzyme preparation released similar material from cell walls of other Bacillus species, spore coats of B. megaterium and coats of autoclaved B. cereus spores. Extracts of freshly harvested B. cereus spores were more active than those from spores which had been stored for several weeks at 2°. Extracts from disintegrated spores of B. megaterium had no enzymic activity; the enzyme system was associated with the insoluble spore coat fraction. The action of the enzyme differed from that of lysozyme or glucosaminidase; the reaction products did not give a significant reaction for N-acetylhexosamine and visible lysis proceeded more rapidly with cell walls of B. cereus than with B. megaterium. Possible functions of the enzyme may be to release ‘ spore peptide ’ from the spore coat during germination and to lyse the sporangium and free the spore during sporulation.
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An Apparatus for the Continuous Culture of Bacteria at Constant Population Density
More LessSUMMARY: A self-regulating continuous culture apparatus is described, designed for studying growth and enzyme production in bacteria which require complex media; it might also be suitable for the cultivation of suspended tissue cells. The growth vessel is a tilted cardioid-shaped flask spinning about its long axis. Aeration and mixing are achieved in such a manner that no frothing occurs and no ‘ anti-foam ’ agents are required. Adherent bacterial colonies do not form on the walls of the growth vessel. Samples are removed rapidly under aerobic conditions. The basal medium can be automatically augmented with special metabolites at constant concentration without contaminating the main medium reservoir. The apparatus is autoclaved in one unit after assembly. Factors governing the design of similar small-scale apparatus are discussed.
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