- Volume 22, Issue 2, 1960
Volume 22, Issue 2, 1960
- Article
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Intracranial Infection of Mice with Bordetella pertussis
More LessSUMMARY: A bacteriological and histological study was made of the sequence of events in unprotected and protected mice, after intracranial challenge with Bordetella pertussis. No difference in the results was obtained whether mice were injected through the parietal bone or through the foramen magnum. Injected material was distributed throughout the subarachnoid space and ventricles. In unprotected mice organisms multiplied continuously until death. Bacterial multiplication was limited to the cranial cavity, in which it was practically confined to the ciliated layer over the ependyma. There was no invasion of the brain substance. The histological changes in the brain included purulent meningitis, choroiditis, polymorphonuclear infiltration of perivasvular spaces and cerebral substance, haemorrhages, hydrocephalus and necrosis of nerve cells. In protected mice there was a brief initial multiplication of organisms and then their rapid elimination. Histologically the brains of immune mice after challenge were characterized by an intense mononuclear-cell response, which persisted for 2-3 weeks, subsiding slowly during the following 10 weeks.
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Fine Structure Mapping by Complete Transduction Between Histidine-requiring Salmonella Mutants
More LessSUMMARY: The growth characteristics, accumulations and genetic tests carried out by means of complete transduction for over 200 independently isolated histidine-requiring (his) mutants of Salmonella typhimurium are described. Sites of mutation engendering the histidine-requiring phenotype all lie within a short chromosomal region. This region is divisible into seven smaller regions, or gene loci, each correlated with a specific physiological phenotype. The gene loci appear to be linearly arranged and are linked in the order E, F, A, B, C, D and G. The five central loci are linked in an order corresponding to the sequence of reactions in the biosynthetic pathway for histidine. The functions of the two terminal loci, E and G, are unknown. The loci are nearly, or truly adjacent. A few of the sites of mutation are accurately mapped within a single gene. All the evidence is consistent with the view that processes involved in intra- and inter-genic recombination are identical. Some characteristics of multisite mutations are described. Several multisite mutations are interpreted as due to deletion of genetic material while one (his-57) is interpreted as due to an inversion, with or without concomitant deletion of an adjacent region. Single-site and multisite mutations exert specific effects on recombination frequencies in nearby regions.
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Complementation Mapping by Abortive Transduction of Histidine requiring Salmonella Mutants
More LessSUMMARY: Determination has been made by the technique of abortive transduction of the functional relationships of over 200 Salmonella typhimurium histidine mutant alleles. The results are compared with those obtained by genetic recombination Hartman, Loper & Šerman, 1960 and enzyme studies Ames, Garry & Herzenberg, 1960. The results indicate that abortive transduction tests can be used to position rapidly sites of mutation on a chromosome map within the region of a single gene. The tests for genetic complementation reveal that some genes behave as single functional units, while other genes are composed of two, and some of four, functional subunits (complementation units). The existence of mutants with overlapping, intra-genic non-complementing patterns may allow crude mapping, solely by complementation, of some mutational sites within certain single genes. In gene D, the ordering of mutational sites by this technique so far conforms with that obtained by the more classic methods of map construction based on inferences derived from recombination analyses. Data are presented bearing on the individuality of the gene and the effect of mutation thereon. It is concluded that some genetic damages, localized within single genes, can secondarily affect the expression of adjacent gene(s) in addition to their primary effect upon the gene in which they are located (position effect).
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The Genetic Control of the Enzymes of Histidine Biosynthesis in Salmonella typhimurium
More LessSUMMARY: The last four enzymes in the pathway of L-histidine biosynthesis have been assayed in various histidineless mutants of Salmonella typhimurium. Three classes of mutants (B, C and D) have been shown to be associated with lack of imidazoleglycerol phosphate dehydrase, imidazoleacetol phosphate transaminase, and histidinol dehydrogenase, respectively. These studies, in conjunction with the genetic work of Hartman, Loper & Šerman (1960), indicate that the sequence of the genes on the chromosome corresponds to the sequence of the enzymes in the pathway of the biosynthesis. Certain mutants, which were shown genetically to behave as multisite mutants, have been shown to be missing these three enzymes. These multisite mutants are also missing a fourth biosynthetic enzyme, histidinol phosphate phosphatase, for which no single site mutants are available. It has been found that the level of activity of the series of enzymes of histidine biosynthesis can be raised about 15-fold over the wild-type level by growing a histidine-requiring mutant on formylhistidine as a source of histidine.
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Concentration and Electron Microscopy of the Characteristic Particle of Foot-and-Mouth Disease
More LessSUMMARY: The infective component of the virus system of foot-and-mouth disease was concentrated and purified by ultracentrifugation and solvent extraction. Infective materials derived from ox, guinea-pig, mouse, chicken, goose and tissue culture were employed. The infective concentrates (D-fractions) and the separated smaller complement-fixing components (U-fractions) were characterized by infectivity, complement-fixing activity and nitrogen content. The D-fractions, of activity up to 1014·5 ID50/g. N, represented less than 0·1% of the initial protein content. Electron micrographs of the D-fractions showed clusters and hexagonal arrays of 25 to 27 mμ ‘spherical’ particles of granular structure. These defined fractions of the virus system were used in a number of subsequent investigations.
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The Complement-Fixation Test in Studies of Components of the Virus System of Foot-and-Mouth Disease and its Antibodies
More LessSUMMARY: The antibody combining properties of the U- and D-fractions of the virus system of foot-and-mouth disease were investigated by complement-fixation methods. The virus fractions were titrated in antibody excess and used as excess antigens in titrations of antisera. The results demonstrate that the complement-fixing activity in antibody excess increased with time of incubation by a threefold or higher factor when the U-fraction was used as antigen than when the D-fraction was used. Titrations of antisera showed that distinct combining activities were involved according to the virus fraction used as antigen in excess. Data in support of this were obtained in adsorption experiments in the absence of complement by titration of the residual antibody activity following sedimentation of the complexes formed with the U- or D-fractions. The reaction in the region of equivalence, in which neither antigen nor antibody is in effective excess, was investigated. A rapid and objective procedure for the treatment of complement-fixation data is presented.
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Complement-fixation Studies of the Specificity of the Interactions between Components of the Virus System of Foot-and-Mouth Disease and its Antibodies
More LessSUMMARY: The type and subtype specificities of the interactions between fractions of the virus system of foot-and-mouth disease and its antibodies were investigated by complement-fixation methods. Specificity is discussed in terms of a cross-fixation ratio. The 25 mμ infective component (D-fraction) combines homotypically with antibody. Thermal degradation of the 25 mμ component produces a smaller component of enhanced activity which combines heterotypically with antibody and resembles the naturally occurring 7 mμ component (U-fraction). The influence of heating upon the 7 mμ component is marked by a sharpened specificity. The specificity and activity of unfractionated starting materials may be interpreted in terms of those of the 25 and 7 mμ components present in the separable fractions. Significant differences between the reactions observed in the long and short incubation procedures emphasize the advantages of the latter in specificity studies. The composition and treatment of the initial antigens and the test procedures employed must be carefully defined in such studies of antigen-antibody combination.
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Tissue Culture on Polythene
More LessSUMMARY: Fragments of solid organs can be squashed, without destroying the viability of the cells, to form a thin sheet of cells which is stuck to polythene film with clotted mouse plasma. The film conveniently floats on a simple culture medium in which embryonic tissues multiply within 24 hr. and adult tissues survive for several days. The tissue on the polythene is thin enough to mount on a microscope slide and examine with phase-contrast illumination and an oil-immersion objective; alternatively, since the polythene is inert to most organic solvents, the tissue can be stained and mounted, using standard histological techniques. The squashed tissue on polythene is more readily infected by viruses and more easily examined than are fragments of tissue embedded in plasma on a coverglass. The method was used to study one virus cytopathogenic for many chick tissues and another virus detected by the haemadsorption phenomenon.
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A Sulphate-Reducing Bacterium from the Sheep Rumen
More LessSUMMARY: A spore-forming, mesophilic, sulphate-reducing bacterium has been isolated from the rumen of hay-fed sheep. The organism is an obligately anaerobic Gram-negative rod which, in the presence of sulphate and 0·1% yeast extract, used only L- or D-alanine, DL-lactate, pyruvate or formate, but not 47 other compounds tested, as hydrogen donor for growth. Under these conditions the sulphate could be replaced by sulphite or thiosulphate. The yeast extract was replaceable by ferrous sulphate + p-aminobenzoic acid + biotin. Evidence is presented that alanine and lactate are metabolized via pyruvate which breaks down to form acetate and formate, the latter then being metabolized to form CO2. The growth of the organisms was inhibited when the concentration of sulphide reached 6–7 μmole/ml. and the maximum dry weight was only 50–60 μg./ml. Washed suspensions of the organism in 2·0% yeast extract utilized gaseous hydrogen for sulphate reduction and H2: H2S ratios of 3·2–4·0 were obtained.
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The Effect of Cold Diluent on the Viable Count of Pseudomonas pyocyanea
More LessSUMMARY: Young actively dividing cultures of Pseudomonas pyocyanea (aeruginosa) at 37° were killed when diluted into cold liquids which were without effect when used above 18°. In general the simpler the composition of the diluent the more lethal it was; distilled water was the most active. The cooling had to be rapid to be effective. Old cultures were only slightly sensitive to this effect. The killed organisms appeared normal when examined under the microscope. Under similar conditions Staphylococcus aureus was resistant to cold shock.
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Application of Quantitative Electron Microscopy to the Study of Mycobacterium lepraemurium and M. leprae
More LessSUMMARY: The standard methods for assessing viability of micro-organisms are not applicable to rat and human leprosy bacilli since neither organism can be grown in vitro. McFadzean & Valentine (1958, 1959) suggested that the electron microscope might provide a quantitative guide to the viability of these organisms by allowing dead forms to be identified. This technique is further investigated and shown to be valid and reasonably accurate for Escherichia coli provided the organisms are not morphologically fixed by the killing agent. It is concluded that the method can assess death of leprosy bacilli in the host when this occurs either naturally or aided by bactericidal drugs, and also loss of viability on storage, but not sudden killing by more violent chemical or physical means. The method has been found useful for following the survival of Mycobacterium lepraemurium in tissue cultures. It is suggested that death in the host but not death occurring on storage can be measured by a simple classification of the bacilli seen with the light microscope after the conventional carbol fuchsin stain. It is indicated that many of the bacilli obtained from untreated human cases of leprosy are dead, while from rats the percentage which is degenerate is low. The significance of various features of the leprosy bacilli seen with the electron microscope is discussed. There is no evidence that the bacilli form spores or capsules.
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The Classification of Fusobacteria from the Human Mouth
More LessSUMMARY: 237 strains of fusobacteria were studied and classified by such characters as cell composition, metabolism and morphology. They are shown to be closely related metabolically and in cell composition, but distinguishable by morphology into two species: Fusobacterium nucleatum Knorr and F. polymorphum Knorr. It is suggested that these organisms are actinomycetes which have lost a number of characters in undergoing adaptation to parasitism.
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The Survival of Washed Suspensions of Mycoplasma
More LessSUMMARY: Washed Mycoplasma organisms died rapidly when suspended in 0·85 % (w/v) NaCl prepared in laboratory ‘distilled’ water (steam condensate) at room temperature; this rapid killing was decreased by adding to the suspensions certain chelating agents, reducing agents or finely ground manganese dioxide. Washed organisms survived well in potassium phosphate (K2HPO4, 0·01 m adjusted to pH 7) dissolved in good quality ‘ deionized’ water at 2–4°. The quality of the water used was very important.
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The Measurement of the Growth of Mycoplasma in Liquid Media
More LessSUMMARY: The growth of Mycoplasma laidlawii strain A in liquid media was estimated by colony count, turbidity, and by measurement of dry weight, total-N and deoxyribonucleic acid (DNA) of washed deposits from centrifuged cultures. Samples from growing cultures were also examined with the phase-contrast microscope. The growth curves of cultures obtained by the colony count method resembled a bacterial growth curve. The other methods became useful only when nearly maximum growth had occurred because of the very small yield of organism per unit volume of culture. The growth of organisms in two different media was compared; the growth curves obtained by the colony count were nearly identical but values for total-N and DNA were different.
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Steroid Growth Requirements and Steroid Growth Inhibitors of Mycoplasma
More LessSUMMARY: Mycoplasma laidlawii strain A did not grow significantly and sometimes died in a peptone + yeast extract (PY) medium; good growth was obtained when 1 % serum or 1 % bovine plasma albumin was added. The addition of cholesterol and some other steroids (at 20μg./ml.) to the basal PY medium gave some growth although less than that given by adding serum or albumin. Certain other steroids (e.g. Δ 1,4 and Δ 1,4,6 -cholesten-3-one) were potent inhibitors of cholesterol-promoted growth at one- tenth the cholesterol concentration. One of these steroids at 2μg./ml. inhibited the growth which was obtained in PY + albumin medium; this inhibition was annulled by 200μg. cholesterol/ml. Two of the inhibitory steroids at c. 300μg./ml. inhibited the growth of M. laidlawii strains A and B, M. mycoides var. capri and M. bomgenitalium in a rich serum-containing medium. The inhibitory effects of one of these steroids was greatly diminished when it was added after growth had begun.
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A Partially Defined Medium for the Growth of Mycoplasma
More LessSUMMARY: The saprophytic Mycoplasma laidlawii strains A and B were grown in a partially defined medium composed of inorganic salts, Casamino acids, vitamins, nucleic acids, glucose and 10 % (v/v) horse serum. The growth was less good than in the complex Edward medium; addition of known growth factors did not improve growth. Addition of yeast extract (0·05 %, w/v) to the partially defined medium allowed growth equivalent to that in Edward medium. The serum could be replaced by 1 % (w/v) crystallized bovine plasma albumin. The suboptimal growth obtained in the albumin-containing medium was not improved by adding cholesterol, Tween 80, lecithin and other growth factors known to be required by other Mycoplasma spp. Riboflavin and nicotinic acids were the only vitamins found to be essential for the growth of M. laidlawii strain A in the albumin medium. The Casamino acids could be replaced by a mixture of known amino acids. The pathogenic M. rnycoides var. capri and M. bavigenitalium grew in the partially defined medium when the concentration of the serum was increased to 20 % (v/v) and yeast extract added to a final concentration of 0·05 % (w/v).
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The Effects of Ribonucleic Acid and Deoxyribonucleic Acid on the Growth of Mycoplasma
More LessSUMMARY: The nucleic acid requirements of several Mycoplasma organisms were examined in a partially denned medium. The saprophytic M. laidlawii strain A did not grow in the basal medium alone Razin & Knight, 1960 a but did when suitable concentrations of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) were added. Too high a concentration of RNA inhibited growth; this inhibition was annulled by increasing the concentration of DNA. Similarly, too high a concentration of DNA inhibited growth, and this inhibition was annulled by RNA. Chemical and enzymic degradations of RNA showed that the growth-promoting effect could be brought about by a ribo-oligonucleotide but not by smaller fragments of the molecule. Similar degradations of DNA showed that the effective moiety was thymidine; thymine was less effective. The degradation of RNA abolished its growth-inhibitory activity. The growth-inhibitory activity of DNA was not affected by its degradation to oligonucleotides, and was only partially diminished by its degradation to nucleotides or nucleosides.
Mycoplasma laidlawii strain B grew in the basal medium when DNA alone was added. This nutritional requirement was also satisfied by thymidine, provided that some RNA was also present. The parasitic Mycoplasma mycoides var. capri resembled M. laidlawii strain B in responding to DNA alone, but differed from the saprophytic strains in its complete indifference to high DNA concentrations. Thymidine replaced DNA only to a certain extent when added together with RNA. A growth-promoting effect of DNA was also found with the L-phase of Streptobacillus moniliformis; thymidine then replaced DNA completely. The RNA/DNA antagonism was found with all the organisms examined. Possible explanations for this phenomenon are discussed.
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Choline Sulphate in Fungi
T. Harada and B. SpencerSUMMARY: The occurrence of choline sulphate in 32 fungi and 9 bacteria was investigated by incorporating Na2 35SO4 into culture media and subsequently identifying radioactive choline sulphate in cell extracts by chromatography and radioautography. The production of choline sulphate appears to be restricted to higher fungi since it was not found in any of the tested Phycomycetes. Of the Ascomycetes, the Endomycetales did not synthesize choline sulphate but the mycelia of the Sphaeriales did accumulate the compound. The Basidiomycetes and all the Fungi Imperfecti, except Torulopsis utilis, were choline sulphate positive. The choline sulphate negative organisms, including all of the bacteria tested, could not be induced to produce choline sulphate by adding large amounts of choline to the culture media or by growing on chemically defined media in which inorganic sulphate was the sole sulphur source. On the other hand, Aspergillus oryzae produced choline sulphate on a minimal medium containing inorganic sulphate, cysteine, cysteic acid or taurine as sulphur sources. The distribution of choline sulphate production has been discussed in terms of the absence or presence of the enzyme choline sulphokinase.
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The Measurement of Bacterial Motility
More LessSUMMARY: Bacterial motility was measured by a simple method which consists of counting the number of organisms which pass across a small aperture in a given time. The count is proportional to the bacterial concentration in the suspension and also to the average speed of the bacteria. A slight increase in the viscosity of the suspending medium above that of a buffer solution has the effect of increasing motility; further increase in viscosity decreases it.
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Flagellar Pattern and Growth of Bacillus spp
More LessSUMMARY: Observations by electron microscopy and by light microscopy of material stained to demonstrate flagella and cell walls, simultaneously, showed that in non-filamentous Bacillus spp. the flagella of growing bacilli were usually fully developed towards one pole, and developed on the other pole as the process of fission advanced. This is similar to the condition in other types of bacteria, previously described, and is in accordance with the theory that bacterial reproduction is not by simple fission, but by production of a bud from a mother cell.
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