Growth In Broth Culture Research Articles

Predicting the aerobic growth of Yersinia enterocolitica on pork fat and muscle tissues

Flasks of Tryptic Soy Broth, (TSB), unacidified (pH 7·2) or acidified with HCl or lactic acid to pH 6·3 or 5·5 and samples of sterile pork fat or muscle tissue, were inoculated with logarithmic phase cultures of a strain of Yersinia enterocolitica which had been isolated from a pork packing plant. The broth cultures were incubated at temperatures between 2 and 25°C, and growth rates determined from increases in the optical densities at 600 nm. The tissue samples were incubated at temperatures between −2·4 and 20·4°C, and growth rates were determined from increases in the viable counts. The organism grew without a lag phase in all broth cultures. At any temperature the rates of growth were lower in media of pH 6·3, and lower again in media of pH 5·5 than in unacidified broth; and growth rates were lower in media acidified with lactic acid than in media of the same pH acidified with HCl. The data for each medium were well described by the regression line of the plot of the square roots of growth rates against temperature. The organism grew on fat tissue, of pH 6·3±0·3, without a lag phase at all temperatures, at rates comparable with the rates of growth in unacidified TSB of pH 7·2; i.e. at rates faster than would be predicted for a medium of pH 6·3. The organism did not grow on muscle tissue of pH 5·6±0·2, at temperatures ≤6°C. At higher temperatures, the organism grew on muscle tissue only after a lag of 24 h or more at rates which were less than those that would be predicted from the rates of growth in TSB acidified to pH 5·5 with lactic acid. It appears that models for the growth of Y. enterocolitica are likely to underestimate the growth on pork fat and to overestimate the growth on pork muscle. Three previously published models would generally predict much faster growth in broth cultures, under all the studied conditions of temperature and pH, than was observed for the strain used in this study.

Population Dynamics of Bacteriophage and Bacillus Subtilis in Soil

The dynamics of the interaction of populations of Bacillus subtilis and a temperate bacteriophage in soil microorganisms were examined. The purpose of the study was to investigate these dynamics in a structured habitat approaching that of their natural habitat in soil, in contrast to previous investigations in broth or chemostats. We addressed three main questions. What are the population dynamics of a phage—bacteria interaction in soil? What role might the heterogeneity of the soil play in shaping the interaction? Are the dynamics controlled more by the population biology of the phage or of the bacteria? The phage used was isolated from Arizona desert soil in which B. subtilis is common. Phage and bacteria were grown separately or together in sterile soil microcosms consisting of autoclaved soil rich in organic material. Densities of phage and bacteria were estimated through repeated sterile sampling of the microcosms by spreading precise dilutions of sampled soil suspensions onto plates of microbiological media. The plates for estimation of phage densities contained in addition a lawn of host bacteria. While the principal focus is on temperate phage ecology, the dynamics of a single example of interaction between B. subtilis and virulent phage is also presented. In all cases an initial epidemic of phage occurred, followed by stable equilibria lasting weeks to months. A threshold host density for phage outbreak occurs at °5 x 106 colony—forming units per gram of soil. At equilibrium the phage, both temperate and virulent, were much less abundant than the bacteria. The temperate phage did not depress the equilibrium host density, while the virulent phage lowered it by a factor of ten. The acidic soil of these experiments caused rapid and permanent inactivation of free phage, making the continuous interaction of phage and host essential for persistence of phage. Low levels of phage resistance (superimmunity or genetic) were typical of host populations. Most properties of the interaction between B. subtilis and phage in soil are quite different from those observed with chemostat populations of Escherichia coli and virulent phages. Potential doubling times and rates of increase in soil for phage and B. subtilis were calculated. At equilibrium, soil slows the interaction of phage and host relative to population growth in broth culture, and possibly also makes it heterogeneous in space and time. The life—history features of B. subtilis and temperate phage are considerably more complex than those of non—sporeforming bacteria and virulent phage. The biotic relationship of temperate phage and host may be distinct from predation or parasitism. Patterns in the ecology and evolution of temperate and virulent phage were explored, leading to the expectation that temperate forms will be more common among the phages of soil bacteria.

Outer membrane proteins of Bacteroides fragilis grown in vivo

The outer membrane protein (OMP) profiles of four different strains of Bacteroides fragilis, as determined by Coomassie blue stained polyacrylamide gels, were compared after growth in broth culture and in the mouse peritoneal cavity. There was no induction of the expression of large quantities of novel OMP after growth in vivo. Mouse immunoglobulin G and albumin were associated with the bacterial OMP, but could be removed by washing.

Effects of isoniazid and of ceforanide against virulent tubercle bacilli in cultured human macrophages

Isoniazid (INH) is said to inhibit tubercle bacilli equally well in vivo and in vitro, and to be mycobactericidal. Ceforanide (CEF) can inhibit tubercle bacilli in vitro but has been found ineffective clinically. These two drugs were tested against virulent tubercle bacilli in cultured human macrophages (MP), partly to compare the results with clinical experience, and partly for a better understanding of antituberculosis activities of these drugs in human beings. INH had the same minimal inhibitory concentration (MIC) against tubercle bacilli in MP as in 7H9 broth cultures. It killed multiplying bacilli in MP but not nonmultiplying bacilli, even at 100 times MIC. It killed both multiplying and nonmultiplying bacilli in broth cultures. It interfered with its own effectiveness against intra-MP bacilli by preventing nonmultiplying bacilli from beginning to multiply and thus become susceptible to killing. These findings help explain why this demonstrably mycobactericidal drug produces relapses of tuberculosis when used alone. It was confirmed that CEF is able to inhibit growth in broth cultures (MIC=10 μg/ml). However, it was not effective against either multiplying or nonmultiplying bacilli in MP at concentrations up to 50 μg/ml. These results with the drugs INH and CEF support the good record of correlation between the human MP model of tuberculosis and clinical experience in antituberculosis chemotherapy.

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1–3 and 7–9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4–6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.

Wolinella curva sp. nov.: "Vibrio succinogenes" of Human Origin

Asaccharolytic, anaerobic vibrios which require formate and fumarate for growth in broth culture have been isolated from humans. Some of these strains resemble Wolinella succinogenes phenotypically, but show no deoxyribonucleic acid (DNA)-DNA homology with either W. succinogenes or Wolinella recta. In this investigation, six unidentified strains were compared with reference and type strains of W. recta and W. succinogenes by using cluster analysis of phenotypic characteristics, enzyme-linked immunosorbent assays, and cell wall ultrastructure. One unidentified strain, a sewage isolate, was W. succinogenes. The remaining unidentified strains showed no DNA-DNA homology with either W. succinogenes or W. recta. Wolinella curva sp. nov. is proposed for four isolates which were distinct, as determined by serological and DNA-DNA homology experiments, from the previously described species. W. curva strains could also be differentiated from W. recta by morphology and by the growth of W. curva in the presence of Janus green (0.1 g/liter), basic fuchsin (0.032 g/liter), sodium deoxycholate (1.0 g/liter), indulin scarlet (0.5 g/liter), oxgall (10 g/liter), safranine (0.5 g/liter), azure II (0.025 g/liter), penicillin (16 μg/ml), and polymixin B (4 (μg/ml). W. curva and W. succinogenes are similar in cell ultrastructure and in other phenotypic features.

Copper-inhibition of the growth of oral streptococci and actinomyces

Copper ions were found to inhibit the rate of growth in broth culture of Streptococcus mitis, Streptococcus mutans, Streptococcus salivarious, Streptococcus sanguis, Actinomyces viscosus and Actinomyces naeslundii. In all cases, 10 −3 M copper inhibited the rate of growth, whereas 10 −4 M and lower concentrations had little or no effect. At the concentrations used in mouthwashes one mode of action of copper ions is to reduce the rate of growth of oral bacteria in vitro.

Hyaluronate capsule prevents attachment of group A streptococci to mouse peritoneal macrophages.

The antiphagocytic properties of the streptococcal hyaluronic acid capsule were explored in a system in which binding of the organism to the phagocyte, but not ingestion, could occur. The capsule was found to be highly effective in preventing attachment of two strains of group A streptococci to mouse peritoneal macrophages. Variation in attachment with phase of growth in broth culture (low in early log phase, high in late-log phase and thereafter) could be accounted for by production and subsequent loss of capsule. Hyaluronidase treatment removed the capsule and increased adherence; treatment with proteolytic enzymes removed M protein and decreased resistance to phagocytosis in whole human blood but did not remove the capsule or increase adherence to the mouse peritoneal macrophages.

Prevention of confluent hypersensitive necrosis in tobacco leaves by a bacterial protein-lipopolysaccharide complex

A protein-lipopolysaccharide (pr-LPS) complex has been selectively detached from washed living cells of Erwinia chrysanthemi NCPPB 454 by treatment with disodium ethylene diamine tetraacetate (EDTA). The material was partially purified by ultrafiltration, then chemically characterized, and its purity was checked by spectrophotometry and gel chromatography. The method yielded a protein-lipopolysaccharide complex of the outer membrane containing about 15% protein. The protein moiety was characterized by a major polypeptide of 40 000 daltons. Dispersions of the complex were injected into interveinal areas of mature tobacco leaves kept at 25°C. A slight chlorosis slowly developed in most treated areas. After 48 h, treated and control areas were injected with 4 × 10 7 cells/ml Pseudomonas syringae NCPPB 2664. Whereas the control areas developed confluent hypersensitive necrosis, the areas pretreated with the complex did not. Prevention of hypersensitivity was consistent at concentrations of 50 to 500 μg/ml; complete or partial prevention was obtained in the range of 3 to 50 μg/ml. Substances immunologically identical to the purified pr-LPS complex were spontaneously released by E. chrysanthemi during growth in broth cultures. The results thus suggest that pr-LPS complexes of bacterial outer membranes may play a rôle in modifying the response of plants to phytopathogenic bacteria.

Properties of some bacteriocins produced by Rhizobium trifolii.

Bacteriocins produced by six strains of Rhizobium trifolii were found to be of the relatively low molecular weight, non-phage type. The molecular weights ranged from approximately 1-8 X 105 to 2-0 X 105. All were of protein composition, as indicated by buoyant density (1-32 to 1-34 g/cm3) in CsC1 and by sensitivity to proteolytic enzymes. They were resistant to RNAase but sensitive to DNAase. The six bacteriocins could further be separated into two subgroups on the basis of sensitivity to extremes of pH, binding to filter membranes, activity spectrum on sensitive strains of R. trifolii, and possibly mode of action on sensitive bacteria. Bacteriocin production occurred spontaneously during the early-to mid-exponential phase of bacterial growth in broth culture.

The Ultrastructure of Mycoplasma hyorhinis in Broth Culture

Summary A pathogenic strain of Mycoplasma hyorhinis in the logarithmic phase of growth in broth culture was examined by electron microscopy and found to have a structure typical of the genus. The organism reproduced by binary fission and by budding to form elementary bodies.

Antigenicity of Myicoplasma mycoides I.—Examination of Body Fluids from Cases of Contagious Bovine Pleuropneumonia

Scrum, plasma, lysed blood cells, urine and pleural fluid or lymph (inflammatory exudate from subcutaneous inoculation) were obtained from cattle naturally and artificially infected with Mycoplasma mycoides. These fluids were examined for antigens of M. mycoides by means of the agar gel precipitin test and the quantitative agar gel precipitin test. They were also examined for antibodies against M. mycoides by means of the complement fixation and slide agglutination tests, and for viable M. mycoides by growth in broth cultures. Hyperimmune sera for use in these tests were prepared in sheep by the intravenous injections of washed organisms. Viable organisms were obtained only from the pleural fluid in natural cases of CBPP, but in the experimental cases the organisms were present in the serum, plasma and lymph. Antibodies against M. mycoides were demonstrated in sera and plasma samples of all cases. Specific precipitating antigens were demonstrated in all the fluids, urine possessing at least 5, lymph and pleural fluid at least 6 and serum and plasma at least 6 and sometimes 7 antigens. The 5 antigens in urine were common to all fluids, while the extra 1 in lymph and pleural fluid was also present in serum and plasma. In addition to these so-called major antigens, minor ones were also demonstrated but they were not studied in detail.

IDENTIFICATION OFMYCOPLASMATACEAEBY THE FLUORESCENT ANTIBODY METHOD

Clark, Harold W. (The George Washington University, Washington, D.C.), Jack S. Bailey, Richard C. Fowler, and Thomas McP. Brown. Identification of Mycoplasmataceae by the fluorescent antibody method. J. Bacteriol. 85:111-118. 1963.-The conditions of the fluorescent antibody reactions were studied in relation to their application to Mycoplasmataceae or pleuropneumonia-like organisms (PPLO). Mycoplasma hominis type 1 and 2 antigens and their homologous antisera were used to determine the activity and specificity of these and other strains. Fluorescein isothiocyanate conjugated antiserum globulin preparations were used in both the direct and indirect fluorescent antibody methods. A direct tube technique was used for the detection and measurement of growth in broth cultures by the addition of conjugated antiserum. The specific fluorescent staining and recognition of hot water fixed M. hominis colonies was presented as a suitable identification standard. The antigenic activity was found to remain in the insoluble residue after exposure of M. hominis strains to sonic vibration (9 kc) for 30 min and centrifugation. Brief 2-min exposures of tissue cells to vibration (9 kc) caused the disruption of tissues, with the release of viable and "bound" nonwashable strains that reacted specifically with fluorescent antibody. It is proposed to apply both the sonic vibration and the fluorescent antibody techniques for the identification of Mycoplasmataceae in human tissues.

Polysaccharide Haptens from Urine of Cattle infected with Mycoplasma mycoides

SERUM, plasma, lysed blood cells, urine and pleural fluid or ‘lymph’ (inflammatory exudate) were obtained from cattle naturally and experimentally infected with Mycoplasma mycoides. These materials were examined for antigens of M. mycoides by means of the agar-gel double diffusion precipitin test and the quantitative agar-gel precipitin test1. They were also examined for antibodies against M. mycoides by means of the complement fixation test2 and the slide agglutination test3, and for viable M. mycoides by growth in broth cultures.