Peptone-yeast Extract Medium Research Articles

Formation of beta-lactamase in Bacteroides fragilis: cell-bound and extracellular activity.

Nine strains of Bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce beta-lactamase. There was a correlation between formation of beta-lactamase and high values of the minimal inhibitory concentration against beta-lactam antibiotics. B. fragilis strain B70 was used for optimizing the production of beta-lactamase. The highest bacterial yield was obtained in a proteose peptone-yeast extract medium. Optimal conditions for growth and beta-lactamase production were obtained at 37 C and pH 7.0. The beta-lactamase was released into the surrounding medium during the growth period to about 50%. Osmotic shock released about 20% of the total activity, and remaining activity was found in the cytoplasmic fraction. Substrate profile studies on four beta-lactamase-producing strains showed that the enzymes were mainly cephalosporinases. They are inhibited by cloxacillin, p-chloromercuribenzoate, and iodine. Analytical isoelectric focusing in polyacrylamide gel gave an isoelectric point of 4.9 +/- 0.2 for three of the strains and 5.6 +/- 0.2 for one. Comparison with beta-lactamases from aerobic gram-negative species with regard to isoelectric points showed no similarities. Also the molecular weight of the beta-lactamase from strain B70 of 43,000 indicates that this is a new class of beta-lactamase.

GLUCOSE METABOLISM OF ORANGE-COLORED STREPTOCOCCUS BOVIS

Experiments with 14C-labeled glucose indicated that the orange-colored variety of Streptococcus bovis assimilated about 12% of consumed glucose in the synthetic medium and 6% in the peptone-yeast extract medium. These values are very much higher than those of ordinary lactic streptococci which show the values of around 0.4%. Although the yield of lactic acid did not exceed 90% of consumed glucose, this species is also a homofermenter if the assimilated glucose is taken into account.Different from ordinary lactic acid bacteria, S. bovis can grow in the medium containing ammonium salt as a sole source of nitrogen but organic nitrogen is preferable even for this species.

Analysis of Lipid Composition of Isolated Human Sebaceous Gland Homogenates After Incubation with Cutaneous Bacteria. Thin-Layer Chromatography

The effects of specific species of skin bacteria on human sebaceous gland lipids in vitro were analyzed. Isolated dissected sebaceous glands were pooled, homogenized, and sterilized, then incorporated into peptone-yeast extract medium and used as substrate for growth of Propionibacterium acnes, P. granulosum, and Staphylococcus epidermidis subgroup II. The sebaceous lipids were analyzed by thin-layer chromatography before and after bacterial growth. The most striking effect of bacteria on sebaceous gland lipid composition was the hydrolysis of sebaceous triglycerides. The degree of hydrolysis varied with bacterial strain but was most complete with P. acnes and P. granulosum. Staphylococci were not effective in hydrolyzing sebaceous triglycerides at pH 4.5 although, when the pH of the medium was raised to pH 6.4, some strains of staphylococci were as effective as the propionibacteria in hydrolyzing sebaceous triglycerides to free fatty acids. Thus minor changes in acidity may play asignificant role in controlling the lipolytic activity of staphylococci on skin. Another effect of bacterial action on sebaceous gland lipids was the esterification of sebaceous cholesterol to cholesteryl esters. Thus, bacterial action must be taken into account in evaluating studies of alterations in cutaneous cholesterol and cholesteryl esters in skin surface lipids in normal and disease states.

Effect of carbon source on the replication and transmission of yeast mitochondrial genomes.

Genetic crosses were made using strains of Saccharomyces cerevisiae which carried cytoplasmically inherited markers conferring resistance to erythromycin, oligomycin and chloramphenicol. The frequency of transmission of these mitochondrial loci to diploid progeny was found to be influenced by the physiological state of the haploid parents, and was not affected by the cis or trans configuration of the three resistance markers. No recombinational polarity was seen in any of the crosses. Growth of a haploid parental strain to stationary phase in a yeast extract peptone medium containing glycerol as a carbon source resulted in a high level of transmission of mitochondrial markers when crossed with a strain grown to stationary phase in the same medium but with glucose as carbon source. When cells were grown under the same conditions as those used in the genetic crosses they were found to contain more mitochondrial DNA relative to nuclear DNA when glycerol was used as a carbon source than when glucose was used. Two criteria were used to determine the amount of mitochondrial DNA present: i) incorporation of radioactive precursors into the different DNA species; and ii) measurement of the mass of DNA from amounts of ultraviolet-absorbing material at the appropriate buoyant densities in isopycnic CsCl gradients. It is proposed that the two to three fold difference in the ratio of mitochondrial DNA to nuclear DNA in stationary phase cells grown in the presence of glycerol compared to glucose reflects an increased number of mitochondrial genomes in derepressed mitochondria. This difference, by a “genome dosage” effect, could account for the variations in the genetic parameter of the frequency of transmission, i.e., strains grown with glycerol as a carbon source contain more mitochondrial genomes than glucose-grown strains and thus will contribute more mitochondrial markers to the zygote.

A cycloheximide-resistant mutant of Tetrahymena pyriformis

A mutant of Tetrahymena pyriformis, syngen 1, resistant to cycloheximide was obtained after mutagenesis (with N-methyl- N′-nitro- N-nitrosoguanidine) followed by a cross (to obtain macro-nuclear expression of the mutant phenotype). A genetic analysis has shown that cycloheximide resistance in the mutant strain is due to a dominant nuclear allele, designated chx-1. Heterozygotes ( chx-1 chx + ) are initially resistant but segregate stable, sensitive cell lines during vegetative growth, demonstrating that allelic exclusion occurs with this determinant, as with many others in syngen 1. This feature, coupled with the selective advantage conferred by the chx-1 allele in the presence of cycloheximide, makes this mutation a useful genetic tool. A strain homozygous for the chx-1 allele exhibits an exponential growth rate identical to that of the wild type in proteose peptone-yeast extract medium in the absence of cycloheximide. In 10 μg/ml of the drug, the resistant cells grow at a somewhat lower rate, after an initial lag and adaptation to the presence of the drug. This concentration causes complete inhibition of growth and eventual lysis of wild-type cells. The cellular basis for cycloheximide resistance and adaptation in the mutant is presently under investigation.

Energetics of growth in a tyrothricin-producing strain of Bacillus brevis.

Summary: The particulate NADH oxidase activity of Bacillus brevis is inhibited by tyro-cidine and the kinetics of inhibition indicate that the NADH dehydrogenase region is the primary site of action. A decrease in NADH oxidase activity of electron transport particles and of NADH oxidase activity of whole organisms occurs in batch culture in the later stages of growth when production of tyrothricin increases. That respiration exhibited by growing cells takes place via the respiratory electron-transport system is indicated by the similarities in levels of activity and cyanide sensitivity of endogenous and NADH oxidation of growing cells. Exponential growth on glycerol asparagine media is diphasic, a slower growth rate commencing as the O2 tension of the culture approaches zero and tyrothricin production increases. Molar growth yield data based on overall measurements of growth related to O2 uptake in batch culture lead to low ATP/O ratios. In an attempt to determine Y 0 and ATP/O ratios under optimum conditions of growth a method is described which was based entirely on the growth and respiratory activity of exponentially growing cells. Such measurements led to higher values for Y 0 and to ATP/O ratios approaching 3, indicating the participation of three sites of phosphorylation during exponential growth. Tyrocidine and DNP inhibited growth, the tyrocidine at concentrations similar to those produced at the end of the exponential growth phase. Y 0 /Y ATP ratios (ATP/O ratios) on glycerol asparagine media were lower than on peptone yeast extract medium which yielded no tyrothricin, a possible indication that some form of uncoupling occurs in organisms grown on glycerol asparagine medium which does not yield tyrothricin.

Effects of cyclic nucleotides and nucleoside triphosphates on stalk formation in Caulobacter crescentus.

Extensive stalk elongation in Skl mutants of Caulobacter crescentus occurs when they are grown in complete medium. This stalk elongation is less pronounced in synthetic medium with glucose as the sole carbon source than in complex peptone yeast extract medium. Addition of exogenous nucleoside triphosphates (adenosine triphosphate [ATP], guanosine triphosphate [GTP], cytidine triphosphate, and uridine triphosphate) inhibits stalk elongation of the Skl mutants, whereas cyclic guanosine 3',5'-monophosphate (GMP) stimulates stalk elongation in the Skl strains grown in synthetic glucose medium. Cyclic GMP also produces stalk elongation in wild-type C. crescentus and concurrently produces a cell division defect resulting in cellular filament formation. Under conditions tested, cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic adenosine monophosphate did not enhance stalk elongation. Endogenous ATP and GTP levels in the mutants are significantly lower than corresponding nucleotide concentrations of the parent wild-type strains. Control of syntheses resulting in stalk formation in C. crescentus appears to be related to intracellular concentrations of nucleotides, with cyclic GMP as a prominent candidate for an important regulatory role in this aspect of morphogenesis.

Endocytosis in Tetrahymena pyriformis: The selectivity of uptake of particles and the adaptive increase in cellular acid phosphatase activity

Summary The cellular acid phosphatase activity of Tetrahymena pyriformis , grown in peptone-yeast extract medium, is markedly increased after edocytosis of yeast suspension, but not by uptake of various sizes of polystyrene or polyvinyltoluene latex particles. This suggests that the adaptive enzyme increase in response to endocytosis and intracellular digestion is not due solely to the act of endocytosis itself and that the quality of the material taken up is itself involved. It is calculated that about ten latex particles (1.95 μ diameter) are taken up per average Tetrahymena per minute. This is equivalent to a volume uptake of 42 μm 3 /min/average cell. An area of membrane greater than the entire surface area of the cell is calculated to have been involved during the 150 min period of endocytosis. Starved Tetrahymena showed increased cellular acid phosphatase activity after endocytosis of yeast suspension or of peptone-yeast extract, but did not endocytize latex particles to any great extent. Microscopic examinations showed that fed Tetrahymena rapidly endocytized useful materials such as peptone, yeast suspension or washed living bacteria and also indigestible materials such as latex particles and ink suspensions. Starved cells on the other hand, took in only the useful materials. Latex particles presented to the starved cells at the same time as peptone were however rapidly taken up. Other results suggest that cells grown in peptone-yeast extract medium take up particles at rates proportional to their relative numerical concentrations and that this uptake is not markedly related to particle size. Digestible particles are not taken up in preference to indigestible ones under these conditions. The results are discused.

Effect of endocytosis upon acid phosphatase activity of tetrahymena pyriformis.

Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.

X-ray-induced mitotic recombination, gene conversion and mutation in Saccharomyces and the radioprotective effect of cysteine.

1. The X-ray-induced frequency of mutation, mitotic recombination and gene conversion was determined in Saccharomyces cerevisiae. 2. X-rays induced only few mutations compared with alkylating nitrosamides but were highly efficient in the induction of mitotic recombination and gene conversion. Therefore, X-rays in spite of their low mutagenicity turned out genetically to be a very harmful agent for diploid yeast cells. 3. A protection against X-ray induced recombination and conversion could be achieved by the well known protecting agent cysteine. The protection factor is about 2. A similar effect could be observed with yeast extract-peptone medium but not with the amino acid serine. 4. Furthermore, the protective effect of cysteine and yeast-extract peptone medium was studied at various concentrations and preincubation times.

Temperature adaptation of the growth and division process of Tetrahymena pyriformis: I. Adaptation phase

Growth rates of Tetrahymena pyriformis GL are reported following temperature shifts and during induction of synchronous cell division. Use of an electronic cell counter permitted the ready determination of cell volume distributions. These distributions were used in determining growth rates. Tetrahymena pyriformis GL was cultured in proteose peptone yeast extract medium at 28.0, 30.8, 32.5, and 33.9 °C. Following a temperature shift from 28 °C to a higher temperature, a brief period of continued cell division occurred followed by a prolonged period of blocked cell division. Division block was permanent at 33.9 °C and was of a temporary nature during the temperature adaptation phase at 30.8 and 32.5 °C. Correlation coefficients of the growth rates suggest that cell growth during temperature adaptation was exponential. Cell volumes were log-normally distributed before the temperature shift and during periods of temperature adaptation. The slope of the log volume class distribution was constant with time during the period of inhibited cell division. During induction of synchronous division, produced by intermittent heat shocks, cell growth was also exponential, cell volumes were log-normally distributed and the slope of the log volume class distribution was constant. This constancy in slope during periods of inhibited cell division indicates that the rate of cell growth is independent of the division cycle during single and multiple temperature adaptation. During periods of inhibited cell division the rate of cell growth at 30.8 °C was significantly different from that at 32.5 °C. However, the rate of growth at 33.9 °C was significantly lower than at 30.8 or 32.5 °C. This indicates that the optimum temperature for the rate of cell replication and growth are different.

Temperature adaptation of the growth and division process of Tetrahymena pyriformis: II. Relationship between cell growth and cell replication

Replication rates and volume distributions of Tetrahymena pyriformis GL determined with a commercially available electronic cell counter are reported for several temperatures. Tetrahymena pyriformis GL was cultured in proteose peptone yeast extract medium at 28.0, 30.8, 32.5, and 33.9 °C. During the period of exponential cell replication at all temperatures, the volume distribution was constant, lognormal and the standard deviation from the median volume was temperature independent. This indicates a steady state of balanced growth at 28.0, 30.8, and 32.5 °C during exponential cell replication. It is demonstrated that the knowledge of volume distribution and generation time is insufficient for calculating rates of cell growth between divisions unless the parameters for the momentary volume distribution can be measured independently. Statistical evaluation of data from the literature confirms previously reported findings which indicate that growth of Tetrahymena over extended periods of the division cycle is approximately exponential and not linear. The previous proposition that control mechanisms for growth of Tetrahymena and Paramecium are different, is thus unnecessary. A new model, which describes the control of cell growth and cell replication is suggested. The model accounts for the lognormal distributions of generation times and volumes as well as the nearly exponential rate of growth between divisions. The mean rate of cell replication which decreases with increasing temperatures above the optimum rather than all temperatures shows a negative correlation with the median log volume of the culture. This is in accordance with the predictions of the proposed growth model and furthermore indicates that the growth rate is to a large extent independent of cell replication.

Use of dialyzing culture technique for high yield of Mycoplasma.

Pollock, Mary E. (University of Minnesota, Minneapolis). Use of dialyzing culture technique for high yield of Mycoplasma. J. Bacteriol. 90:1682-1685. 1965.-The saprophytic pleuropneumonia-like organism, Mycoplasma laidlawii, type A, was cultivated within a dialyzing membrane suspended in a reservoir of uninoculated medium. Generation time was not decreased by this method, but the period of active growth was prolonged, and the final yield was 10-fold that attained by the usual cultural methods. A defined mixture, which alone would not support growth of Mycoplasma, could replace the complex soy peptone-yeast extract medium in the inner membrane vessel of the dialyzing culture flask with no decrease in growth rate or yield.

病原性好塩菌に関する研究-I

Much attention has been paid on the Takikawa's so-called pathogenic halophilic bacteria, a new type of bacteria suspected to cause food poisoning. It has been only a short period since the investigation started and there are still many problems to be solved, in which pathogenicity of the organisms is still obscure due to the unavailability of susceptible experimental animals. In the present work, effect of various culturing conditions of the organisms upon the lethality for mice has been studied. The results may be summerized as follows: 1. The organisms grew well and gave a high lethality for mice when cultured in a 3% NaCl containing broth which was prepared with casein peptone and fish meat extract. The organisms grew rapidly in such 3% NaCl containing liquid media as the broth composed of meat peptone and fish meat extract, plain peptone water, or peptone-yeast extract medium, nevertheless, the lethality of the cultures were rather low as compared with the above-men-tioned broth culture. 2. As to the NaCl requirement of the organisms, the fastest growth rate was observed in a 3% NaCl broth, while there was no appreciable difference in the lethality for mice between the concentration 2 to 4%. 3. Growth of the organisms and their lethality for mice were varied according to the reaction of medium. Whithin the range of pH 6-8, the higher the pH value, the more rapid in growth or higher in lethality. 4. Presence of glucose in the medium enhanced the growth of the organisms. It was noted that the addition of more than 0.5% of glucose in the medium resulted in decrease in the lethality. 5. In the case of Matsuda strain (serotype XIII), higher lethality was observed in the early stage of culture of the organisms. The organisms lost their lethal toxicity for mice gradually by prolongating the incubation period. 6. Within a range 24° to 37°C., the highest lethality was recorded when the organisms were incubated at 24°C.

Factors affecting aspartase activity.

Depue, Robert H. (Hahnemann Medical College, Philadelphia), and Albert G. Moat. Factors affecting aspartase activity. J. Bacteriol. 82:383-386. 1961.-Cells of Escherichia coli grown in a glucose-mineral salts medium contain about one-fifth the amount of aspartase activity observed in cells grown in a yeast extract-peptone medium. The aspartase activity of the cells grown in glucose-salts medium would appear to be too low to provide a mechanism for synthesis of amino groups. Aspartase was purified approximately eightfold by ammonium sulfate precipitation and column chromatography of cell-free extracts. The purified preparation was specific for l-aspartic acid and contained no fumarase activity. A divalent metal ion requirement was demonstrated, this requirement being satisfied by cobaltous or manganous ions. The enzyme activity was found to be dependent upon free sulfhydryl groups. Biotin did not appear to be directly involved in the aspartase reaction since high concentrations of avidin did not alter the reaction rate. The Michaelis constant for aspartase with aspartic acid as substrate was determined to be 0.033 m.

An evaluation of the standards used in the study of the yeast nucleus

1. In the brewery yeast BY 1, the nucleus is visible in 1–2% of the cells from 6–8-day cultures in barley malt wort and 0·5–1% of the cells from 10–12-day cultures in Galactose Peptone Yeast extract medium.