pH Of Growth Medium Research Articles

Proton Motive Potential Difference Across the Plasma Membrane of Plant Cells: a Comparison of Cultured Cells and Protoplasts From Italian Ryegrass (Lolium multiflorum Lam.) Endosperm

The electrochemical proton gradient (ΔμH+) was measured across the plasma membranes of ryegrass (Lolium multiflorum Lam.) endosperm cells grown in suspension culture. The pH gradient (ΔpH) and the transmembrane electrical potential (ΔΨ) of cells and protoplasts were compared. The pH gradient (ΔpH) was measured from the distribution of acetylsalicylic acid and 5,5-dimethyloxazolidine and the membrane potential (ΔΨ) was calculated from the distribution of the lipophilic tetraphenylphosphonium cation (TPP+) between the cells and medium. The protoplasts and cells maintained a similar ΔpH. At the external pH of the growth medium (pH 5.5), the cytoplasmic pH was about 7. Under these conditions the ΔΨ was -60 mV (negative inside) for cells and -46 mV for protoplasts. ΔpH and ΔΨ have values which vary reciprocally depending on the external pH. The main contributor to ΔμH+ at pH 4.0 is ΔpH whereas, at pH 7.0, ΔΨ is the main component. The external K+ concentration influences the cytoplasmic pH significantly only at the higher external pH values.

OPTIMIZATION OF NUTRIENTS AND CULTIVATION CONDITIONS IN GLUCOAMYLASE PRODUCTION

Initial adjustment of pH of growth medium in the range of 3.0–5.0 was found to be necessary for optimal production of glucoamylase by a newly isolated strain of Aspergillus niger. Optimum temperature of incubation under stationary conditions was found to be 26–28° C, although incubation at higher temperatures viz. 30 and 37° C led to faster fungal growth rate and increased biomass. In complex medium, the optimum glucose and peptone concentrations were 2 and 3% respectively, and the presence of trace elements was found to be essential for maximal enzyme yield.

Reconstruction of the gill from single-cell suspensions of the eel, Anguilla japonica

Minute gill arches, about 2 mm in length and with 17-20 pairs of filaments, were reconstructed from single-cell suspensions of the adult eel gill. Mixed populations of the dissociated cells were cultured with growth medium (pH 7.2-7.4) either in a Rose chamber or in a roller tube at 20 degrees C. Single cells began to aggregate within 1 h into small cell clusters, which increase in size with selective adhesion of free cells. Melanin cells were frequently observed in the aggregates. Cell aggregates containing melanin cells transformed progressively into a gill barlike structure, and then, filamentlike structures grew from the bar. Secondary lammelalike structures were observed on the filaments. With growth of the gill bar, the filaments increased in both number and length, and after about 1-2 wk minute gill arches were reconstructed with original histological properties and circulatory system.

Immune responses in human colon cancer: I. Microcytotoxicity assay for measuring killing of adherent human colon cancer cell lines

A short term microcytotoxicity assay system using radioisotope release as an index of target cell damage has been developed to evaluate immune killing of cultured adherent human colon cancer cell lines. Using this assay system, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and spontaneous cell-mediated cytotoxicity of adherent human colon cancer cell lines can be assessed in 4 hr or less. An essential step in the development of this system was the successful 51Cr-labeling of human colon cancer target cells with subsequent low spontaneous release. This was achieved through careful attention to cellular growth phase and medium pH during the labeling and assay period. In this microsystem, labeled colon cancer cells spontaneously released 51Cr at a mean rate of 2% per hr during the assay, a level low enough not to obscure specific cytotoxic responses. Complement-dependent cytoxicity was measured most conveniently over a 2-hr period, whereas antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity were optimal when measured over a 4-hr period.

Factors affecting the aggregation of Actinomyces naeslundii during growth and in washed cell suspensions.

Various factors affecting the aggregation of Actinomyces naeslundii strain 12104 were studied. When the pH of glucose-supplemented growth medium fell below 5.5, the cells aggregated and formed microbial masses which tenaciously adhered to the culture vessels. When the organism was cultured in the same medium in the absence of glucose, maximum growth was reduced and the final culture pH values remained above 6.5, but the cells were more dispersed and nonadherent. Adjusting the final pH of these cultures to below 5.5 with HCl caused the cells to aggregate. Cells from unsupplemented cultures with final pH values of 6.7 were washed by centrifugation, dispersed by vigorous shaking, and suspended in buffer at pH values ranging from 4.5 to 8.0. Aggregation (expressed as the percent reduction of optical density at 520 nm after incubation at 37 degrees C) occurred rapidly at pH values below 6.0 but did not readily occur at higher pH values. Aggregation of strain 12104 in washed cell suspensions was induced by low pH and influenced by cell concentration and ionic strength of the environment. Low pH values also induced aggregation in washed cell suspensions of Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, and Actinomyces viscosus.

Disturbance of the machinery for the gene expression by acidic pH in the repressible acid phosphatase system of Saccharomyces cerevisiae.

When the pH of growth medium containing a limited amount of inorganic phosphate is kept below 3.0, cells of Saccharomyces cerevisiae produce repressible alkaline phosphatase but no repressible acid phosphatase. The same cells produce acid phosphatase immediately on shifting the medium pH to 4.0 or above. Like intact cells, spheroplasts prepared from cells grown at pH 3.0 or 4.5 in medium with a limited amount of inorganic phosphate in suspension begin production of acid phosphatase immediately after pH shift from below 3.0 to 4.0 whereas sheroplasts from cells grown in inorganic phosphate-rich medium showed a prolonged lag period (3 h). The enzyme formation on the pH shift was sensitive to cycloheximide. No significant differences could be detected in cellular growth or in incorporation of 3H-L-lysine or 14C-adenine between cells cultivated at pH 3.0 and 4.5. These results along with the fact that the expression of structural genes of repressible acid and alkaline phosphatases is controlled by a common genetic regulatory system, at least in part, indicate that the genetic regulatory system operates to express the structural genes even at low pH, though the expression of repressible acid phosphatase is interrupted. Coupled experiments of temperature and pH shifts with the temperature-sensitive mutants of the regulatory genes suggest that the acidic pH affects the function of the cytoplasmic products of those genes in the expression of the structural gene. Based on these observations, a revised model involving the simultaneous functioning of the regulatory factors was suggested for the genetic regulation of repressible acid phosphatase synthesis.

Effects of X-Irradiation on Cell Division, Oxygen Consumption, and Growth Medium pH of an Insect Cell Line Cultured in Vitro

Cultured Trichoplusia ni cells in exponential growth were administered x- ray doses of 10,000 R and then subcultured. The untreated cell population began exponential growth within a few hours after subculture, eventually reaching stationary growth phase 96 hr later at a cell density of 2.08 x 10$sup 6$ cells/ ml, whereas the irradiated cell population did not change for 24 hr after irradiation and then began exponential growth at a rate similar to that of control cells, also reaching stationary phase at 96 hr, but at a cell density of 0.93 x 10$sup 6$ cells/ml, which is less than half the maximum density of controls. From 24 to 96 hr after treatment, the x-irradiated cells were characterized by an increased consumption of oxygen that was nearly twice the amount utilized by control cells. The pH of the cell growth medium increases over 96 hr from 6.3 to 6.6 for irradiated as well as for untreated cultures, but since the number of x-rayed cells is less than half the number of untreated cells, the pH increase, per cell, of medium from irradiated cultures is about twice that of medium from control cultures. (auth)

Cefoxitin, a Semisynthetic Cephamycin Antibiotic: Antibacterial Spectrum and Resistance to Hydrolysis by Gram-Negative Beta-Lactamases

The in vitro activity of cefoxitin, 3-carbamolyloxymethyl-7-alpha-methoxy-7[2-(2-thienyl)acetamido]-3-cephem-4-carboyxlic acid, was investigated. Activity against gram-positive organisms was less than that of cephalothin and cephloridine. It was highly active against gram-negative bacilli, with activity against Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae equal to that of currently available cephalosporins. In addition, it was active against certain Enterobacter strains, Serratia marcescens, indole-positive Proteae and Herellea. The strains of these latter bacteria were strains susceptible to carbenicillin and ticarcillin. Pseudomonas aeruginosa and other Pseudomonas species were resistant. Changes in pH, inoculum size, and type of growth medium had no significant effect on the activity of the antibiotic. Cefoxitin was highly resistant to hydrolysis by various types of gram-negative beta-lactamases. The precise role of resistance to beta-lactamase hydrolysis varied from strain to strain. Bacterial resistance to cefoxitin was not necessarily related to hydrolysis of the antibiotic. However, the resistance of cefoxitin to hydrolysis did contribute to its activity. Cefoxitin could function as an inducer of beta-lactamase activity and effectively bound to purified beta-lactamases.

Structure and function of the lysosomes of human fibroblasts in culture: dependence on medium pH.

Extract: Histochemical and ultrastructural consequences of pH variations in growth media were studied using three normal human fibroblast strains. Histochemical staining for acid phosphatase indicated increased amounts of enzyme activity in cells grown at higher pH. The ultrastructural consequences of an increase in growth medium pH was dramatic. A picture resembling storage disease developed in these cells within a few days. The cells became filled with bodies which could roughly be divided into two types. Type I had a homogeneous matrix, stained slightly with ruthenium red, was usually not membrane bound, and exhibited no acid glycerophosphatase activity. The nature and origin of these bodies is unclear. Type II bodies contained whorles of membranes and inhomogeneous matrix which was acid phosphatase positive but did not stain with ruthenium red. This type was always membrane bound and qualifies as lysosomes. Normal cells thus grown at higher pH were morphologically similar to cells cultured at neutral pH from patients with I-cell disease. These morphologic changes did not affect cell viability or cell growth. Speculation: A morphologic picture of severe storage disease can be induced in normal fibroblasts simply by increasing the medium pH. This is in agreement with our earlier observation that the lysosomal function of mucopolysaccharide degradation is progressively inhibited as pH of the growth medium increases. When attempting to diagnose genetic disease in cultured cells it is important to realize that intracellular lysosomal function is sensitive to extracellular pH. The observation of such pH sensitivity might also be a useful tool in studies on the general nature of lysosomal function.

Formation of a histidine-peroxide adduct by H2O2 or ionizing radiation on histidine: chemical and microbiological properties.

We find that small amounts of hydrogen peroxide in the presence of histidine strongly inhibit the growth of Salmonella typhimurium in glucose medium (pH 70). Other amino acids and analogues, e.g. imidazole, alanine, and histamine, are relatively or completely inactive under the same conditions. We also find that gamma-irradiated oxygenated solutions of histidine inhibit bacterial growth to an extent proportional to the amount of H2 0 2 produced radiolytically. In the glucose-containing microbiological growth medium (pH 70, t = 37°c), the presence of histidine greatly retards the normal rate of disappearance of added H202. These and other chemical and microbiological observations reported here suggest that histidine and H202 readily form a stable adduct. A histidine-peroxide crystalline adduct has been prepared and chemically characterized (Dirscherl and Mosebach 1954). Subsequently the preparation of other amino-acid-peroxide adducts has been described (Dirscherl and Moersler 1964).

Growth and Protelytic Activity of Pseudomonas fluorescens in Eggs and Egg Productsa

SUMMARYThe growth characteristics and proteolytic activities of 3 strains of Pseudomonas fluorescens were determined in blended fresh egg white (initial pH 7.8–8.0), egg white adjusted to pH 7.4, egg white with added yolk, 30‐day‐old egg white (pH 9.0), and various egg media at the 2% protein level. In all cases the number of viable cells increased markedly during the first l–2 weeks of incubation. Under certain conditions of incubation, strains 5 and 17 showed proteolytic activity in blended fresh egg white, and strain 10 in egg white adjusted to pH 7.4 and in egg white with added egg yolk. Growth and proteolytic activity were also observed in the 30‐day‐old egg white (pH 9.0). All strains showed growth and proteolytic activity in the spray‐dried egg white growth medium (pH 7.4, autoclaved). Proteolysis was also determined on solid media with various egg constituents as substrate.

Observations on Porcine Melioidosis in Malaya

This work was aimed to evaluate six qualitative and quantitative methods (hemolytic activity, cetyltrimethylammonium bromide agar plate method, oil spread method, drop collapse method, surface tension measurement and emulsification index) to study biosurfactant production by sixty-nine bacterial isolates which were obtained from petroleum crude contaminated soil and water samples. Among all isolates, Pseudomonas aeruginosa NCIM 5514 was evaluated as the most potent isolate producing rhamnolipid. Effectiveness of growth medium pH, inoculum size and concentration of NaCl on surface active properties and biomass & rhamnolipid production in fermentation medium were studied. Highest surface activity was obtained at 1% (v/v) inoculum at initial pH of the medium 7.2, which resulted 4.389 ± 0.019 and 3.146 ± 0.087 g/l biomass and rhamnolipid, respectively. Notable surface activity of rhamnolipid produced by P. aeruginosa NCIM 5514 makes it feasible to be used for industrial application.

Chelating agents for selective growth of bacteria.

THE use of pH buffers for stabilizing the pH of growth media has become common bacteriological practice. There do not appear to be any reports, however, of the similar use of chelating agents for controlling the concentration of metal ions as ‘metal buffers’1,2. With metal ions there is a more complex situation, of course, since there are few chelating agents selective for one particular metal. An observed effect on bacterial growth can, therefore, be due to simultaneous sequestration of several metal ions and this must be allowed for in designing experiments.

培養基のpHと細菌発育について

In order to explain the effect on the enzyme activities of bacteria to glucose and succinate on the reaction by resting cells under different pH from that of culture media, the author observed the enzyme activities of Sal. 57 S under a various pH of reaction media by means of conventional WARBURG's manometer. Sal. 57 S used in this study were cultured at the pH of 5.4, 7.2 and 8.3 respectively. The effect on the enzyme activity by a sudden change of pH during the reaction was also studied. The results obtained were as follows.1) Concerns to the enzyme activity to glucose, the bacterial cells showed an adaptation to the different pH of reaction media by short time incubation of resting cells in that media and gave the proper reaction rate to the pH of reaction media. By the change of the pH of reaction media to the identical pH of culture media during the reaction, this adaptation to the reaction media was gradually lost and the reaction rate approached to the rate found at the same pH as growth media. But the bacterial cells cultured at the pH of 5.4 adapted to the pH of reaction media in alkaline by long time incubation in that and the reaction rate retained for long in spite of the change of pH to the pH of growth media.2) In the case of the activity to succinate, the bacterial cells did not show an adaptation to the different pH of reaction media by such incubation as above. The enzyme activity of either culture was found to be highest at the pH of 5.4.