Showing papers on "Growth medium published in 1995"

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Abstract: Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.

Production and pH-Dependent Bactericidal Activity of Lactocin S, a Lantibiotic from Lactobacillus sake L45.

Abstract: The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 deg C. When Lactobacillus sake L45 was grown in a fermentor at pH 5, it produced 2,000 to 3,000 bacteriocin units per ml, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity was observed during fermentation at pH 6.0. When 1% ethanol was included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aerating the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.

Increased protein secretion and adherence to HeLa cells by Shigella spp. following growth in the presence of bile salts.

Abstract: Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanced the bacterial invasion of HeLa cells. Growth in the presence of other structurally similar bile salts or detergents had little or no effect. Deoxycholate-enhanced invasion was not observed when bacteria were exposed to deoxycholate at low temperatures or when chloramphenicol was added to the growth medium, indicating that bacterial growth and protein synthesis are required. Increased invasion is associated with the presence of an intact Shigella virulence plasmid and is correlated with increased secretion of a set of proteins, including the Ipa proteins, to the outer membrane and into the growth medium. The increased invasion induced by the bile salts appears to be due to increased adherence. The enhanced adherence was specific to Shigella spp., since the enteroinvasive Escherichia coli strains tested did not exhibit the effect in response to growth in bile salts.

Antibiotic effect of linolenic acid from Chlorococcum strain HS-101 and Dunaliella primolecta on methicillin-resistant Staphylococcus aureus

Abstract: Methanol extracts fromChlorococcum strain HS-101 andDunaliella primolecta strongly inhibited the growth of a strain of methicillin-resistantStaphylococcus aureus (MRSA), which is causing serious problems in Japanese hospitals. So that the anti-MRSA substance(s) could be purified and identified, the growth medium was improved for antibiotic production. When the two strains were cultured in their improved media, antibiotic production byChlorococcum strain HS-101 was 1.8-fold that in the standard BG-11 medium, and production byD. primolecta was 2.3-fold. The activity pattern of fractions eluted by silica-gel or gel-permeation chromatography suggested that both strains produced two antibiotic substances. Identification of the purified substances by NMR and GC-MS showed that one of the active substances in both strains wasα-linolenic acid. Ten fatty acids from other sources were tested, and it was found that unsaturated fatty acids had antibiotic activity against MRSA, with the highest activity that of γ-linolenic acid.

Studies on carbon material requirements for bacterial proliferation and spore germination under stress conditions: a new mechanism involving transmission of physical signals.

Abstract: The growth of bacteria is often enhanced by addition of carbon materials such as graphite or activated charcoal to the growth medium. In this work, bacterial strains that strictly require such carbon materials under the ordinarily lethal stress caused by high concentrations of salt were isolated. The organisms were gram-positive, spore-forming, sugar-nonfermenting aerobic bacilli and were provisionally designated "Bacillus carbophilus" Kasumi after examination of their phenotypic traits. The growth- and germination-promoting effects of graphite and activated charcoal were demonstrated either quantitatively on agar plates containing fine crystals of the carbon materials mixed with a nonpermissive growth medium or qualitatively on agar plates on nonpermissive growth media half-covered with fine carbon particles. Further experiments demonstrated a novel feature of the phenomenon; i.e., the ability to induce colony formation on the nonpermissive plate was transmissible through the air, as well as through plastic or glass barriers. The mechanism probably involves transmission of physical signals regulating cell growth.

Enrichment, isolation and counting of soil microorganisms

Abstract: Publisher Summary The chapter discusses enrichment, isolation, and counting of soil microorganisms. A great number of morphological and physiological types of microorganisms can be found in soils. Enrichment and isolation of soil microorganisms are based on the cultivation of these microorganisms in liquid or agar media. To obtain anaerobic growth conditions for facultative anaerobes, it is sufficient to prevent the oxygen diffusion in the growth medium. This can be achieved by slowly passing a high-purity gas over a hot column. If necessary, clean the gas further by passing it over a cold catalyst or Oxisorb. The isolation of strictly anaerobic bacteria by plating methods poses special problems. Cellulose is mainly degraded by fungi in soils. In contrast, most bacteria can only be cultivated on partially hydrolyzed cellulose. Pseudomonaceae are gram-negative bacteria that are found in soil, water, and air. Oligotrophic bacteria include taxonomically and physiologically different microbial groups. Until the early 1970s, the classical concept of aerobic diazotrophs—that is, bacteria able to use molecular nitrogen (N 2 ) as their sole nitrogen source for growth—referred only to bacteria able to grow under atmospheric oxygen concentrations. Soils and water may contain up to 10 8 –10 12 bacterial cells per gram of soil or milliliter of water, if determined by microscopic techniques. Since 1970, epifluorescence microscopy has become the major technique for direct enumeration of bacteria and fungi in soil. Cyanobacteria are prokaryotic microorganisms containing chlorophyll a and producing oxygen as a by-product of their photosynthetic activity. Cultures of cyanobacteria can be preserved after growth and should not be maintained in refrigerator-like other prokaryotes.

Cell cultures of and cell culturing method for nontransformed pancreatic, thyroid, and parathyroid cells

Abstract: The present invention provides a method for producing an expanded, enriched, non-transformed human cell culture of human pancreatic, thyroid or parathyroid endocrine cells and other types of cells which comprises (1) preparing partially purified, minced tissue that includes a desired type of cells; (2) concentrating the desired cells; (3) resuspending the concentrated cells in a growth medium which selects in favor of the desired cells and in which those cells are proliferated without being transformed and differentiated functions are retained through periodic passaging; (4) culturing the resuspended cells in the growth medium to effect sustained cell division; and (5) passaging the cultured cells periodically to expand the culture. The present invention further provides clonal strains of cells derived from the above-mentioned cell culture and procedures to form matrix-embedded aggregated and non-aggregated cells for providing pseudotissues and products such as matrix-embedded pancreatic islets (pseudoislets). Growth medium and conditioned medium is provided for the culturing of the cells and clonal strains, the growth medium comprising a suitable basal medium supplemented with effective concentrations of hypothalamus and pituitary extracts, serum and other ingredients, which growth medium selects in favor of desired human cells and against passenger cells including fibroblast, macrophage, and capillary endothelial cells such that the desired cells are selectively proliferated without being transformed and an expanded cell culture is provided of functionally differentiated, expanded, non-transformed human cells that is substantially free of such passenger cells.

Factors affecting the electroporation of Bacillus subtilis

Abstract: Bacillus subtilis 168 trp- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies. Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 x 10(3) transformants per microgram plasmid DNA. Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used. The cell walls of B. subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly. The response of a range of other B. subtilis strains to electroporation by the method produced was found to be variable. In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.

Effects of cadmium and of YAP1 and CAD1/YAP2 genes on iron metabolism in the yeast Saccharomyces cerevisiae.

Abstract: Summary: Saccharomyces cerevisiae was more resistant to cadmium when the growth medium contained excess iron. Cadmium reduced the amount of iron taken up by cells during growth, and the cell ferrireductase activity was also strongly inhibited. These effects depended on the YAP1 and CAD1/YAP2 gene dosage. The growth rate of cells in iron-deficient conditions and their ferrireductase activity in the absence of added cadmium were also strongly affected by the dosage of YAP1 and CAD1/YAP2 genes. Our results suggest an indirect influence of these genes on iron metabolism, possibly via modification of the cell redox status.

A defined medium for rumen bacteria and identification of strains impaired in de novo biosynthesis of certain amino acids

Abstract: A completely defined growth medium has been developed to determine the nitrogen requirements for several species of ruminal bacteria, and has revealed two strains which are impaired in de novo biosynthesis of certain amino acids. Using NH4Cl as a sole nitrogen source, the medium supported growth of Butyrivibrio, Selenomonas, Prevotella and Streptococcus species. One strain of B. fibrisolvens (E14) and one strain of P. ruminicola (GA33) did not grow in the presence of NH4Cl until the medium was supplemented with amino acids or peptides. For B. fibrisolvens strain E14, methionine was identified as the specific growth-limiting amino acid although methionine alone did not support growth in the absence of NH4Cl. For P. ruminicola strain GA33, any individual amino acid other than methionine or cysteine could supplement the medium and support growth. Enzyme assays confirmed a lack of NADH and NADPH-dependent glutamate dehydrogenase (GDH) activities in this strain.

Enzymatic properties of lipase and characteristics production by Lactobacillus delbrueckii subsp. bulgaricus.

Abstract: Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl2 to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.

Association with MDCK epithelial cells by Salmonella typhimurium is reduced during utilization of carbohydrates.

Abstract: Association of Salmonella typhimurium with MDCK epithelial cells in monolayers, represented primarily by intracellular bacteria after 30 min of contact, with centrifugation followed by vigorous washing, was measured during aerobic and anaerobic growth of the bacteria in brain heart infusion broth. Cell association was greatest during a short period in the late log phase of growth under aerobic conditions. At this time, the pH of the growth medium was changing from acid to alkaline and glucose (0.2% initially) was exhausted. Addition of excess glucose (0.5%) to brain heart infusion broth, which was not exhausted before the bacteria entered the stationary phase of growth, in which cell association dropped sharply, resulted in repression of cell association by the bacteria. The repressive effect of glucose on cell association could not be reversed by exogenous cyclic AMP in the bacterial growth medium. Under anaerobic conditions, the effect of glucose on cell association by the bacteria was not as great and the glucose was not exhausted before the bacteria entered the stationary phase. When S. typhimurium was grown in a rich but carbohydrate-free medium, cell association by the bacteria increased earlier in the growth cycle under both aerobic and anaerobic conditions. The addition of glucose and certain other utilizable carbohydrates to this medium caused a repression of cell association by S. typhimurium that was greater under aerobic growth conditions. These results show that cell association by S. typhimurium, which is accompanied by rapid internalization (cell invasion), is the same under aerobic and anaerobic conditions if the bacteria are grown to the log phase in a carbohydrate-free medium. This suggests that prior reports of greater cell invasion by S. typhimurium during anaerobic growth may have arisen from the use of media containing carbohydrates which were found to be more repressive during aerobic growth of the bacteria.

Growth of a mutant defective in a putative phosphoinositide-specific phospholipase C of Schizosaccharomyces pombe is restored by low concentrations of phosphate and inositol.

Abstract: A mutant (plc1-1) of Schizosacharomyces pombe unable to grow on a minimal medium containing high amounts of phosphate was selected. On yeast-extract agar its growth is temperature sensitive. Tests in liquid synthetic medium show that growth of the mutant is partially restored by lowering the phosphate and inositol concentrations in the growth medium. The growth defect is fully suppressed by a plasmid encoding a putative protein having the structural features of phosphoinositide-specific phospholipases C (PI-PLC). This protein, of 899 amino-acids, contains the characteristic X and Y domains found in all PI-PLCs of higher and lower eucaryotes and reveals, in addition, an EF-hand motif (putative Ca2+-binding site). Like the corresponding enzyme from Saccharomyces cerevisiae, the S. pombe PI-PLC is most similar to the δ form of PI-PLC isoenzymes. The cloned gene integrates at the plc1 site indicating that plc1 codes for a putative PI-PLC. Plc1 physically maps on the left arm of chromosome II between rad11 and mei3.

A Modified Growth Medium for Bacillus thuringiensis

Abstract: A published medium for the cultivation of Bacillus thuringiensis was modified for use in high-density fed-batch fermentations. This medium was diagnosed to have a complex nutrient limitation in batch and continuous cultivations. By selective additions of different medium constituents to a chemostat culture, it was established that the limitations were alleviated only by the addition of yeast extract or corn steep liquor ; neither the addition of any salt nor that of individual amino acids influenced the culture behavior. In shake flask cultures, a reduction in glucose concentration and addition of corn steep liquor made the medium carbon-limited ; enrichment with corn steep liquor alone showed dual limitations. These modified media resulted in higher cell densities and showed an increase in the amounts as well as the potency of insecticidal protoxins.

Microbial adaptation to iron: a possible role of phosphatidylethanolamine in iron mineral deposition

Abstract: Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with iron(III) (5 mm) complexed to citrate, the sole source of carbon, with no apparent diminution in cellular mass. Atomic absorption studies of different cellular fractions and supernatant at various growth intervals revealed that the trivalent metal was initially internalized. At approximately 41 h of incubation, the soluble cellular extract contained 9.5% of the iron originally found in the growth medium. However, as bacterial multiplication progressed, most of the metal was deposited as an extracellular insoluble gelatinous residue. Phosphatidylethanolamine appeared to be an important organic constituent of this precipitate. X-ray fluorescence and diffraction studies revealed that iron(III) was deposited as amorphous hydrated oxide. Scanning electron microscopy and energy dispersive X-ray microanalysis of the pellet aided in the identification of irregular shaped bodies rich in iron and oxygen that were associated with carbon-containing elongated structures. Examination of the bacterial cells by a transmission electron microscope equipped with an electron energy loss spectrometer indicated the deposition of iron within the cells.

A process for the reduction of the nucleic acid content of a fungus imperfectus

Abstract: The RNA content of Fungi Imperfecti, for example Fusarium graminearum can be reduced with low protein loss by heating it in the presence of its growth medium to a temperature above 68 °C and separating growth medium from it.

High-yield production of diphtheria toxin mutants by high-density culture of C7(β) tox+ strains grown in a non-deferrated medium

Abstract: A high-density growth approach was utilized to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheria: C7 (beta)(tox-201,tox-9) and C7 (beta)(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are CRM9 and CRM107; both of them carry the mutation in their binding site and, as a result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. Since iron inhibits diphtheria toxin production, the traditional approach has been to grow the bacteria in a very low iron concentration. The procedure described here involved the use of a modified, non-deferrated, growth medium that provided fast and high-density growth of the bacteria, and which, when associated with simultaneous depletion of glucose and iron, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15-20 g/l dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM products, which remained stable following microfiltration and ultrafiltration, could be easily purified using a two-step chromatography procedure.

UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase is amplified in tunicamycin-resistant soybean cells.

Abstract: A tunicamycin-resistant soybean cell line was developed by gradually increasing the concentration of tunicamycin in the growth medium. At the final stage, the resistant cells could survive in media containing 60 μg/ml of tunicamycin, whereas normal cells show a greatly retarded growth rate at 0.5 μg/ml of antibiotic. The tunicamycin-resistant cells had a greater than 40-fold increase in the activity of the enzyme UDP-GlcNAc:dolichyl-P GlcNAclP transferase, a 2–3-fold increase in the activity of dolichyl-P -mannose synthase, but no increase in the activities of other enzymes of the lipid-linked saccharide pathway such as dolichyl-P -glucose synthase or mannosyl transferases. There was also no change in the activities of the glycoprotein-processing enzymes, glucosidase I or glucosidase II, as compared to wild-type cells. The increase in GlcNAclP transferase was due to an increased production of enzyme, as seen by a dramatic increase in the amount of a 39–kDa protein, which is presumed to be this enzyme protein. The GlcNAclP transferase from tunicamycin-resistant cells was equally sensitive to tunicamycin as was the wild-type enzyme, but was considerably more labile to temperatures above 30°C. The activity in tunicamycin-resistant cells was greatly stimulated by exogenous dolichyl-P. The spectrum of oligosaccharides from labeled lipid-linked oligosaccharides was similar in wild-type and tunicamycin-resistant soybean cells, but the resistant cells had significantly greater amounts of the shorter and much lower amounts of the larger-sized oligosaccharides.

Regulation of endo-1,4-β-glucanase secretion fromTermitomyces clypeatus by carbon catabolic product(s)

Abstract: Secretion of CMCase byTermitomyces clypeatus was only observed in the presence of a gluconeogenic amino acid, a citrate-cycle acid, maleate, subinhibitory concentrations of glucosamine, or fluoride in the medium. The enzyme was not secreted in the presence of caffeine or IBMX or theophylline, and these phosphodiesterase inhibitors lowered the secretion of CMCase by glutamate. The presence of both glucosamine and glutamate in a cellulose medium were, however, antagonistic to CMCase secretion. In a growth medium, xylose and glucose were equivalent carbon source for the fungus while succinate was a poor source and strongly repressed growth at higher concentrations. Growth ofT. clypeatus was highly favored in media containing xylose/glucose with succinate/glutamate. During growth ofT. clypeatus in a glucose medium, the intracellular glucose level was stabilized by the presence of succinate, glutamate or glucosamine in the medium. All these observation suggested that a negative cellular regulation, mediated by carbon catabolic product(s), existed inT. clypeatus which regulated the secretion of CMCase. A transient but significant increase of intracellular cAMP and cGMP levels was observed at the onset of mycelial growth in glucose and glucose/maleate media, respectively.

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

Abstract: A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T0) or at T3, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.

Action of retinoic acid on human glioblastoma-astrocytoma--14 cells in culture.

Abstract: Monolayer and agar culture techniques were used to examine the antiproliferative activities and morphological alterations of glioblastoma-astrocytoma (G1-As-14) cells induced by 20 mumol retinoic acid (RA). RA treated cells assumed flattened appearance and formed multilayers no longer. Most of the cells formed cross-bridges with one another. RA treatment caused time-dependent, dose-dependent and cell seeding-dependent reduction of growth in both monolayer and in agar cultures. RA-induced growth inhibition was also affected by concentration of fetal bovine serum in the culture medium. All these effects could be reversed within 48 h after withdrawal of RA from the growth medium. The results demonstrated that the respective cell line was sensitive to RA-induced growth inhibition and morphological alterations which were generally associated with reduced expression of malignant phenotype.

Callus culture from hypocotyls of Kosteletzkya virginica (L.) seedlings

Abstract: It was shown that callus established from Kosteletzkya virginica (L) Presl (Malvaceae) can grow in salinities higher than 200 mM NaCl if previously accomodated stepwise Callus lines developed from seedlings of different harvests or of the same harvest at different times, all showed the same pattern of growth and sensitiviy to salinity The absorption of Na+ into the callus increased with increasing external NaCl concentration In the callus, Na+ was apparently distributed outside and inside a cellular membrane (possibly the plasmalemma) This membrane was, apparently, capable of regulating the Na+ concentration in the protoplast Outside this membrane Na+ accumulated to concentrations higher than in the external growth medium Exogenously supplied proline or glycine-betaine did not affect the growth of the callus Externally applied ABA stimulated growth under saline conditions and increased the accumulation of proline Growth and proline content were positively correlated in callus exposed to salinity, but in the presence of ABA they were negatively correlated ABA was involved in both growth and proline accumulation, but there was no clear relationship between these two effects Both ABA and proline, if added to the growth medium, improved the appearence of the callus

Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells

Abstract: Batch suspension cultures of chicory cells (Cichorium intybus L. var. Witloof) possess a NADH-specific nitrate reductase activity that peaks on day 3 of a 10 d growth cycle. When both nitrate and ammonium are used as nitrogen sources, chicory cells absorb nitrate first. Amonium uptake becomes predominant at day 3, even though NO 3 - , was still present in the medium. Although abscisic acid impairs growth as well as 15 NO - 3 uptake and reduction, it promotes nitrate reductase activity as measured both in vivo and in vitro. Specific activity is 50% higher in ABA-treated cells than in controls. These conflicting data may be explained either in terms of nitrate reductase levels or by the availability of reducing power and energy. Since NRA is generally controlled by the availability of the reducing power, the energy status of the cell, the adenylate nucleotide pools, were measured simultaneously with the carbohydrate levels within the cell and the growth medium. The energy charge was not modified during the growth cycle, regardless of the growth conditions. Yet ABA modified the intracellular carbo-hydrate metabolism and inhibited the acidic invertase, the sucrose synthase and the sucrose phosphate synthase activities. Modified assimilation rates of nitrate in chicory cells grown in the presence of ABA, were probably correlated to modified carbohydrate metabolism pathways leading to increased availability of reducing power, energy and C-skeletons.

Effect of carbon source and pH of the growth medium on spore germination in Phycomyces blakesleeanus

Abstract: Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD+-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.

Chinese hamster V79 cells harbor potentially lethal damage which is neither fixed nor repaired for long times after attaining maximal survival under growth conditions

Abstract: The kinetics of the repair and fixation of potentially lethal damage (PLD) was studied in log-phase Chinese hamster V79 cells. The postirradiation (10 Gy) survival of cells treated with hypertonic saline increased when these cells were incubated further in conditioned medium but not in growth medium, indicating that damage which is neither fixed by hypertonic saline nor amenable to repair in growth medium is nonetheless repaired in conditioned medium. Recovery of X-irradiated cells incubated in growth medium or in conditioned medium was maximal by about 70 min and was two times higher in conditioned medium than in growth medium. Cells incubated in growth medium for 70-120 min postirradiation continued to repair damage when subsequently shifted to conditioned medium only. Thus PLD is not fixed by the time the recovery plateau has been attained in growth medium, and this unfixed PLD can still be repaired when cells are shifted to conditioned medium. To study the kinetics of fixation of PLD (without hypertonic saline), the survival of cells incubated in growth medium for up to 9 h postirradiation was compared with that for cells incubated in conditioned medium. These results show that the damage was neither fixed nor misrepaired in growth mediummore » but rather remained unrepaired for up to 2 h, and that damage fixation in growth medium does not begin until after 2 h and is completed by 6 h postirradiation. 21 refs., 4 figs., 1 tab.« less

Cell cultures of and cells culturing method for nontransformed parotid cells

Abstract: The present invention provides a method for producing an expanded non-transformed cell culture comprising the steps of: (1) preparing partially purified, minced tissue; (2) concentrating the resulting cells and tissue pieces; (3) resuspending the concentrated tissue cells and pieces in a culture medium capable of supporting sustained cell division that is contained in a culture vessel; (4) incubating the cells; and (5) passaging the cells periodically. The present invention further provides clonal strains of cells derived from the above-mentioned cell culture, medium and conditioned medium designed for the culturing of parotid cells and other glandular cells such as pancreatic, thyroid, and parathyroid, and cells, and the use of cultured pancreatic cells to form pancreatic pseudotissues composed of matrix-embedded aggregated (pseudoislets) or individual cells, to treat blood sugar disorders in mammals, and to test for cytotoxicity and autoimmune activities with reference to pancreatic endocrine cells. The nontransformed cells are cultured in a growth medium comprising a suitable basal medium supplemented with effective concentrations of hypothalamus and pituitary extracts, and serum.