Showing papers on "Growth medium published in 1994"

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

Abstract: The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.

Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells.

Abstract: Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.

Transcriptional induction kinetics from the promoters of the catabolic pathways of TOL plasmid pWW0 of Pseudomonas putida for metabolism of aromatics.

Abstract: We determined, under several growth conditions, the kinetics of mRNA synthesis from the four Pseudomonas putida pWW0 plasmid promoters involved in degradation of xylenes and methylbenzyl alcohols via toluates. Transcription by XylS of the meta-cleavage pathway operon promoter (Pm) for the metabolism of alkylbenzoates was stimulated immediately after the addition of an effector, both in Luria-Bertani (LB) medium and in minimal medium. Activation of the sigma 54-dependent upper-pathway operon promoter (Pu) and the xylS gene promoter (Ps) by effector-activated XylR was dependent on the growth medium used: on minimal medium, activation of transcription from Pu and Ps occurred immediately after the addition of a XylR effector; in contrast, activation appeared only after several hours when cells were growing on LB medium. When Pm was induced through the physiological overexpression of XylS, mediated by XylR when this regulator was activated by upper-pathway effectors, the kinetics of transcription from Pm was similar to that of Pu and Ps: maximum values were reached after delays of several hours in rich medium and after several minutes in minimal medium. The delay in the induction of transcription of sigma 54-dependent promoters reflects catabolite inhibition exerted by LB components, since the addition of yeast extracts, Casamino Acids, or several combinations of amino acids dramatically inhibited the synthesis of XylR-controlled sigma 54-dependent promoters. Expression from xylR gene tandem promoters occurred independently of the growth medium used.

Growth-promoting factors for Bifidobacterium longum

Abstract: The ability of various biological materials to promote growth of Bifidobacterium Iongum (ATCC 15708) was tested using Bacto B12 assay medium (Difco Corp.). Supplements included yeast extract, beef extract, malt extract, α-lactalbumin, β-lactoglobulin, trypticase soy broth, phytone-peptone and unknown factors from Escherichia coli spent broth. Growth of B. longum 15708 was monitored by measuring turbidity and pH. Yeast extract, α-lactalbumin and β-lactoglobulin were the best growth promoters. Growth in the presence of E. coli spent broth was maximal; however, fresh enzymatically hydrolyzed E. coli broth was as effective. Beef extract and trypticase soy broth were effective to some extent. Malt extract and phytone-peptone did not significantly enhance growth. All materials lost growth promoting activity when their disulfide bonds were reduced-alkylated.

Regulation of Periplasmic Carbonic Anhydrase Expression in Chlamydomonas reinhardtii by Acetate and pH

Abstract: The effects of mixotrophic growth with acetate and growth medium pH on expression of extracellular carbonic anhydrase (CA) in Chlamydomonas reinhardtii were evaluated. Addition of 10 mM acetate to the culture medium resulted in reduction of CA activity that was parallel to the reduction generated by growth of the algae in high external CO2 concentrations. This reduction in activity is a consequence of lower level of the CA protein as determined by western analysis. Transcript abundance of cah-1, the gene encoding the low CO2-induced CA, is also reduced by the addition of acetate as verified by northern analysis. Measurements of photosynthesis and respiration suggest that the acetate-induced reduction of CA expression is not a function of lowered photosynthetic capacity, but may be the result of increased internal CO2 concentration generated by high, acetate-stimulated respiratory rates. Growth medium pH can also influence extracellular CA expression. The induction of CA activity, protein abundance, and transcript levels by exposure to limiting inorganic carbon (Ci) concentrations is much more pronounced at higher than at lower pH values. The relationship between pH regulation of CA expression and its role in the Ci-concentrating mechanism are discussed.

Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism

Abstract: The levels of pyruvate kinase (PYK1), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast-to-hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25 degrees C to 37 degrees C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25 degrees C to 37 degrees C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late-exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape.

Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue

Abstract: This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400µg/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB3 derived from cells transfected with pSV3neo, and 2RB5 derived from cells transfected with pSV3neo, revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.

Enhanced Yields of Iron-Oxidizing Bacteria by In Situ Electrochemical Reduction of Soluble Iron in the Growth Medium

Abstract: An electrochemical apparatus for culturing chemolithotrophic bacteria that respire aerobically on ferrous ions is described. Enhanced yields of the bacteria were achieved by the in situ electrochemical reduction of soluble iron in the growth medium. When subjected to a direct current of 30 A for 60 days, a 45-liter culture of Thiobacillus ferrooxidans grew from 6 × 107 to 9.5 × 109 cells per ml. Growth of the bacterium within the electrolytic bioreactor was linear with time. A final cell density corresponding to 4.7 g of wet cell paste per liter was achieved, and a total of 320 g of wet cell paste was harvested from one culture. The apparatus was designed to deliver protons concomitantly with electrons; therefore, the pH of the culture remained stable at 1.6 ± 0.1 for the duration of growth. This laboratory-scale apparatus may be readily adapted to pilot or production scale. It is thus anticipated that abundant numbers of iron-oxidizing bacteria may be obtained for both fundamental and applied studies.

Antagonistic effect of nickel on the fermentative growth of Escherichia coli K-12 and comparison of nickel and cobalt toxicity on the aerobic and anaerobic growth.

Abstract: The facultative anaerobic enterobacterium Escherichia coli requires the activity of nickel-containing hydrogenase for its anaerobic growth. Deficiency of the specific nickel transport system led to a hydrogenase-minus phenotype and slowed down the fermentative growth in the nik mutant. Addition of 300 pM nickel to the growth medium could restore the hydrogenase activity. This restoration resulted in the recovery of anaerobic growth. A fur- ther increase of nickel concentration inhibited growth. Thus nickel shows an antagonistic effect on the anaerobic growth of E. coli. To study the mech- anism of nickel toxicity, two classes of nickel-resistant mutants were isolated. The nkr mutant was obtained by selecting colonies grown on nickel-containing minimal plate. It acquired simultaneously the resistance to cobalt. A nonspecific magnesium transport mutant corA was isolated on cobalt-containing plate. The corA mutant was also resistant to nickel. When analyzing the influence of nickel and cobalt on the bacterial growth, we obtained two interesting observations. First, anaerobic growth was less sensitive than aerobic growth to cobalt toxicity. In contrast, nickel toxicity did not vary from the growth conditions. Second, cobalt seems to abolish the growth, while nickel appears to slow down the growth rate under the condition used. Environ Health Perspect 1 02(Suppl 3):297-300 (1 994).

Effect of glucose concentration on citric acid production by Yarrowia lipolytica

Abstract: Citric acid production by Yarrowia lipolytica ATCC 20346 was studied in a laboratory-scale fermenter and schematically subdivided into three different phases, appropriately characterised by unstructed kinetic models. As the sugar level in the growth medium was increased from 40 to 108 g dm−3; a negative effect on the kinetic growth rate constant of the trophophase was obtained, where both the yield coefficients for glucose and nitrogen were found to be approximately constant. By using a production medium devoid of nitrogen, a citrate lag phase was observed whilst the yeast reduced its intracellular nitrogen fraction from 7–8% (w/w) to a new equilibrium value as low as 2·3–4·4%. Finally, the idiophase was found to be a non-growth associated process, the citric acid formation rate being dependent on the cellular nitrogen concentration only. The yield factors for citric, isocitric and fumaric acids appeared to be independent of the initial concentration of glucose in the production medium (Sop). However, the yield factors for α-ketoglutaric, malic, pyruvic and cis-aconitic acids exhibited a negative correlation with respect to Sop, being virtually equal to zero for Sop values greater than 160–175 g dm−3. The strain in question was capable of equalling the best mutant strains of Aspergillus niger used industrially in citric acid productivity (ca. 1·05 g dm−3 h−1), but not in selectivity, since citric acid represented only 85·5% of total citric and isocitric acid and 83·7% of the total acid.

Polysaccharide production in Pseudomonas cepacia

Abstract: The effects of glucose, osmolarity, temperature and mode of growth on exopolysaccharide production in Pseudomonas cepacia was studied in batch culture using a chemically defined growth medium. Polymer production was maximal under conditions of a 2% (w/v) glucose supplement, 0.4 M NaCl and an incubation temperature of 35 degrees C. In addition, polysaccharide composition and molecular weight varied with mode of growth. On agar culture there was a decrease in pyruvate and rhamnose content yet an increase in the amount of acetate compared to the polymer isolated from broth culture equivalents. The clinical implications of these results are discussed in relation to the potential pathogenicity of P. cepacia in cystic fibrosis patients.

Manipulation of polyunsaturated, branchedchain and trans-fatty acid production in Shewanella putrefaciens strain ACAM 342

Abstract: Shewanella putrefaciens strain ACAM 342 produced a range of polyunsaturated fatty acids (PUFA) including 18:2ω6, 18:3ω3, 18:3ω6, 18:4ω3 and 20:5ω3 (EPA). Under culture in Zobell's broth at 15 °C exponential-phase cultures produced 3.2 + 0.3% EPA, or 1.2 + 0.2 mg (g bacterial dry wt)-1. Alteration of growth phase, addition of biotin to the medium or a 10-fold decrease in nutrient levels had no significant effect (P>0.05) on levels of EPA. Growth in 7.0% NaCl medium markedly decreased the overall degree of fatty acid unsaturation (P<0.01), while growth on acetate as the sole carbon source increased the level of 18:2.6 from 0.4 + 0.1% to 7.1 % of total fatty acids through inhibition of branched-chain fatty acid synthesis. Addition of desaturase cofactors to the growth medium increased the proportion of EPA from 3.2 + 0.3% to 4.6%, but decreased the quantitative yield from 1.2. 0.2 to 0.9 mg (g bacterial dry wt)-1. This bacterium represents a model organism to study bacterial PUFA production. The trans-monounsaturated fatty acid 16:1ω7t was also produced under all culture conditions, together with four other trans-isomers, namely 14:1ω5t, 15:1ω6t, 17:1ωt and 18:1ω7t. Although the relative levels of trans-acids also changed under various culture conditions, there was no evidence that they were produced as a starvation or stress response.

Effect of medium composition on 1-octen-3-ol formation in submerged cultures of Pleurotus pulmonarius

Abstract: The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O2 to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium.

Regulation of phosphatidylinositol:ceramide phosphoinositol transferase in Saccharomyces cerevisiae.

Abstract: Maximal phosphatidylinositol:ceramide phosphoinositol transferase activity was measured in yeast cells harvested during the exponential phase of growth. The addition of inositol to the growth medium resulted in a twofold increase in IPC synthase activity in cells grown in the presence or absence of exogenous choline. Enzyme activity was not regulated in yeast inositol biosynthesis regulatory mutants by the addition of inositol to the growth medium.

Stimulatory effects of aluminum on the growth of cultured roots of tea

Abstract: The effect of Al on the growth of excised tea (Camellia sinensis L.) roots differentiated from stem segments in vitro was investigated in shaking liquid culture. 1) The optimum initial pH for tea root growth in modified MS medium was 5.5. Growth of main and lateral roots was affected by the difference in NAA concentrations. Main root growth was stimulated by 5×10-7 and 1×10-6 M NAA, and lateral root growth was stimulated by 5 × 10-6 and 1 × 10-5 M NAA. 2) Main root growth showed a progressive increase for 3 weeks, then became stationary. Lateral roots began to grow at 2 weeks, with a progressive increase over the following 6 weeks. 3) The growth of the tea roots in the main or lateral root growth medium was stimulated by the supply of Al and P together, but was not affected when Al and P were supplied separately. 4) The pH of the culture medium decreased gradually to about 4.0 during the shaking culture, and remained stable. Growth of roots supplied with Al was stimulated after the decrease of me...

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium.

Abstract: Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa3 as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa3 and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.

Modulation of biological functions of Naegleria fowleri amoebae by growth medium

Abstract: Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri, LEE, and a highly pathogenic strain, LEEmpC1, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri, LEE and LEEmpC1, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.

Antibiotic production by Streptomyces aureofaciens using self-cycling fermentation.

Abstract: The self-cycling frementation (;rSCF) technique was applied to culture of Streptomyces aureofaciens. SCF is a method of continuous fermentation in which the metabolism of a microorganism is monitored by a measurement such as dissolved oxygen. These data are sent to a computer to allow it to control the system. Tetracycline production was observed only at exceedingly low iron concentrations in the growth medium. Repeatability of cycles was found to be dependent upon the presence of tetracycline in the fermentation broth as well as the strain of microorganism grown in the fermentor. Tetracycline was produced by an improved specific rate when compared to results in the literature for this organism grown using the batch method.

Growth medium containing cyclodextrin and low concentration of horse serum for cultivation of Helicobacter pylori.

Abstract: The growth of Helicobacter pylori, a Gram-negative microaerophilic bacterium, is often difficult and requires complex media with the supplementation of 5% to 10% blood or blood derivatives. We have found that Brucella broth supplemented with 1% heated horse serum and 0.1% beta-cyclodextrin supports the good growth of H. pylori. The degree of growth and production of urease and vacuolating cytotoxin in this medium were equal to those in the medium supplemented with 5% horse serum. This medium was found to be suitable for both the routine laboratory culture and primary isolation of H. pylori from biopsy samples.

Catharanthus roseus cell cultures: Growth, alkaloid synthesis and antidiabetic activity

Abstract: Suspension cultures of Catharanthus roseus were established from three different cell lines namely, CWS, CWS-A and CWS-G on MS medium supplemented with 2,4-D (0.4 ppm). The three cell lines were cultured in growth and production media. Cell line CWS grown in production medium showed a PCV (packed cell volume) of 70% in 21 days. The cells synthesized 0.10% ajmalicine in the production medium and the cell extract caused a 71% decrease in blood sugar in diabetes induced rats. In growth medium the cells showed a PCV of 97% in 21 days and produced trace amounts of alkaloids. The cell extracts did not show any antidiabetic activity. The CWS-A cell line showed a PCV of 98% in 21 days and synthesized 0.036% ajmalicine in production medium. The extracts had no hypoglycaemic effect. In growth medium the cells showed a 98% PCV in 21 days and produced trace amounts of alkaloids. The cell extract caused an 86% decrease in blood sugar. The CWS-G cell line grown in flasks failed to synthesize significant levels of alkaloids both in growth and production media. The cell extracts did not show any antidiabetic activity.

Factors Influencing Histamine Formation by Psychrotrophic Luminous Bacteria Photobacterium phosphoreum.

Abstract: The present work was carried out to investigate factors influencing histamine formation by washed cell suspensions or cell-free extracts and growth in each condition on Photobacterium phosphoreum isolated from mackerel when stored at 0°C. In the study of the effects of the reaction temperature on histamine formation and histamine forming activity, the optimum was at 35-40°C, and this was also observed at 50°C. Also while the growth was optimum at 25°C growth did not occur at 35°C. In regard to the effect of the reaction pH value, the activity was optimum at pH 5.5-6.5 and did not occur at pH 8.0, while growth was good at pH 5.5-8.0. In regard to the reaction NaCI concentration, the activity of the washed cell suspensions was optimum in 2-4% and low in 1 and 5%, and the activity of the cell-free extracts was optimum in the absence of NaCI and decreased with an increase of the concentration, while the growth was similar to the trend obtained on the activity of the washed cell suspensions. In regard to the reaction histidine concentration, activity increased quantitatively in the concentration above l00μg/ml and did not vary in that under 10μg/m/. In regard to the culture age, the activity was low in the early logarithmic phase, became maximal in the latest logarithmic phase, and decreased with the age of the culture. In regard to the presence of glucose in the growth medium, the activity increased in the presence of glucose and the activities with incubation time were different between the presence and the absence of glucose, while the pH value of the culture decreased with an increase of the activity. In regard to the oxygen tension of the culture medium, the activities were higher in anaerobic culture than in aerobic one, while growth was higher in the aerobic one.

Cell culturing method and medium

Abstract: The present invention provides a method for producing an expanded non-transformed cell culture of human liver cells comprising the steps of: (1) preparing partially purified, minced human liver tissue, (2) concentrating the resulting cells and tissue pieces, (3) resuspending the concentrated tissue cells and pieces in a growth medium, (4) culturing the resuspended cells in the growth medium for a time and under conditions to effect sustained cell division, and (5) passaging the cultured human liver cells periodically to expand the culture. The growth medium comprises a combination of a basal medium and ingredients to provide a medium in which the cultured human liver cells are selectively proliferated without being transformed, providing an expanded culture of proliferated, functionally differentiated human liver cells that is substantially free of fibroblast, macrophage and capillary endothelial cells. Also provided is the improvement of harvesting cells of the expanded culture at a selected PDL preferably>5, providing a high density cell suspension of such proliferated human liver cells, and incubating such high density cell suspension in a calm-down medium to induce a mitotically quiescent state and, using a culture procedure which encourages aggregation, making the cells adhere tightly to form a three-dimensional cell organization typical of the organ of origin, thereby forming organoids.

The influence of growth medium on serum sensitivity of Bacteroides species.

Abstract: The susceptibility of 12 different Bacteroides strains (representing nine species) to the bactericidal effect of human serum complement was investigated. When grown in nutrient-rich proteose peptone-yeast extract medium, all 12 strains were, to varying degrees, sensitive to serum. However, when grown in Van Tassell and Wilkins's minimal medium, six of the 12 strains became markedly more serum resistant. Five of these six strains became totally resistant to serum when grown in heat-inactivated (56 degrees C, 30 min) sheep serum. By Percoll discontinuous density centrifugation and light microscopy, the ratio of bacteria with large and small capsules was found to vary with the growth medium used. Lipopolysaccharide (LPS) was extracted with aqueous phenol after growth in the three media. Polyacrylamide gel electrophoresis (PAGE) and silver staining of the LPS showed some differences in LPS profiles in all strains tested. Therefore, variation of growth conditions results in alterations of both the expression of surface structures and, in some cases, sensitivity to serum. The biochemical basis for these changes requires further investigation.

Characterization of membrane-bound ATPase activity of Leuconostoc oenos: growth conditions

Abstract: A protocol for Leuconostoc œnos plasma membrane isolation, which was improved for studying lipidic and proteic constitution according to growth conditions, allowed us to determine some kinetic properties of membrane-bound ATPase activity: optimum pH, 5.6; the Michaelis constant (K m) for ATP, 1.1 mm; Mg2+-dependence. L. œnos membrane-bound ATPase activity was regulated according to cell growth phase, growth medium and extracellular conditions. The regulation mode of this activity was not investigated but it was very rapid: differences in level of activity were observed in less than 1 h when extracellular pH was modified or when ethanol was added. Wine induced a large decrease in ATPase activity of L. œnos. This may explain the loss of cell viability after direct inoculation into wine, when malolactic fermentation was not spontaneously obtained.

Tolerance of lespedeza Bradyrhizobium to acidity, aluminum, and manganese in culture media containing glutamate or ammonium

Abstract: Lespedeza juncea (sericea lespedeza) is a perennial forb-legume often planted on acid disturbed lands to fix N 2 . The abilities of 14 lespedeza Bradyrhizobium strains (cowpea classification group) to grow in a defined medium (glutamate as the N source) were determined at different pH values with varied amounts of Al and Mn. Growth response times were determined based on days required for strains to achieve a detectable amount of turbidity. Growth was not significantly affected at pH 4.5 or by 500 μM Mn compared to pH 6.7. Addition of 50 μM Al at pH 4.5 significantly ( P ⩽ 0.05) decreased growth of half of the strains in comparison to controls at pH 4.5. A pH of 4.3 significantly ( P ⩽ 0.05) decreased growth of 93% of the strains. Addition of 50 and 80 μM Al at pH 4.3 significantly decreased growth of most strains compared to pH 4.3 controls. Most strains did not become turbid at pH 4.1, the most inhibitory treatment. Based on growth response times, bacterial growth may be sufficient in soils at or above pH 4.5 to insure adequate nodulation. Strains isolated from acid disturbed lands were generally more tolerant of inhibitory treatments than other strains, including commercial strains. Plate count experiments indicated that differences in growth response times were the result of sustained decreased growth rates although initial lag time periods for biological adjustment also occurred. These results agreed with those obtained by a new method using a less pH-buffered growth medium (ammonium as the N source) in which the bacteria decreased the pH as they multiplied, theoretically to a stable inhibitory value (i.e. tolerance value).

Establishment of human parotid pleomorphic adenoma cells in culture: Morphological and biochemical characterization

Abstract: This study reports the establishment of alpha-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP1) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP1 produce low levels of alpha-amylase and relatively high levels of alpha-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP1 are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation of alpha-amylase in these cells.

Sucrose transport and hydrolysis in Rhizobium tropici

Abstract: The Rhizobium tropici strain CFN 299 was maintained on PY medium and was grown in minimal medium (MM) with sucrose, glucose, fructose and glutamate (or their combination) as carbon sources. Bacteria were able to simultaneously use different carbon sources and, with a combination sucrose and glutamate, the growth rate was faster than with either carbon source alone. Sucrose transport was induced by sucrose and partially repressed by glucose and glutamate if they were included in MM as additional carbon sources. The transport of sucrose was active because both an uncoupler (dinitrophenol, DNP) and inhibitors of terminal oxidation (KCN, NaN3) severely reduced sucrose uptake. Sucrose transport was also sensitive to a functional sulfhydryl reagent but was much less sensitive to EDTA and arsenate. We obtained nonlinear Lineweaver-Burk plots for the uptake of sucrose (by sucrose-grown bacteria), and this implied the existence of at least two uptake mechanisms. Invertase (EC 3.2.1.26) is the main enzyme for sucrose hydrolysis in this organism. This enzyme was induced by sucrose and had high activity in mid-log phase cells when sucrose was the sole carbon source (0.2%). Invertase activity was not detected in growth medium. In general, the results obtained support the idea, that R. tropici is adapted to sucrose utilization and to multicarbon nutrition during its interaction with plants.

A simple medium without blood modified for successful isolation & cultivation of LD bodies.

Abstract: A simple growth medium for primary isolation and subsequent cultivation of Leishmania donovani promastigotes without using whole blood is described. This medium is modified from Aljeboori's biphasic medium (used originally only for cultivation), containing only beef extract, peptone, sodium chloride, bactoagar and foetal calf serum (FCS). We have modified the medium by adding glucose and ascertaining the pH in the solid phase and by drastically reducing (91 %) FCS in the liquid phase. The medium helps in isolation of L. donovani promastigotes from kala-azar patients, in addition to luxurious growth of parasites. The medium is simple, reliable, reproducible and convenient, with minimal interference in using the parasitic cells for immunological, molecular and isoenzyme studies