Showing papers on "Growth medium published in 1999"

An efficient microbiological growth medium for screening phosphate solubilizing microorganisms

Abstract: A novel defined microbiological growth medium, National Botanical Research Institute's phosphate growth medium (NBRIP), which is more efficient than Pikovskaya medium (PVK), was developed for screening phosphate solubilizing microorganisms. In plate assay the efficiency of NBRIP was comparable to PVK; however, in broth assay NBRIP consistently demonstrated about 3-fold higher efficiency compared to PVK. The results indicated that the criterion for isolation of phosphate solubilizers based on the formation of visible halo/zone on agar plates is not a reliable technique, as many isolates which did not show any clear zone on agar plates solubilized insoluble inorganic phosphates in liquid medium. It may be concluded that soil microbes should be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers.

Modulation of Plasma Membrane H+-ATPase Activity Differentially Activates Wound and Pathogen Defense Responses in Tomato Plants

Abstract: Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells. A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity. Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L. peruvianum cell cultures and to induce wound response genes in whole tomato plants. Conversely, an activator of the H+-ATPase (fusicoccin) acidified the growth medium of L. peruvianum cell cultures and suppressed systemin-induced medium alkalinization. Likewise, in fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed. However, fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes. Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane. In addition, alkalinization of the L. peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase. Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes. The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed.

Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis

Abstract: Recent studies have shown that a number of bacteria, including lactococci, possess an inducible acid tolerance response (ATR); that is, they acquire the ability to survive otherwise lethal acid concentrations following preexposure to mildly acidic conditions (6, 8, 16, 20, 21, 23). Exposure of log-phase Lactococcus lactis subsp. cremoris NCDO 712 cells to an extracellular pH (pHo) of 5.0 for 1 h resulted in a 100-fold increase in survival after a 2-h challenge with acetic acid at pHo 4.0 (21). The cells acquired acid tolerance by a mechanism which involved protein synthesis. The magnitude of the ATR was dependent on the degree of acidification of the growth medium, as indicated by its pHo. However, it was clearly established that pHo participated in the ATR through its effect on the pH of the cell cytoplasm (pHi). Several factors could affect the pHi of cells and in so doing might be involved in induction of the ATR. Nannen and Hutkins (18) showed that during log-phase growth of a number of strains of lactic acid bacteria, including strains of lactococci, the pHi decreased as the pHo decreased due to the production of lactic acid. Usually, more than 95% of either lactose or glucose is converted to l-lactic acid when lactococci are grown anaerobically in batch culture. However, a number of reports have shown that at low growth rates in chemostat cultures the spectrum of fermentation end products changes (4, 25). Thomas et al. (25) demonstrated that when growing anaerobically at low growth rates in glucose-limited chemostat cultures, strains of lactococci switched their fermentation pathway from homolactic fermentation to mixed-acid fermentation and that the end products were formic, acetic, and lactic acids. Formic acid has a pKa similar to the pKa of lactic acid, but acetic acid has a higher pKa than lactic acid and at equimolar concentrations is more effective at reducing the pHi of cells (24). There is good evidence from both batch (13) and chemostat (1) culture studies that the membrane H+-ATPase plays a key role in regulating the pHi of lactic acid bacteria and may be the most important mechanism involved in pHi regulation in these bacteria. In the present study we were mainly concerned with changes in the ATR induced by changes in the growth rate and the pHo of glucose-limited chemostat cultures of L. lactis. We observed that as the pHo of batch cultures decreased, the level of the ATR increased. However, the growth rate also decreased as the pHo decreased. In addition, in batch cultures the level of the ATR increased from early in the log phase to the stationary phase (see Table ​Table1).1). Chemostat cultures allowed us to vary the pHo and the growth rate of a culture independently, and therefore, the role of each of these parameters in the induction of an ATR in L. lactis subsp. cremoris NCDO 712 could be assessed independently. Variations attributable to the phase of growth were also eliminated in chemostat cultures. In addition, other factors, such as the cytoplasmic ATP levels, the rate of ATP generation, the levels of H+-ATPase, and the rate of acid production, were examined for possible correlations with changes in the pHi of L. lactis. TABLE 1 Acid resistance of L. lactis subsp. cremoris NCDO 712 at various stages of growth in batch cultures at several constant pHo values

Salt stress in Mesembryanthemum crystallinum L. cell suspensions activates adaptive mechanisms similar to those observed in the whole plant

Abstract: A salt-tolerant stable cell-suspension culture from the halophyte Mesembryanthemum crystallinum L. has been established from calli generated from leaves of 6-week-old well-watered plants. Optimal cell growth was observed in the presence of 200 mM NaCl, and within 7 d cells were able to concentrate Na+ to levels exceeding those in the growth medium. Accumulation of Na+ was paralled by increases in the compatible solute pinitol and myo-inositol methyl transferase (IMT), a key enzyme in pinitol biosynthesis. Increasing concentrations of NaCl stimulated the activities of tonoplast and plasma-membrane H(+)-ATPases. Immunodetection of the ATPases showed that the increased activity was not due to changes in protein amount that could be attributed to treatment conditions. A specific role for these mechanisms in salt-adaptation is supported by the inability of mannitol-induced water stress to elicit the same responses, and the absence of enzyme activity and protein expression associated with Crassulacean acid metabolism in the cells. Results demonstrate that these M. crystallinum cell suspensions show a halophytic growth response, comparable to that of the whole plant, and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response.

Continuous axenic cultivation of Pneumocystis carinii.

Abstract: Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle’s salt supplemented with S-adenosyl-l-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, l-cysteine and l-glutamine, and horse serum. Incubation is in room air at 31°C. The pH of the medium begins at 8.8 and rises to ≈9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.

Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow.

Abstract: Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy. Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH. In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium. Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1. When C. cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition. In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited. These data indicate that C. cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose. This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C. cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing.

GlaA Promoter Controlled Production of a Mutant Green Fluorescent Protein (S65T) by Recombinant Aspergillus niger during Growth on Defined Medium in Batch and Fed-Batch Cultures

Abstract: The first successful expression of the Aequorea victoria green fluorescent protein (GFP) gene in Aspergillus niger is described. When the wild-type GFP gene was expressed in A. niger, neither the fluorescence nor the full translation product of the wtGFP gene was detectable. However, the expression of a mutant form of the green fluorescent protein (S65TGFP) gene resulted in the formation of a functional fluorescent polypeptide. The synthesis of S65TGFP was used to study glaA promoter controlled heterologous gene expression by recombinant A. niger in batch and fed-batch cultures using a defined growth medium. Cells were grown on xylose as noninducing carbon source, and the production of S65TGFP was accomplished by the addition of maltose. The recombinant protein accumulated up to 10 or 25 mg of S65TGFP g-1 cell dry weight using either a maltose pulse for induction or continuous addition of the inducing carbon source, respectively. Irrespective of the induction protocol, the recombinant protein started to accumulate 2 h after addition of the inducing carbon source and reached its maximum specific concentration 10 h after induction. Bright green fluorescing fungal pellets were first detectable by fluorescence microscopy 4-5 h after the onset of maltose addition.

An improved growth medium for Flavobacterium psychrophilum

Abstract: Supplementing cytophaga agar and broth with 0.5 g l-1 each of D(+) galactose, D(+) glucose, L-rhamnose and skimmed milk gave a dramatic improvement in the isolation of the fish pathogen Flavobacterium psychrophilum. By means of spread-plating, approximately double the number of colonies of larger size were obtained on the improved medium compared to cytophaga agar alone. In supplemented cytophaga broth, growth of Fl. psychrophilum was more rapid and generated greater biomass.

Inhibitory Effect of Fusarium Mycotoxins on Growth of Brewing Yeasts. 2. Deoxynivalenol and Nivalenol

Abstract: The effect of the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) on growth of Saccharomyces cerevisiae lager and ale strains has been studied The toxins were added into the growth medium in low and high concentrations Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods The inhibitory effect of both DON and NIV on yeast growth was dependent on toxin concentration Additionally, when the extent of inhibition of yeast growth caused by high concentrations of both toxins was observed, it was subject to yeast strain, length of incubation and method used to assess yeast growth The lowest concentrations of mycotoxin causing significant inhibition on growth of brewing yeasts were: 100 μg/ml DON for the lager strain and 50 μg/ml for the ale strain, and 50 μg/ml NIV for the ale strain

Semisynthetic culture medium for growth and dihydroxyacetone production by Gluconobacter oxydans

Abstract: Only three vitamins (pantothenate, p-amino benzoic acid, nicotinic acid) and two amino acids (serine, glutamine) were required in the growth medium for Gluconobacter oxydans which allowed the concentration of yeast extract to be reduced to 5–10% of the previous concentration. When compared with data from cultivations with complex media, the new medium gave a lower yield (about 0.02 g biomass per g glycerol) and comparable growth rate (0.24 to 0.38 h−1) but a higher productivity (10.3 g dihydroxyacetone/gh).

Inhibitory Effect of Fusarium Mycotoxins on Growth of Brewing Yeasts. 1 Zearalenone and Fumonisin B1

Abstract: The Fusarium mycotoxins zearalenone (ZEA) and fumonisin B 1 (FB 1 ) added to the growth medium in low and high concentrations, were investigated as a possible cause of inhibition of growth of Saccharomyces cerevisiae lager and ale strains. Toxic effects were assessed by measurement of dry weight or growth relative to controls, cell number, viability and conductance changes of the growth medium using indirect and direct methods. The Fusarium mycotoxins studied affected growth on brewing yeasts, but the inhibitory effect was dependent on concentration. Low concentrations (0.1-2 μg/ml) had no significant effect on growth compared to controls. Although high concentrations of both mycotoxins strongly affected growth, the inhibitory effect depended on toxin concentration and type, yeast strain, length of incubation and method used to assess growth. The lowest concentrations of mycotoxin causing significant inhibition on growth of these brewing yeasts were 50 μg/ml ZEA for both yeast strains, and 10 μg/ml FB 1 for the lager strain and 50 μg/ml for the ale strain.

Effect of growth medium composition on psilostachyinolides and altamisine production

Abstract: Callus cultures of Ambrosia tenuifolia were established from sub-apical leaves. The explants were grown on basal media MS (Murashige and Skoog) supplemented with 10 μM kinetin, 1 μM 2-4 dichlorophenoxyacetic, ascorbic acid and cysteine and cultivated either in light or darkness. To determine the effect of each individual salt component on the growing rate of the callus and the psilostachyinolides and altamisine production in the culture, the concentrations of each nutrient was tested at different levels, ranging from 50% above to 50% below the standard medium. Interestingly, increased concentration of boron in the medium, resulted in a four fold increase in the production of sesquiterpene lactones by the callus. However, positive production of psilostachynolides and altamisine was only detected when the basal concentrations of the others components of the basal media were used. In addition, production of altamisine was highly sensitive to the variations of nutrients. No statiscally effect on terpenoide production by the callus was detected by varying light exposure. These results indicates that conditions for the optimal production of these terpenoids can be enhanced by modifying nutrients of the MS basal media.

Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro

Abstract: Efficacy of silver thiosulfate (STS) in reducing ethylene-induced culture abnormalities during minimal growth conservation of microplants was studied in seven potato (Solanum tuberosum L.) genotypes. Different concentrations of STS (0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 μg ml–1) were tested in minimal growth medium based on MS medium supplemented with 20 g l–1 mannitol and 40 g l–1 sucrose. STS improved the microplant growth and reduced the culture abnormalities during prolonged maintenance of potato shoot cultures in vitro. The beneficial effect of STS was most prominent for number of green leaves per microplant and leaf senescence. After 16 months of storage, desirable microplant growth was observed in cultures conserved in medium containing 6.0–9.0 μg ml–1 STS. The profile of the peroxidase isozymes of conserved cultures did not show any apparent genetic variation due to the presence of STS in the conservation medium.

Accumulation of arsenic in a unicellular alga Chlamydomonas reinhardtii

Abstract: The unicellular alga Chlamydomonas reinhardtii accumulated and biomethylated arsenic efficiently. A wall-less cell strain (CW-15) of Chlamydomonas reinhardtii proliferated in a low level arsenic-containing medium (0.01-0.1 mmol dm -3 ) more than that in an arsenic-free medium. Although the growth of the algal cells was only slightly more inhibited in a growth medium containing arsenic at a concentration of 1.0 mmol dm -3 than that in an arsenic-free medium, it was completely inhibited at concentrations of 10 and 100 mmol dm -3 . Furthermore, transformed strains were obtained by random introduction of plasmid pJD67, carrying an Arg + gene, into a wall-less cell Arg - mutant CC425 strain. Finally we selected a strain, named AS1, among the transformed CC425 of the arsenic-sensitive group. The accumulation of arsenic by the AS1 strain was about three-fold higher than that by the CW-15 strain and 80-90% of the inorganic arsenic was transformed into a dimethylarsenic compound. It is suggested that the AS1 strain is a suitable model for investigation of the accumulation and biomethylation of arsenic by microalgae in freshwater environments.

Evaluation of agar and agarose gels for studying mechanical impedance in rice roots

Abstract: Agar and agarose gels were evaluated as systems to mechanically impede roots of rice (Oryza sativa L.). Two-layer gels were used so that seedlings established in a layer of weak gel (0.35% weight/volume) and then grew downwards to encounter a treatment gel of up to 5.0% (w/v). Agarose gels were stronger than agar gels of the same concentration, reaching a maximum penetrometer resistance of 1.2 MPa at a concentration of 5.0%, compared to 0.3 MPa with agar. The 5.0% agar gel stimulated elongation of the seminal axis by 40% in seedlings of variety TN1 (compared with elongation in the 0.2% gel), but decreased it by 15% in the variety Lac 23. Although increasing agarose concentration decreased seminal axis elongation in both varieties, the seminal axis did not reach the lower layer of treatment gel when the concentration of the treatment gel was greater than 2.0%. The decreased root elongation was therefore a non-mechanical inhibition. In experiments conducted using a different batch of agarose, these inhibitory effects were not seen and strong agarose gels stimulated seminal axis elongation. It was concluded that the agar and agarose gel systems studied were unsuitable for studying the effect of mechanical impedance on the elongation of rice roots and that great care should be taken in interpreting the results of experiments using gels as a growth medium.

Transformation and morphological changes of murine L cells by transfection with a mutated form of beta-catenin.

Abstract: To shed light on the oncogenic nature of mutant beta-catenin, we introduced a form of the cDNA that lacked an entire exon 3 into L cells derived from murine s.c. tissue. Aberrant beta-catenin protein accumulated in the cytoplasm and nuclei of these cells (designated L-MT), whereas in L cells transfected with wild-type beta-catenin (designated L-N), normal beta-catenin protein was expressed at a level similar to that of parental cells. L-MT cells also changed morphologically from a fibroblast-like appearance to a more cuboidal shape. Their rate of proliferation was the same as that of L cells and L-N cells, but the saturation density of L-MT cells appeared to increase in association with a multilayer growth pattern. Furthermore, L-MT cells required a lower concentration of serum in the growth medium than did parental cells. These alterations in cell growth and morphology suggested that mutated beta-catenin was stabilized in the transfected cells and induced the oncogenic phenotype.

Cell culturing method and medium for producing proliferated, normal, differentiated human liver cells

Abstract: The present invention provides improvements in 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, the improvement comprising the steps 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 organisation typical of the organ of origin, thereby forming organoids. Methods of gel-embedding the single cells, aggregates and organoids produced by the method; the growth medium and calm-down medium; various different methods for use of the normal, proliferated human liver cells produced by the method, are also provided.

Stimulated production of diosgenin in Dioscorea galeottiana cell suspension cultures by abiotic and biotic factors

Abstract: Dioscorea deltoidea cell suspension cultures were established in modified Murashige and Skoog medium The diosgenin production increased from 010 g−1 to 398 g−1 dry cell weight when cells were cultivated in the light and in a growth medium limited in phosphate and sucrose The addition of 13 g of autoclaved fungal mycelium of Alternaria tenuis per litre of cell culture growing in the dark induced the production of 004 mg diosgenin g−1 dry cell weight In both cases, the production of diosgenin was preceded by a transient induction of isopentenyl diphosphate isomerase activity

Fermenting Escherichia coli is able to grow in media of high osmolarity, but is sensitive to the presence of sodium ion.

Abstract: Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na+ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Na+ were shown to extrude Na+ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231–237). The growth rate and Na+ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na+. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Na+ extrusion activity.

Influence of growth medium composition on the lipolytic enzyme activity of Ophiostoma piliferum (CartapipTM)

Abstract: Production of esterases (carboxyl esterase EC 3.1.1.1) and lipases (glycerol ester hydrolase EC 3.1.1.3) by Ophiostoma piliferum (CartapipTM), a fungus commercialized to decrease fatty acyl esters in wood, is described. The influence of various combinations of carbon and nitrogen sources, in the growth medium, was examined. Medium containing yeast extract as a nitrogen source and olive oil as a carbon source was found to be optimal for extracellular esterase (221 U dm−3) and lipase (152 U dm −3) activities. Further increases in those enzyme activities were achieved by decreasing medium pH from 6.5 to 5.5 (esterase 508 U dm−3; lipase 415 U dm −3) and increasing medium calcium content from 8 m mol dm−3 to 160 m mol dm−3 (esterase 4084 U dm−3; lipase 508 U dm −3) © 1999 Society of Chemical Industry

Kinetics, stereospecificity, and expression of the malolactic enzyme.

Abstract: Mass spectrometric measurement of carbon dioxide production was used to study malolactic fermentation (MLF) in Lactobacillus collinoides isolated from cider. The kinetics and stereospecificity of the malolactic enzyme (MLE) were studied, and the stoichiometry of the reaction sequence was investigated. The optimum pH for activity of the MLE was 4.9. MLF was more rapid (in both intact cells and cell extracts) when L-malic acid was used than when D-malic acid or the racemic mixture was added. The enzyme was found to be constitutively present in L. collinoides. Addition of L-malic acid (37 mM) to the growth medium resulted in increased MLE activity; addition of the D isomer alone or the racemic mixture resulted in lower activities. Addition of the main sugars in apple juice (fructose, sucrose, and glucose) to the growth medium in the presence of malic acid repressed production of MLE to similar extents in all three cases; in the absence of malic acid, instead of inhibiting MLF, addition of sugars to the growth medium somewhat increased the residual MLE activity.

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii.

Abstract: A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K m values obtained for acetaldehyde, ethanol, NADH2, and NAD+ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol.

Stability of chemical parameters of tissue culture medium (pH, osmolarity, electrical conductivity) as a function of time of growth

Abstract: Changes in medium pH, osmolarity (OS), and electrical conductivity (EC) were studied as a function of time in Murashige and Skoog (MS) liquid proliferation and rooting medium Microshoots of wild pear (Pyrus syriaca), bitter almond, and ‘Spunta’ potato were targeted Results indicated an acidic drift in pH in the different species on both proliferation and rooting medium The EC was increased significantly with time whereas solution OS was decreased Different species caused different effects on EC or OS depending on growth medium The decline in OS in the proliferation medium was higher when pear and almond were grown, while on rooting medium the decline was sharper with potato and almond The EC of proliferation medium was increased more when almond was grown; while on the rooting medium all species had similar effects Under these saline conditions, low nutrient elements availability, poor plant growth, and nutrient element imbalance are expected This study illustrates that the change in the

Preferential Use of Acetate over Glucose Involves Acetate-Mediated Inhibition of Glucose Uptake during Diauxic Growth of Carrot Cells

Abstract: It has been reported that suspension-cultured rice cells grown on mixed carbon sources of glucose (Glc) and acetate exhibited diauxic growth in which acetate was the preferred carbon source (Lee and Lee 1996). Carrot (Daucus carota L.) suspension cells, showing a diauxic growth very similar to that of rice cells, were used to delineate the mechanisms underlying this preferential use of acetate over Glc. Uptakes of both Glc and 3-O-methylglucose (3-OMG), a non-metabolizable Glc analogue, were similarly inhibited when acetate or butylate, weak acids which are capable of transporting protons into the cytosol, were present in the uptake assay mixture containing cells harvested during the Glc-utilizing second growth phase. Inhibition of Glc uptake by these weak acids was similar when equivalent experiments were carried out with isolated plasma membranes. It was further shown that Glc uptake, which requires a proper proton gradient across the plasma membranes, was inhibited during the first growth phase by acetate-mediated alkalization of growth medium and/or simultaneous acidification of cytosol. This study strongly suggests that Glc utilization in plant cells is inhibited by co-presenting carbon source(s) which can alter the proton gradient across the plasma membrane.

Mitogenic activity of fetal bovine serum, fish fry extract, insulin-like growth factor-I, and fibroblast growth factor on brown bullhead catfish cells - BB line

Abstract: Bioassays were performed to assess the effects of different levels of growth medium supplementation with fetal bovine serum (FBS), fish fry extract (FE), combinations of FBS and FE, and addition of insulin-like growth factor I (IGF-I) and fibroblast growth factor (FGF) on the proliferation of brown bullhead catfish cells (BB line). Treatments (n = 4) were: 2.5, 5, 10, and 15.0% FBS or FE and 5/2.5, 5/5, 10/2.5, and 10/5 of a FBS/FE combination as supplement to the growth medium, or the addition of 0.1, 1, 2.5, 10, 25, and 75 ng/ml of either IGF-I or FGF to the growth media. Initial cell density was 1.1 x 10(6) cells per well on uncoated 24-well plates. Incubation temperature was 29.5 +/- 0.7 degrees C. Six hours after plating, initial culture medium was removed, plates rinsed with Dulbecco's phosphate buffered saline, treatment media added, and cells allowed to proliferate for 24 hours. Another bioassay was performed with rat myoblast omega cells (RMo) using the same levels of growth medium supplemented with FBS, FE and FBS/FE. Base growth medium was Dulbecco's MEM. The initial cell density was 7.2 x 10(6) cells per well, and the bioassay was carried out at 36.0 +/- 0.5 degrees C, on a 95% air, 5% CO2 incubator. Increasing levels of FBS had a positive effect (P < 0.05) on the proliferation of both BB and RMo cells. Increasing levels of FE had a negative effect (P < 0.05) on the proliferation of BB cells and totally inhibited the proliferation of RMo cells at any level of supplementation. Higher levels of FE on the FBS/FE combinations presented a negative effect on the proliferation of both BB and RMo cells (P < 0.05). Insulin-like growth factor I had a positive quadratic effect (P < 0.05) on the proliferation of BB cells. Apparently, mammalian growth factors slightly stimulated mitogenic activity in fish cells, while FE contained factors which inhibited the mitogenic activity of RMo and BB cell lines.

Method for determining antibiotic sensitivity of bacteria

Abstract: A method and apparatus for determining the minimum inhibitory concentration of an antibiotic for a target microorganism are provided. The method includes the steps of: (a) providing a microorganism growth medium (14, 34); (b) providing a sensible reagent (17, 36), which includes an antibiotic mixed with a marker, the marker having a signal with a magnitude proportional to the marker's concentration; (c) incorporating the reagent into the growth medium, in a manner that creates a gradient of concentrations (18, 40) of the antibiotic and marker within the growth medium; (c) inoculating the growth medium with the target microorganism; (d) incubating the inoculated growth medium for a period of time sufficient for the target microorganism to grow a detectable amount on a first section (20, 42) of growth medium; (e) determining a growth boundary (28, 48) between the first section of growth medium having detectable target microorganism growth and a second section (24, 46) having substantially no detectable target microorganism growth; (f) measuring the signal magnitude at the growth boundary; and (g) determining a minimum inhibitory concentration of the antibiotic using the measured signal magnitude.