Influence Of Culture Medium Composition Research Articles

Modulation of Siderophore Production by Pseudomonas fluorescens Through the Manipulation of the Culture Medium Composition.

Pseudomonas fluorescens has the ability to produce the siderophore pyoverdine, a biotechnologically significant iron chelator, which has a wide range of potential applications, such as in agriculture (iron fertilizers) and medicine (development of antibiotics). The present work aimed to evaluate the influence of culture medium composition on the production of siderophores by P. fluorescens DSM 50090, an industrial relevant strain. It was found that the bacterium grown in minimal medium succinate (MMS) had a higher siderophore production than in King B medium. The replacement of succinate by glycerol or dextrose, in minimal medium, originated lower siderophore production. The increase of succinate concentration, the addition of amino acids or the reduction of phosphate in the culture medium did not improve siderophore production by P. fluorescens. The results obtained strongly suggest that (i) MMS is more appropriate than King B for large-scale production of siderophores; (ii) the modification of the culture medium composition, particularly the type of carbon source, influences the level of siderophore secreted; (iii) the production of siderophore by P. fluorescens seems to be a tightly regulated process; once a maximum siderophore concentration has been reached in the culture medium, the bacterium seems to be unable to produce more compound.

Influence of Culture Medium Composition and Light Conditions on the Accumulation of Bioactive Compounds in Shoot Cultures of Scutellaria lateriflora L. (American Skullcap) Grown In Vitro

Methanolic extracts from in vitro grown Scutellaria lateriflora shoots cultured on five Murashige and Skoog (MS) medium variants supplemented with different combinations of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) under different light conditions (monochromatic light, white light and no light) were analysed by HPLC for three groups of metabolites: flavonoids (26 compounds), phenolic acids and their precursors (19+2) and phenylethanoid glycosides (2). The analyses revealed the presence of baicalein, baicalin, wogonin, wogonoside, 3,4-dihydroxyphenylacetic acid and verbascoside. There was clear evidence of the influence of plant growth regulators and light conditions on the accumulation of the analysed groups of secondary metabolites. The amounts of the compounds changed within a wide range—for the total flavonoid content, 30.2-fold (max. 1204.3 mg·100 g−1 dry weight (DW)); for 3,4-dihydroxyphenylacetic acid, 5.5-fold (max. 33.56 mg·100 g−1 DW); and for verbascoside, 1.5-fold (169.15 max. mg·100 g−1 DW). The best medium for the production of most of the compounds was the Murashige and Skoog variant with 1 mg l−1 BAP and 1 mg l−1 NAA. For verbascoside, the best ‘productive’ medium was the MS variant supplemented with 0.5 mg l−1 BAP and 2 mg l−1 NAA. The accumulation of the metabolites was stimulated to the greatest extent by blue light, under which the extracts were found to contain the highest total amount of flavonoids and the highest amounts of flavonoid glucuronides, baicalin and wogonoside, as well as of verbascoside. Their amounts were, respectively, 1.54-, 1.49-, 2.05- and 1.86-fold higher than under the control white light.

Production of Bioactive Compounds with Antitumor Activity Against Sarcoma 180 by Pleurotus sajor-caju

This work studied the influence of culture medium composition and pH on exopolysaccharide (EPS) production by Pleurotus sajor-caju and validates the antitumor activity of the produced EPSs and of the mycelial biomass (intracellular polysaccharides [IPSs]) against Sarcoma 180 (S180) cells. The effect of the initial concentrations of (NH₄)₂SO₄, yeast extract and soy peptone on EPS production by P. sajor-caju was studied in shake flasks. A bioreactor was used to evaluate the pH values and the initial CaCO₃ and glucose concentrations. Extracts of EPSs (PE1) and IPSs obtained through two different separation processes (PM1 and PM2) were tested on mice inoculated with S180 cells. A medium containing 2.5, 1.0, and 1.0 g/L of (NH₄)₂SO₄, yeast extract and soy peptone, respectively, provided the highest EPS concentration (0.6 g/L). The use of pH 4.0, 1.0 g/L CaCO₃ and 20 g/L initial glucose concentration enhanced EPS productivity (3.84 g/L per hour). The PE1 extract promoted the highest reduction of S180 growth (86%), followed by the PM2 extract (80%); growth reduction was dose-independent for both substances. This work provides information about culture medium and conditions that enhanced the production of extracellular polysaccharides by P. sajor-caju. The results can contribute to the search for new bioactive products bringing novel aspects to the medical and pharmaceutical areas.

THE INFLUENCE OF SOME FACTORS ON THE FORMATION OF BIOFILM BY TOXIGENIC AND NON-TOXIGENIC VIBRIO CHOLERAE EL TOR STRAINS

The early stages of biofilm formation on polystyrene surface were studied in 14 Vibrio cholerae El Tor strains, all of which carried genes of vps cluster, mshA, and hapR genes. 13 Vibrio cholerae El Tor strains were shown to be capable of adsorption on the walls of microtiter plate wells and of ring formation at the "polystyrene-medium" interface. One of the Vibrio cholerae El Tor strains tested was defective in this ability. The use of different media - modified M1 medium and 1 per cent peptone - failed to reveal sufficient influence of culture medium composition on the ability of Vibrio cholerae to form multilayer biofilm at the phase boundary "polystyrene-medium". Temperature reduction from 37°C to 25°C also didn t result in statistically significant enhancement of monolayer formation in both media. Quantitative evaluation of biofilm formation showed that this ability was clearly marked only in two toxigenic and two non-toxigenic strains of Vibrio cholerae El Tor. Monolayer comparison in ELISA demonstrated that only two toxigenic and two non-toxigenic strains possessed the ability for exopolysaccharide production.

Influence of Culture Medium Composition on Relative mRNA Abundances in Domestic Cat Embryos

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality.

Critical Influence of Culture Medium and Cr(III) Quantification Protocols on the Interpretation of Cr(VI) Bioremediation by Environmental Fungal Isolates

The influence of culture medium composition on chromium(VI) quantification according to diphenylcarbazide (DPC) colorimetric determination was evaluated. Considering the eventual biospeciation of Cr(VI) as a mechanism of microbial bioremediation, the possibility to quantify Cr(III) in culture medium was also explored. Yeast nitrogen base (YNB) was identified as the least interferent culture medium for Cr(VI) quantification by DPC and it was applied to compare different strategies for Cr(III) oxidation. The most appropriate oxidation protocol consisted in the reaction with 80 mM KIO4 at room temperature for 30 min prior to DPC. Parameters like basal culture medium (vitamins + salts + oligoelements), C and N source were systematically evaluated, either independently or in combination. Results demonstrated that C source was the most interferent culture medium component, being the use of sucrose preferable to glucose. A medium arbitrarily named as YNB′ (YNB without amino acids and ammonium sulfate plus 50 g L−1 sucrose and 0.6 g L−1 (NH4)2SO4) was defined for Cr(VI)-amended fungal cultures. Kinetics of growth, Cr(VI) removal, and nutrient consumption for isolates A. pullulans VR-8, filamentous fungus PMF-1, and Lecythophora sp. NGV-1 were obtained. The order of Cr(VI) removal efficiency was as follows: A. pullulans VR-8 > Lecythophora sp. NGV-1 > filamentous fungus PMF-1, and a similar trend was observed for biomass yield and nutrients consumption. Studies on biospeciation by means of the selected Cr(III) oxidation protocol were unsuccessful, leading to Cr(VI) values much lower than expected. It revealed that this kind of protocols should be cautiously evaluated when studying microbial Cr(VI) bioremediation.

Influence of culture medium composition, dissolved oxygen concentration and harvesting time on the production of Serratia entomophila, a microbial control agent of the New Zealand grass grub

The bacterium Serratia entomophila (Enterobacteriaceae) has been developed as a commercially available biopesticide for control of the pasture pest Costelytra zealandica. The influence of culture medium composition, dissolved oxygen (DO) concentration and harvesting time were investigated in order to optimise the production of S. entomophila. In batch fermentations, highest yields were achieved using sucrose (40 g L−1) as the carbon source, followed closely by fructose and molasses. The effect of yeast extract (YE), marmite and bakery yeast as cell growth enhancers was also examined in both batch and fed-batch mode. Culture medium containing 20 g L−1 of YE (fed-batch) produced the highest cell density. No significant effect on cell yield was detected when cultures were supplemented with bakery yeast or marmite. The DO concentration influenced biomass production: a 5-fold increase in cell density was achieved when the concentration of DO was maintained in the range of 20–50% (5.7×1010 CFUs mL−1) in comparison with 1% (1.2×1010 CFUs mL−1). In cultures maintained at 1 and 20% DO concentration, cells harvested from the exponential growth phase survived for less than 2 weeks when stored at 4°C. In contrast, high cell survival (85–100%) was achieved when cells were harvested after they had entered the stationary growth phase. Recommendations are provided for the production of robust, high cell density cultures of S. entomophila.

Rat hepatocytes cultured on a monkey kidney cell line: Expression of biotransformation and hepatic metabolic activities

The present study deals with the expression of cytochrome P-450 and two monooxygenase activities (aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation) by hepatocytes cultured on feeder layers of MS cells, in an attempt to investigate the influence of culture medium composition on phase I activities of hepatocytes. When compared with culture medium I (serum-free, hormone-supplemented Ham's F12), 4-day MS hepatocyte cultures supplemented with nicotinamide (medium II) allowed better retention by hepatocytes of cytochrome P-450 and phase I activities (aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation). A moderate additive effect was found after increasing branched-chain amino acid composition (medium III). Increasing the concentration of all amino acids, galactose and pyruvate (medium IV) resulted in higher levels of cytochrome P-450, aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation which, after 8 days in culture, were at 33, 49 and 43% of day 0 values, respectively. A further increase of branched-chain amino acid concentration produced only a moderate effect (medium V). Under these culture conditions, hepatocytes also expressed basic metabolic functions. After 10 days, MS hepatocyte cultures retained 43, 71 and 65% of ureogenesis, triglyceride secretion and albumin production measured at day 0. In contrast, pure hepatocytes cultured under the same conditions expressed these three hepatic functions only until the fifth or sixth day of culture.

In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, compared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (7.49 +/- 3.3) were higher than the cell numbers of those developing in ECM (55.1 +/- 2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOSM, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage.

The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line

When grown in the standard Dulbecco's medium the human liver derived Hep G2 hepatoma cell line shows only 10–20% of the cytochrome P-450-dependent mixed function oxidase (MFO) activity of freshly isolated human adult hepatocytes. However, the MFO activities and, to a lesser extent, the activities of UDP-glucuronyltransferase and glutathione-S-transferase can be increased by altering the composition of the growth medium. Modified Earle's medium was more effective in this respect than Williams' E medium and increased the O-dealkylations of ethoxyresorufin, benzyloxyresorufin and pentoxyresorufin 50-, 30- and 10-fold, respectively.

Hämolysewirkungen durch Listeria innocua auf Schaferythrozyten

The influence of culture medium composition on hemolytic effects produced by Listeria innocua on sheep blood agar has been investigated. Neither α- nor β-hemolysis could be observed around L. innocua colonies grown on sheep erythrocyte agar lacking glucose. However, green zones of hemolytic phenomena were observed around colonies grown on such agar which contained 0,6 % (w/v) glucose. By using L. innocua cell suspensions and culture filtrates this glucose-associated hemolysis only occurred in media which were weakly buffered (1 m M Na-PBS). This hemolytic phenomenon was attributed to the resulting acidification of the culture medium caused by the metabolic breakdown of glucose by Listeria. The results of this study demonstrate the necessity of including adequate buffer conditions when assaying the hemolytic property of a Listeria strain. It was found that the addition of 20 mM Na-PBS to the assay medium was sufficient to eliminate artificial “acidic-hemolysis”. Moreover, the hemolysis observed with known hemolytic Listeria strains was unaffected by this buffering, as was in fact the case even when conditions of higher buffering capacity were employed. By testing in this manner, no evidence has been found which would suggest the existence of an hemolysin, either intra- or extracellular, produced by Listeria innocua. An accurate method for the determination of hemolysis caused by strains of the genus Listeria is proposed. This method of assaying hemolysis in a liquid grown medium has proven effective in determining the hemolytic properties of strains which appeared negative or questionable on blood agar as well as strains which in virulence tests were negative. The ability to accurately assess the hemolytic properties of Listeria strains, is essential in determining the association of Listeria hemolysin with pathogenicity of this genus. Together with current investigations on the genetics of hemolysin production by Listeria the determination of hemolytic activities will eventually allow to understand better the pathogenic principle of hemolytic strains of Listeria monocytogenes.

The influence of culture medium composition on the incidence of chromosomal breakage.

In patients with chromosomal instability and healthy subjects, significant differences were observed in the chromosomal breakage incidence in simultaneous lymphocyte cultures set up with TC medium 199, Eagle's Minimum Essential Medium, or RPMI, or RPMI 1629. The importance of the choice of culture medium for mutagenicity testing and studies of so-called spontaneous breakage is shown. Cultures incubated with TC Medium 199 showed the highest chromosomal breakage incidence.