Showing papers on "Growth medium published in 1983"

High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium.

Abstract: A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar. Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum. Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum. Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency. Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%.

Hyaluronic acid from bacterial culture

Abstract: Hyaluronic acid, a polysaccharide, is prepared in high yield from streptococcus bacteria by fermenting the bacteria under anaerobic conditions in a CO2-enriched growth medium, separating the bacteria from the resulting broth and isolating the hyaluronic acid from the remaining constituent of the broth. The bacteria may be grown free of endotoxins by filtering all ingredients through a 10K Millipore (Reg. Trademark) filter prior to inoculation of the medium and subsequently maintaining pyrogen-free conditions. Separation of the microorganisms from the polysaccharide is facilitated by killing the bacteira with trichloroacetic acid. After removal of the bacterial cells and concentration of the higher molecular weight fermentation products, the hyaluronic acid is isolated and purified by precipitation, resuspension and reprecipitation.

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis

Abstract: A NaCl-tolerant cell line which was selected from ovular callus of `Shamouti9 orange ( Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na + and Cl − uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K + and Cl − accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl − . (d) Removal of Ca 2+ from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change.

Freezing Injury and Root Development in Winter Cereals

Abstract: Upon exposure to 2°C, the leaves and crowns of rye ( Secale cereale L. cv `Puma9) and wheat ( Triticum aestivum L. cv `Norstar9 and `Cappelle9) increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to −11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of −20°C while root growth was reduced at −15°C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions. Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed. Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (

Developmental regulation of the Aspergillus nidulans trpC gene.

Abstract: We have cloned the trifunctional trpC gene from Aspergillus nidulans by hybrid phage lambda complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. Four different phages sharing a 4.3-kilobase region were obtained. Plasmid subclones containing this region also complemented the E. coli trpC mutant. We determined that a 1.8-kilobase DNA fragment was minimally required for complementation. The fragment hybridized with two poly(A)+ RNAs, 3.0 and 3.2 kilobases in length. We infer that these transcripts are Aspergillus trpC mRNAs and that the entire Aspergillus trpC gene is not required for complementation in E. coli. Levels of both trpC transcripts in poly(A)+ RNA are regulated by growth medium composition. They were highest when cells were grown in minimal medium containing nitrate as the nitrogen source and lowest when cells were grown in medium containing yeast extract. The concentrations of the transcripts are also regulated during conidiophore development. Conidiating cultures grown on medium containing yeast extract had significantly higher levels of both transcripts than did hyphae grown in minimal medium containing nitrate. Levels of the transcripts in mature spores were equivalent to those found in hyphae grown in minimal medium containing nitrate. Results from nutritional experiments with an A. nidulans trpC mutant suggest that developmental regulation of trpC mRNA levels may be related to a high requirement for tryptophan or a compound derived from tryptophan during conidiation.

Influence of Water Stress on the Vacuole/Extravacuole Distribution of Proline in Protoplasts of Nicotiana rustica

Abstract: Tobacco (Nicotiana rustica) plants were stressed by addition of polyethylene glycol solution (−20 bar) to the growth medium. The proline contents and concentrations in total protoplasts, vacuoles, and extravacuolar fractions of these plants have been determined and compared with protoplasts and cell fractions of well-watered plants. As compared to the control, the stress treatment of intact plants results in a 7-fold increase of the proline content in the extravacuolar fraction while the vacuolar content was enriched only 2.6-fold. In protoplasts of control plants, a proline concentration ratio (extravacuole to vacuole) of 1 was measured. In protoplasts of stressed plants, this ratio was nearly 3. Thus, water stress seems to have an effect on a tonoplast-localized transport system for proline.

Characteristics of Escherichia coli grown in bay water as compared with rich medium.

Abstract: Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.

Whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus.

Abstract: A two-step batch fermentation-bioconversion of vanillin (4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hydroxy-3-methoxybenzoic acid) was developed, utilizing whole cells of Streptomyces viridosporus T7A In the first step, cells were grown in a yeast extract-vanillin medium under conditions where cells produced an aromatic aldehyde oxidase In the second step, vanillin was incubated with the active cells and was quantitatively oxidized to vanillic acid which accumulated in the growth medium Vanillic acid was readily recovered from the spent medium by a combination of acid precipitation and ether extraction at greater than or equal to 96% molar yield and upon recrystallization from glacial acetic acid was obtained in greater than or equal to 99% purity

Regulation of expression of Escherichia coli pilus K99.

Abstract: An immunoassay demonstrated that the assembled K99 pilus on the surface of Escherichia coli grown in minimal medium appeared during the logarithmic phase of growth, but the synthesis of K99 subunits, as measured by nonequilibrium two-dimensional gel electrophoresis, occurred throughout the life cycle of the cell. Contrary to other reports, the addition of glucose to the growth medium did not affect K99 pilus assembly or subunit synthesis, although in a K99+ adenyl cyclase (cya) mutant, subunit synthesis was reduced. There was no reduction in the amount of assembled K99 on the cell surface of the cya mutant compared with the wild-type parent. The addition of L-alanine to minimal medium repressed K99 synthesis. However, if L-threonine or L-isoleucine was also included in the growth medium, the effect of L-alanine was reduced. Chloramphenicol caused a complete inhibition of K99 subunit synthesis, but assembly proceeded normally. Growth at 18 degrees C inhibited both subunit synthesis and pilus assembly. Approximately 92% of all cellular K99 was associated with the outer membrane, and 4% was associated with the inner membrane. No K99 was detected in the cytoplasm.

Enhanced somatic embryogenesis using maltose

Abstract: Methods and compositions are provided for the growth of cultured plant cells which include the addition of maltose as a carbohydrate source. Additional improvements in the plant cell growth medium can be obtained by adding a source of ammonium ion to the medium to obtain a synergistic effect with the maltose.

Pulse labeling of yeast cells and spheroplasts.

Abstract: Publisher Summary This chapter focuses on the pulse labeling of yeast cells and spheroplasts. Pulse-labeling experiments are technically simple however several variables must be carefully controlled. These variables are discussed in this chapter. It is always useful to know the amino acid composition of the polypeptide to be studied. The specific radioactivity of the completed polypeptide depends not only on the specific radioactivity of the amino acid pool in the cell, but also on the abundance of that amino acid in the polypeptide chain. To utilize as much of the added radioactive tracer as possible, pulse-labeling should be performed under conditions where protein synthesis is rapid. One important factor is to transfer the cells or spheroplasts from growth medium to labeling medium rather quickly; only 3–4 minutes are necessary for centrifugation and resuspension. The labeling medium contains no amino acids, however small amounts may be carried over from the growth medium of spheroplast buffer because the cells and spheroplasts are not washed before suspending in labeling buffer. These small amounts do not significantly compete with [ 35 S]methionine for incorporation into protein, however may provide pools of other amino acids necessary to sustain protein synthesis.

Regulation of prostaglandin production and ectoenzyme activities in cultured aortic endothelial cells

Abstract: Prostaglandin production, angiotensin-converting enzyme, and 5′-nucleotidase were measured in porcine aortic endothelial cells in situ (with a multiwell template on an opened aorta), in primary culture and in subcultures. Changes during culture were monitored and the effects of culture conditions were investigated by growing cells on a biological matrix or on plastic, by adding different sera to the growth medium, and by harvesting cells enzymically or mechanically. Prostacyclin production by endothelium in primary culture is highest immediately after cell isolation and subsequently declines; this pattern is repeated each time the cells are subcultured. The level at which production stabilises is ∼ 200 pgċ106 cells−1ċh−1. Detaching cells by physical means stimulates production much more than enzymic dispersion; the type of serum or the presence of a biological matrix does not alter prostaglandin production. The relative amount of prostaglandin E produced increases with time, from ∼20% of the prostacyclin production shortly after isolation to > 100% in subcultured cells. None of the culture conditions that we tested altered this trend. Angiotensin-converting enzyme activity decreases during primary culture, but activity can be sustained by including homologous serum (from whole blood or from platelet-free plasma) in the culture medium. The method of harvesting cells, or the presence of a matrix, did not affect enzyme activity. 5′-Nucleotidase also declines during culture, with a progressive decrease in both Km and Vmax from template to primary culture to subcultures. None of the variations in culture conditions prevented this change. Ectoadenosine-deaminase activity, not detectable in cultured cells, can be measured in the template. Part of this activity was released by the vascular wall and could be due to plasma diffusing from the interstitial space.

Extracellular lysine production from hydrocarbons byArthrobacter globiformis

Abstract: A bacterium isolated from Burdwan (India) soil was found to accumulatel-lysine in the growth medium and was identified asArthrobacter globiformis. The strain grew and accumulated lysine in a purely synthetic medium. Supplementation of the synthetic medium stimulated growth but did not improve the yield. The entire fermentation period could be divided into a growth phase and a production phase, which could be prolonged by adjustment of pH to the neutral range. Among the different hydrocarbon and nitrogen sources tested SR gas oil at 4 % and ammonium sulphate at 0.4 %, respectively, were found most to be suitable. Different vitamins and antibiotics stimulated growth and lysine yield; inoculum of 7 % (V/V) of the medium was found to be optimal. The yield of lysine under optimal conditions was 3.4 g per litre medium. Lysine was isolated in crystalline form from the fermented broth by IEC and found to be a purel-isomer.

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar.

Abstract: The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.

Lactic acid bacteria which do not decarboxylate malic acid and fermentation therewith

Abstract: Bloating of brine fermented cucumbers can be greatly reduced by using lactic acid bacteria which do not decarboxylate malic acid and therefore do not produce carbon dioxide during fermentation. Also, certain high acid wines can be improved by fermenting fruit with bacteria which do decarboxylate malic acid. A method has been discovered of differentiating between species of lactic acid bacteria which do and do not decarboxylate malic acid. This method comprises growing a lactic acid bacterium in a suitable malic acid-containing nutritive growth medium under conditions suitable for growth and monitoring the pH of the medium during growth. The pH will decrease only when a lactic acid bacterium is present which does not decarboxylate malic acid. When malic acid is decarboxylated, a proton is removed from the solution in accordance with the following equation: ##STR1## Therefore, the pH of the medium either remains the same by neutralizing lactic acid produced by the fermentation of the carbohydrate source or increases when insufficient lactic acid is present. The above method is particularly fast and easy for screening bacteria obtained from purposely mutated species of lactic acid bacteria. Many strains of bacteria can be tested together by streaking them on a single layer agar plate.

Continuous production of patulin by immobilized cells ofPenicillium urticae in a stirred tank reactor

Abstract: Production of the mycotoxin, patulin, by immobilized cells ofPenicillium urticae was chosen as a model system to study the formation of secondary metabolites on a long term basis. Spores of the fungus were immobilized in carrageenan beads and allowed to germinate in situ by incubation in a growth medium. Production of the antibiotic by the populated biocatalyst was then followed in a continuous stirred tank reactor (CSTR) under three different regimes of nutrient feed. Using a nitrogen free feed, antibiotic production gradually declined but the cells could be re-activated by addition of a diluted growth medium. Prolonged production of patulin (up to 440 h) was observed when a source of nitrogen (ie. yeast extract) was supplied to the feed medium.

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium.

Abstract: Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.

On characterization of the growth of Escherichia coli in batch culture.

Abstract: Several growth monitoring parameters, including adenine nucleotide contents, were measured during Escherichia coli K12 batch cultivation in mineral medium with glucose The adenylate energy charge with its mean value of 083 remained roughly stable during growth The total adenylate and ATP pools (nmol/mg dry weight), and also the individual cell volume changed with a pattern of two maxima at approximately 4 and 10 h of cultivation After the exhaustion of the glucose from the growth medium the adenylate energy charge, the pools of ATP and total adenylate started to decrease marking the onset of the stationary growth phase Our data indicate that actually there was only a limited period within the logarithmic growth phase during which the growth might have been balanced: during this period of 15 h different growth monitoring parameters (optical absorbance, cell number, total cell volume, and ATP content per ml) increased with almost equal rates Moreover, as ATP pool and median cell volume during this short period were approximately constant, the culture might have been even in the steady state

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Abstract: Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free "attachment medium" was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation.

Amylase inhibits Neisseria gonorrhoeae by degrading starch in the growth medium.

Abstract: Highly purified salivary alpha-amylase inhibited the growth of fresh isolates of Neisseria gonorrhoeae on GC agar base medium supplemented with 2% IsoVitaleX (BBL Microbiology Systems). Hydrolysis of starch in the medium by amylase resulted in a negative starch-iodine test. However, purified amylase did not inhibit gonococcal growth on agar plates that contained hemoglobin (chocolate agar). This effect was not caused by inhibition of amylase, since amylase activity was the same in the presence or absence of blood products. Moreover, survival of N. gonorrhoeae in buffered saline was not affected by amylase. These results suggest that amylase inhibited the growth of N. gonorrhoeae on GC agar base plates by hydrolyzing starch.

INDUCIBLE POSTREPLICATION REPAIR IS RESPONSIBLE FOR MINIMAL MEDIUM RECOVERY IN UV‐IRRADIATED Escherichia coliK–12

Abstract: — Ultraviolet (UV)-irradiated Escherichia coliK–12 uvrA cells showed higher survival if plated on minimal growth medium rather than on rich growth medium, i.e., they showed minimal medium recovery (MMR). A 2-hour treatment of UV-irradiated cells with rifampicin inhibited the subsequent expression of MMR, and produced a large reduction in survival. We have recently isolated a new mutant (mmrA1) that does not show MMR. The mmrA mutation protected UV-irradiated uvrA cells from the effect of rich growth medium on survival, but not from the effect of rifampicin on survival. DNA daughter-strand gap (DSG) repair in UV-irradiated (4 J/m2) uvrA cells was inhibited to the same degree whether rich growth medium was added immediately after irradiation or after 10 min of postirradiation incubation in minimal growth medium. However, chloramphenicol added immediately after irradiation greatly reduced this repair; there was less reduction if it was added 10 min after UV irradiation. These findings suggest that MMR is an inducible repair phenomenon, and that rich growth medium inhibits this repair process itself rather than its induction.

Induced changes in the surface of Neisseria gonorrhoeae.

Abstract: Growth of Neisseria gonorrhoeae strain F62 on medium containing pyruvate and a high ratio of cysteine to cystine resulted in functional and structural changes that are consistent with phenotypic changes in lipopolysaccharide. Both transparent (O−) and moderately opaque (O+) variants became more sensitive to killing by normal human serum and more resistant to killing by pyocin G, a bacteriocin from Pseudomonas aeruginosa. Electrophoresis of outer membranes in the presence of sodium dodecyl sulfate demonstrated differences also dependent upon the growth medium. When gels were treated with periodic acid and stained with silver, lanes containing outer membranes obtained after growth in the modified medium demonstrated two bands in addition to those independent of the growth medium. The enhancement of these additional bands by periodate treatment indicated that they represent material containing carbohydrate. The mechanism by which the changes in the growth medium affected the surface of N. gonorrhoeae is not ...

Physiological and Nutritional Studies with an Isolate of Leptolegnia Sp. from the Freshwater Nematode Neomesomermis Flumenalis

Abstract: The basic physiological responses and nutritional requirements for mycelial growth of an isolate of Leptolegnia sp. were determined. The optimum temperature was 19 C, and the optimum pH range was 4.7 to 5.7. The optimum level of vitamin-free casamino acids in the presence of glucose was 20 g/l, and the optimum glucose concentration was 8 g/1l. When organic nitrogen sources were supplied either individually or as a mixture (vitamin-free casamino acids), L-proline, L-arginine, L-histidine, L-threonine, L-serine, L-glutamic acid, glycine, L-leucine, L-alanine, L-glutamine, L-valine, and L-aspartic acid were utilized. L-phenylalanine, L-tyrosine, L-isoleucine, L-lysine, and L-a-amino-n-butyric acid were utilized only from the mixture and not as individual sources. Ammonium chloride functioned as a nitrogen source but neither potassium nitrate nor potassium nitrite were utilized. L-methionine (optimum concentration of 50 ppm) did not serve as either a carbon or a nitrogen source, but of the sulfur sources tested, it supported the best growth. Glucose, maltose, starch, glycogen, and sucrose were readily utilized as carbon sources. When glucose was omitted from a growth medium containing casamino acids, L-threonine, L-alanine, L-isoleucine, L-leucine, L-lysine, L-tryptophan, urea, L-serine, glycine, cysteic acid, L-aspartic acid, L-glutamic acid, and Lornithine were utilized earlier than in the presence of glucose, and, thus, probably served as carbon sources. A requirement for a high level of organic nitrogen was also found when individual organic nitrogen sources were used in the presence of glucose. L-proline and Lalanine had optimum concentrations of 20 g (2400 mg N/l) and 7.5 g (1200 mg N/l), respectively. The optimal concentration for an inorganic nitrogen source was much lower. A basal medium adjusted to pH 5.2 and composed of glucose, methionine, potassium phosphate, a standard complement of micronutrients and either L-proline, L-alanine, or ammonium chloride was devised.