The Influence of Calcium or Manganese on the Resistance to EDTA, Polymyxin B or Cold Shock, and the Composition of Pseudomonas aeruginosa Grown in Glucose- or Magnesium-depleted Batch Cultures

Cultures were batch grown in simple salts media in which growth was limited either by depletion of glucose and magnesium (C/Mg-dep) or by glucose alone (C-dep). Cultures were also grown in these media supplemented by calcium and/or manganese.
All cultures grown in the C-dep media were sensitive to ethylenediaminetetraacetic acid (EDTA), polymyxin and also to cold shock but were relatively resistant to ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA). Inclusion of calcium or manganese in the growth medium enhanced lysis by EDTA. Cultures grown in the basic C/Mg-dep media were resistant to EDTA, EGTA, polymyxin and to cold shock. Sensitivity to these agents was retained by cultures grown in C/Mg-dep media supplemented with Ca²⁺ and/or Mn²⁺. Cells grown in C/Mg-dep media with added Mn²⁺ were more sensitive to EDTA and polymyxin than those from the unsupplemented C/Mg-dep media but still resistant compared with C-dep cultures. All cultures from supplemented C/Mg-dep media were more sensitive to EGTA than those from any of the C-dep media.
Whole cells and cell walls from these various media had differing amounts of cell wall, phosphorus, amino sugar, carbohydrates, readily extractable lipid (REL), total phospholipid (PL), and especially differences in cell wall divalent metal cation content.
The differences in PL, REL and amino sugars and carbohydrate did not correlate with the response of C-dep and C/Mg-dep bacteria to EDTA, EGTA or polymyxin. The results are discussed in relation to the hypothesis that the sensitivity of Pseudomonas aeruginosa to polymyxin and EDTA is more dependent on outer membrane cation content rather than on other components, e.g. PL and lipopolysaccharide.     

Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa.

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

Sucrose concentratidns in the growth medium influence the membrane lipids of Streptococcus mutans

Streptococcus mutans was cultivated in media containing sucrose (10–40%, w/v) and the sucrose induced changes in chemical and physical properties of its membrane lipids were investigated. The degree of unsaturation in the fatty acids of both total lipid and glycolipid fractions decreased when the sucrose concentration was increased. An electron spin resonance spectroscopic study revealed the reduction of membrane lipid fluidity by adding sucrose to the growth medium. Liposomes prepared from membrane lipids of bacteria grown with sucrose showed less osmotic volume changes than those of bacteria grown without sucrose. These results suggest that modification of membrane lipid composition, fluidity and osmosis-resistance have an important role in the ability of Streptococcus mutans to grow in sucrose at high concentrations.     

Nitrofurantoin Media for the Isolation of Pseudomonas aeruginosa

Selective and enrichment media containing nitrofurantoin were compared with media containing cetrimide in the isolation of Pseudomonas aeruginosa from contaminated specimens. The nitrofurantoin media were as sensitive as cetrimide media, were easier to prepare and use and constant in performance.     

Cystine-glutamate transport interactions in rat renal cortical tubules,brushborder vesicles,and cultured renal tubule cells

Glutamate had no significant effect on the uptake of 0.025 mM cystine by isolated rat renal cortical tubules and brushborder membrane vesicles in contrast to lysine which significantly inhibits cystine transport. Glutamate, however, markedly inhibited cystine uptake by rat renal tubule cells grown in a serum-free, hormonally defined media for 5 days. Lysine also inhibited cystine transport in these cultured renal tubule cells.     

Lipid metabolism and cell composition of the oleaginous yeast Apiotrichum curvatum grown at different carbon to nitrogen ratios

Apiotrichum curvatum ATCC 20509, an oleaginous yeast that can accumulate up to 60% of its cellular dry weight as intracellular lipid when grown with excess carbon, was grown in nitrogen-limited, balanced, and lactose-free medium with asparagine as nitrogen source and lactose as carbon source. Biomass and lipid accumulation were measured, cell composition was analyzed, and catalase activity was followed as marker enzyme for peroxisomes. The organism accumulated 54% of its dry weight as total cellular lipid when grown under nitrogen limitation and accumulated only 20-25% of its dry weight as lipid when grown in balanced medium. When starved for carbon, cells utilized endogenous lipid and carbohydrate as carbon and energy sources; the intracellular contents of lipid and carbohydrate decreased by 31 and 26%, respectively. Intracellular carbohydrates also seemed to be used as intermediates for lipid accumulation and lipid turnover. Catalase activity was strongly induced (over 10-fold increase in specific activity) when cells metabolized endogenous lipid. The lipid content of cells was inversely related to catalase activity and to intracellular protein or total nitrogen content. Lipid content showed no correlation with intracellular carbohydrate content.     

Biochemical mechanism of combined effect of ethambutol and sparfloxacin against Mycobacterium smegmatis

M. smegmatis cells grown in the presence of combination of ethambutol (EMB) and sparfloxacin (SPX) had decreased level of total cellular lipids as compared to control as well as cells grown in the presence of sub-inhibitory concentration (MIC50) of individual drugs. Amongst various phospholipids analyzed, maximum decrease was observed in the content of phosphatidylinositolmannosides (PIMs) of the cells grown in combination of EMB and SPX. In contrast, the subcellular distribution of phospholipids revealed a significant increase in PIMs content of both cell wall and cell membrane of the cells grown in the presence of combination of drugs as compared to control as well as individual drugs. Mycolic acids of M. smegmatis cells were found to be main targets as combination of drugs resulted in significant decrease in total cellular as well as cell wall mycolic acids as compared to control and individual drugs. Changed lipid composition of M. smegmatis cells grown in the presence of MIC50 of EMB, SPX and combination resulted in significant surface changes as was evident from decreased limiting fluorescence (Fmax) intensity of 1-anilinonaphthalene-8-sulfonate (ANS). Thus, the results of this study suggested that ethambutol and sparfloxacin in combination exerted their antimycobacterial effect principally due to their action on phosphatidylinositolmannosides (PIMs) and mycolic acids, which form the permeability barrier of mycobacteria.     

Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures

We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.     

Influence of food quality on growth and reproduction in Daphnia

SUMMARY. 1. Growth and reproduction were measured for Daphnia pulex (Leydig) fed Scenedesmus obliquus (Turp.) Kutz. grown with nitrogen-sufficient and nitrogen-deficient media. The incorporation of carbon into the body of D. pulex was traced using ¹⁴C-labelled algae and biochemical fractionation.
2. Scenedesmus cultured on N-defkient media allocated more carbon to lipid and less to protein than when cultured on N-sufficient media.
3. In a short-term feeding experiment, Daphnia accumulated more lipid when fed N-deficient algal cells high in lipid content than when fed algae grown on N-sufficient media that were lower in lipid.
4. Animals grew faster and produced larger broods when fed algae grown on an N-sufficient medium over an 8-day period. Daphnids fed N-deficient algae had higher quantities of body lipid, suggesting it was not a lack of energy which limited their growth and reproduction.
5. Daphnia fed algae grown on N-deficient media allocated more lipid to eggs, and their offspring lived longer under starvation conditions.     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Influence of culture medium of the fatty-acid profile in enteric bacteria.

Enteric bacteria having a high content of cyclopropane fatty acids steeply increase their synthesis when grown on insufficiently propitious culture media (meat-peptone agar or modified Drobot'ko synthetic medium) as compared with bacteria grown under more favourable conditions (meat-peptone broth). Simultaneously, a decrease in monounsaturated fatty acids and increase in palmitic acid are observed. One of the main factors underlying the change in the proportion of fatty acids in bacteria grown on synthetic medium is an increase in medium pH in the process of their growth. Enteric bacteria containing minute amounts/or not containing cyclopropane fatty acids at all (under the experimental conditions used) change their fatty-acid profile little if the culture medium is changed. When grown under insufficiently favourable conditions, these bacteria mainly display an enhanced content of palmitic acid and a lowered content of octadacenoic acid as compared with bacteria grown under more favourable conditions. Of the culture media used, meat-peptone broth, which affords the most favourable conditions for eneteric bacteria growth, is the most suitable medium for obtaining data of taxonomic value.     

Effects of Lead on the Lipid Compusition of Micrococcus luteus Cells

Micrococcus luteus cells cultivated in medium containing lead salts exhibited a sequence of changes in the quantity of total cellular lipids with essentially no changes from normal cellular yields. The lipid composition of cells cultivated one to four times was moderately decreased (phase I) whereas that of cells cultivated five to six times was reduced by as much as 50% (phase II). Cells cultivated more than six times in lead-containing media had progressively greater quantities of lipid (phase III) approaching that found in control cells. These cells with reestablished lipid contents showed no further effects from more prolonged exposure to lead salts. Chromatographic studies of total lipids of cells of each lipid phase revealed relatively complete lipid compositions. These results indicated that lead is apparently affecting a common biochemical parameter in the biosynthesis of lipids of lipid phase II cells. Changes in the relative quantities of individual components were observed in both the nonpolar and polar lipids in each lipid phase. The most notable changes were the decrease in aliphatic hydrocarbons with concomitant increases in the diglycerides and components identified as a complex family of ketones. Microscopy examinations of control and lead-treated cells revealed electron dense inclusion bodies in membrane fragments in only lead-treated cells.     

A novel approach for medium formulation for growth of a microalga using motile intensity

Motile intensity of the cells, defined as the specific mean kinetic energy, was measured by image analysis and used to formulate a suitable medium for the cultivation of a motile microalga. Nitrogen source at 60 mg/L was used as the target component. The cells grown in a medium containing urea showed the highest motile intensities during cultivation when compared to the cells grown with NH(4)Cl or (NH(4))(2)SO(4) as the nitrogen source. The urea culture gave the highest biomass yield and lipid content of 1.06+/-0.12 g/L and 22.34+/-2.56%, respectively after 150 h of cultivation. These results indicated a strong dependence of motile intensity on the nitrogen source used in the growth medium. This concept, which requires only a small droplet of the sample, can find potential application in the design and optimization of culture media.     

The influence of conditioned media and nonessential amino acid supplementation on the growth of cells in vitro

The growth enhancing effect of media conditioned by cells from established lines (BHK and L60) is comparable to that of media conditioned by cells of primary origin (chick embryo). However, the properties of the conditioned media from these two systems show marked differences: the growth enhancing factors in the former are dialyzable and heat-stable, in contrast to the non-dialyzable and heat-labile factors in the latter. Media conditioned for only four hours by BHK or L60 cells stimulated cell growth. Amino acid analyses revealed that non-essential amino acids had appeared in these conditioned media. To verify this as the metabolic basis of conditioning by cells from established lines, media containing dialyzed serum were supplemented with each of six non-essential amino acids, and assayed on BHK and L60 at various population densities. Serine was the most stimulatory and alanine the most inhibitory of the amino acids tested. Mixed supplementation of the medium showed that when low levels of alanine and serine were added simultaneously, cell growth was enhanced but any increase in the level of alanine required an increase in the level of serine also to achieve growth stimulation.     

Identification of sulfoquinovosyl diacylglycerol as a major polar lipid in Marinococcus halophilus and Salinicoccus hispanicus and substitution with phosphatidylglycerol

The sulfonolipid sulfoquinovosyl diacylglycerol normally associated with photosynthetic membranes was identified as a major lipid in Marinococcus halophilus, Salinicoccus hispanicus ("Marinococcus hispanicus"), and Marinococcus sp. H8 (Planococcus sp. H8). Phosphatidylglycerol and 0%-10% cardiolipin accounted for the remaining polar lipids in these moderately halophilic, Gram-positive bacteria. Negative-ion fast atom bombardment mass spectrometry was used to quantify these three polar lipids from cells grown in media containing 0.03 to 4 mol NaCl/L. All strains revealed dramatic shifts in the ratio of sulfonolipid to phospholipid dependent on the salinity of the growth media, when grown in media with low phosphate content. Highest sulfonolipid content occurred during best growth in 0.5-2 mol NaCl/L, approaching 80%-90% of the total polar lipids. It was demonstrated that growth of M. halophilus in the presence of elevated phosphate and low sulfate blocked the shift to decreased phospholipids most notably during growth in 0.5-2 mol NaCl/L, without significant influence on growth. The data suggest that in low-phosphate media the influence of NaCl concentration on growth rate (and resulting demand for phosphate by competing pathways) is the primary factor responsible for exchange between phospholipid and sulfonolipid. We conclude that sulfoquinovosyl diacylglycerol, by substitution with phospholipids, contributes to the ability of these Gram-positive cocci to adapt to changing ionic environments. A comparison of 16S rRNA established a close similarity between Planococcus sp. H8 and M. halophilus.     

Lipid accumulation in the rat liver: a histological and biochemical study.

The sequential pattern of lipid accumulation and associated biochemical changes were studied in two commonly used experimental models of nutritional fatty liver in rats. Female rats were maintained for 8 weeks on high fat, low protein diets containing adequate methionine and choline, and drinking water ad libitum (Diet 1), or deficient in methionine and choline and containing 20% ethanol as a substitute for drinking water (Diet 2). Histologically, there was a progressive increase in liver lipids, mainly in the periportal areas. Occasional foci of liver cell necrosis with lipogranuloma formation occurred in areas of severe fatty change. These changes appeared earlier and were more marked in rats maintained on Diet 2. Electron micrographs revealed large lipid droplets in the liver cells, which sometimes contained myelin figures. The mitochondria were enlarged, distorted and appeared as amorphous structures with disorientated cristae in rats on Diet 1, whereas they had a condensed conformation in rats maintained on Diet 2. Rough endoplasmic reticulum was fragmented and degranulated particularly in rats on Diet 1, and smooth endoplasmic reticulum showed hyperplasia and vesiculation in rats on Diet 2. There was a progressive increase in the total liver lipids and triglycerides in both the groups of rats. This fatty change was accompanied by a significant increase in hepatic 3-hydroxybutyrate, acetoacetate, malate, 2-oxoglutarate, citrate, lactate, ammonia, glutamate, alanine and aspartate, and a significant decrease in oxaloacetate, urea and glucose concentrations. The mass action ratios for alanine aminotransferase, aspartate amino transferase, and glutamate dehydrogenase, generally moved in a parallel direction. Hepatic ATP content was considerably reduced accompanied by a decrease in [ATP]/[ADP] ratios and a significant increased in [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] ratios. There was a corresponding decrease in the [NAD+]/[NADH] ratios both in the cytoplasmic and mitochondrial compartments. These biochemical changes were particularly severe in rats maintained on Diet 1 and Diet 2 for 8 weeks. There was a very good relationship between impaired mitochondrial and endoplasmic reticulum functions, redox and phosphorylation states, and the relevance of their changes to the fate of fatty liver cells.     

Fatty Acid and Aliphatic Hydrocarbon Composition of Sarcina lutea Grown in Three Different Media

Sarcina lutea was grown in Trypticase Soy Broth, Nutrient Broth, and a chemically defined medium. Gas chromatographic analysis of lipid components demonstrated that the composition of the medium had an effect on the relative per cent composition of the aliphatic hydrocarbons and fatty acids present in the cells. The branched olefinic hydrocarbons from the organisms grown in Trypticase Soy Broth showed no predominance or only a slight predominance of odd-numbered carbon chains, whereas the hydrocarbons from cells grown in the other two media showed an obvious predominance of odd-numbered carbon chains. The monocarboxylic fatty acid content and distribution showed only minor differences, with all normal saturated fatty acids present in relatively small quantities for cells grown in Nutrient Broth and in a chemically defined medium.     

Initiation, Growth and Nuclear Characteristics of Tissue Cultures of Paeonia suffruticosa

Tissue cultures of the garden paeony, Paeonia suffruticosa have been established using explants of etiolated stems. Callus formation was induced on agar-solidified media containing ammonium ions or amino acids together with the hormones 2,4-dichlorophenoxyacetic acid and kinetin, but not on media lacking the reduced nitrogen component. Attempts to induce callus from explants from green plants were completely unsuccessful and were characterized by the production of intense brown colorations, both of the explant and the medium. Subcultured tissue without the added hormones produced roots, both on solid and liquid media. Growth was tested on a range of liquid media, SH/2, SH, SH × 2 and SH—M, containing 1250, 2500, 5000 and 2500 mg/l potassium nitrate. The SH—M medium also contained 1650 mg/l ammonium nitrate. Growth measured as increased fresh weight was best in the SH/2, SH and SH—M media and was curtailed in the SH × 2 medium. Soluble protein content was highest at the lowest nitrogen concentration. A histochemical comparison of tissue grown on the SH/2, SH—M and SH—M lacking hormones showed that the cells in all the cultures remained diploid while differences were established in total nuclear protein, measured using the ninhydrin-Schiff procedure. Nuclei from SH—M grown cells have a higher protein content than those from the SH/2 medium while cells from the SH—M medium lacking hormones show a further increase in nuclear protein. This raises the question whether this change in nuclear protein is related to the morphogenesis of roots which occurs in this medium.     

Effects of hyperbaric oxygen on the growth and properties of Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa in 2 atmospheres absolute of 100% oxygen at 37 degrees C produced two types of abnormal colonies--stunted, rough colonies, termed dwarfs, and large, domed, mucoid colonies, termed giants. The occurrence of these variants depended upon the partial pressure of oxygen and the inoculum size. Subculture of dwarf or giant colonies produced a mixture of both colony types after incubation in hyperbaric oxygen, and colonies of normal appearance after incubation in air. Electron micrographs of ultrathin sections showed that cells from dwarf colonies had a more clearly defined envelope region than cells from normal colonies. Giant colony and normal colony-derived cells were of similar appearance. Whole cells from giant colonies contained more carbohydrate, readily extractable lipid, neutral lipid and free fatty acid than cells from normal colonies; the two cell types showed similar contents of 2-keto,3-deoxyoctonic acid and total phospholipid, but different proportions of individual phospholipids. Cells from dwarf, giant and normal (air-grown) colonies were incubated in air on nutrient agar containing either polymyxin, tetracycline or phenoxyethanol. Relative to cells from normal colonies, cells from dwarf colonies showed enhanced resistance to all three agents and cells from giant colonies showed enhanced resistance to polymyxin and tetracycline only. The resistance of cells from variant colonies was lost following a single subculture in air in the absence of antibacterial agents. It was concluded that the envelopes of cells from dwarf and giant colonies differed both from each other and from those of normal cells. These differences, and the formation of variant colonies, appeared to result from bacterial adaptation to hyperbaric oxygen rather than from mutation.