The Differing Effects of Cetyltrimethylammonium Bromide and Cetrimide B.P. upon Growing Cultures of Escherichia coli NCIB 8277

Growing cultures of Escherichia coli NCIB 8277, when treated with low concentrations of Cetrimide B.P., decrease in turbidity more markedly than when equivalent concentrations of cetyltrimethylammonium bromide (CTAB) are applied. Treatment with higher concentrations of CTAB but not of cetrimide evokes a prompt, extensive rise in turbidity. It is suggested that in growing cultures CTAB tends to cause cell aggregation whereas cetrimide tends to cause cell lysis, and that the lytic effect is related to the presence of impurities in cetrimide. The possibility that other antimicrobial surfactants promote lysis is considered.     

Cage Culture Turbidostat: a Device for Rapid Determination of Algal Growth Rate

The present cage culture turbidostat consists of a growth chamber and a control unit. The microorganisms (photoautotrophic algae) are kept in the growth chamber by porous membranes (pore size 1 to 3 μm) which retain the algae but allow efficient exchange of the growth medium. Flow rate and composition of the medium can therefore be varied independently of algal population density. A reciprocating pumping mode of the medium is introduced to obtain more gentle clearance of membranes than that provided by rotation or stirring in other membrane fermentors. Pulsed light and a light-emitting diode/light-sensitive transistor couple are used to monitor the turbidity of the culture, independent of external light needed for growth. The control unit keeps the turbidity constant by frequent activation of the dilution pump. Theoretical analysis of growth in the turbidostat shows that integrated activation time of the dilution pump is proportional to the growth rate of the organism. Theoretical analysis was also used to determine minimum flow-rate and nutrient concentration of medium to cover the requirement of the algae. Experiments with three different marine diatoms were carried out, and they demonstrated that the growth rate could be determined every hour and that the cultures could be kept at constant turbidity over 10 to 14 days at least.     

THE EFFECT OF SELENITE ON THE PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE BLUE-GREEN ALGA PHORMIDIUM LURIDUM VAR. OLIVACEA1

Phormidium luridum cultures were treated with sodium selenite in concentrations ranging from 10^?6 M to 10^?2 M. In contrast to the increasing culture turbidity of control and 10^?6 M selenite cultures, the turbidity of the other selenite cultures declined in proportion to time and selenite concentration. Chlorophyll extraction revealed similar results. Photosynthetic activity was inhibited within 6 hr in all cultures except control and 10^?6 M selenite. Phormidium at concentrations greater than 10^?6 M selenite showed a gradual loss of the bright green color and turned semitransparent. Cell-associated granules of reduced selenium were observed at higher selenite concentrations. Other structural changes observed were the presence of intracellular and intercellular spaces, spheroplast formation, and gradual cell lysis. Protein analyses of total cell samples and supernatant fractions confirmed cellular breakdown of selenite-treated algal cells.     

Effect of Diquat (1,1′-Ethylene-2,2′-Dipyridylium Dibromide) on the Photosynthetic Growth of Rhodospirillum rubrum

Diquat (2 x 10(-4)m) inhibited both aerobic and anaerobic growth of Rhodospirillum rubrum. With photosynthetic cultures, diquat affected the synthesis of bacteriochlorophyll more readily than cell mass (turbidity). Diquat retarded the synthesis of bacteriochlorophyll and some protein more readily than that of other cellular constituents such as ribonucleic acid, deoxyribonucleic acid, and cell mass. With cells deficient in phosphate, diquat inhibited the uptake-conversion of inorganic phosphate completely only when 3-(3,4-dichlorophenyl)-1,1'-dimethyl urea and ascorbate were also present.     

Estimation of Staphylococcus aureus growth parameters from turbidity data: characterization of strain variation and comparison of methods

Turbidity methods offer possibilities for generating data required for addressing microorganism variability in risk modeling given that the results of these methods correspond to those of viable count methods. The objectives of this study were to identify the best approach for determining growth parameters based on turbidity data and use of a Bioscreen instrument and to characterize variability in growth parameters of 34 Staphylococcus aureus strains of different biotypes isolated from broiler carcasses. Growth parameters were estimated by fitting primary growth models to turbidity growth curves or to detection times of serially diluted cultures either directly or by using an analysis of variance (ANOVA) approach. The maximum specific growth rates in chicken broth at 17 degrees C estimated by time to detection methods were in good agreement with viable count estimates, whereas growth models (exponential and Richards) underestimated growth rates. Time to detection methods were selected for strain characterization. The variation of growth parameters among strains was best described by either the logistic or lognormal distribution, but definitive conclusions require a larger data set. The distribution of the physiological state parameter ranged from 0.01 to 0.92 and was not significantly different from a normal distribution. Strain variability was important, and the coefficient of variation of growth parameters was up to six times larger among strains than within strains. It is suggested to apply a time to detection (ANOVA) approach using turbidity measurements for convenient and accurate estimation of growth parameters. The results emphasize the need to consider implications of strain variability for predictive modeling and risk assessment.     

Estimation of Staphylococcus aureus Growth Parameters from Turbidity Data: Characterization of Strain Variation and Comparison of Methods

Turbidity methods offer possibilities for generating data required for addressing microorganism variability in risk modeling given that the results of these methods correspond to those of viable count methods. The objectives of this study were to identify the best approach for determining growth parameters based on turbidity data and use of a Bioscreen instrument and to characterize variability in growth parameters of 34 Staphylococcus aureus strains of different biotypes isolated from broiler carcasses. Growth parameters were estimated by fitting primary growth models to turbidity growth curves or to detection times of serially diluted cultures either directly or by using an analysis of variance (ANOVA) approach. The maximum specific growth rates in chicken broth at 17°C estimated by time to detection methods were in good agreement with viable count estimates, whereas growth models (exponential and Richards) underestimated growth rates. Time to detection methods were selected for strain characterization. The variation of growth parameters among strains was best described by either the logistic or lognormal distribution, but definitive conclusions require a larger data set. The distribution of the physiological state parameter ranged from 0.01 to 0.92 and was not significantly different from a normal distribution. Strain variability was important, and the coefficient of variation of growth parameters was up to six times larger among strains than within strains. It is suggested to apply a time to detection (ANOVA) approach using turbidity measurements for convenient and accurate estimation of growth parameters. The results emphasize the need to consider implications of strain variability for predictive modeling and risk assessment.     

The effects of aeration and agitation on the measurement of yeast biomass using a laser turbidity probe

The paper reports the measurement of biomass concentration using a laser turbidity probe. A suspension of Bakers’ yeast (0.5?50?gl^-1) was subjected to various conditions of agitation and aeration in a stirred tank reactor and the turbidity measured using the probe. Both agitation and aeration were found to influence the turbidity. At any constant biomass concentration, the effect on the turbidity measurement of changing agitation or aeration rate independently was linear, while at any constant conditions of agitation and aeration rate, the relationship between turbidity and biomass concentration was non-linear. The results indicate that, in a bioprocess with non-particulate medium, it is possible to correct for the effects of aeration and agitation on turbidity measurement using a multivariate calibration model. A procedure for calibration and correction of measurements for the effects of agitation and aeration is presented and is verified using experimental data. This procedure may be generalised for other applications.     

On-line measurement and control of cell concentration of Saccharomyces cerevisiae using a laser turbidimeter

Summary On-line measurement and control of cell concentration of Saccharomyces cerevisiae using a laser turbidimeter was carried out. Effects on the turbidity measurement of operating parameters such as agitation speed, aeration rate, and the concentration of antifoam agent were investigated. Correlations between the on-line-measured turbidity and the off-line dry cell weight concentration were made at various operating conditions. Using the correlations the cell concentration was successfully estimated in a range of up to 8 g/L in batch cultures. The on-line monitoring and regulation of the cell concentration in a range of up to 35 g/L were also satisfactory in continuous and turbidostat cultures with cell recycle.     

Effects of medium composition on penicillin-induced hydrolysis and loss of RNA and culture turbidity in group A streptococci.

Exposure to penicillin G of exponentially growing cultures of group A streptococci growing in chemically defined medium (CDM) can lead to extensive loss of culture turbidity. Significant reductions in culture turbidity did not accompany comparable treatments of group A streptococci growing in Todd-Hewitt broth (THB). Studies with THB and a high-molecular-weight (greater than 12,000) fraction of THB demonstrated that components in this complex medium inhibited the efflux of RNA hydrolysis products from otherwise intact cells. Hydrolysis products accumulated intracellularly and inhibited the extensive hydrolysis of RNA and consequently the loss of culture turbidity. Results of survival studies with cultures of group A streptococci exposed to penicillin G in THB demonstrated that this treatment protocol produces conditions of phenotypic tolerance relative to exposure in CDM. In combination, these findings provide further support for the hypothesis of RNA hydrolysis as the bactericidal mechanism of penicillin G action in this nonlytic death phenotype.     

A computer model for the growth and differentiation of a fungal colony on solid substrate

Mold growth and differentiation are closely related to the formation of secondary products. In solid-substrate fermentations this interrelationship is often more completely realized than in submerged cultures. Solid substrate reactions are used commercially in a limited manner in the western world, but are relatively common in Asia. Basic studies in solid-substrate fermentation should yield results applicable to all types of commercial mold fermentations for the production of a secondary product. This paper presents a relatively simple model for the growth of a mold colony on a solid surface with a defined medium utilizing glucose. Unlike submerged cultures the model must account for both cellular differentiation and the spatial heterogeneity in the system. Model parameters were estimated independently using literature values. The results of the simulation studies suggest that mass transfer limitations are at least partially responsible for the proliferation of differentiated structures on solid substrates as compared to liquid cultures. Since the concentration profile depends on the depth of the substratum, conditions that enhance conidia production can be achieved by controlling the depth of the solid medium.     

Effects of turbidity on life history parameters of two species of Daphnia

SUMMARY. 1. Life table experiments were carried out in a range of turbidities on D. pulex , representing a 'clear-water', and on D. barbata , representing a 'turbid-water' species.
2. In contrast to predictions based on seasonal occurrence patterns, D. pulex had a higher intrinsic rate of increase ( r _c)s over most of the turbidity range than D. barbata. Consistent differences in life history between the two species were found, but life history characteristics were rather insensitive to turbidity levels. Although turbidity per se appears to have little direct effect on life history parameters, it could influence the seasonal succession of these two species by interacting with other factors.
3. It is suggested that visual predation by fish rather than turbidity per se probably influenced the seasonality of Daphnia species. During the warmer months, selective removal of D. pulex by visually foraging fishes may allow D. barbata to gain dominance. Effects of temperature and nutrition also merit further study.     

Mathematical modelling of a mixed culture cultivation process for the production of polyhydroxybutyrate

Mixed cultures submitted to acetate "feast" and "famine" cycles are able to store intracellularly high quantities of polyhydroxybutyrate (PHB). It was demonstrated in a previous study that the intracellular PHB content can be increased up to 78.5% (g HB/gVSS) of cell dry weight in a sequencing batch reactor (SBR) with optimised operating conditions. The specific PHB formation rate was also shown to be higher for mixed cultures than for pure cultures. Such high intracellular PHB contents and specific productivity open new perspectives for the industrial production of polyhydroxyalkanoates (PHA) using mixed cultures instead of pure cultures. The main goal in this work was to develop a mathematical model of mixed cultures envisaging the optimisation of PHB production. A relatively simple two-compartments cell model was developed based on experimental observations and other models proposed in the literature. A convenient experimental planing allowed to identify the kinetic parameters and yield coefficients. Experiments were performed with and without ammonia limitation enabling the analysis of PHB formation independently of the cell growth process. The experimental true yields partially confirm the theoretical values proposed in the literature. The final model exhibited high accuracy in describing the process state of most experiments performed, thus opening good perspectives for future model-based optimisation studies.     

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.     

Effects of thermal stratification and mixing on reservoir water quality

In this study, the effect of thermal stratification on water quality in a reservoir has been investigated by field observations and statistical analysis. During the summer period, when stratification is evident, field observations indicate that the observed dissolved oxygen concentrations drop well below the standard limit of 5 mg l⁻¹ at the thermocline, leading to the development of anoxia. The reasons for variations in the dissolved oxygen concentrations were investigated. Variations of air temperature and other meteorological factors and lateral flows from side arms of the lake were found to be responsible for the increase of dissolved oxygen concentrations. It was also observed that turbidity peaked mostly in the thermocline region, closely related to the location of the maximum density gradient and thus low turbulence stabilizing the sediments in the vertical water column. Relatively cold sediment-laden water flowing into the lake after rain events also resulted in increased turbidity at the bottom of the lake. Nondimensional analysis widely used in the literature was used to identify the strength of the stratification, but this analysis alone was found insufficient to describe the evolution of dissolved oxygen and turbidity in the water column. Thus correlation of these parameters was investigated by multivariate analysis. Fall (partial mixing), summer (no mixing), and winter (well mixed) models describe the correlation structures between the independent variables (meteorological parameters) and the dependent variables (water-quality parameters). Statistical analysis results indicate that air temperature, one day lagged wind speed, and low humidity affected variation of water-quality parameters.     

Density-dependent regulation of growth in 1 cell suspension cultures : II. Synthesis of total protein and collagen in presence of rapidly declining oxygen tensions

High density L cell suspension cultures were previously shown to remain viable for prolonged periods of time and to exhibit stability of population and regulation of DNA synthesis and mitosis comparable to growth-inhibited, dense attached cultures of fibroblasts. The present data demonstrate that this similarity extends to the regulation of total protein and collagen synthesis in spite of a media pO₂ decline to virtually undetectable levels within 8 h after resuspension of the cells. Possible mechanisms accounting for this are discussed as well as evidence suggesting comparable pO₂ changes in the cellular microenvironment of dense attached cultures. It is concluded that, for the parameters so far investigated, cell to cell contact is not a universal prerequisite of density-dependent inhibition of growth.     

Unimportance of counterflux in the energetics of "downhill" transport

Adam Kepes suggested that the cellular transport and hydrolysis of orthonitrophenyl-beta-d-galactopyranoside is powered by the counterflux of the d-galactose resulting from beta-galactosidase action within the cell. His explanation would rationalize the unique insensitivity of this galactoside transport to energy poisons such as azide. But contrary to the predictions of this hypothesis, (i) there is no initial large inhibition that progressively lessens as galactose is produced. This was shown with a double wavelength stopped-flow spectrophotometer developed to eliminate interference from turbidity transients. (ii) The azide sensitivity does not increase with an external concentration of galactose sufficient to reverse the thermodynamic gradient. (iii) Mutation in galactose utilization or growth on highly catabolite-repressing regimens did not increase the azide sensitivity, and induction of galactose transport and metabolism did not decrease azide sensitivity. It was found that Kepes measurements must have contained two artifacts. One is that the control rate of hydrolysis decreases with time as the dense cell suspension becomes anaerobic. The other is that azide causes turbidity changes for some time after its introduction. If the former is avoided by magnetic stirring and the latter by double wavelength spectrophotometry or controls without substrate, the inhibition is constant from the earliest time that can be measured. It is therefore concluded that energy-unstarved cells, exposed to azide, still have adequate energy reserves to couple to the downhill transport, although their potential is not adequate to drive accumulation against a concentration gradient.     

The effect of temperature on lipid-n-alkane interactions in lipid bilayers

The relationship between age-related alterations in the lipid composition of cultured rat-heart fibroblasts and several biochemical and biophysical parameters was investigated. Aged (14-15-day-old) cultures displayed higher mole ratios of sphingomyelin to phosphatidylcholine, as well as elevated cholesterol levels. A concomitant increase was observed in the total protein content of the cells and in the Vmax values of both membranal and cytoplasmic marker enzymes. Fluorescence photobleaching recovery was employed to study the lateral mobility of the lipid probe NBD-phosphatidylethanolamine and of membrane glycoproteins that bind succinylated concanavalin A. The mobile fractions of both probes were higher in aged cultures, while the lateral diffusion coefficients were lower. To further demonstrate the dependence of the above parameters on the cellular lipid composition, we have manipulated the lipid composition of old cultures by treatments with liposomes (small unilamellar vesicles) of specific compositions. Treatments which reversed the lipid composition towards that of young (5-6-day-old) cultures caused a concomitant reversal of the measured biochemical and biophysical parameters to the values observed in young cultures. These findings suggest that alterations in the organization and mobility of cell membrane constituents are involved in mediating changes in cellular functions. In view of our previous findings on cultures of rat-heart myocytes (Yechiel, E., Barenholz, Y. and Henis, Y.I. (1985) J. Biol. Chem. 260, 9132-9136), it appears that the modulation of cellular properties through the membrane lipid composition may be a general phenomenon in many cell types.     

Aging of rat heart fibroblasts: relationship between lipid composition,membrane organization and biological properties

The relationship between age-related alterations in the lipid composition of cultured rat-heart fibroblasts and several biochemical and biophysical parameters was investigated. Aged (14–15-day-old) cultures displayed higher mole ratios of sphingomyelin to phosphatidylcholine, as well as elevated cholesterol levels. A concomitant increase was observed in the total protein content of the cells and in the V_max values of both membranal and cytoplasmic marker enzymes. Fluorescence photobleaching recovery was employed to study the lateral mobility of the lipid probe NBD-phosphatidylethanolamine and of membrane glycoproteins that bind succinylated concanavalin A. The mobile fractions of both probes were higher in aged cultures, while the lateral diffusion coefficients were lower. To further demonstrate the dependence of the above parameters on the cellular lipid composition, we have manipulated the lipid composition of old cultures by treatments with liposomes (small unilamellar vesicles) of specific compositions. Treatments which reversed the lipid composition towards that of young (5–6-day-old) cultures caused a concomitant reversal of the measured biochemical and biophysical parameters to the values observed in young cultures. These findings suggest that alterations in the organization and mobility of cell membrane constituents are involved in mediating changes in cellular functions. In view of our previous findings on cultures of rat-heart myocytes (Yechiel, E., Barenholz, Y. and Henis, Y.I. (1985) J. Biol. Chem. 260, 9132–9136), it appears that the modulation of cellular properties through the membrane lipid composition may be a general phenomenon in many cell types.     

Metabolic profiling reveals that time related physiological changes in mammalian cell perfusion cultures are bioreactor scale independent

Metabolic profiling was used to characterize the time course of cell physiology both in laboratory- and manufacturing-scale mammalian cell perfusion cultures. Two independent experiments were performed involving three vials from the same BHK cell bank, used to inoculate three laboratory-scale bioreactors, from which four manufacturing-scale cultures were initiated. It was shown that metabolomic analysis can indeed enhance the prime variable dataset for the monitoring of perfusion cultures by providing a higher resolution view of the metabolic state. Metabolic profiles could capture physiological state shifts over the course of the perfusion cultures and indicated a metabolic “signature” of the phase transitions, which was not observable from prime variable data. Specifically, the vast majority of metabolites had lower concentrations in the middle compared to the other two phases. Notably, metabolomics provided orthogonal (to prime variables) evidence that all cultures followed this same metabolic state shift with cell age, independently of bioreactor scale.