Comparison of properties of collected cells and cells from the culture vessel during continuous culture of Streptococcus mutans Ingbritt

In continuous-culture studies chemostat effluents are usually collected into a receiving flask in an ice bath to obtain enough cells for an experiment. It is assumed that the properties of these are not significantly different from those of the culture in the chemostat vessel. This assumption has been tested for the dental pathogen Streptococcus mutans Ingbritt. Collected supernatant fluid and cells were compared with supernatant fluid and cells taken directly from the culture vessel, for four major groups of culture properties: viability and biomcss, concentrations of metabolites and nutrients, activities of selected enzymes, and glycolytic rates. The assumption held true except for glycolytic rate during endogenous metabolism. It is suggested that comparison of collected and culture vessel cells is an important control which should be done in all continuous culture studies of microbial physiology and biochemistry, but that the properties of Strep, mutans cells collected on ice up to 16 h do reflect those of cells actively growing in the chemostat.     

Characteristics of the chemostat culture of murine leukemia L 1210 cells

Mouse leukemia L 1210 cells were cultivated under glucose limitation in a chemostat. More than 20 steady-states were established over 9 different dilution rates ranging from 0.20 day⁻¹ (cell doubling time 83 h) to 2.0 days⁻¹ (cell doubling time 8.3 h). The steady-states were characterized by: a constant cell number, constant cell volume, constant concentrations of DNA, RNA, and L-lactate (in the culture supernatant), a constant percentage of cells labelled by autoradiography, and constant rate of incorporation of [³H]TdR, [³H]uridine, and ¹⁴C-labelled amino acids into cellular acid-precipitable material. Individual steady-states were maintained for periods up to 600 h continuous operation of the chemostat. A maximum output of 66.4 × 10⁶ cells/h was obtained at a dilution rate of 1.3 day⁻¹. The glucose substrate constant was determined as 0.0063 mg/ml. The relationships between dilution rate and the steady-state cell concentration, glucose concentration, and output of L 1210 cells from the chemostat, were in general agreement with the theoretical curves. It was found that the principles of continuous culture derived from the study of microorganisms are to a large extent applicable to the cultivation of animal cells.     

Properties and performance of microorganisms in chemostat culture

In the design of new processes for large-scale production of microbial cells and their products, fermentation technologists may well be led to consider the utility of continuous-flow culture systems, with their inherent high productivity potential. However, the properties and performance of microorganisms in chemostat culture are markedly different from those in batch culture and this must be taken into account. This review illustrates, with selected examples, the range of properties that are expressed differently (sometimes uniquely) in chemostat culture and points to the possible ways in which these chemostatinduced changes in microbial behaviour may be exploited.     

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.     

Release into culture medium of membrane-associated, tumor-specific antigen by B-16 melanoma cells.

Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57Bl/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57Bl/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.     

The effect of lowering the pH on the composition and metabolism of a community of nine oral bacteria grown in a chemostat

Nine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0.05 h-1 (mean generation time 13.9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7.0, pH control was discontinued. The pH then decreased until it stabilized at pH 4.1 after 9 d (16 generations), while the Eh rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7.0, 'Streptococcus mitior', Veillonella alcalescens and S. sanguis were most numerous while at pH 4.1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4.6. The survival at pH 4.1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7.0, the community growing at pH 4.1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.     

The biochemical composition and calorific content of a rotifer and its algal food: comparison of a two stage chemostat and batch culture

It is often assumed that the use of a two-stage chemostat yields algal food with a well-defined nutritional composition that can maintain herbivores in a steady state of growth. In this study I investigated two bacteriafree culture techniques, continuous flow chemostats and batch cultures, to determine whether the biochemical composition of the rotifer Encentrum linnhei differed in the two cultures. Changes in the biochemical composition and calorific content of the algal food were also examined. In the rotifer reaction vessel only the lipid content of the algal food increased significantly with dilution rates, while significant decreases in protein and carbohydrates were detected at increasing algal densities. A different pattern was observed in the response of the unused algal cells to variables such as dilution, algal input and algal densities in the sump of the rotifer chemostat. In the chemostat the biochemical composition of the rotifers varied as expected with dilution rates, algal input and food availability but significant differences were found in the biochemical composition of the animals growing in the reaction vessel and those collected from the sump. In contrast, the biochemical content of batch-grown E. linnhei varied with time in a way that depended upon food availability and also on the biochemical state of the algal food. However, at the end of the exponential phase of growth, when maximum densities had been achieved, batch-grown rotifers were more biochemically nutritious than chemostat-grown animals in their steady-state phase.     

The cyclone column culture vessel for batch and continuous,synchronous or asynchronous,culture of microorganisms

A simple inexpensive apparatus with a working volume of 10 liters of culture is described. Details of construction and procedures for operation of the cyclone column vessel are given. The vessel is self-contained, so that experimental parameters of temperature and aeration are individually controlled; homogeneous mixing and representative sampling of the culture, besides control of foam without need for antifoam, are obtained. The vessel may be used in single or multistage systems for aerobic or anaerobic cultivation of organisms in batch, chemostat, or phased cultures.     

High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10⁷ cells/ml, when the specific perfusion rate was set to 2.3 vol day^-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.     

An automatic apparatus for continuous suspension culture with a concentrating device

A chemostat in which mammalian cells can be raised in continuous suspension culture is described. It is constructed from commercially available parts. This apparatus has the advantage over earlier models in that the medium can be pumped off free of cells, thus suddenly increasing the cell concentration in the culture. The apparatus has been successfully used in studies on contact inhibition.     

Adaptive acid tolerance response of Streptococcus sobrinus

Streptococcus mutans and Streptococcus sobrinus are the bacteria most commonly associated with human dental caries. A major virulence attribute of these and other cariogenic bacteria is acid tolerance. The acid tolerance mechanisms of S. mutans have begun to be investigated in detail, including the adaptive acid tolerance response (ATR), but this is not the case for S. sobrinus. An analysis of the ATR of two S. sobrinus strains was conducted with cells grown to steady state in continuous chemostat cultures. Compared with cells grown at neutral pH, S. sobrinus cells grown at pH 5.0 showed an increased resistance to acid killing and were able to drive down the pH through glycolysis to lower values. Unlike what is found for S. mutans, the enhanced acid tolerance and glycolytic capacities of acid-adapted S. sobrinus were not due to increased F-ATPase activities. Interestingly though, S. sobrinus cells grown at pH 5.0 had twofold more glucose phosphoenolpyruvate:sugar phosphotransferase system (PTS) activity than cells grown at pH 7.0. In contrast, glucose PTS activity was actually higher in S. mutans grown at pH 7.0 than in cells grown at pH 5.0. Silver staining of two-dimensional gels of whole-cell lysates of S. sobrinus 6715 revealed that at least 9 proteins were up-regulated and 22 proteins were down-regulated in pH 5.0-grown cells compared with cells grown at pH 7.0. Our results demonstrate that S. sobrinus is capable of mounting an ATR but that there are critical differences between the mechanisms of acid adaptation used by S. sobrinus and S. mutans.     

Estimation of steady-state culture characteristics during acceleration-stats with yeasts

Steady-state culture characteristics are usually determined in chemostat cultivations, which are very time-consuming. In contrast, acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed with a constant acceleration rate are not so time-consuming, especially at high acceleration rates. Therefore, the A-stat could be advantageous to use instead of the chemostat. However, the highest acceleration rate, meaning the fastest A-stat that can be applied for estimating steady-state culture characteristics, is not known yet. Experimental results obtained with Zygosaccharomyces rouxii, an important yeast in soy sauce processes, showed that the culture characteristics during the A-stat with an acceleration rate of 0.001 h(-2) were roughly comparable to those of the chemostat. For higher acceleration rates the deviation between the culture characteristics in the A-stat and those in the chemostat obtained at the same dilution rate generally started to increase. The source of these deviations was examined by simulation for Saccharomyces cerevisiae. The simulations demonstrated that this deviation was not only dependent on the metabolic adaptation rate of the yeast, but also on the rate of change in environmental substrate concentrations during A-stats. From this work, it was concluded that an A-stat with an acceleration rate of 0.001 h(-2) is attractive to be used instead of chemostat whenever a rough estimation of steady-state culture characteristics is acceptable.     

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides.

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.     

Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

Investigation of prostaglandins into the culture supernatant of chronic lymphocytic leukaemia (CLL) cells

Prostaglandins have been proposed as intercellular humoral mediators in the immune response and characterised as regulatory agents in the control of intracellular metabolism. The aim of this work was to determine PGE and PGF2 alpha concentrations in the blood plasma and in the supernatant of 96 hour PHA stimulated and unstimulated leukaemic cell cultures of CLL patients. 62 patients with CLL classified in the 1st or 4th stage according to RAI and 23 healthy individuals were investigated. The proliferation degree of the culture cells was tested by incorporating tritiated thymidine. The prostaglandin concentrations was estimated by the isotopic method using RIA-kit. In the 4th stage of CLL a low value of blastogenic transformation was observed, whereas in the 1st stage the value were similar to those of the control group. It was shown that in the 4th stage of the disease an increase in the PGE concentrations occurs in the blood plasma and the culture supernatant without PHA together with a significant decrease in the PGF2 alpha in the culture supernatant, whereas in the 1st stage a significant decrease in the PGE in the culture supernatant with PHA as compared with those of the control group is noted. These results may indicate on antagonistic action of PGE and PGF2 alpha in leukaemic cell proliferation.     

Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA

Abstract Streptococcus mutans GS-5 synthesizes an exo-β-d-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans . These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria. The control of degradation of plaque fructans by modulation of the release of the fructanase enzyme from S. mutans may play a critical role in the temporal and spatial separation of the synthesis and degradation of dental plaque fructans.     

Presence of Trypanosoma cruzi antigen on the surface of both infected and uninfected cells in tissue culture

LLC-MK2 cell monolayers infected with Trypanosoma cruzi were shown by immunofluorescence to present parasite antigens on the surface of both parasitized and non-parasitized cells after completion of the first intracellular cycle and rupture of infected cells. The cell-culture supernatant fluid at this stage, as well as the supernatant fluid of parasites left overnight in culture medium were concentrated and contained antigen capable of binding to uninfected cell monolayers. The origin of this antigen, as well as its eventual role in the pathogenesis of Chagas' disease, are discussed.     

Cell wall metabolism in Bacillus subtilis subsp. niger: accumulation of wall polymers in the supernatant of chemostat cultures

Cell wall polymers were measured both in the cells and in the cell-free medium of samples from steady-state chemostat cultures of Bacillus subtilis, growing at various rates under magnesium or phosphate limitation. The presence of both peptidoglycan and anionic wall polymers in the culture supernatant showed the occurrence of wall turnover in these cultures. Variable proportions of the total peptidoglycan present in the culture samples were found outside the cells in duplicate cultures, indicating that the rate of peptidoglycan turnover is variable in B. subtilis. Besides peptidoglycan, anionic wall polymers were detected in the culture supernatant: teichoic acid in magnesium-limited cultures and teichuronic acid in phosphate-limited cultures. In several samples, the ratio between the peptidoglycan and the anionic polymer concentrations was significantly lower in the extracellular fluid than in the walls. This divergency was attributed to the occurrence of direct secretion of anionic polymers after their synthesis.     

Protein expression by planktonic and biofilm cells of Streptococcus mutans

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.