Maximum Cell Population Research Articles

Liquid medium for growth of Legionella pneumophila.

The medium described is a simple yeast extract broth capable of growing large number of Legionella neumophila, the causative organism of Legionnaires disease. Filtration was chosen as a means of sterilization, since medium that was autoclaved did not support growth without the presence of Norite A. The filtered medium gave rapid cell growth and maintained the initial antigen production. The observed generation time was 99 min with a maximum cell population of 2 X 10(2) COLONY-FORMING UNITS PER ML IN APPROXIMATELY 40 H.

A regenerating computer model of the thymus

This computer model simulates the cell population kinetics of the development and later degeneration of the thymus. Nutritional factors are taken into account by the growth of blood vessels in the simulated thymus. The stem cell population is kept at its maximum by allowing some stem cells to divide into two stem cells until the population reaches its maximum, thus regenerating the thymus after an insult such as irradiation. After a given number of population doublings the maximum allowed stem cell population is gradually decreased in order to simulate the degeneration of the thymus. Results show that the simulated thymus develops and degenerates in a pattern similar to that of the natural thymus. This simulation is used to evaluate cellular kinetic data for the thymus. The results from testing the internal consistency of available data are reported. The number of generations which most represents the natural thymus includes seven dividing generations of lymphocytes and one mature, nondividing generation of small lymphocytes. The size of the resulting developed thymus can be controlled without affecting other variables by changing the maximum stem cell population allowed. In addition, recovery from irradiation is simulated.

The quantitative requirements of human diploid cells (strain MRC-5) for amino acids, vitamins and serum.

The uptakes of all essential amino acids, vitamins (except riboflavin), glucose and serum during growth of human diploid cells (MRC-5) were determined. The amino acid uptakes varied considerably with the conditions of culture. The glucose requirement is several times greater than that for mouse LS or human HeLa cells. These analytical results were used to modify the medium so as to ensure that an excess of all defined medium constituents was present and pH was not limiting during study of the serum requirements. It was then found that maximum cell populations were directly proportional to the serum concentration. Hence the growth was limited by the supply of an unknown growth factor in serum. The serum growth factor was not replaced by a mixture of over 60 vitamins, co-enzymes, hormones and other organic and inorganic compounds considered to be possible growth factors, although this mixture did not lower the growth rate and somewhat (22%) increased the yield from the serum growth factor. The unit of serum growth factor is precisely defined in terms of the amount in a standard batch of calf serum. This standard contains 10 units/ml whereas the other batch of serum used contained only 5 units/ml.

The effect of naturally occurring organic substances on the growth of a red tide organism

The effect of soil extracts on the growth of the Florida red tide organism. Gymnodiniam brere. was investigated to determine the potential effect of organic matter in rivers on red tide outbreaks. Soil from a peat bog was extracted with water, filtered through a membrane filter (0.22 μm), then fractionated into four molecular-weight fractions (0–500; 500–1000; 1000–10,000; and 10,000) by means of Amicon Diaflo ultramembranes. Fractions were analyzed for organic carbon, iron and manganese. The effect of different amounts of the last three fractions on the rate of growth and maximum cell population of G. breve cultures was determined. No substantial change in the growth constant ( K e ) with change of concentration of added fraction was noted, nor was there a marked variation from one fraction to another. The maximum cell population increased with an increase in the amount of added organic extract; and using the second and third fractions, the increase was related to the amount of iron present (linear correlation coefficient, r = 0.994, P = 0.001); the last molecular-weight fraction showed a similar relationship, though the effectiveness in increasing N max was less (on the basis of amount or iron added). The results were compared with the effect of adding gibberellic acid, which is a naturally occurring hormone that reportedly reduces the time for maximum cell populations to be attained.

The response of Gymnodinium breve to municipal waste materials

The response of the unarmored dinoflagellate Gymnodinium breve, which is the causative organism in catastrophic fish kills along the Florida Gulf coast, to enrichment with selected inorganic nutrients, municipal waste materials, and various detergent components has been determined. The biostimulatory effects of the various enrichments were determined by a modification of the Provisional Algal Assay Procedure of the Joint Industry/Government Task Force on Eutrophication. Inorganic nutrients (orthophosphate, nitrate, and ammonia) were added individually and in combination, and the results were compared to equivalent enrichments with the effluent from a secondary sewage-treatment plant. The maximum cell population, Nmax, attained could be increased 3-fold by the sew-age-treatment plant effluent or by the equivalent combination of inorganic nutrients; individually, however, the inorganic nutrients had no pronounced effect on maximum cell population, Nmax (except for a 30% increase produced by slight orthophosphate enrichment). The results of these studies indiccate that, at concentrations of orthophosphate typical of Florida coastal waters (ca. 0.10 ppm), the growth-promoting potential (as reflected by Nmax) of the medium was a linear function of the ammonia-nitrogen concentration (0.01 to 0.11 ppm). The sewage-treatment plant effluent was presumably low in detergent phosphate, having been obtained from a treatment plant some 6 months after the enactment of a ban on phosphate-containing detergents. Additions of orthophosphate or detergent-phosphate to the treatment-plant effluent did not significantly increase the observed biostimulatory effect of the waste material.

Effect of osmotic pressure, Na+-K+ ratio and medium concentration on the enzyme activity and growth of L cells in suspension culture.

The effect of changes in osmotic pressure and in the Na+/K+ ratio on the doubling time, maximum cell population, enzyme activity, and isoenzyme distribution pattern in suspension cultures of L cells was determined. The growth of viable cells is relatively flat over a rather wide range of osmotic pressures (220 to 440 mOsm per kg). The presence of extra salt or sucrose beyond that needed to reach the minimum osmotic pressure (220) is detrimental to cell growth as reflected by a delay in the onset of logarithmic growth, a slower growth rate, a decreased maximum population, and accelerated death phase. Excessive K+ ion is toxic, but the level at which it is toxic interacts with osmotic pressure of the medium. Enzyme activity and isoenzyme distribution patterns for those enzymes studied did not vary as a function of osmotic pressure, ionic ratios, or medium concentration.

The glucose, insulin and glutamine requirements of suspension cultures of HeLa cells in a defined culture medium.

ABSTRACT The serum supplement in a defined medium for the growth of HeLa cells could be replaced by protamine-zinc-insulin (0·2 u./ml). Insulin (0·4 u./ml) replaced the growth-stimulatory properties of protamine-zinc-insulin, whilst protamine sulphate (5 μg/ml) was found to be toxic to the cells. The addition of insulin to cultures depleted of insulin increased both cell growth rates and maximum cell populations. In the defined medium, HeLa cells could only utilize glutamate when a small amount of glutamine was included. Glucose, at a level of 2 mg/ml, was shown to limit maximum cell populations. The growth yield from glucose was 295 μg cell dry weight/mg glucose. When the medium glucose concentration was increased to 4 mg/ml, HeLa cell populations in excess of 16×105 cells (i.e. 640 μg dry weight)/ml were routinely achieved in the defined medium supplemented with insulin. Growth is then limited by the amino acid supply. Increasing the amino acid concentration of the medium by 50% raised the maximum cell population to 23·5 × 105 cells (i.e. 940 μg dry weight)/ml.

Improvements in a chemically defined medium for the growth of mouse cells (strain LS) in suspension.

ABSTRACT In the medium used in previous work, both growth rate and maximum cell population density were reduced in protein-free medium. This was due to iron limitation. In a medium supplemented with iron, the average minimum population doubling time was reduced from 78 to 37 h and the average maximum cell population density increased from 1·6×106 to 3·3 × 106/ml. In the improved medium described, sodium pyruvate, α-ketoglutaric acid, methylcellulose and polyvinylpyrrolidone are no longer required although methylcellulose prevents an initial fall in population which occurs in polymer-free medium. The present study indicates that amino acids now limit maximum cell population density. By increasing the concentration of the relevant amino acids we have obtained maximum cell populations in excess of 5 × 106 cells/ml without medium change. Finally we describe a method for measuring the growth of the LS cell in the denned medium by an opacity method.

The choline and serum protein requirements of mouse fibroblast cells (strain LS) in culture.

ABSTRACT The maximum cell population density of mouse fibroblast (strain LS) cells growing in static suspension culture was found to be directly proportional to the dialysed calf serum concentration. This was due to choline limitation and the fact that serum protein was a major source of choline. The growth yield (Y) was 3·2 × 105 cells/μg choline chloride. Studies on the role of serum in the presence of excess choline showed the following. When protein was omitted from the medium, cell death occurred. Whole serum protein could be replaced by either (1) bovine serum albumin fraction V, or (2) crystalline bovine serum albumin + sodium pyruvate and α-ketoglutarate, or (3) polyvinylpyrrolidone + methylcellulose + pyruvate and a-ketoglutarate. The population doubling time was 24 h in the presence of whole serum protein and increased considerably with the substitutes (1—3). The increase in maximum cell population density (without medium changes) exceeded 2·9 × 106 cells/ml with either whole serum protein or substitutes (1) and (2). With serum substitute (3) the maximum increase in population density was reduced to 1·6 × 106 cells/ml.