Development of a defined minimal medium for the growth of Neisseria gonorrhoeae

This investigation describes the development of a solid and a liquid medium (Gonococcal Genetic Medium; GGM) which support the rapid growth of 41 gonococcal clinical isolates and laboratory strains with a minimum number of nutritional components. The complete medium contains minimal salts, eight amino acids, two nitrogen bases, vitamins, coenzymes, key metabolic intermediates, and some miscellaneous components. Results indicate that GGM can be modified and simplified even further than we described. In liquid GGM, several gonococcal strains grew logarithmically after a 2- to 3-h lag period with generation times ranging from 72 to 115 min, reaching optical densities of 175 to 320 Klett units in the presence of seven amino acids and in the absence of a CO(2) atmosphere. The development of a solid and a liquid defined minimal medium such as GGM should greatly broaden the avenues of experimentation for biochemical genetic studies with N. gonorrhoeae, especially gonococcal genetic transformation. N. gonorrhoeae can be classified into eight major and minor phenotypic groups, depending on its growth responses on GGM to just five amino acids: cysteine and cystine, arginine, proline, isoleucine, and serine. Such results demonstrate the feasibility of using GGM as a simple, sensitive, rapid probe for investigating the epidemiological patterns of gonorrhea.     

Effect of culture medium composition on inhibition of growth ofNeisseria gonorrhoeae by lactobacilli

The role of genital microorganisms in resistance to gonococcal infection is usually based on their in vitro inhibition of gonococcal growth. Three different culture media (GC, DSA, and MRS) were evaluated for their ability to support the growth of 23 lactobacilli strains and the detection of the antigonococcal activity of these bacteria. The MRS medium was the most suitable medium for the growth of lactobacilli since it favored a good growth of all the lactobacilli strains tested, but it was inhibitory toNeisseria gonorrhoeae. Decreasing the concentration of Tween 80, ammonium citrate, and sodium acetate to one-tenth of their original concentrations yielded a modified MRS medium which still supported good growth of the lactobacilli and was no longer inhibitory to the gonococci. While GC medium did not allow any detection of the production of antigonococcal activity by the lactobacilli, both modified MRS and DSA media allowed the detection of this activity by the agar overlay technique. The use of modified MRS medium is recommended since it is less selective than DSA medium for the growth of lactobacilli.     

A chemically defined fermentation medium for the growth of Micromonospora purpurea

A chemically defined medium for Micromonospora purpurea has been devised, consisting of glucose, a nitrogen source, calcium carbonate, magnesium sulfate, dibasic potassium phosphate, and the required trace quantities of iron, copper, zinc, and manganese. Using washed cell inocula, dry mycelial weights of more than 16 mg./ml. were obtained in 7-day shaken-flask fermentations. Nutrient requirements for M. purpurea are discussed and growth data presented. Sucrose, maltose, starches and dextrins could be substituted for glucose, and resulted in good growth of the organism. A number of amino acids and inorganic nitrogen-containing compounds were capable of utilization as sole nitrogen sources. Weekly serial transfers of the culture in defined medium have shown no diminution in mycelial weights over a four-month period.     

An improved heat-stable glutamine-free chemically defined medium for growth of mammalian cells

A heat-stable chemically defined medium, free of glutamine, is described for the growth of mammalian cells in suspension culture. The presence of L-alanine in the defined medium permitted the omission of glutamine. A 22-fold increase in the population of a substrain of mouse L cells was obtained (3.4 × 10⁶ cells/ml) in six days with no medium replenishment during incubation. Maximum yields (27 × 10⁶ cells/ml) were obtained by daily medium replacement and venting of cultures. Growth was also improved in a line of cat kidney cells and HeLa cells, and in another substrain of L cells.     

Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined growth medium

Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH4Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids. A. succinogenes growth trends and end product distributions in AM3 and rich medium fermentations were compared. The effects of NaHCO3 concentration in AM3 on end product distribution, growth rate, and metabolic rates were also examined. The A. succinogenes growth rate was 1.3 to 1.4 times higher at an NaHCO3 concentration of 25 mM than at any other NaHCO3 concentration, likely because both energy-producing metabolic branches (i.e., the succinate-producing branch and the formate-, acetate-, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the A. succinogenes metabolic map, the reasons for A. succinogenes glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or alpha-ketoglutarate. The inability to synthesize alpha-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes.     

Development of competence for genetic transformation of Streptococcus mutans in a chemically defined medium

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS(11-17)) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS(+) (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS(11-17).     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

Development of an improved chemically defined minimal medium for Listeria monocytogenes.

A chemically defined minimal medium for Listeria monocytogenes has been developed by modification of Welshimer's medium. The growth factors required by L. monocytogenes Scott A are leucine, isoleucine, arginine, methionine, valine, cysteine (each at 100 mg/liter), riboflavin and biotin (each at 0.5 micrograms/ml), thiamine (1.0 micrograms/ml), and thioctic acid (0.005 micrograms/ml). Growth was stimulated by 20 micrograms of Fe3+ per ml as ferric citrate. Glucose (1%) and glutamine (600 mg/liter) are required as primary sources of carbon and nitrogen. Glucose could not be replaced by various organic acids or amino acids. Of several sugars tested, fructose, mannose, cellobiose, trehalose, maltose (weak), glycerol (weak), and the amino sugars glucosamine, N-acetylglucosamine, and N-acetylmuramic acid supported growth in the absence of glucose. Evidence was found that chitin and cell walls of starter bacteria (Lactococcus lactis) supported survival of L. monocytogenes, which suggests that the pathogen may obtain carbon and energy sources during colonization of some foods, such as cheeses, by assimilating bacteria or molds that are present.     

Development of a chemically defined diet for ants

A chemically defined diet is a useful tool for the study of nutritional physiology of organisms. We have developed such a diet for Camponotus carpenter ants to facilitate experiments on nutritional requirements of these ants. Worker colonies of Camponotus floridanus were fed with a chemically defined diet, containing all essential minerals, amino acids, vitamins, growth factors and sucrose in an agar matrix. After 13 weeks, neither the number of raised pupae, their dry weight, nor the mortality of workers in subcolonies fed with this diet differed significantly from control colonies fed with Bhatkar-Whitcomb-agar, in addition to cockroaches and diluted honey. Therefore, this diet is adequate for a normal brood production and a maximal growth rate of C. floridanus larvae, at least for a period of three months. This diet should be suitable for ants that are able to feed on agar-based food resources in general. Received 6 September 2006; revised 5 October 2006; accepted 18 October 2006. An erratum to this article is available at .     

Development of a new chemically defined sporulation medium for the yeasts in the genus Lipomyces

A chemically defined sporulation medium (AF medium) for the yeasts belonging to the genus Lipomyces was developed. The chemical composition was derived from chemical analyses of soybean extract. Some chemical modification of the AF medium indicated that the nitrogen sources (aspartic and glutamic acids) and zinc ion were essential for sporulation. The significance of medium pH was discussed.     

Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium

Willett, Norman P. (University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pa.), and Guy E. Morse. Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium. J. Bacteriol. 91:2245-2250. 1966.-A chemically defined medium was developed for Streptococcus agalactiae which supported growth comparable to that obtained in complex medium. The effects of long-chain fatty acids on growth of the organisms were determined turbidimetrically. The order of activity of the fatty acids was dependent upon whether complete inhibition or median response (50% inhibition point) was used as a parameter of activity. When complete inhibition of growth was used as a measure, the degree of unsaturation of C(18) acids enhanced antimicrobial activity. However, when the median response was used as an index, this order was reversed. Increase in carbon chain from C(12) to C(18) did not correlate with either complete inhibition or median response points. Antimicrobial activity of unsaturated and saturated fatty acids was reversed by bovine serum albumin and other compounds, suggesting a bacteriostatic action.