Monoxenic cultivation of the rotifer Brachionus calyciflorus in a defined medium

Summary Techniques are described for the initiation and maintenance of axenic cultures of Euglena gracilis strain Z and monoxenic cultures of Brachionus calyciflorus variety pala with the Euglena, using in both the same defined, buffered medium. The medium, which is inorganic—except for the citrate chelating agent, the buffer, and vitamins B₁ and B₁₂ — has been used for the axenic cultuvation of the Euglena for more than 13 months. The monoxenic Brachionus cultures, established by inoculating rotifers into Euglena cultures, have been maintained for more than 8 months. Contamination tests on the rotifer cultures were performed frequently in three different test media.Mictic females, males, and resting eggs of Brachionus were observed in the monoxenic cultures, and considerable variation in the length of the posterolateral spines was noted.The compatibility of a rotifer to a defined medium which sustains the axenic culture of its food organism is a feature of this system which is convenient, useful, and unique to date in synxenic rotifer culture work.Supported by National Science Foundation Grant No. GB 7717.     

Comparative growth of Porphyromonas gingivalis strains in a defined basal medium

Porphyromonas gingivalis is an asaccharolytic bacterium whose metabolism is dependent on the uptake of small peptides and amino acids. The aim of this work was to study the growth of P. gingivalis in a defined basal medium (DBM) supplemented with various sources of proteins. The strain 49417 as well as other virulent isolates could grow in DBM containing 1% bovine serum albumin (BSA). Cells cultivated under this condition showed a slightly modified protein profile, and expressed hemagglutinating as well as proteolytic activities. Other natural proteins under investigation could not support the growth in the DBM. On the other hand, the strain 33277 as well as other avirulent strains of P. gingivalis could not use BSA as a substrate. The ability of P. gingivalis to grow in DBM-BSA is not entirely dependent on its ability to degrade the protein substrate as strain 33277 was able to extensively hydrolyse the molecule. Differences in either metabolic enzymes or peptide transport mechanisms may explain the distinctive behavior between virulent and avirulent strains. Data from this work suggest a relationship between nutritional requirements and virulence of P. gingivalis in an animal model. The DBM-BSA may represent a more appropriate medium for studies on the physiology of P. gingivalis.     

Methanogenesis in rumen ciliate cultures ofEntodinium caudatum andEpidinium ecaudatum after long-term cultivation in a chemically defined medium

The methanogenic activity in the presence of Entodinium caudatum and Epidinium ecaudatum was well preserved after long-term cultivation. Microscopic observation revealed that methane production in the presence of E. caudatum was probably caused by their intracellular methanogenic activity, while methane production in the presence of E. ecaudatum f caudatum et ecaudatum could be attributed to both the methanogenic bacterial fraction of their external surface and their intracellular activity. Methane production per protozoan cell of E. caudatum and E. ecaudatum was 2.1 nmol per cell per d and 6.0 nmol per cell per d, respectively. E. caudatum was responsible for almost the entire methane production in the culture. The activity of free methanogens constituted approximately 50% of the total methane production in the E. ecaudatum culture. Decrease of digestibility of substrates and differences in the fermentation end products accompanied the inhibition of methanogenesis in both cultures by penicillin G, streptomycin, chloramphenicol, 2-bromoethanesulfonate, and pyromellitic diimide. E. caudatum appeared to be more sensitive than E. ecaudatum to the compounds tested. Hydrogen recoveries based on both volatile fatty acids and methane production suggested that the methanogenic population appeared not to be fully able to consume hydrogen produced in the protozoan cultures. The culture conditions tested were found to be suitable for experiments on the relationship between rumen ciliates and rumen bacteria.     

Leishmania donovani: a chemically defined medium suitable for cultivation and cloning of promastigotes and transformation of amastigotes to to promastigotes

A chemically defined medium using commercially available alpha-MEM supplemented with hemin, HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 x 10(7)/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients' bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16-20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.     

Studies on the cultivation of mammalian cell lines in a serum-free,chemically defined medium

Summary An improved chemically defined, serum-free medium for the cultivation of a variety of continuous cell lines has been developed. Eight lines of human origin, three lines of nonhuman primate origin, and five lines of rodent origin have been cultured serially for as long as a year in the medium. Growth rates of several serial lines resulted in as much as 20- to 30-fold increases per week. Hormones such as insulin, cortisol, and thyroxine significantly improved growth of cultures in the defined medium. Vitamin B₁₂ and biotin were required for growth. Lipids such as oleic acid, lecithin, and cholesterol also promoted growth of several cell lines. Virtually all continuous cell lines tested grew well upon initial transfer into the serum-free defined medium. Most cell lines could be serially subcultured rapidly with little evidence that selection of rare cell types was necessary for growth in the defined medium. However, a few cell lines such as the BHK-21 (hamster) cell and the AKR (mouse embryo) cell required prolonged periods (4 to 8 weeks) of culturing before rapid growth occurred. Primary cell cultures and other diploid cells such as human fibroblast (strain WI-38) could not be subcultured successfully in the present medium.     

The growth of Paracoccus halodenitrificans in a defined medium

A synthetic medium, consisting of inorganic salts and any of a number of carbon sources, supported the aerobic growth of Paracoccus halodenitrificans when supplemented with thiamine. The same medium plus an appropriate nitrogenous oxide supported anaerobic growth when additionally supplemented with methionine. The observation that vitamin B12 or betaine replaced methionine suggested that P. halodenitrificans had a defect in the cobalamin-dependent pathway for methionine biosynthesis, as well as the inability to synthesize betaine when growing anaerobically.     

Innovative serum-free medium for in vitro cultivation of promastigote forms of Leishmania species

We described a comparatively simple medium formula (CML) using common, available and reasonably priced ingredients that could be used in place of medium that requires calf serum enhancement for cultivation of Leishmania promastigote forms. This medium equivalently supported the growth of parasites at rates comparable with those obtained with serum supplemented RPMI-1640 medium. Leishmania promastigotes reproduced in CML exhibited moderate to high infectivity capacities when tested against J774 macrophage cell line. No significant difference was noted between Leishmania strains cultivated in the newly modified medium and those grown in RPMI-1640 medium in their cells infectivity and replication potentials. The use of new CML can easily take the place of other biphasic or liquid media because of its easy preparation and instantaneous use, reasonable price, availability of ingredients, and its long shelf life, which is 30–45 days. The fact that this medium is similar to other culture media as far as durability and quantity of produced parasites might give it an advantage over the other currently used media.     

Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1). MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.     

Coenzyme Q production by metabolic engineered Escherichia coli strains in defined medium

Bioprocess and Biosystems Engineering - Coenzyme Q (CoQ) plays an important role as an electron transporter in the respiratory chain. It is formed from a benzoquinone ring and an isoprenoid chain...     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium.

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

Protozoan hemoflagellates Leishmania are causative agents of leishmaniases and an important biological model for study of host-pathogen interaction. A wide range of methods of Leishmania cultivation on both biphasic and liquid media is available. Biphasic media are considered to be superior for initial isolation of the parasites and obtaining high promastigote infectivity; however, liquid media are more suitable for large-scale experiments. The aim of the present study was the adaptation and optimization of the cultivation of Leishmania promastigotes on a biphasic SNB-9 (saline-neopeptone-blood 9) medium that was originally developed for Trypanosoma cultivation and combines the advantages of biphasic and liquid media. SNB-9 medium is characterized with a large volume of the liquid phase, which facilitates the manipulation with the culture and provides parasite yields comparable to parasite yields on such liquid medium as Schneider's Insect Medium. We demonstrate that SNB-9 very considerably surpasses Schneider's Insect Medium in in vitro infectivity of the parasites. Additionally, we show that the ratio of apoptotic parasites, which are important for the infectivity of the inoculum, in Leishmania culture in SNB-9 is higher than in Leishmania culture in Schneider's Insect Medium. Thus, we demonstrate that the cultivation of Leishmania on SNB-9 reliably yields highly infective promastigotes suitable for experimental infection.     

Selection of a chemically defined medium for submerged cultivation of Streptomyces aureofaciens with high extracellular caseinolytic activity

A chemically defined medium was developed for the submerged cultivation of Streptomyces aureofaciens with a high secretion of caseinolytic activity. The medium composition is: 40 g/liter maltose; 1.640 g/liter L-leucine (0.0125M); 1.765 g/liter L-lysine (0.0125M); 6.976 g/liter K2HPO4 (0.04M); 4 g/liter CaCO3; 0.2 g/liter MgSO4.7H2O; 0.01 g/liter ZnSO4.7H2O; 0.01 g/liter FeSO4.7H2O: 0.01 g/liter MnSO4H2O, and 0.005 g/liter CoSO4.7H2O. Quantitative correlations were established between the concentrations of nutrients in the medium and the secretion of proteolytic activity. In this medium the secretion of proteolytic activity parallels growth, reaching a maximum after 70 hr at 30 degrees C in shaker cultures. The secretion appears to be an active process and to require aerobic conditions.