Growth of Pathogenic Leptospira in Chemically Defined Media

A protein-free chemically defined medium for cultivation of pathogenic Leptospira was developed. The medium permitted continued serial subculturing of 9 serogroups (52 strains) of the 12 serogroups (61 strains) tested. Growth was initiated from small inocula, and the growth rate and maximal cell yields were similar to those on serum-containing media. The nutritional requirements of serogroups L. canicola, L. pomona, and L. grippotyphosa were studied in a basal medium composed of inorganic salts, a fatty acid, vitamin B(12), and thiamine. All strains tested utilized ammonium chloride as the sole nitrogen source. A fatty acid, vitamin B(12), and ferrous ions were essential. Growth was stimulated by thiamine, potassium, and calcium ions.     

Tissue Culture of Cassava on Chemically Defined Media

Defined media that promote the initiation and undifferentiated growth of callus derived from stem explants of four cultivars of cassava, Manihot esculenta Crantz, are described. Growth rates and yields of cassava callus after 4 weeks of culture are shown to be comparable to those of callus of Nicotiana tabacum L. cv. Wisconsin No. 38. Nitrogen sources of ammonium nitrate or of ammonium chloride plus succinate supported growth of all four cultivars. Sucrose was superior to glucose as a carbon source. The cassava cultivars differed in their response to increasing concentrations of sucrose between 0.5% (w/v) and 3%, two of them increasing in dry matter with increasing sucrose concentrations of up to 3%. When cultured in the light on defined media that contained higher ratios of cytokinin to auxin, callus of the latter two cultivars turned green. Roots but not shoots differentiated from the callus of all cultivars. The influence of hormone concentrations, sucrose level, and nitrogen source on greening and root formation is summarized.     

Growth of Bacillus coagulans in Chemically Defined Media

A nutritional study was made of five strains of Bacillus coagulans obtained from various culture collections. These five strains were descendants of two original isolates; three had been derived from one parent culture in years past and the other two were transfers from another parent culture. Therefore, the five cultures should have represented two distinct groups of genetically identical cultures. Three of the strains obtained from one culture collection had become methyl red-negative and sorbitol-negative and had gained abilities to hydrolyze gelatin and ferment arabinose. Nutritional requirements of the five cultures, determined at 37, 45, and 55 C, differed considerably among strains; however, thiamine and biotin were required by all cultures at all temperatures. Aspartic acid was stimulatory at 37 C and was required at 45 C; folic acid, basic amino acids, and certain other nutrilites were required at 55 C. Adenine supplementation was necessary for two strains at 55 C to prevent autolysis; this phenomenon is discussed. The response of these organisms to both serine and the basic amino acids at the three growth temperatures seems especially significant. The media devised for the growth of the five strains of B. coagulans used in this study permit excellent growth at three incubation temperatures.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

Growth of Clostridium tertium and Clostridium septicum in Chemically Defined Media

Defined media for the growth of Clostridium tertium and Clostridium septicum are described. The requirements for growth of these two species are compared with each other and with those of Clostridium perfringens.     

Growth of Suspension Cultures of Sugarcane Cells in Chemically Defined Media

A chemically defined medium is desirable for nutritional studies and is frequently necessary for biochemical investigations. Several defined media are available for use with tissue and cell cultures from dicotyledonous plants. A fully defined medium has now been developed for cell suspension cultures from sugarcane. Prior to this, the only medium successfully used for cell cultures of monocotyledonous plants was a modification of Straus' synthetic medium (used to grow cell suspensions of corn). Cell suspension cultures from sugarcane stalk parenchyma, originally established in complex media containing coconut milk or yeast extract, can be grown in this synthetic medium, which consists of inorganic salts, vitamins, sucrose, 2.4-dicliloropheuoxyacetic acid, and a mixture of 13 amino acids. The most important of the amino acids are arginine aspartic acid, and glutamic acid. This simplified medium wilt aid in the investigation of the unusual and important role of arginine in sugar-cane growth and metabolism.     

Growth of Physarum flavicomum and Physarum rigidum in Chemically Defined Minimal Media

Physarum flavicomum, P. polycephalum, and P. rigidum grew at pH 4.2 in a medium composed of mineral salts, glucose, biotin, thiamine, hematin, and four amino acids. Important differences in pH tolerance were noted among the species. The minimal medium of P. flavicomum and P. polycephalum contained the amino acids methionine, glycine, and arginine, but valine was also required by P. rigidum. Starting with an inoculum of about 0.3 mg of protein per 25 ml of minimal medium, P. flavicomum and P. polycephalum grew to 23 mg and P. rigidum to 12 mg of protein per 25 ml in 3, 2, and 4 weeks, respectively. P. flavicomum and P. polycephalum grew with valine or leucine replacing arginine in the minimal medium but the growth yields and growth rates were decreased. All three species utilized homocysteine thiolactone in the minimal media in place of methionine. Serine adequately replaced glycine for P. rigidum but was inhibitory in the minimal medium of P. flavicomum or P. polycephalum unless homocysteine thiolactone also replaced methionine. Growth rates of all three organisms were increased in the presence of seven amino acids (original four plus leucine, lysine, and isoleucine).     

小鼠和大鼠的胚胎培养基及若干相关问题

哺乳动物早期胚胎体外培养所用各种化学成份明确的培养基是研究生命科学中一项重要技术。它已被常规用于研究胚胎早期发育,多种动物及人类辅助生殖技术,转基因动物,动物克隆等领域。介绍了用于移植前胚胎培养基的研究和发展历史,当前所用化学成份明确胚胎培养基的主要组成,特别是针对小鼠和大鼠移植前胚胎所用各种培养基及其成份,讨论了这类培养基发展前景和研究方向。     

Sporulation and Heat Resistance of Bacillus stearothermophilus Spores Produced in Chemically Defined Media

The effect of amino acids on sporulation is discussed. Heat-resistant spores were produced in a chemically defined medium.     

Cultivation of Leishmania donovani and Leishmania braziliensis in Defined Media: Nutritional Requirements

SYNOPSIS Nutritional requirements of promastigotes of Leishmania donovani and Leishmania braziliensis were studied in modifications of a simple defined culture medium. "Continuous growth," considered as propagation through 10 successive passages, was supported by inorganic salts, 14 l -amino acids (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine), glucose, adenosine, and a mixture of 11 vitamins and related growth factors. Purified defatted bovine serum albumin proved beneficial. The nutritional needs of the above species of Leishmania differ from those of 2 other hemoflagellate species, Leishmania tarentolae and Crithidia fasciculata , for which glucose, proline and glutamine were found to be nonessential. It is suggested that lower hemoflagellates may be capable of synthesizing these substrates de novo. Leishmania donovani and L. braziliensis required higher levels of folic acid than L. tarentolae , probably due to the fact that folates are involved as cofactors in the biosyntheses of pyrimidines and serine. Although the mixtures reported here cannot be regarded as "minimal essential" media, they are considerably less complex than the ones employed so far for cultivating hemoflagellates, and are therefore well suited for studies related to nutrition and biosynthetic capabilities of Trypanosomatids.     

Chemically Defined Medium for the Growth of Clostridium perfringens

A defined medium which supports growth of Clostridium perfringens at low inoculum levels was developed. Generation time for strain 8797 was 1.5 times greater than previously reported for growth in purged fluid thioglycolate medium.     

Leishmania donovani: A Chemically Defined Medium Suitable for Cultivation and Cloning of Promastigotes and Transformation of Amastigotes to Promastigotes

A chemically defined medium using commercially available α-MEM supplemented with 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 × 10⁷/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.     

Development of Chemically Defined Media Supporting High-Cell-Density Growth of Lactococci, Enterococci, and Streptococci

Lactococcus lactis IL1403 was used as an experimental strain to develop a chemically defined medium for study of the physiology and metabolic pathways of lactococci. An experimental leave-one-out technique was employed to determine the necessity of each of the 57 chemical components used in medium development. A statistical experimental design approach including three fractional factorial designs and a central composite design was used to optimize the fermentation process with 21 variables composed of 19 nutritional factors grouped from the 57 components and two environmental factors (initial pH and temperature). For L. lactis IL1403, the maximum biomass concentrations obtained with the two optimal chemically defined media developed in this study (ZMB1 and ZMB2) were generally 3.5- to 4-fold higher than the maximum biomass concentrations obtained with the previously described best synthetic media (SA) and 50% to 68% higher than the maximum biomass concentrations obtained with M17, a complex medium commonly used for lactococci. The new chemically defined media support high-cell-density growth of numerous strains of L. lactis, Enterococcus faecalis, and Streptococcus thermophilus.     

Neisseria gonorrhoeae Auxotyping: Differentiation of Clinical Isolates Based on Growth Responses on Chemically Defined Media

A system is described for differentiating clin?cal isolates of Neisseria gonorrhoeae based on their growth or absence of growth on a set of 11 chemically defined agar media. The complete medium, NEDA, contains all of the compounds required for gonococcal growth; but isolates differ in their ability to grow on NEDA from which selected compounds are individually omitted. The differential compounds include L-proline, L-arginine, L-ornithine, L-methionine, hypoxanthine, uracil, thiamine, and thiamine pyrophosphate. A distinctive pattern of growth responses on the standard media defines an auxotype. Twenty auxotypes were found among a group of 251 gonococci which were isolated from patients examined in the clinics of one city during a 3-month span of time. Another collection of 74 strains from several different countries yielded two additional auxotypes. The stability of the nutritional requirements on which the auxotyping depends was verified in two ways. Cultures isolated from different anatomic sites of a patient or from sexual partners represented the same auxotype, as did cultures which were repeatedly isolated from cases of presumptive treatment failures. Also, the auxotypes of gonococci remained the same after numerous subcultures. The reproducibility of results and the variety and number of auxotypes indicate the potential value of the auxotyping system as an epidemiological tool.     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Continuous Cultivation for Apparent Optimization of Defined Media for Cellulomonas sp. and Bacillus cereus

Steady-state continuous culture was used to optimize lean chemically defined media for a Cellulomonas sp. and Bacillus cereus strain T. Both organisms were extremely sensitive to variations in trace-metal concentrations. However, medium optimization by this technique proved rapid, and multifactor screening was easily conducted by using a minimum of instrumentation. The optimized media supported critical dilution rates of 0.571 and 0.467 h⁻¹ for Cellulomonas and Bacillus, respectively. These values approximated maximum growth rate values observed in batch culture.     

Burkholderia pseudomallei Requires Zn(sup2+) for Optimal Exoprotease Production in Chemically Defined Media

The effects of various medium components on exoprotease production by Burkholderia pseudomallei were studied in order to understand the production of virulence factors by this pathogenic organism. FeSO(inf4), NaCl, and MgSO(inf4) do not significantly affect exoprotease activity, and CaCl(inf2) has only a slight influence. Conversely, ZnCl(inf2) plays a fundamental role since it drastically increases exoprotease production.     

Chemically Defined Medium for Growth and Sporulation of Clostridium perfringens

A chemically defined medium was developed that could support sporulation and growth of Clostridium perfringens strains ATCC 12916 and H9. This medium consisted of a modification of the basal medium of Boyd et al. plus 0.1% sodium thioglycolate and 0.5% monosodium glutamate. Five other strains grew, but did not sporulate, in this medium. With the addition of more vatamins into the medium, two more strains grew but did not sporulate. The effects of glucose, monosodium glutamate, ammonium glutamate, and sodium thioglycolate on growth and sporulation of C. perfringens ATCC 12916 in the defined medium was investigated.