Observation on complete schizogony of Plasmodium vivax in vitro

Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media. Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites. The RPMI 1640 and Waymouth media suitable for the cultivation of P. falciparum were also used with markedly less success. Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium.     

Observation on Complete Schizogony of Plasmodium vivax in Vitro1

Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media. Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites. The RPMI 1640 and Waymouth media suitable for the cultivation of P. falciparum were also used with markedly less success. Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium.     

Minimal Growth Requirements for Clostridium perfringens and Isolation of Auxotrophic Mutants

The minimal growth requirements for two strains of Clostridium perfringens were defined, and both synthetic and semisynthetic plating media were developed. Plate counts of the wild-type strains on both of these minimal media were equivalent to those on complex media. A number of auxotrophic mutants of each strain were isolated, and their phenotypes were defined.     

Definition of physical integrity of synaptosomes and myelosomes by enzyme osmometry

Isotonic requirements for synaptosomes were shown to vary with the concentration of sucrose or mannitol in the isolation medium, as well as with their differential permeability to polyols and ions. The technique of enzyme osmometry, which permits quantitation of the osmotic integrity in a heterogeneous population, was used to defined the osmotic requirements for synaptosomes and myelosomes in a variety of ionic and non-electrolyte media. Important differences, observed in the rank order of permeability of synaptosomal and myelosomal membranes to electrolyte media, were consistent with the known channel density/electrical activity of the corresponding plasma membranes.     

Strongyloides fülleborni: development in axenic culture.

Exogenous Strongyloides fülleborni were cultured axenically in a variety of media. The best media contained freshly prepared chick embryo extract. Sulfhydryl reducing compounds and a low oxygen-increased carbon dioxide gas phase were required. Only a few individuals developed to adults; males having well-developed gonads, while females had segmented eggs. Other individuals terminated as filariforms and a large number remained as immature rhabditoid larvae. In the second generation, the majority of larvae developed to filariforms, only three becoming females. The nutritional and environmental requirements of the rhabditoid phase are those of a parasite and not of a free-living nematode.     

Biological indicators for low temperature steam formaldehyde sterilization: effect of defined media on sporulation, germination index and moist heat resistance at 110 degrees C of Bacillus strains

Choice of a biological indicator depends upon selecting a strain with the optimum balance of desirable properties. Screening 20 strains of Bacillus spp. for sporulation on three defined media has shown the wide variation that occurs in requirements for sporulation and properties of the resultant spores. Comparison of germination index and moist heat resistance of resultant spores suggest that a combination of high germination index, high heat resistance and linear inactivation may not be possible.     

Biological indicators for low temperature steam formaldehyde sterilization: effect of defined media on sporulation, germination index and moist heat resistance at 110dEC of Bacillus strains

Choice of a biological indicator depends upon selecting a strain with the optimum balance of desirable properties. Screening 20 strains of Bacillus spp. for sporulation on three defined media has shown the wide variation that occurs in requirements for sporulation and properties of the resultant spores. Comparison of germination index and moist heat resistance of resultant spores suggest that a combination of high germination index, high heat resistance and linear inactivation may not be possible.     

Substrate utilization in defined media

Substrate utilization in defined media for two flower spiroplasmas (S. floricola and FS SR-3) and honeybee spiroplasma (HBS AS-576) was investigated. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, HBS (AS-576) could ferment trehalose; FS (SR-3), sucrose; and S. floricola, trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in growth requirements for amino acids. Only S. floricola utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and BSA were essential to all three spiroplasmas. Palmitic acid, which was non-essential, promoted growth significantly.     

The role of iron in the growth of human leukemic cell lines

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.     

Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and L?wenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.     

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.     

Simplified media for the growth of Haemophilus influenzae from clinical and normal flora sources.

The nutritional requirements of 43 strains of Haemophilus influenzae isolated from clinical and normal flora sources were investigated. Two defined minimal media were developed by modifying the medium of Herriott et al. (1970):74% of the strains could grow on the minimal media and Herriott's medium; the remaining strains could not grow on any of these media.     

Insect cell nutrition: Emphasis on sterols and fatty acids

Summary The requirements for, or the utilization of, vitamins, sugars, amino acids, and lipids, as determined with established insect cell lines, are reviewed. Also discussed is the importance of inorganic salt balance and osmotic pressure. Nutrient requirements, as determined in experiments with established cells, are compared to media formulations based on the analysis of insect hemolymphs and to the dietary requirements of insects. It is suggested that culture media formulated in strict agreement with hemolymph analysis may be unnecessarily complex and do not ensure maximum growth.     

Response surface methods for optimizingSaccharopolyspora spinosa,a novel macrolide producer

Summary Strain A83543, recently identified asSaccharopolyspora spinosa, was cultured in a variety of media to optimize macrolide titer. Response surface methodology (RSM) was used to improve the fermentation medium and to characterize the microorganism's response to systematic variations in medium composition. Three sequential RSM studies on wild-type A83543 and two high macrolide-producing mutants showed that each strain produced maximum titers in nearly identical fermentation media. No obvious differences in nutrient requirements were evident in the three strains indicating little interaction between mutational change and medium composition through at least two cycles of mutagenesis. The overall increase in macrolide titer starting from the wild-type organism in the original fermentation medium to the second-generation mutant in the optimized medium was over 25-fold.     

Effects of preservation media on in vitro maturation of porcine oocytes

The effects of preservation media for ovaries on in vitro maturation of porcine oocytes was studied. The cumulus-oocyte complexes (COCs) obtained from ovaries that had been preserved in three different media at various temperatures for different time intervals were cultured in the M199 maturation medium. The preservation media used were 0.9% saline solution, BCS (Braun-Collins solution) and Dulbecco's phosphate buffered saline solution (PBS). Mature oocytes obtained from the ovaries preserved in three preservation media for 8 h were electrically activated. The activated oocytes were then cultured in the NCSU23 embryo culture medium for 16 h to observe activation, or for 144 h to observe embryo development. It was found that the preservation temperature significantly affected maturation of the porcine oocytes. A preservation temperature of about 25 degrees C showed an optimal maturation rate for a preservation time of 8 h for the three preservation media. Although the preservation temperature was a major factor influencing the maturation rate, different preservation media at 25 degrees C for 8 h also significantly affected the maturation rate, activation rate and embryo development. Among these three preservation media, PBS exhibited the highest cleavage rate indicating that PBS should be a better preservation medium for porcine ovaries at 25 degrees C for 8 h or longer periods.     

Production and characteristics of hemolysins of Escherichia coli

Snyder, Irvin S. (University of Iowa, Iowa City), and Nancy A. Koch. Production and characteristics of hemolysins of Escherichia coli. J. Bacteriol. 91:763-767. 1996.-Filterable and nonfilterable hemolysins were produced by Escherichia coli in beef-heart infusion, casein hydrolysate, and chemically defined media. Differences were found among the three media in the time of appearance and in the relationship between production of these two hemolysins. The nonfilterable hemolysin produced in the three media, as well as the filterable hemolysin produced in the beef-heart infusion medium, were destroyed in 1 hr at 56 C. The filterable hemolysin produced in the casein hydrolysate and chemically defined media was unaffected by exposure to 56 C for 1 hr. Formalin inactivated the hemolysins produced in all three media. The optimal pH for activity of the nonfilterable hemolysin varied with time of production, whereas the optimal pH for the filterable hemolysin was constant. Certain carbohydrates were required for the production of filterable hemolysin by E. coli grown in casein hydrolysate and chemically defined media.     

Development of a Bioprocess for Murine Dimeric IgA Production

Monoclonal IgA was produced under serum free conditions by a murine hybridoma cell line (ZAC3) in a hollow fibre, a continuous stirred tank and a fluidized bed reactor. Differences in the antibody adsorption to DEAE chromatography matrices, an essential step in downstream processing, were related to the production systems. Chromatography on hydroxyapatite was used to separate monomeric, dimeric and polymeric IgA. This method was successfully applied to IgA produced by all three reactor configurations. The binding of dimeric and polymeric IgA to antigen was tested by ELISA. Using 2-dimensional SDS-PAGE analysis, dimeric IgA from the three sources was shown to be identical with respect to isoelectric points of alpha, kappa and J-chain but showed marked differences in purity. Finally the efficiency of the three bioprocesses was assessed by comparing the product yields after purification. This included the reactor specific production rates, media and time requirements and time consumption for the production of 1 g purified dimeric IgA.     

Optimization of mouse embryo culture media using simplex methods

Culture media were developed for pronuclear-stage mouse embryos using simplex optimization, which has the benefit of being able to optimize several components simultaneously. Initially, several different media were generated. All media contained the same components, yet each medium was characterized by having a different component at a high concentration. The simplex procedure identified 4 components (NaCl, pyruvate, KH2PO4 and glucose) which at high concentrations were detrimental to embryo development, compared to the other components tested. For example, all embryos cultured in a medium with high NaCl blocked at the 2-cell stage. The optimization method then adjusted each medium by lowering the concentration of the component or removing it entirely, which resulted in a significant increase in development. In an experiment comparing 8 media generated from the simplex optimization, along with 7 other media, removal of KH2PO4 resulted in the largest increase in development; 88% of embryos were greater than or equal to 4 cells on Day 3 after hCG, and 53% developed into blastocysts by Day 5. Another experiment compared 4 of the best media generated from the simplex optimization. In 3 out of the 4 media, 90% or more of the embryos were greater than or equal to 4 cells on Day 3. In 3 of the media, approximately 60% or more of the embryos developed into blastocysts. The simplex optimization procedure is an efficient method for developing culture media and determining requirements for development in vitro.     

Batch- and continuous-culture studies of a methane-utilizing mixed culture

A methane-utilizing mixed culture isolated from activated sludge by selective enrichment at 45°C was found to consist of three interacting species: a methaneutilizing bacterium, a citrate-utilizing bacterium, and a methanol-utilizing bacterium. All three species grew well at 45°C. Three different stable mixed cultures were reconstituted by various combinations of these pure cultures. The nutritional requirements and substrate ranges for each pure culture were determined. The nutritional requirements and substrate ranges for each pure culture were determined. The saturation constant for the methane-utilizing bacterium on methane (K) and for the methanol-utilizing bacterium on methanol (K) were 1.73 × 10^?6M and 4.51 × 10^?7M, respectively. The volumetric mass transfer coefficient for methane (K_L a) was determined to be 65.6 hr^?1.     

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.