Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.     

Evaluation of culture medium for nisin production in a repeated-batch biofilm reactor

In this study, a biofilm reactor with plastic composite support (PCS), made by high-temperature extrusion of agricultural products and polypropylene, was evaluated for nisin production using L. lactis strain NIZO 22186. The high-biomass density of the biofilm reactor was found to contribute to a significantly shorter lag time of nisin production relative to a suspended-cell reactor. In comparison to glucose (579 IU/mL), sucrose significantly increased the nisin production rate by 1.4-fold (1100 IU/mL). However, results revealed that high levels of sucrose (8% w/v) had a suppressing effect on nisin production and a stimulating effect on lactic acid production. A high concentration of MgSO4.7H2O at 0.04% (w/v) was found to reduce the nisin production, while concentrations of KH2PO4 of up to 3% (w/v) did not have any significant effect on growth or nisin production. The best of the tested complex media for nisin production using the PCS biofilm reactor consisted of 4% (w/v) sucrose, 0.02% (w/v) MgSO4.7H2O, and 0.1% (w/v) KH2PO4. Nisin production rate in the biofilm reactor was significantly increased by 3.8-fold (2208 IU/mL) when using the best complex medium tested.     

Evaluation of a low-cost calorimetric approach for rapid detection of tuberculosis and other mycobacteria in culture

Aims: The objective of this study was to evaluate the effectiveness of microcalorimetry in rapid detection of mycobacterium species using an inexpensive Isothermal microcalorimetry (IMC) instrument. In addition, we compared microcalorimetry with conventional monitoring techniques. Methods and Results: Isothermal microcalorimetry measures heat production rate and can provide rapid detection of living mycobacteria in clinical specimens. Using liquid medium showed that bacterial activity measured by IMC using a TAM Air^® agreed with the triphenyl tetrazolium chloride (TTC) assay. Using solid medium to enhance growth, fast‐growing mycobacteria detection was achieved between 26 and 53 h and slow‐growing mycobacteria detection was achieved between 54 and 298 h. In addition, the calorimetric data were analysed to estimate the growth rate and generation time of the mycobacteria monitored. Significance and Impact of the Study: Infections caused by mycobacteria are severe and difficult to treat. With 9·27 million new cases of tuberculosis in 2007, developing countries experience severe health and economic consequences owing to the lack of an affordable, fast detection method. Research‐grade IMC instruments are too expensive to use in developing countries. Our study demonstrates that less‐expensive instruments such as the TAM air ^® are adequate for mycobacteria detection and therefore establishes a clear proof of concept.     

Assessment of the LOVO device for final harvest of novel cell therapies: a Production Assistance for Cellular Therapies multi-center study

Background aimsThe final harvest or wash of a cell therapy product is an important step in manufacturing, as viable cell recovery is critical to the overall success of a cell therapy. Most harvest/wash approaches in the clinical lab involve centrifugation, which can lead to loss of cells and decreased viability of the final product. Here the authors report on a multi-center assessment of the LOVO Cell Processing System (Fresenius Kabi, Bad Homburg, Germany), a cell processing device that uses a spinning filtration membrane instead of centrifugation.MethodsFour National Institutes of Health Production Assistance for Cellular Therapies cell processing facilities (CPFs) assessed the LOVO Cell Processing System for final harvest and/or wash of the following three different cell products: activated T cells (ATCs), tumor-infiltrating lymphocytes (TILs) and bone marrow-derived mesenchymal stromal cells (MSCs). Each site compared their current in-house, routinely used method of final cell harvest and/or wash with that of the LOVO device.ResultsFinal harvest and/or wash of ATCs, TILs and MSCs using the LOVO system resulted in satisfactory cell viability and recovery with some substantial improvement over the in-house methods of CPFs. Processing time was variable among cell types/facilities.ConclusionsThe LOVO Cell Processing System provides an alternative to centrifuge-based technologies. The system employs a spinning membrane filter, exposing cells to minimal g-forces compared with centrifugation, and is automated and closed. This small multi-center study demonstrated the ability of the LOVO device to yield satisfactory cell viability and recovery of T cells and MSCs.     

Pulsed column reactor as a novel tool for continuous enzymatic synthesis of peptide in an aqueous/organic biphasic medium

We propose a novel effective method for a continuous peptide synthesis in an aqueous/organic biphasic medium using a pulsed column reactor. N-Formyl-l-aspartyl-l-phenylalanine methyl ester was enzymatically synthesized continuously. With this extractive method using a pulsed column reactor, we can synthesize peptides with a stable performance even if a peptide (or a peptide-amino acid complex) is precipitated due to its high hydrophobicity.     

Basal medium development for serum-free culture: a historical perspective

The evolution of basal synthetic formulations to support mammalian cell culture applications has been facilitated by the contributions of many investigators. Definition of minimally-required nutrient categories by Harry Eagle in the 1950's spawned an iterative process of continuous modification and refinement of the exogenous environment to cultivate new cell types and to support emerging applications of cultured mammalian cells. Key historical elements are traced, leading to the development of high potency, basal nutrient formulations capable of sustaining serum-free proliferation and biological production. Emerging techniques for alimentation of fed batch and continuous perfusion bioreactors, using partial nutrient concentrates deduced from spent medium analysis, can enhance medium utilization and bioreactor productivity.     

Enhanced biodegradation of phenanthrene in a biphasic culture system

Degradation of phenanthrene byPseudomonas aeruginosa AK1 was examined in (i) an aqueous mineral salts medium to which phenanthrene particles of varying size (i.e. diameter) were added, and (ii) an aqueous/organic biphasic culture system consisting of mineral salts medium supplemented with 2,2,4,4,6,8,8-heptamethylnonane (HMN) as the phenanthrene-carrying organic phase. In both systems, the rate of phenanthrene biodegradation could be significantly enhanced by manipulations leading to improved phenanthrene mass transfer into the aqueous phase. With crystalline phenanthrene, the rate of biodegradation was found to be directly correlated to the particle surface area, whereas in the biphasic system the rate of biodegradation of the dissolved phenanthrene was mainly governed by the HMN/water interface area. In the latter system, exponential growth with a doubling time t _d of 6–8 hours has been achieved under conditions of intensive agitation of the medium indicating that phenanthrene degradation by strain AK1 is limited mainly by physicochemical parameters. Addition of selected surfactants to the culture medium was found to accelerate phenanthrene degradation by strain AK1 only under conditions of low agitation (in the presence of HMN) and after pretreatment of phenanthrene crystals by ultrasonication (in the absence of HMN). Evidence is presented that the stimulating effect of the surfactants was primarily due to improved dispersion of phenanthrene particle agglomerates (in the aqueous mineral salts medium supplemented with phenanthrene crystals) or of the phenanthrene-carrying lipophilic solvent drops (in the aqueous/organic biphasic culture system) whereas the solubilizing activity towards phenanthrene was neglectible. Under conditions of intensive mixing of the culture medium (i.e. if a high particle surface area or HMN/water interface area, respectively, is provided), the addition of surfactants did not enhance phenanthrene biodegradation.     

COMBO: a defined freshwater culture medium for algae and zooplankton

In order to conduct experiments on interactions between animals and food organisms, it is necessary to develop a medium that adequately supports the growth of both algae and zooplankton without the need to alter the medium to accommodate either the algae or the animals. We devised a freshwater medium, named COMBO, that supports excellent growth of both algae and zooplankton. Two types of algae, Ankistrodesmus falcatus and Stephanodiscus hantzschii, were reared in COMBO and their growth rates were not significantly different from those of algae grown in a reference medium (WC). One of these algae, A. falcatus, was then fed to a cladoceran, Daphnia pulicaria, which was also cultured in COMBO, and the resulting fecundities of D. pulicaria were compared to those of animals reared in natural surface water. We also determined whether the value of COMBO as a medium for D. pulicaria was affected by modifications in nitrogen or phosphorus concentration to evaluate whether the new medium will be useful in nutritional research. Lowering the N or P content of COMBO did not affect the reproductive performance of D. pulicaria. Other researchers have also reported excellent growth and reproduction by numerous algae and zooplankton reared in COMBO. Our results suggest that COMBO is an effective artificial, defined culture medium capable of supporting robust growth and reproduction of both freshwater algae and zooplankton. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

The in vivo effects of a culture medium. II. Influence of culture medium administered prior to irradiation on hemopoietic recovery of gamma-irradiated mice.

The culture medium administered to C57Bl/6 mice 18 h and 8 h before a single irradiation (9 Gy) had a radioprotective effect and clearly influenced postirradiation changes in haemopoiesis. Haemopoiesis recovery appeared to be faster in culture medium-pretreated animals than in those irradiated without such pretreatment. By 12-15 days after irradiation, the thymus cortex appeared to be repaired, on day 21 a multiple increase in extramedullary erythropoiesis, myelopoiesis and megakaryocytopoiesis in the red pulp was found and later, on day 28, the lymphopoiesis in the white pulp of spleen was restored. The rate of haemopoiesis proliferation of predominantly myeloid cells which reached a control level on day 28 following irradiation. Consequently, the regenerative processes in blood-forming organs were accompanied by considerable reticulocytosis and complete recovery of neutrophil and platelet counts in the peripheral blood as seen on day 21. Despite a slower rate complete recovery of the total leukocyte count was reached by day 180 after irradiation.     

Immobilized serotonin: a novel substrate for cell culture

Baby hamster kidney cells, bovine aortal endothelial cells, bovine smooth muscle cells, and chick embryo fibroblasts were all observed to attach and grow on serotonin which had been immobilized by covalent coupling to agarose beads. While growth and morphology of cells on immobilized serotonin appeared normal, a change in cell function may have occurred since the pattern of polypeptides expressed by these cells was different from that of cells grown on two other substrates: immobilized fibronectin and tissue culture plastic. By changing the composition of the fetal calf serum proteins in the growth medium it was shown that cells attach directly to immobilized fibronectin without mediation by medium components. In contrast, cells were found not to attach directly to immobilized serotonin but to attach indirectly via factors absorbed onto immobilized serotonin from fetal calf serum. The major component of this cell attachment activity was shown not to be fibronectin and was identified following separation by SDS-PAGE, electroblotting, and cell binding on nitrocellulose filters. The cell attachment activity compromises a major protein species of Mr 70,000 which is the molecular size of the recently identified serum spreading factor also called vitronectin.     

Photolysis in a culture medium for Tetrahymena pyriformis

Considerable variability has been found in the yield of cells in batch cultures of Tetrahymena pyriformis grown axenically in 1% tryptone/0.05% yeast extract. This variability has been traced to the photolysis by visible light of the flavin mononucleotide and thiamine components of yeast extract.     

Hyperlipidemic rabbit serum reduces recovery of acidic glycosaminoglycans from tissue culture medium

Hyperlipidemic rabbit serum and its lipid extract were found to impair the precipitation of glycosaminoglycans by cetylpyridinium chloride. The inhibition was 60–80% in the case of sulfated glycosaminoglycans and 75–85% in the case of hyaluronic acid. The interfering compounds could be removed by extracting the samples twice with a six-fold volume of ethanol-ether (2:1) for 1 h.     

Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster

Cold resistance in insects has traditionally been measured in terms of survival following a stress, but alternative methods are increasingly being used because of their relevance to the ecology of organisms and their utility in characterizing variation among species, populations and individuals. One such method capable of discriminating among Drosophila species and conspecific Drosophila populations from different environments is adult chill coma recovery time, the time taken for adults to become active again after being knocked down by a cold stress. Here we characterized the chill coma response of D.melanogaster in detail. Adults were exposed to a range of temperatures and stressful periods prior to measuring recovery. Recovery from chill coma in D.melanogaster was biphasic; as flies were stressed under cooler temperatures, recovery times leveled off and then decreased before sharply increasing again as mortality starts to occur. This biphasic response has previously been observed in D.subobscura where it has a somewhat different shape. A second mechanism therefore acts at relatively lower temperatures to ameliorate the effects of the cold stress. When D.melanogaster were reared at 19 and 25 °C for two generations, the shape of the curve relating temperature to recovery time was similar, but flies from the warmer temperature had longer recovery times and showed responses that leveled off and then decreased at relatively higher temperatures. As exposure time to cold stress was increased, recovery times also increased except at mild stress levels. Chill coma recovery in D.melanogaster is a complex trait and likely to reflect multiple underlying components.     

A novel modified culture medium for enhancing redifferentiation of chondrocytes for cartilage tissue engineering applications

The dedifferentiation of articular chondrocytes during in vitro expansion deteriorates the hyaline cartilage regeneration. Many approaches have been developed to enhance the redifferentiation of chondrocytes. In this study, a new and effective protocol to improve the redifferentiation of porcine chondrocytes in a pellet form was established. Pellets were initially treated in the modified culture media containing ternary mixtures, binary mixtures, or single reagents of sodium citrate (SCi), sodium chloride (SCh), and ethylenediaminetetraacetic acid (EDTA) at varied concentrations during the first 3 days of culture, followed by a normal culture medium until 21 days. Viability, proliferation, cartilaginous gene expression, extracellular matrix formation, and morphology of treated cell pellets were comparatively examined. Chondrocytes exposed to SCi, SCh, and EDTA individually or in combinations of two or three chemicals were non-cytotoxic when the concentration ranges of the chemicals were 1.83–2.75, 5.00–7.50, and 1.00–1.50 mM, respectively. Cells treated with the modified media containing EDTA alone and EDTA-containing mixtures enhanced glycosaminoglycan production as well as upregulated cartilaginous gene expression, despite their low proliferation rates. Overall, when all three reagents were in use, a pronounced synergistic effect on the activations of glycosaminoglycan accumulation and type II collagen production was explicitly observed at most, particularly when cells were cultured in the medium containing SCi, SCh, and EDTA at concentrations of 2.20, 6.00, and 1.20 mM, respectively. With a use of this protocol, the redifferentiation of articular chondrocytes for regeneration of hyaline cartilage for tissue engineering applications could be readily achieved.