Electrochemical changes in media due to microbial

Microbial metabolism affected the electrical impedance parameters of a two terminal-measuring cell-containing growth media. The relationship between microbial growth and relative changes in both The capacitive and resistive parts of impedance was examined. Both components of impedance were shown to be indicative of bacterial growth. In low conductivity media the change in the conductance of the media (G_sol) clearly correlated to bacterial growth. In more conductive media the relative changes in G_sol were smaller, and in these media measurements of the changes of polarization capacitance (C_pol) were useful for monitoring bacterial growth.Yeast growth in two media resulted in large changes in C_pol (20–100%) while the changes in G_sol were very small (1–4%). This result indicated that, for some combinations of microorganisms and media, measuring C_pol might be preferable over G_sol for the detection of microbial growth.Microbial metabolism resulted in a change of 2–2.5 units in pH. This pH change resulted in a 40% change in C_pol but less than a 14% change in G_sol.     

Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass

Sequential-co-culture technique was investigated in this study for the production of bioethanol from relatively cheaper lignocellulosic biomass of Kans grass (Saccharum spontaneum). The consortium of Pichia stipitis and Zymomonas mobilis was used to develop a suitable sequential-co-culture system. The Kans grass biomass was hydrolyzed in such a manner that the two sugar fractions, xylose rich and glucose rich were generated (a separate study). The P. stipitis cells and respective fermentation media (xylose rich) were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media were fed to the same vessel. Different strategies have been followed and experiments were conducted initially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentation media and Kans grass hydrolysate media to compare the kinetic parameters. The sequential addition of cultures with their respective media and imposed process conditions, showed better utilization of total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic condition for Z. mobilis fermentation were found significant. The average ethanol yield (Y_p/s) and overall volumetric productivity (r_po) were found as 0.453 g_p/g_s and 1.580 g/l/h respectively for Kans grass hydrolysate media and 0.474 g_p/g_s and 2.901 g/l/h respectively for synthetic fermentation media.     

Excretion products of algae: Identification of biogenic amines by gas-liquid chromatography and mass spectrometry of their trifluoroacetamides

The trifluoroacetyl derivatives of 12 biogenic amines were separated by gas-liquid chromatography and identified by mass spectrometry. Vacuum sublimation was used to separate nanomole quantities of amines from algal media and natural waters that contained algae. Trimethylamine, 1-aminopropane, 2-aminopropane, and ethanolamine were found in the culture medium of Ochromonas. Chlamydomonas-enriched backwater contained butanolamine. 1-Aminopropane was found in the media of Nitzschia, Ankistrodesmus, and Microcystis. Concentrations were approximately 300 μg/liter for Ankistrodesmus and 240 μg/liter for Microcystis media.     

Escape of Salmonellae from Chlorination during Ingestion by Pristionchus lheritieri (Nematoda: Diplogasterinae)

Salmonella typhi and S. wichita survived 15 min in 10 ppm free chlorine solution during ingestion by the free-living nematode, Pristionchus lheritieri, and were viable when defecated in suitable media. S. typhi and S. wichita were recovered in 13.5 and 46.6% of the trials on five different media.     

Development of a Solid Medium for Growth and Isolation of Axenic Microcystis Strains (Cyanobacteria)

Solid media on a base of B-12 or CB medium with agarose or agarose of low melting temperature were developed for the cultivation of Microcystis species. The media with 0.4% gel showed the highest number of CFU, and increasing the gel concentration resulted in a reduction of the number of CFU. There was no difference in the numbers of CFU between pour and spread plates made of the solid media. By using the solid media, 31 clones of Microcystis species were isolated from natural blooms in Lake Kasumigaura, and 5 axenic strains (1 of M. wesenbergii and 4 of M. aeruginosa) were established from the clones.     

Effects of water potential and solute on the growth and interactions of two fungal symbionts of the mountain pine beetle

We investigated the effect of water potential (WP) on the growth of, and interaction between, two ophiostomatoid fungi, Grosmannia clavigera and Ophiostoma montium, associated with the mountain pine beetle (Dendroctonus ponderosae). The WP of malt extract agar was amended by adding potassium chloride (KCl) or sucrose. Growth of both fungi decreased with WP on KCl-amended media. Growth of G. clavigera also decreased with WP on sucrose-amended media, although growth was stimulated on these media compared to unamended treatments. Growth of O. montium remained relatively constant on sucrose-amended media, confounding the effect of WP on this species. Both fungi were able to colonize media occupied by the other species, but at a slower rate than on unoccupied media, indicating competition. In most treatments, G. clavigera grew faster than O. montium and colonized a greater area when the two fungi were inoculated concurrently but distant to one another on a Petri dish. However, when each fungus was inoculated adjacent to a 10-d-old well-established colony of the other species, O. montium colonized occupied media more effectively than G. clavigera considering the growth rate of each species alone. Thus, G. clavigera dominated primary (uncolonized) resources on most media, whereas O. montium was more effective in colonizing secondary (occupied) resources. The differential response of the two fungi to sucrose indicates that they may use different carbon sources, or use different carbon sources at different rates, in the tree. Fine-scale resource partitioning, differences in primary and secondary resource capture abilities, and the non-equilibrium dynamics in an attacked tree over time, could all act to promote the co-existence of two unit-restricted dispersers on a discontinuous resource.     

Babesia bovis: Effect of Albumax II and orotic acid in a low-serum in vitro culture

Bovine serum is an essential factor for the continuous in vitro growth of Babesia bovis parasites. Culture media typically contain 40% (v/v) of bovine serum. In the present study assays with low-serum media were performed. The growth of B. bovis Mo7 was successively lower in media with 30%, 20% and, principally, with 10% of serum. Without serum, the parasites were not able to propagate. In media with 10% of serum, the supplementation with Albumax™ II improved visibly the growth of B. bovis at the end of each cycle. Regarding the addition of orotic acid, no considerable effect was observed in media with 20% or 10% of serum, with or without Albumax™ II. B. bovis parasites cultured in vitro in all these media maintain their typical morphology during the intraerythrocytic stages.     

Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli

In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l⁻¹.     

Isubgol as an Alternative Gelling Agent for Microbial Culture Media

Isubgol, the mucilaginous husk derived from the seeds of Plantago ovata, has been successfully used as a gelling agent for microbial culture media. As illustrative examples, fast growing symbiotic bacterium, Rhizobium meliloti and saprophytic fungi, Aspergillus flavus and Penicillium chrysogenum were cultured on media gelled with either Isubgol or agar. All the three microbes employed in the study exhibited normal growth when cultured on their respective media gelled with Isubgol. Rather, Isubgol gelled medium appears to promote the growth of bacterial cultures as the colonies on this medium were denser than the corresponding ones on the medium gelled with agar. Likewise, on Isubgol gelled medium, sporulation in both the fungi took place earlier than on the medium gelled with agar, thus indicating the promotive influence of the former gelling agent.     

Mcph1-Deficient Mice Reveal a Role for MCPH1 in Otitis Media

Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1^{tm1a /tm1a}) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute''s Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1^{tm1a /tm1a} mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1^{tm1a /tm1a} mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1^{tm1a /tm1a} mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.     

Diversity in the Ammonia-Oxidizing Nitrifier Population of a Soil

Multiple genera of ammonia-oxidizing chemoautotrophic nitrifiers in a soil were detected, isolated, and studied by means of modified most-probable-number (MPN) techniques. The soil examined was a Waukegon silt loam treated with ammonium nitrate or sewage effluent. The genera Nitrosomonas and Nitrosospira were found to occur more commonly than the genus Nitrosolobus. Three different MPN media gave approximately the same overall ammonia oxidizer counts within statistical error after prolonged incubation but differed markedly in ratios of Nitrosomonas to Nitrosospira. Selectivity and counting efficiency of MPN media were studied by observing the growth response of representative pure cultures isolated from the soil. Selectivity was evident in each medium with respect to all strains tested, and the media differed greatly in incubation times required to obtain maximum counts.     

Evaluation of growth and biomass production of Arthrospira (Spirulina) fusiformis in laboratory cultures using waters from the Ethiopian soda lakes Chitu and Shala

The cost of nutrient media is the major challenge for biomass production of Spirulina. Although much effort has been made to use enriched seawater for the cultivation of this microalga, little attention has been given to the potential of water of soda lakes. In this study, growth (μ, day^?1) and biomass production (B) of Arthrospira fusiformis cultivated using waters of the soda lakes Chitu and Shala with or without supplementation were evaluated. Comparable μ and B values were achieved in both Lake Chitu water-based media (CBM) and Lake Shala water-based media (SBM), with slightly higher values in the latter. Both CBM and SBM supplemented with the standard Spirulina medium (SM) by 25 % and 50 % supported considerably higher μ and B. The pH and salinity of the cultures showed significant variations (P?μ and B. The observed higher μ and B were probably associated with the reduction in pH and salinity of the supplemented media due to addition of bicarbonate–carbonates and dilution, and provision of the limiting nutrient nitrogen. The higher μ and B in SBM may have resulted from some of their aggregate chemical parameters, which were closer to those in the SM, and abundant PO₄-P. This seems to suggest that Lake Shala water is more conducive to Arthrospira. We contend that 25 % and 50 % supplemented Lake Shala water can be preferably used to produce Arthrospira biomass, thereby reducing the cost of nutrients by 75 % and 50 %, respectively.     

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in Escherichia coli

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk⁻ppx⁻ or ppk⁻ppx⁻/ppk⁺ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.     

Aspartate facilitates mitochondrial function,growth arrest and survival during doxorubicin exposure

Genomic screens of doxorubicin toxicity in S. cerevisiae have identified numerous mutants in amino acid and carbon metabolism which express increased doxorubicin sensitivity. This work examines the effect of amino acid metabolism on doxorubicin toxicity. S. cerevisiae were treated with doxorubicin in combination with a variety of amino acid supplements. Strains of S. cerevisiae with mutations in pathways utilizing aspartate and other metabolites were examined for sensitivity to doxorubicin. S. cerevisiae cultures exposed to doxorubicin in minimal media showed significantly more toxicity than cultures exposed in rich media. Supplementing minimal media with aspartate, glutamate or alanine reduced doxorubicin toxicity. Cell cycle response was assessed by examining the budding pattern of treated cells. Cultures exposed to doxorubicin in minimal media arrested growth with no apparent cell cycle progression. Aspartate supplementation allowed cultures exposed to doxorubicin in minimal media to arrest after one division with a budding pattern and survival comparable to cultures exposed in rich media. Aspartate provides less protection from doxorubicin in cells mutant in either mitochondrial citrate synthase (CIT1) or NADH oxidase (NDI1), suggesting aspartate reduces doxorubicin toxicity by facilitating mitochondrial function. These data suggest glycolysis becomes less active and mitochondrial respiration more active following doxorubicin exposure.     

In vitro studies of Coelomomyces punctatus from Anopheles quadrimaculatus and Cyclops vernalis

Attempts to grow mycelium of Coelomomyces punctatus from Anopheles quadrimaculatus larvae were made using more than 50 combinations of known vertebrate and invertebrate tissue culture media and microbiological media. Growth and/or differentiation of mycelium into sporangia were observed in several media. Significant growth of hyphal fragments and differentiation into young resting sporangia occurred in conditioned Mitsuhashi-Maramorosch insect tissue culture medium. This medium was conditioned by growth for 3 weeks in it of Varma's Anopheles stephensi tissue culture cells and was supplemented with 20% heat-inactivated fetal bovine serum and a synthetic tripeptide, glycyl-histidyl-lysine. Limited growth and elongation of lateral hyphal branches and subsequent development into resting sporangia with typical outer wall markings and pigmentation of mature forms were observed in a modified brain-heart infusion medium. Some media stimulated hyphae to develop into smooth-walled, spherical bodies of size and appearance typical of young sporangium initials but with no further maturity. In most media, no growth or development of mycelium occurred, but the fungus remained alive for 2–4 weeks. Mycelium of C. punctatus dissected from Cyclops vernalis did not grow and develop in any of the media utilized. However, in one case the mycelium differentiated into gametes shortly after dissection into modified brain-heart infusion medium.     

Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae)

Mtitchell J. S., Halton D. W. and Smyth J. D. 1978. Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae). International Journal for Parasitology8: 389–397. Cotylurus erraticus metacercariae obtained from around the heart of rainbow trout were excysted and grown in vitro and in vivo to egg-producing adults. For in vitro development, tissue culture media M199 or NCTC 135 was used, together with varying amounts of chicken serum. Worms grown in media containing the highest concentration of serum (80% per volume) showed the fastest rate of development, measured by the time taken for the first eggs to appear in the uterus. The testes, ovaries and vitellaria of these worms were comparable in structure and histochemistry with those of worms reared in gulls. Eggs were produced by worms in all media containing chicken serum, but the eggs had abnormal shells and failed to embryonate.     

Methyl Violet: a Selective Agent for Differentiation of Klebsiella pneumoniae from Enterobacter aerogenes and Other Gram-Negative Organisms

Three selective media for differentiation of Klebsiella pneumoniae from Enterobacter aerogenes on the basis of colonial morphology were evaluated. Using methyl violet 2B as a selective agent, strains of K. pneumoniae isolated from urine, fresh water, and fresh produce were tested against other members of Enterobacteriaceae in addition to strains of Aeromonas hydrophila and Pseudomonas aeruginosa. Comparison of colonial morphology showed K. pneumoniae produced larger, smoother colonies than other bacteria tested. These media were developed to aid in presumptive separation of K. pneumoniae from E. aerogenes in the monitoring of bacterial quality of water.     

CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi,especially Metarhizium acridum,from soil

The selective media most commonly used for isolating hyphomycetous species of entomopathogenic fungi from non-sterile substrates rely on N-dodecylguanidine monoacetate (dodine) as the selective fungicide. Although these media are effective for isolating many species of Metarhizium and Beauveria from soil, they are inefficient media for isolation of an important Metarhizium species, Metarhizium acridum, from non-sterile soil. Our current study was directed to formulating a dodine-free selective medium that is efficient for isolating naturally occurring Beauveria spp. and Metarhizium spp., especially M. acridum, from soil. The selective medium (designated CTC medium) consists of potato dextrose agar plus yeast extract (PDAY) supplemented with chloramphenicol, thiabendazole and cycloheximide. In comparisons with selective media previously reported in the literature, the CTC medium afforded colonies that were larger and had both earlier and more abundant conidiation of entomopathogenic fungi, features which greatly facilitated identification of the emerging entomopathogenic fungi. In addition to efficient re-isolation of M. acridum, this medium also is an effective tool for selective isolation of Metarhizium brunneum, Metarhizium robertsii, Beauveria bassiana and Beauveria brongniartii from non-sterile field-collected soil samples inoculated (spiked) with fresh conidia in the laboratory.     

Enhancement of tPA production under hyperoxic conditions by BHK cells in serum-free and serum-containing cultures

Effects of a wide range of dissolved oxygen concentration (DO, 0.5–28 mg/l) on anchorage-dependent BHK cell growth, metabolism and tPA production were examined with both serum-containing and serum-free media. In the range of DO from 0.5 to 5 mg/l, tPA production increased with an increase in DO in both media. Cell growth was higher at 5 mg/l DO than that at 0.5 or 2 mg/l DO in serum-containing medium, but it did not vary in serum-free medium in this DO range. Further investigation under hyperoxic conditions (DO > 6.8 mg/l) revealed that specific rates of tPA production were enhanced by 2-fold in serum-containing and 1.7-fold in serum-free media, although cell growth depressed above 5 mg/l of DO. Slight increases in specific rates of lactate accumulation and glucose consumption were observed in both media under hyperoxic conditions. In serum-free medium, cells were found to be less tolerant to hyperoxic conditions than those in serum-containing medium. A DO shift-up with shifting time of 4 h in serum-containing medium was found to influence significantly both cell growth and tPA production.