A low-cost antifoam additive for agar-based culture media

A highly effective, low-cost silicone-based antifoam emulsion is described which, at a final concentration of 100 ppm, prevents bubble formation during the preparation and dispensing of agar media. The compound is heat-stable, has a long shelf-life and offers considerable savings in cost by reduction in wastage of time and materials. It has no demonstrable deleterious effect on the growth of a wide range of pathogenic and commensal bacteria, or on antibiotic disc susceptibility testing, when examined using a range of different media.     

Partial purification and characterization of chorionic gonadotrophin in plasma and in culture medium of trophoblast cells from the marmoset monkey (Callithrix jacchus)

A biologically active gonadotrophin has been purified from the media of long-term cultures of trophoblast cells of the common marmoset monkey by a combination of precipitation and chromatography. Marmoset chorionic gonadotrophin (CG) is a glycoprotein which binds Concanavalin A and wheat germ agglutinin. The protein purified from culture media exists as several isoelectric species with pI in the range pH 3.5-4.5. On gel filtration it eluted with an apparent molecular weight of 68-72,000 but on PAGE migrated as if it was 58-65,000. A glycoprotein with similar characteristics has been recovered from plasma samples of pregnant marmosets. Biological activity of partly purified CG from media, as determined by a mouse testicular cell bioassay, was 1-3 i.u./mg protein.     

A thermostable cocktail for the liquid scintillation counting of heterogeneous media

Considerable analytical errors arise in the liquid scintillation counting of heterogeneous media as a consequence of gel instability. With large sample numbers, a major causative factor of this instability is temperature changes during the counting period. An emulsifier-scintillation cocktail has been designed to provide stable counting conditions for heterogeneous media over a temperature range of 10–30°C, i.e., the wide range of temperature likely to be encountered in liquid scintillation counters lacking sample cooling facilities. A comparison was made with a conventional commercially available emulsifier-scintillator.     

New detection systems of bacteria using highly selective media designed by SMART: selective medium-design algorithm restricted by two constraints

Culturing is an indispensable technique in microbiological research, and culturing with selective media has played a crucial role in the detection of pathogenic microorganisms and the isolation of commercially useful microorganisms from environmental samples. Although numerous selective media have been developed in empirical studies, unintended microorganisms often grow on such media probably due to the enormous numbers of microorganisms in the environment. Here, we present a novel strategy for designing highly selective media based on two selective agents, a carbon source and antimicrobials. We named our strategy SMART for highly Selective Medium-design Algorithm Restricted by Two constraints. To test whether the SMART method is applicable to a wide range of microorganisms, we developed selective media for Burkholderia glumae, Acidovorax avenae, Pectobacterium carotovorum, Ralstonia solanacearum, and Xanthomonas campestris. The series of media developed by SMART specifically allowed growth of the targeted bacteria. Because these selective media exhibited high specificity for growth of the target bacteria compared to established selective media, we applied three notable detection technologies: paper-based, flow cytometry-based, and color change-based detection systems for target bacteria species. SMART facilitates not only the development of novel techniques for detecting specific bacteria, but also our understanding of the ecology and epidemiology of the targeted bacteria.     

The application of chromogenic media in clinical microbiology

Since 1990, a wide range of chromogenic culture media has been made commercially available providing useful tools for diagnostic clinical microbiology. By the inclusion of chromogenic enzyme substrates targeting microbial enzymes, such media are able to target pathogens with high specificity. Examples of target pathogens include Staphylococcus aureus, Streptococcus agalactiae, Salmonella spp. and Candida spp. The inclusion of multiple chromogenic substrates into culture media facilitates the differentiation of polymicrobial cultures, thus allowing for the development of improved media for diagnosis of urinary tract infections and media for the enhanced discrimination of yeasts. The purpose of this review is to provide some insight into how such media work and appraise their utility in routine clinical diagnostics, in comparison with conventional media.     

In vitro cloning of tumor stem cells in semi-solid media containing agar and agarose

Summary The formation of tumor stem cell colonies in vitro has been studied by comparing the growth of three mouse teratocarcinoma derived cell lines and one human teratocarcinoma derived cell line in semi-solid media containing either agar or agarose. We show that agaroses should be used in higher concentrations than agar to obtain comparable results. The maximum number of colonies were obtained in agarose over a broader range of concentrations (1%–4% for SeaPrep and 0.5%–2% for SeaPlaque agarose) than in agar, which allowed anchorage-independent growth of tumor cells only over a narrow concentration range (0.3%–0.5%). Overall, the preparation of media containing agarose was less cumbersome than preparation of agar-containing media, primarily because agaroses gelled more slowly and remained liquid in the physiological temperature range. Furthermore, the transfer of colonies from semi-solid media containing agarose to solid surface tissue culture dishes was much more efficient than the transfer of colonies from agar. The stock solutions of SeaPrep agarose could be kept ready for use for extended periods of time. All these features show that the low melting point agarose has considerable advantages over agar for preparation of semi-solid media for anchorage-independent tumor cell growth.     

Experimental adaptation to high and low quality environments under different scales of temporal variation

We investigated the role of the scale of temporal variation in the evolution of generalism in populations of the bacterium Pseudomonas fluorescens. Replicate populations were propagated as batch cultures for approximately 1400 generations (192 days), in either high quality media only, low quality media only, or were alternated between the two at a range of temporal scales (between 1 and 48 days). Populations evolved in alternating media showed fitness increases in both media and the rate of alternation during selection had no effect on average fitness in either media. Moreover, the fitness of these populations in high quality media was the same as for populations evolved only in high quality media and likewise for low quality media. Populations evolved only in high or low quality media did not show fitness improvements in their nonselective media. These results indicate that cost-free generalists can evolve under a wide range of temporal variation.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Effect of substrate on the production of antifungal volatiles from Bacillus subtilis

An antibiotic-producing strain of Bacillus subtilis has been shown to produce potent antifungal volatiles (AFV). These volatiles are active against a range of fungal species and are produced on a range of growth media and in loam-based compost. In vitro antifungal volatile activity on nutrient agar is enhanced with the addition of D-glucose, complex carbohydrates and peptones. The addition of L-glucose led to significantly less AFV activity than comparable levels of D-glucose. Growth studies in liquid culture revealed that B. subtilis failed to grow in response to L-glucose. Further growth studies on solid media showed no clear correlation between enhanced bacterial growth and increases in in vitro AFV activity in response to supply of substrates. Low level AFV activity was also detected from oilseed rape roots inoculated with B. subtilis . Gas chromatography mass spectrometry headspace analysis of B. subtilis cultures grown on various substrates revealed common similarities between substrates promoting AFV activity, although it was not possible to isolate individual antifungal compounds.     

Requirement of chelating compounds for the growth of Corynebacterium glutamicum in synthetic media

Summary The effects of various iron-complexing substances on the growth of Corynebacterium glutamicum in synthetic medium were investigated. The data obtained indicate that C. glutamicum has an absolute requirement for the presence of an iron-complexing compound as a growth factor for rapid and abundabnt growth in synthetic media. This requirement can be met by adding low concentrations (10^–5 M) of certain dihydroxyphenols (catechol, protocatechuate) or relatively high concentratuions (0.1%) of citrate to the medium or by autoclaving a small amount of glucose together with other media components. The addition of catechol or protocatechuate to synthetic media has advantages over media preparation with citrate or autoclaved glucose. In the described synthetic broth supplemented with catechol or protocatechuate growth is largely independent of the iron content of the medium in a range between 0.037 and 0.37 mM.Offprint requests to: W. Liebl     

Enthusiastic portrayal of 3D bioprinting in the media: Ethical side effects

There has been a surge in mass media reports extolling the potential for using three‐dimensional printing of biomaterials (3D bioprinting) to treat a wide range of clinical conditions. Given that mass media is recognized as one of the most important sources of health and medical information for the general public, especially prospective patients, we report and discuss the ethical consequences of coverage of 3D bioprinting in the media. First, we illustrate how positive mass media narratives of a similar biofabricated technology, namely the Macchiarini scaffold tracheas, which was involved in lethal experimental human trials, influenced potential patient perceptions. Second, we report and analyze the positively biased and enthusiastic portrayal of 3D bioprinting in mass media. Third, we examine the lack of regulation and absence of discussion about risks associated with bioprinting technology. Fourth, we explore how media misunderstanding is dangerously misleading the narrative about the technology.     

Cell culture medium improvement by rigorous shuffling of components using media blending

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.     

Parameter sensitivity analysis and improvement of a two-layer arterial wall model

The nonlinear two-layer arterial wall model introduced by von Maltzahn, et al. [11] is subjected to a rigorous parameter sensitivity and range of validity analysis. The model is based on the assumption that in large muscular conduit arteries the two mechanically significant layers are media and adventitia. Using curve-fitting techniques, the media is determined to be isotropic and the adventitia to be anisotropic. As a result of the range of validity analysis, the polynomial relationship for the energy density function of the media is changed to an exponential relationship. This leads to new coefficients for the polynomial of the adventitia. All coefficients have specific mechanical meanings. The parameter sensitivity analysis demonstrates convincingly that all model parameters are significantly important.     

Detection of Glu-Glu-tagged proteins in mammalian cell culture media by dot immunoblotting

A dot immunoblotting technique has been developed to estimate the relative expression levels of tagged recombinant human proteins in mammalian cell culture media. Variations in sample denaturation, blocking agents and membrane composition and treatment were used to optimize the signal-to-noise ratio of the defined procedure. The method is rapid, with sensitivity extending to the low nanomolar range for a number of recombinant proteins. This technique should have general utility for antibody-based measurements of other tagged and non-tagged proteins in cell culture media or in biological fluids.     

Plant regeneration from different explants in Helianthus smithii Heiser

Leaf, rhizome, and stem explants of Helianthus smithii, a wild relative of the cultivated sunflower, have been tested for their morphogenic potential on media containing a range of hormonal combinations including α-naphthaleneacetic acid and 6-benzylaminopurine (0–2 mg/l). Somatic embryos appeared on the majority of the tested media incorporating auxins, while shoots were observed on media containing exclusively 6-benzylaminopurine. Fertile plants were recovered from all explant types under a wide range of conditions, albeit with different efficiencies. The induction mechanism of the observed shoots is discussed. Received: 12 July 1996 / Revision received: 13 September 1996 / Accepted: 5 October 1996     

The effect of pH on the population growth of three species of duckweed: Spirodela oligorrhiza, Lemna minor and Wolffia arrhiza

Three species of duckweed, Spirodela oligorrhiza, Lemna minor and Wolffia arrhiza were grown under aseptic conditions on both buffered and unbuffered solutions of Jacob's media. Media with manually regulated pH levels were also used. Growth on unbuffered media is initially rapid but eventually inhibited, probably by increased pH levels. On buffered media growth is poor and effects of buffers cannot be separated out. These media give inadequate pictures of the species’ responses to changes in pH. Growth is most successful on media with regulated pH where sustained logarithmic population increases were achieved. Spirodela and Lemna rates are symmetrical about an almost neutral, optimal pH, declining fairly rapidly away from the optimum. Wolffia has an optimum at pH 5 and growth declined with increasing pH. All three species have optima at, or below, the neutral point. The range of tolerance of duckweeds is broader than has previously been suspected. Estimated lower limits, optimum and upper limits for each species are: Wolffia, pH 4·5–0·10, Lemna pH 4–6·2–10, Spirodela pH 3·7–0·10. Growth rate along a pH gradient is best described by means of polynomial equations: second-degree equations are sufficient for Spirodela and Lemna but a fifth-degree equation is required for Wolffia. Rates of population growth are similar for all species. In decreasing order they are: Wolffia, Lemna, Spirodela. However, in biomass units Lemna grew more than six and Spirodela seventeen times faster than Wolffia.     

Rapid estimation of glucose and amylase in fermentation culture broth with test strips by reflectance photometry

Summary The applicability of clinical multilayer test strips based on dry reagent chemistries for the quantitative analysis of fermentation media constituents was evaluated. For glucose and amylase determinations a significant correlation to conventional wet chemical methods was obtained. Bacterial biomass in the range of 0.5–5 g/l has no interfering effect. It is a rapid and cost-effective method for fermentation process monitoring and control.     

An antimicrobial removal device as an alternative to membrane filtration in the sterility testing of antibiotics

An Antimicrobial Removal Device (ARD) is proposed as an alternative to membrane filtration in the sterility testing of antibiotics. The device consists of a glass vial containing adsorbent resins which completely removed the antibacterial activity of a range of antibiotics without affecting the recovery of contaminating bacteria. The method is simple to use and has several advantages compared to membrane filtration including easier aseptic handling and lack of antibiotic carry-over to subculture media. A problem with resin turbidity in subculture media was overcome by the use of an indicator medium or by subculture into fresh medium.     

Kinetics of lipase-catalyzed esterification in supercritical CO(2)

This study compares two solvents for enzymatic reactions: supercritical carbon dioxide (SCCO(2)) and organic solvent (n-hexane). The model reaction that was chosen was the esterification of oleic acid by ethanol catalyzed by an immobilized lipase from Mucor miehei (Lypozyme). The stability of the enzyme appeared to be quite good and similar in both media but was affected by the water content. Partition of water between solvents and immobilized enzyme has been calculated from experimental adsorption isotherms. The water content of the solid phase has a dramatic influence on the activity of the enzyme and its optimum value for activity was about 10% (w/w) in both media. A kinetic study enabled a Ping-Pong Bi-Bi reaction mechanism with inhibition by ethanol to be suggested. Despite some differences in kinetic constants, activity was in the same range in both media. Hypotheses for explaining the kinetic constant variations have been proposed and particular attention has been paid to the pH effects.     

Liquid scintillation counting of 14C- and 3H-labeled samples on solid supports: a general solution of the problem

The data presented demonstrate the potential of surfactant-fortified scintillation cocktails in overcoming many of the problems encountered in the quantitation of radioactivity on solid supports. Using a broad range of representative ionic and polar biochemical compounds, the in vial elution and quantitative recovery of ³H and ¹⁴C-labeled components from a wide assortment of common solid support media has been demonstrated. This methodology in combination with zero elution counting systems and in some circumstances gelled suspension counting should combine to overcome most of the problems associated with determination of isotopic activity on such solid support media.