Stability of tissue culture medium pH as a function of autoclaving,time, and cultured plant material

Autoclaving is a standard procedure for sterilizing nutrient media for plant tissue cultures. Most tissue cultures are grown at pH 5.2 to 5.8 with pH adjustments being made prior to autoclaving. This paper reports that there are significant differences between initial pH levels and pH levels following autoclaving, particularly in the pH range of 5.7 to 8.5. This effect is noted with and without agar. In addition, we report that with time the pH of the medium drifts into the acid range. When Cucumis callus was added to the medium, the pH was changed significantly within 48 hours. The amount and direction (increase or decrease of pH) was significantly correlated with the original pH. This suggests that researchers should be wary of the true pH situation in their medium. In addition, in publications authors should specify whether their medium pH value was determined before or after autoclaving.     

Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

Ion movement through gramicidin A channels. Studies on the diffusion-controlled association step

The permeability characteristics of gramicidin A channels are generally considered to reflect accurately the intrinsic properties of the channels themselves; i.e., the aqueous convergence regions are assumed to be negligible barriers for ion movement through the channels. The validity of this assumption has been examined by an analysis of gramicidin A single-channel current-voltage characteristics up to very high potentials (500 mV). At low permeant ion concentrations the currents approach a voltage-independent limiting value, whose magnitude is proportional to the permeant ion concentration. The magnitude of this current is decreased by experimental maneuvers that decrease the aqueous diffusion coefficient of the ions. It is concluded that the magnitude of this limiting current is determined by the diffusive ion movement through the aqueous convergence regions up to the channel entrance. It is further shown that the small-signal (ohmic) permeability properties also reflect the existence of the aqueous diffusion limitation. These results have considerable consequences for the construction of kinetic models for ion movement through gramicidin A channels. It is shown that the simple two-site-three-barrier model commonly used to interpret gramicidin A permeability data may lead to erroneous conclusions, as biionic potentials will be concentration dependent even when the channel is occupied by at most one ion. The aqueous diffusion limitation must be considered explicitly in the analysis of gramicidin A permeability characteristics. Some implications for understanding the properties of ion-conducting channels in biological membranes will be considered.     

Growth of Mycobacterium lepraemurium in Cultures of Mouse Peritoneal Macrophages

Successful growth of Mycobacterium lepraemurium was observed in cultures of mouse peritoneal macrophages. The optimal host cell maintenance medium was composed of 40% horse serum, 50% of the chemically defined medium NCTC 109, and 10% of a 1:5 dilution of beef embryo extract, supplemented with both liver extract and ferric nitrate. Multiplication of the bacilli was observed in 1 week and maximal growth in 6 to 7 weeks. All macrophages were filled with tens to hundreds of the organisms in cultures showing maximal growth. Glycerol caused an increase in the normal length of M. lepraemurium, without a corresponding increase in the number of the bacilli. Elongation of M. lepraemurium was observed 3 or 4 days after infection. Rapid and uniform growth of M. lepraemurium was achieved in serially transferred cultures (subcultures). The cumulative increase of the number of intracellular bacilli was 1.4 x 10(20)-fold in 14 transfers over a period of 68 weeks in one series, and 10(17)-fold in 12 transfers over a period of 56 weeks in another series. The generation time of M. lepraemurium was 7 days, a growth rate which approximates the fastest growth of the organisms in vivo. Organisms harvested from cultures at various stages of growth produced murine leprosy in mice, but showed no growth in bacteriological media. The present model offers an opportunity for studies on the host-parasite relationship without the complication of extracellular growth of the parasites.     

A Monitor for Airborne Bacteria

A unique agar drum sampler is described which indicates, continuously, the number of viable, bacterial particles per unit volume of air at the time and point of sampling. By selection of the timer and the sampling rate the sampler is suitable for quite a wide range of concentration and time. An impaction line of 484 in. greatly increases the capacity of this device over slit samplers and other instruments designed to give time-concentration data for viable airborne particles. This sampler should prove useful for: (i) monitoring airborne bacteria in hospitals, public places, and food and industrial plants; (ii) decay rate studies of bacterial aerosols; (iii) evaluation of aerial germicides; (iv) determination of effectiveness of air conditioning systems in removing airborne bacteria; and (v) many other studies in aerobiology.     

The effect of whey protein hydrolyzates on the lactic acid fermentation

Summary The batch fermentation of whey permeate to lactic acid was improved markedly by the addition of enzymehydrolyzed whey protein. Acid concentrations greater than 90 g/l were achieved at a productivity of 4.3 g/l per h and a 98% substrate use. Cell mass concentration reached 6 g/l. The acid productivity achieved is somewhat higher than that typical for fermentation of whole whey. The process economics, based on in-house hydrolyzate preparation, look promising. Presented in this paper are the experimental results showing the effects of hydrolyzate concentration on acid and cell mass production.