Factors in the Membrane Filtration of Enteroviruses

The filtration of two species of enteroviruses through membranes of porosity ranging from 50 to 220 mμ was studied. It was shown that extensive or total losses of virus may attend filtration at these porosities, apparently owing to adsorption of the virus to the membrane matrix. This could be minimized by the incorporation of serum into the virus suspension at the time of filtration, or by pretreating the membrane with serum or with a gelatin solution. It was also found that the first few drops of filtrate, even under optimal conditions, were likely to be virus-free, so that the filtration of too small a volume of virus suspension would result in a relatively great loss of titer. The degree to which these factors were critical was found to decrease with increasing pore diameter.     

Tetrazolium Reduction as a Measure of Metabolic Activity for Glass-Adherent Mycoplasma pneumoniae

The reduction of tetrazolium was used to assay the metabolic capability of developing Mycoplasma pneumoniae cultures on glass. Generally, the amount of tetrazolium reduced correlates with the amount of growth as measured by protein. Until a culture enters the late phase of the growth cycle, the drop in pH of culture medium provides similar information. In this last stage of growth, protein appears to be leveling. The pH continues to fall, but tetrazolium reducing activity decreases. Thus, considering the entire M. pneumoniae growth cycle, formazan production is a more reliable measure of metabolic capability of the organisms than either protein or pH. The reduction of tetrazolium provides a quantitative means of assessing enzymatic activities of glass-adherent M. pneumoniae.     

Factors Affecting the Activity of Phenolic Disinfectants

Low challenge phenol coefficient and high challenge use-dilution tests were made on a neutral cocoanut oil soap emulsion of o-phenylphenol and aqueous solutions of sodium o-phenylphenate prepared in the laboratory from the phenol using a stoichiometric amount of NaOH as well as with increasing amounts of excess NaOH. The phenol had considerably greater activity in both test methods when emulsified with the neutral soap than when converted to the phenate and dissolved in water. Use dilution test results against Salmonella choleraesuis with both the phenol and the phenate were within the range which would have been predicted from the Salmonella typhosa coefficient results employing the conventional conversion multiple of 20 to determine the maximal number of parts of water to which one part of germicide could be added. With the emulsified phenol this was also true where Staphylococcus aureus was employed in both procedures. With the aqueous solution of the phenate the maximal safe use-dilution by the phenol coefficient found for S. aureus and the same conventional conversion procedure was roughly five times higher than the maximal safe use-dilution found by the use-dilution method. Results with aqueous solutions of the phenate to which increasing amounts of excess NaOH were added showed no significant differences in the phenol coefficient method with either S. typhosa or S. aureus. In the use-dilution method, significant decreases in activity were found as the excess NaOH was increased to 4% with both S. choleraesuis and S. aureus. Although the pH values of aqueous solutions of the phenate were raised as the amount of free NaOH was increased, the decreases in pH observed as the dilution with water was increased were such that only small differences existed at the high critical killing dilutions found in the low challenge phenol coefficient method, whereas rather large differences existed at the lower critical killing dilutions in the high challenge use-dilution method.     

Factors Affecting the Cellulolytic Activity of Rumen Contents

The cellulolytic activity of rumen contents was assayed by measuring losses in weight and tensile strength of cotton yarn incubated in rumen contents in the presence of dietary additives (barley, tallow) and at different pH values. The addition of barley depressed cellulolysis and the titer of filter paper-degrading bacteria only if the pH was allowed to fall. Lowering the pH from 7.0 to 6.0 by addition of HCl almost completely inhibited attack of cotton and greatly reduced the titer of filter paper-degrading bacteria. The layering of tallow on cotton inhibited attack of cotton but did not decrease the titer of filter paper-degrading bacteria. The results are discussed with special reference to the importance of the study of cellulosic substrates, which require a known enzyme or mixture of enzymes for attack.     

Polarographic Determination of Respiratory Activity with Clark Oxygen Electrode

The authors have developed a polarographic technique in which Clark oxygen electrode (stationary platinum electrode covered with a polyethylene membrane) is employed for the determination of respiratory activity. The procedure of the operation is very simple, yet it was highly sensitive to the changes of the oxygen concentration in the solution, and the value of current was proportional to the oxygen concentration. The respiratory activity and control of rat liver mitochondria could be determined satisfactorily by this method.     

An Evaluation of Three New-Generation Tetrazolium Salts for the Measurement of Respiratory Activity in Activated Sludge Microorganisms

XTT (3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and WST–1 (4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate) are tetrazolium salts that have become commercially available only in relatively recent years; they differ from earlier such compounds in that their reduction gives rise to a formazan product that is water soluble. We have established the sites in the prokaryotic respiratory chain at which each of the dyes is reduced to its corresponding formazan and have evaluated the suitability of each for the colorimetric estimation of electron transport system activity in populations of activated sludge microorganisms. Reduction of all three tetrazolium salts was shown to be proportional to cell biomass and oxygen uptake and to be susceptible to low levels of the reference toxicant 3,5-dichlorophenol. XTT, which was not inhibitory at concentrations of up to 2 mM and was reduced by 91% of isolates from a sample of culturable activated sludge bacteria, was chosen for further assay development. XTT-formazan production was found to be stimulated by the availability of an exogenous carbon and energy source, and by the presence of the electron-coupling agent phenazine methosulfate. Less than 3% of XTT reduction by an activated sludge sample was abiotic. An assay based on this compound could be a valuable and simple tool for the routine monitoring of the performance of wastewater treatment systems.     

Factors Affecting the Methanogenic Activity of Methanothrix soehngenii VNBF

Methane production by Methanothrix soehngenii VNBF grown on acetate (50 mM) as the sole carbon and energy source was influenced by the addition of Fe, trace elements, and pesticides. The addition of Fe and trace elements significantly enhanced the rate of CH₄ production. The addition of pesticides in the early growth phase caused complete inhibition. However, less inhibition was noted when pesticides were added during early exponential growth phase. Addition to culture tubes of Co, Ni, or Mo at 2 μM produced 64, 41, or 17%, respectively, more CH₄ than that produced in tubes lacking the corresponding trace element. A concentration of more than 5 μM of these trace elements in the medium resulted in decreased CH₄ production, presumably because of toxic effects.     

Factors Affecting the Antimicrobial Activity of Vitamin K5

Pure cultures of Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphylococcus aureus, and Pseudomonas fluorescens were used in this investigation. The bactericidal concentrations of vitamin K(5) required for E. coli, B. subtilis, P. vulgaris, S. aureus, and P. fluorescens; the effect of an absence of oxygen; the effect of contact time with E. coli and S. aureus; and the effect of initial counts per milliliter of E. coli were studied. The bactericidal concentrations ranged from 60 ppm of K(5) for S. aureus to 220 ppm for E. coli, with an initial count of 160,000 to 200,000 cells per milliliter and a contact time of 12 hr in nutrient broth. The gram-positive bacteria tested were more susceptible to the antimicrobial activity of vitamin K(5) than the gram-negative bacteria. In the studies conducted under nitrogen atmosphere, the per cent inhibition showed an inverse relationship to the bactericidal concentrations required for complete inhibition in studies conducted under air atmosphere. This finding suggested that there might be different factors responsible for inhibition depending on the species of bacteria being tested, and it also might help explain the difference in concentrations necessary for inhibition. Cells of E. coli and S. aureus were not inhibited immediately on coming into contact with vitamin K(5); 50% inhibition occurred after 25 and 32 min, respectively. A rapid inhibition rate was maintained until approximately 90% inhibition occurred, after whch a rapid decrease in the rate was noted.     

Flow Cytometric Analysis of 5-Cyano-2,3-Ditolyl Tetrazolium Chloride Activity of Marine Bacterioplankton in Dilution Cultures

The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not “activate” a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.     

Chemical Factors Affecting Palmelloid-Forming Activity of Chloroplatinic Acid on Chlamydomonas eugametos

In order to elucidate the physiological mechanism of palmelloid formation of the unicellular green alga Chlamydomonas eugametos by chloroplatinic acid, the factors causing inhibition of the palmelloid formation were studied in various media. Chloroplatinic acid lost its palmelloid-forming effect in the presence of nitrite ion and amines having a second functional group. Nitrate, potassium, and sodium ions and monofunctional amines were without influence on the effect of chloroplatinic acid. These results indicate that the active form of platinum (IV) may undergo ligand substitution, the resulting complex being unable to cause palmelloid formation.     

絮凝-膜法制备皂荚皂苷工艺研究

对皂荚种皮皂苷进行水法提取,采用阳离子瓜尔胶对提取液进行絮凝,再通过超滤、纳滤进行分离,制备获得的皂苷纯度为87.3%.研究认为,提取液中的皂苷与多糖、蛋白等杂质结合紧密,而阳离子瓜儿胶对杂质具有良好的选择性,因而絮凝处理是保证膜法分离过程的关键,其二者联用显示出良好的应用前景.     

Factors Affecting Activity and Stability of the Kluyveromyces lactis Killer Toxin

A number of physical parameters determining the activity and stability of the killer toxin produced by the yeast Kluyveromyces lactis have been investigated. The toxin was active over a relatively narrow pH range of 4.4 to 5.8, with a maximum at the lower end of the range. However, it was stable up to at least pH 8.0 but appeared to be irreversibly inactivated below pH 4.4. The toxin was stable at 40°C but rapidly inactivated at 50°C. Strong agitation caused the inactivation of the toxin in one medium but not another; this seemed to be due to oxygen-mediated disulfide bond formation, which could be prevented by sulfhydryl protecting agents.     

Toxic Effects on Bacterial Metabolism of the Redox Dye 5-Cyano-2,3-Ditolyl Tetrazolium Chloride

The monotetrazolium redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used as a vital stain of actively respiring bacteria for several years. In this study, inhibitory effects on bacterial metabolism of this redox dye have been examined in a brackish water environment (Kiel Fjord, Germany) and a freshwater environment (Elbe River, Germany). As the results from time series experiments (1 to 10 h) show, bacterial growth and respiration of the investigated natural communities were clearly reduced by CTC supply. Compared with untreated controls (100%), CTC-treated samples showed distinctly lower heterotrophic bacterial plate counts (0 to 24 and 11.8 to 23.7%, respectively), bacterial production (0.9 to 14.1 and 1.1 to 9.6%, respectively), bacterial respiration (4.1 to 9.4 and 6.8 to 43.8% for several concentrations of (sup14)C-labeled glucose), and [(sup14)C]glucose incorporation (0.2 to 4.2%). Additionally, toxicity of CTC was demonstrated by luminescence in a Microtox bioassay. CTC concentrations of 0.1 and 5.0 (mu)M required only 15 min for decreases of approximately 50 and 100%, respectively. The suppression of CTC on several bacterial metabolic processes suggests that determination by the CTC technique underestimates the actual number of active cells distinctly. This conclusion is confirmed by the comparison of generation times calculated on the basis of thymidine uptake data and active bacterial counts determined by the CTC assay and microautoradiography. While unrealistic short generation times (0.5 to 5 h) resulted from the CTC assay, the generation times calculated according to microautoradiography ranged within values (7 to 21 h) reported elsewhere for comparable aquatic environments. The inhibitory effect of CTC demonstrated in our experiments is an aspect with regard to the application of this tetrazolium dye for the estimation of active bacteria in natural aquatic environments which hitherto has not been considered.     

Factors Affecting the Activity of Cellulases Isolated from the Rumen Digesta of Sheep

Sodium phosphate buffer was used to extract cellulases from the plant solids fraction of rumen contents. The mixed cellulase preparation had maximal activity at pH 6.9 and 49°C. The V_max and the apparent K_m for wheaten hay cellulose were 19.8 glucose units/min and 6.35 mg/ml, respectively, and for microcrystalline cellulose (Sigmacell) at the same enzyme concentration, they were 33 glucose units/min and 27.5 mg/ml, respectively. For these assays a glucose unit was defined as nanomoles of glucose plus twice the nanomoles of cellobiose. Consideration of thermodynamic and kinetic data suggested that the hydrolysis of a relatively labile arabino-xylan comprising 3% of the wheaten hay cellulose was dependent on prior removal of the protecting β-1,4-glucose chains at the outer surface of the cellulose preparation. Sequential removal of structural polysaccharides from the plant cell wall rendered the latter more susceptible to cellulase activity. Cellulase activity was stimulated by increasing the concentration of phosphate from 5 to 50 mM. The stimulation was magnified in the presence of cell-free rumen fluid. Cellulase activity was not stimulated by calcium, magnesium, iron, zinc, manganese, copper, or cobalt ions and was unaffected by the chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. O-phenanthroline inhibited activity by 30 to 50%, but this may have been due to nonchelate properties. Anaerobic conditions or thiol protective agents were not essential for either the activity or stability of the cellulases during assay. An ultrafiltrable inhibitor of cellulase activity was detected in cell-free rumen fluid.     

Efficient Filtration and Sizing of Viruses with Membrane Filters

Untreated membrane filters retain viruses by adsorption, as well as by physical restriction which occurs when the pore diameter of the filter is smaller than that of the virus particle. As originally recommended by Elford, membranes had to be pretreated with proteinaceous material to preclude virus adsorption. However, coating materials that prevent adsorption of certain viruses do not necessarily prevent adsorption of other viruses. In contrast to proteins, salts enhance virus adsorption. Viruses treated with sodium lauryl sulfate to reduce the surface tension, or purified viruses in distilled water, are not adsorbed to membranes. A procedure is recommended by which viruses may be passed through membranes with a porosity twice the diameter of the virus. Such filtrates, which contain 50 to 100% of the initial virus concentration, should be used for sizing viruses by subsequent filtration through smaller pores. The determination of virus size would then be based on the major population of particles in the virus suspension. In the past, as little as 0.1 to 0.001% of the initial virus population was the basis for size determination, because more than 99.9% of the virus was often lost by adsorption to membranes during the clarifying procedures.     

Factors Affecting the Activity and Stability of Alkaline Phosphatase in a Marine Pseudomonad

Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.     

Factors Affecting the Antibacterial Activity of the Ruminal Bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h⁻¹) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2–1.2 h⁻¹) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h⁻¹) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h⁻¹) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml⁻¹) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application. Received: 6 February 2002 / Accepted: 27 March 2002     

Glutamate Catabolism of Rickettsia rickettsi and Factors Affecting Retention of Metabolic Activity

Glutamate catabolism and the factors contributing to metabolic stability of purified suspensions of Rickettsia rickettsi were investigated. By use of (14)C-glutamate, it was shown that CO(2) was produced from all carbons of glutamate and that (14)CO(2) production was reduced by the addition of most of the unlabeled intermediates of the citric acid cycle and pyruvate. Oxalacetate, added in various concentrations, did not stimulate glutamate utilization. When the cells were suspended in bovine plasma albumin (BPA), CO(2) production from glutamate proceeded at a nearly uniform rate for 8 hr at 32 C and for 24 hr at 15 C. When BPA was used, the cells retained their metabolic activity at 0 or 30 C regardless of cell concentration, and were not influenced by the addition of varoius metabolites. Without BPA, metabolic stability was directly related to concentration. Of the stabilizers tested on low concentrations of rickettsiae, reduced glutathione was the most effective, provided that the gas phase contained predominantly N(2). Under these conditions of low partial pressure of O(2), glutamate further stabilized metabolic activity and was actively metabolized. The cells were also stabilized by oxidized glutathione in a gas phase of air, but under these conditions glutamate was utilized at a more moderate rate and it impaired metabolic stability.