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.     

Reduction of a Tetrazolium Salt in Determining Growth Activity of Yeast-Phase Histoplasma capsulatum

A colorimetric method using a tetrazolium compound, 3,4,5-dimethylthiozalil-(1 or 2),2,5-diphenyltetrazolium bromide (TTBr), was developed for studying the growth activity of yeast-phase Histoplasma capsulatum. Materials extracted in phosphate buffer, pH 7.0, from cells at different stages of growth reduced TTBr. Colorimetric changes were correlated with enzymatic activity. Under standardized conditions specified herein, the optical density of the reduced tetrazole was an index of the growth activity of the organism.     

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.     

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.     

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.     

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.     

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.     

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.     

影响血清中GPI-PLD活性检测的因素

采用在碱性条件下正丁醇抽提的人胎盘膜上的碱性磷酸酶（ＡＬＰ）作底物, 检测血清中糖基磷脂酰肌醇－ 特异性的磷脂酶Ｄ （ＧＰＩ－ＰＬＤ） 的活性水平． 这种ＡＬＰ 含疏水的ＧＰＩ锚定膜结构（ａｎｃｈｏｒ）, 与血清保温后能被其中的ＧＰＩ－ＰＬＤ降解成亲水的不含ＧＰＩ－锚定的ＡＬＰ． 采用Ｔｒｉｔｏｎ Ｘ－１１４ 二相分离法和梯度凝胶电泳法来分离含ＧＰＩ的ＡＬＰ和不含ＧＰＩ的ＡＬＰ, 计算出转化率（％ ）, 用来表示ＧＰＩ－ＰＬＤ酶活性． 对这两种方法进行比较后, 表明在一般实验室条件下, 二相分离法更为简便, 并对其影响因素进行了全面探讨．     

Some Factors Affecting the Activity of Diethylpyrocarbonate as a Sterilant

Quantitative data indicated logarithmic death in 5 degrees Brix Concord grape juice when concentrations of cells under 10(7)/ml were exposed to diethylpyrocarbonate (DEPC). Species differed considerably in their resistance; e.g., 50 ppm reduced the viable count of Saccharomyces cerevisiae over nine log(10) cycles, whereas 200 ppm reduced the count of Byssochlamys fulva ascospores by only about 1 log. DEPC lethality was enhanced by higher temperatures; destruction at 40 C was 10- to 100-fold greater than at 20 C. Studies on death rates showed that most yeasts and fungal spores were killed during the first hour of exposure, whereas 24 h or longer was needed for maximal destruction of several lactic acid bacteria. Repair of DEPC-induced damage was believed responsible for the slower death rates of the lactics.     

Membrane Filtration of the Reiter Treponeme

A membrane filtration technique with commercially available membrane filters (Millipore Corp.) was effective for the removal of Reiter treponemes from liquids such as fluorescent-antibody conjugates, to which the organisms are added for adsorption. Reiter treponemes from an 8-day culture were not microscopically detectable in filtrates through membranes with a pore diameter of 0.45 μm, but treponemes were demonstrated in the filtrate by cultural methods. No organisms of the 8-day culture passed through a membrane filter having a pore size of 0.22 μm, as determined by microscopy and culture. Culture data indicated that a filter with a pore size of 0.1 μm was necessary to prevent passage of treponemes from 4-day cultures. It is recommended that a membrane filter with a pore size of 0.22 μm or smaller be used for the removal of Reiter treponemes from suspensions and that the age of the culture be considered in choosing filter pore size.     

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.     

Some Factors Affecting the Hill Reaction Activity in Cotton Chloroplasts

A method of plant culture was developed for growing large leaves of glandless cotton on single stems. Chloroplasts isolated from these leaves actively reduced ferricyanide when assayed for the Hill reaction. Hill reaction activity increased 133% when the 0.5 m sucrose isolation medium was replaced with 10% (w/v) polyethylene glycol, both buffered at pH 7.6. The presence of 2 or 5% (w/v) bovine serum albumin in the sucrose buffer did not increase Hill activity. Ferricyanide reduction in the dark occurred in all assays, and the possibility of gossypol as the reductant is discussed. Half-life of the chloroplasts stored in 10% glycerol at -23 C was 23 days. The ammonium ion at 0.01 m enhanced Hill reaction activity up to 171%. Leaves containing chloroplasts with the highest Hill reaction activity were found near the 8th node below the apex. Leaf water potentials less than -28 bars reduced the activity about 50%. Daylight conditions during the winter months in the greenhouse reduced the activity about 30%.     

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.     

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.