The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

The effect of calcium concentration on the toxicity of copper, lead and zinc to yolk-sac fry of brown trout, Salmo trutta L., in soft, acid water

Yolk-sac fry of brown trout were exposed to three levels of single trace metals (Cu, 20,40,80 nmol 1^-1; Pb, 12·5,25,50 nmol 1^-1; Zn, 75,150,300 nmol 1^-1) typical of concentrations reported for acid soft waters, in flowing, artificial, soft water media maintained at pH 4·5 and [Ca] of 20 or 200 μmol 1^-1for 30 days.
Mortalities were high in fry subjected to all levels of the three trace metals at [Ca] 20 μmol 1^-1, with 80% of the total deaths occurring between days 11 and 15 of the experiment. 25% mortality occurred at low [Ca] and pH 4·5 in the absence of trace metals, with only one death recorded at [Ca] 200 μmol1^-1'(Cu, 80 nmol 1^-1). At high [Ca] all three levels of Cu and Pb impaired net Na and K uptake; Cu was the only metal to reduce the uptake of Ca. The Zn treatments had no significant effect on mineral uptake. Calcification of centra was reduced by all three Cu treatments at [Ca] 200 μmol 1^-1. The lowest Zn concentration (75 nmol 1^-1) was the only other treatment to impair skeletal development. In the absence of trace metals, low [Ca] significantly reduced Ca, Na and K uptake, skeletal calcification and dry mass at pH 4·5.
The deleterious effects of low Cu, Pb and Zn concentrations at low pH and low [Ca], and the ameliorative effect of higher ambient [Ca], are discussed in relation to fishery status in soft, acid waters.     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

Effect of bifidobacteria on nitrites and nitrosamines

The effects of six different bifidobacteria strains were studied on two procarcinogens: nitrite and nitrosamines. Growth of bifidobacteria was not affected by nitrite concentrations below 50 μmol 1^-1. At nitrite concentrations greater than 2000 μmol 1^-1, total growth inhibition was observed. Nitrite elimination by a non-enzymic mechanism was noted for six strains of bifidobacteria. Acids produced by the bacteria seem to be involved in nitrite elimination. Nitrosamines tested had no effect on growth of bifidobacteria. Only one strain ( Bifidobacterium longum BB 536) was able to metabolize nitrosamines by an intracellular mechanism.     

2,4-Dichlorophenoxyacetic acid inhibition of potassium transport in barley seedlings

Growth, potassium uptake and translocation as well as transpiration rates were measured in intact low-salt barley seedlings ( Hordeum vulgare L. cv. Union) in the presence of different 2,4-D concentrations at pH 6.5. Growth was only affected at 10^-3 M .
Above 10^-7 M 2,4-D both uptake by the roots and transport to the shoots were inhibited. The inhibition at 10^-5 M remained constant for at least 24 h. Furthermore inhibition of uptake was measurable within 1 h. Excised roots and roots of intact plants showed the same uptake pattern.
It is suggested that the observed effects were caused by 2,4-D-induced changes in uptake and translocation systems in the roots. Pre-treatment with 10^-5 M 2,4-D had no effect upon subsequent potassium uptake. Transpiration was reduced within 1 h in 10^-4 or 10^-3 M 2,4-D, probably due to changes in water transport or root permeability.     

Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate

Barley plants were grown in nutrient solutions, which were maintained at either 0 (-P) or 15 μ M orthophosphate (+P). After 11 days phosphate influx into the intact roots of the -P plants began to increase by comparison with +P plants. During this period differences became apparent between the treatments in absolute growth rates, as well as in the root:shoot ratios. Phosphate influx in the -P plants continued to increase as a function of time, to a maximum value of 2.4 μmol (g fresh wt)^-1h^-1 at 16 days after germination. This rate was 6 times higher than influx values for +P plants of the same age. During the period of enhanced uptake phosphate was strongly correlated (r²= 0.77) with root organic phosphate concentration. – The enhancement of inorganic phosphate influx into intact roots of -P plants was rapidly reduced by the provision of 15 μ M orthophosphate. Typically, within 4 h of exposure to this concentration of phosphate, influx values fell from 1.80 ± 0.20 to 0.75 ± 0.03 μmol (g fresh wt)^-1 h^-1, while inorganic phosphate concentrations of the roots increased from 0.12 to 1.15 μmol (g fresh wt)^-1 during the same period. Hill plots of the influx data obtained during this period, treating root inorganic phosphate as an inhibitor of influx, gave Hill coefficients close to 2. The rapidity of the reduction of influx associated with increased root inorganic phosphate together with the Hill plot data provide evidence for an allosteric inhibition of influx by internal inorganic phosphate.     

Isolation and properties of free and immobilized β-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

A study of the β-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4·45. Optimum pH was 7·25. Maximal activity was observed at 50°C and activation energy was estimated to be 39·1 kJ mol^-1. Lactose enhanced thermal stability. Using α-nitrophenyl-β-D-galactopyranoside as the substrate, the K _m was 11 μmol 1^-1 and V _max was 85 U mg^-1 protein. β-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. K _m was 47 μmol 1^-1 and V _max was 96 U mg^-1 protein.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

The respiration of young coho Oncorhynchus kisutch at gradually declining oxygen levels and during recovery from oxygen deprivation

The respiration of coho salmon, Oncorhynchus kisutch , weighing between 15 and 50 g was measured at gradually declining oxygen levels and at temperatures ranging between 14 and 17°C. The maximum and minimum oxygen concentrations tested were 250 and 40 μmol L⁻¹, respectively. Respiration rates were measured for 1 h periods before oxygen concentration was lowered by 12.5 or 25.0 μmol oxygen L⁻¹. At the end of these endurance tests the oxygen level was returned to normoxic conditions and respiration rates were determined for the recovery period. Under normoxic conditions (> 200 μmol L⁻¹) the respiration of coho levelled around 5.1 μmol g⁻¹ wet weight h⁻¹. At intermediate levels between 150 and 200 μmol oxygen L⁻¹, the average rate increased to 5.8 μmol g⁻¹ h⁻¹, which could be attributed to higher spontaneous activity of the test animals. At low oxygen levels (< 150 μmol⁻¹) average respiration rates dropped to values between 5.5 and 5.7 μmol g⁻¹ h⁻¹, reaching a minimum of 3.8 μmol g⁻¹ h⁻¹ at oxygen levels below 50 μmol L^μ. First mortality was observed in this range. After exposure to reduced oxygen levels the fish maintained a higher respiration rate when again exposed to normoxic oxygen levels above 200 μmol L⁻¹. Increased respiration rates were observed for a recovery period of 6 h.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

Sterilization of soybean cotyledons by boiling in lactic acid solution

F. RUÍZ-TERÁN AND J.D. OWENS. 1996. The effect of pH on the heat resistance of Bacillus stearothermophilus spores at 100°C in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 sodium phosphate buffer was examined. At pH values of 7.0 and 6.0 spores survived 60 min exposure unharmed but at pH 4.3 and 3.0 they died with decimal reduction times (DRTs) of 27 min and 2.8 min, respectively. Death rates were similar in the presence or absence of hydrated soybean cotyledons. In the presence of phosphate buffer and cotyledons at mean pH 3.6 the DRT was 118 min but in the presence, in addition, of lactic acid it was 11 min. It is suggested that the enhanced death rate was due to toxic effects of undissociated lactic acid. Rhizopus oligosporus NRRL 2710 grew well on cotyledons, having pH values from 7.0 to 3.7, prepared by boiling for 60 min in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 phosphate buffer.     

The occurrence and ecological importance of dissolved ATP in fresh water

SUMMARY. Dissolved ATP, defined as ATP which passes through 0.2 μm filters, was found in fresh water. During the spring diatom bloom in two eutrophic Danish lakes, concentrations of dissolved ATP varied between 0.1 and 3.8 μgl⁻¹, constituting 14–76% of the total ATP (particulate plus dissolved ATP). The kinetics of the light emission obtained from mixing firefly enzyme with dissolved ATP demonstrated that the major proportion of the dissolved ATP was in fact ATP. Despite some variations, the seasonal changes in dissolved ATP paralleled the changes in the increasing phytoplankton population during the rise of the diatom blooms. The dissolved ATP increased after the diatom peak, indicating that release of ATP from the phytoplankton due to mortality may be a major source of dissolved ATP.
Consumption of dissolved ATP was evaluated in uptake experiments using ³H-ATP. Rates of uptake of ³H-ATP by micro-organisms (diameter 0.2–0.6 μm) proved to be close to the rates for ³H-D-glucose uptake. The variations in ³H-ATP uptake during the diatom blooms showed non-systematic changes and ranged between 1.0 and 15.8% h⁻¹ (mean = 4.9% h⁻¹) of the quantity added. Turnover rates for dissolved ATP varied between 12 and 730 ng l⁻¹ h⁻¹ (mean = 175 ng l⁻¹). These rather high rates of turnover suggest that dissolved ATP is an important compound in the metabolism of freshwater bacteria.     

Oxygen Perception and O2 Toxicity in the Freshwater Ciliated Protozoon Loxodes

ABSTRACT. Loxodes reached peak abundance close to the oxic-anoxic boundary (O₂ 5% atm) in two lakes, in test tube cultures, and in glass chambers with horizontal O₂ gradients. Vertical profiles of CO₂, pH, sulfide, and Fe²⁺ in a lake were not closely related to Loxodes abundance. In a laboratory experiment, Loxodes followed a retreating source of O₂ and was repelled by a high pO₂. This behavior was sustained when cells simultaneously swam up or down gradients of both CO₂ and pH. Aggregation of cells was abolished by KCN (10^-4-10^-6 M). Sodium azide (10^-1-10^-4 M) had no effect and 2,4-DNP sharpened the aggregation. Rotenone, Antimycin A, and HOQNO had no obvious effect. Cytochrome oxidase is probably the oxygen receptor. Loxodes striatus contained low activities of superoxide dismutase and catalase. Extracellular production of superoxide (O_-²) and hydrogen peroxide (H₂O₂) were probably not responsible for the exclusion of Loxodes from water with a high pO₂. Continuous exposure of Loxodes to oxygen at normal atmospheric pressure at 10°C led to 50% mortality in 10 days. Cells left free to swim in an oxygen gradient doubled their number in the same period. Light exacerbated the toxic effects of O₂. Behavioral responses to the dissolved oxygen tension probably controlled the spatial distribution of Loxodes.     

Involvement of potassium ions in the action of various antineoplastic drugs on the growth of Saccharomyces cerevisiae

The involvement of potassium ions in the action of some antineoplastic drugs on the growth of Saccharomyces cerevisiae was studied by incubating yeast cells in the presence of drugs at various concentrations and KC1 at concentrations of 50 mmol 1^-1 and 100 mmol 1^-1. The presence of 6.25–50 μg m1^-1 amsacrine or melphalan alone in the culture medium had no significant effect on yeast growth. Addition of KC1 significantly increased the sensitivity to these drugs. On the contrary, incubation of yeast cells with KC1 had no effect on the cytotoxic action of doxorubicin, methotrexate or 5-fluorouracil.     

Some effects of low pH and calcium on the growth and tissue mineral content of yearling brown trout, Salmo trutta

Yearling brown trout, Salmo trutta , were exposed to low mineral content water (nominal concentrations of 20μmol 1⁻¹ magnesium, 7.7 μmol 1⁻¹ potassium, 44 μmol 1⁻¹ sodium) over a pH range of 4.0–5.2 with ambient calcium concentrations of 2.5–60 μmol 1⁻¹. All fish died at pH 4.0 and 4.2 irrespective of ambient calcium concentration and also at pH 4.4 with only 2–3 μmol 1 ⁻¹ calcium (that is calcium-free water except for that leached from the diet or excreted by the fish). Good growth rates were obtained over the remaining treatments which extended down to pH 4.4 with as little as 7 μmol 1⁻¹ calcium. When starved, weight loss was inversely correlated with pH. Effects on plasma chloride, percentage dry weight and calcium, potassium sodium, and phosphorus contents of skin, muscle and bone tissue were also investigated. These demonstrated pH effects on mineral metabolism in starved fish, but no effects were detected in fed fish.     

Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.