Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural water.

A technique is described for the determination of bacterial numbers and the spectrum of actively metabolizing cells on the same microscopic preparation by a combined autoradiography/epifluorescence microscopy technique. Natural bacterial populations incubated with [(3)H]glucose were filtered onto 0.2-mum Nuclepore polycarbonate membranes. The filters were cut into quarters and fixed on the surface of glass slides, coated with NTB-2 nuclear track emulsion (Kodak), and exposed to the radiation. After processing, the autoradiographs were stained with acridine orange. A combination of overstaining on the slightly alkaline side and gradual destaining on the acid side of neutrality gave the best results. Epifluorescence microscopy revealed bright-orange fluorescent cells with dark-silver grains associated against a greenish-to-grayish background. Based on the standardization curves, detection of actually metabolizing cells was optimal when cells were incubated with 1 to 5 muCi of [(3)H]glucose per ml of sample for 4 h and the autoradiographs were exposed to NTB-2 emulsion at 7 degrees C for 3 days. In water samples taken immediately above sandy sediments at beaches of the Kiel Fjord and the Kiel Bight (Baltic Sea, FRG), between 2.3 and 56.2% (average, 31.3%) of the total number of bacteria were actually metabolizing cells. Spearman rank correlation analysis revealed significant interrelationships between the number of active bacteria and the actual uptake rate of glucose.     

High Motility Reduces Grazing Mortality of Planktonic Bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 μm s⁻¹ as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 μm s⁻¹) and a highly motile strain (>45 μm s⁻¹) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 μm³. Grazing mortality was lowest for cells of >0.5 μm³ and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (≤0.1 μm³, >50 μm s⁻¹) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Selective Inhibition by 2-Bromoethanesulfonate of Methanogenesis from Acetate in a Thermophilic Anaerobic Digestor

The effects of 2-bromoethanesulfonate, an inhibitor of methanogenesis, on metabolism in sludge from a thermophilic (58°C) anaerobic digestor were studied. It was found from short-term experiments that 1 μmol of 2-bromoethanesulfonate per ml completely inhibited methanogenesis from ¹⁴CH₃COO⁻, whereas 50 μmol/ml was required for complete inhibition of ¹⁴CO₂ reduction. When 1 μmol of 2-bromoethanesulfonate per ml was added to actively metabolizing sludge which was then incubated for 24 h. it caused a 60% reduction in methanogenesis and a corresponding increase in acetate accumulation; at 50 μmol/ml it caused complete inhibition of methanogenesis and accumulation of acetate. H₂, and ethanol.     

Frequency of Dividing Cells as an Estimator of Bacterial Productivity

It has recently been proposed that the frequency of dividing bacterial cells (FDC) can be used to predict growth rates of natural aquatic bacterial assemblages. We have examined the relationship between FDC and growth rate in bacteria from southern-temperate, coastal marine waters by using incubation under conditions of manipulated nutrient availability and exclusion of bacterivores. The regression of the natural logarithm of bacterial instantaneous growth rate (μ) on FDC resulted in a better fit than regression of untransformed μ on FDC. The regression equation was ln μ = 0.299FDC − 4.961. The coefficient of variation for predicted ln μ at mean FDC was 7%. The range of FDC-estimated bacterial instantaneous generation times for coastal Georgia waters was 12 to 68 h, and range of calculated bacterial production rates was 0.6 to 17.6 mg of C·m⁻³· h⁻¹. Unresolved problems of and suggested improvements on the FDC method of predicting growth rate are discussed.     

Carbon- and Nitrogen-to-Volume Ratios of Bacterioplankton Grown under Different Nutritional Conditions

Carbon- and nitrogen-to-volume (C/V and N/V) ratios were determined for freshwater bacterial assemblages grown in lake water filtrate or in water enriched with nutrients (aqueous extract of lake seston, glucose, arginine, phosphate, or ammonium). Biovolume was measured by epifluorescence microphotography, and carbon and nitrogen biomasses were measured with a CHN analyzer. Despite large variations of nutritional conditions (i.e., the composition and concentration of the dissolved organic carbon) and different mean cell sizes of the bacterial assemblage (0.17 to 1.8 μm³ per cell), the C/V, N/V, and carbon-to-nitrogen weight ratios varied little (C/V ratio, 0.14 pg of C per μm³ [standard deviation, 0.057; n = 15]; N/V ratio, 0.027 pg of N per μm³ [standard deviation; 0.011, n = 15]; carbon-to-nitrogen weight ratio, 5.6 [standard deviation, 2.2, n = 15]). An average C/V ratio of 0.12 pg of C per μm³ that was derived from natural and cultured bacterial assemblages is proposed as an appropriate conversion factor for estimation of the biomass of freshwater bacteria.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10¹⁰ to 2.6 × 10¹² (average, 7 × 10¹¹) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10¹² cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10¹⁰ to 1.5 × 10¹² (average, 4 × 10¹¹) cells per g (dry weight) of slime was obtained.     

Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters

The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm³. Cell volume ranged from 0.072 to 0.096 μm³ at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm³ in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from ³H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria.     

Measurement of Glucose Utilization by Pseudomonas fluorescens That Are Free-Living and That Are Attached to Surfaces

The assimilation and respiration of glucose by attached and free-living Pseudomonas fluorescens were compared. The attachment surfaces were polyvinylidene fluoride, polyethylene, and glass. Specific uptake of [¹⁴C]glucose was determined after bacterial biomass was measured by (i) microscopic counts or (ii) prelabeling of cells by providing [³H]leucine as substrate, followed by dual-labeling scintillation counting. The glucose concentration was 1.4, 3.5, 5.5, 7.6, or 9.7 μM. Glucose assimilation by cells which became detached from the surfaces during incubation with glucose was also measured after the detached cells were collected by filtration. The composition of the substratum had no effect on the amount of glucose assimilated by attached cells. Glucose assimilation by attached cells exceeded that by free-living cells by a factor of between 2 and 5 or more, and respiration of glucose by surface-associated cells was greater than that by free-living bacteria. Glucose assimilation by detached cells was greater than that by attached bacteria. Measurements of biomass by microscopic counts gave more consistent results that those obtained with dual-labeling, but in general, results obtained by both methods were corroborative.     

Changes in Bacterial Community Composition and Dynamics and Viral Mortality Rates Associated with Enhanced Flagellate Grazing in a Mesoeutrophic Reservoir

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-μm treatment) or enhance (<5-μm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the “grazer-free” (0.8-μm-filtered) treatment, subject only to a relatively low mortality rate (~17% day⁻¹) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-μm filtrate), grazing mortality was ~200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.     

Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria

Iodide (I⁻)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive ¹²⁵I⁻. When they were grown in a liquid medium containing 0.1 μM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 μM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 ± 3.6 (mean ± standard deviation) pmol of iodide per mg of dry cells and 5.5 × 10³, respectively. In the presence of much higher concentrations of iodide (1 μM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 μM and 0.55 pmol min⁻¹ mg of dry cells⁻¹, respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO₃⁻), was not transported.     

Change in Size of Chromatium minus Cells in Relation to Growth Rate, Sulfur Content, and Photosynthetic Activity: A Comparison of Pure Cultures and Field Populations

The size frequency distribution of planktonic cells of purple sulfur phototrophic bacteria was measured at several depths in a bacterial layer of Lake Cisó (Spain). The bacterioplankton was dominated by Chromatium minus (87 to 94% of the total biomass). The largest cells of C. minus were found in the top part of the bacterial layer. In addition, the in situ and potential specific photosynthetic activity (CO₂ fixation and acetate uptake) and specific pigment content were measured in relation to several key environmental parameters that determine the activity of cells. Potential growth rates were estimated from production rates and biomass. A maximal specific growth rate of 0.074 h⁻¹ was found for the top part of the bacterial layer. Photosynthesis versus light and versus sulfide curves among field samples indicated that light was the main limiting factor controlling the activity of C. minus in Lake Cisó. The specific bacteriochlorophyll a content was very high in all samples (0.27 to 0.36 μg μg of C⁻¹). Results of laboratory experiments performed with pure cultures indicated that the average cell volume changes from 5.9 to 20.0 μm³ and that differences in growth rate, breakdown, or synthesis of sulfur and glycogen and degradation of the photosynthetic apparatus are the main factors accounting for the observed changes in cell volume across the bacterial layer.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N2O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N₂O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg⁻¹. Without added C, peak NO emissions of 4 μg of N kg⁻¹ h⁻¹ were increased to 15 μg of N kg⁻¹ h⁻¹ by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 μg of N kg⁻¹ h⁻¹ in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 μg of N kg⁻¹ h⁻¹ after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N₂O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N₂O produced during nitrification in soil.     

Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures

The administration of inorganic Cd and Hg in vivo has been shown to result in markedly different metal concentrations in rat liver. Primary cultures of rat hepatocytes were utilized to gain insight into the dispositional differences between these chemically similar metals. Hepatocyte monolayer cultures were exposed to several concentrations of Cd or Hg (3, 10 and 30μm) in serum-containing medium for 30min. The cells were then washed and incubated in fresh medium for the remainder of the experiment. Hepatocytes exposed to Cd accumulated significantly more metal than hepatocytes exposed to equimolar concentrations of Hg. In cells exposed to 3μm-Cd there was an initial loss of Cd from the hepatocytes when placed in fresh medium, followed by a gradual re-uptake of metal, concomitant with increased binding to metallothionein. In hepatocytes exposed to 3 and 10μm-Cd, 87 and 77% of the intracellular Cd was bound to metallothionein within 24h. Loss of Hg from hepatocytes pulsed with 30μm-Hg was also observed upon the addition of fresh medium and continued for the duration of the experiment. No time-dependent increase in Hg binding to metallothionein was observed. A maximum of about 10% of the intracellular Hg was found associated with metallothionein in hepatocytes exposed to 30μm-Hg. Studies utilizing [³⁵S]cysteine incorporation indicated significant increases in the amount of metallothionein synthesized in hepatocytes exposed to 3 and 10μm-Cd (300% of control value) and 30μm-Hg (150% of control value) 24h after metal pulsing. Time-course studies revealed a 6–12h lag in metallothionein synthesis, followed by a significant elevation in [³⁵S]cysteine incorporation into metallothionein between 12 and 24h. These studies suggest that (a) isolated hepatocytes differentiate between Cd and Hg and preferentially accumulate the former, and (b) Cd strongly stimulates the induction of, and preferentially binds to, metallothionein, whereas Hg induces weakly, and does not preferentially bind to, metallothionein.     

Evidence for a Terpene-Based Food Chain in the Gulf of Alaska

A mixture of ¹⁴C-terpenes was prepared from conifer seedlings and introduced into fresh seawater samples taken near Seward, Alaska. Initial rates of oxidation by the indigenous bacteria were linear and faster than the rates of toluene oxidation. Turnover times were 4 to 19 days. Autoradiographic measurements with ³H-terpenes indicated that at least 10% of the 0.6 × 10⁹ to 2.7 × 10⁹ bacteria per liter present could catabolize terpenes. The rate of terpene oxidation, 24 μg of terpenes per g of cells per h with 3 μg of terpenes added per liter, was a constant function of bacterial biomass. The specific affinity of the process was estimated to be between 8.1 and 81 liters/g of cells per h, indicating a high state of induction and the probable presence of terpenes. Terpene-oxidizing bacteria were grown on [¹⁴C]alanine and added to fresh seawater samples. Transfer of the bacterial radioactivity into larger particles at a rate of 146 pg/liter per h from the 2.3 × 10⁹ organisms added indicated that any terpenes present would participate in the food chain.     

Relationships between Biovolume and Biomass of Naturally Derived Marine Bacterioplankton

Microscopic estimation of bacterial biomass requires determination of both biovolume and biovolume-to-biomass conversion. Both steps have uncertainty when applied to the very small bacteria typically found in natural seawater. In the present study, natural bacterioplankton assemblages were freshly collected, passed through 0.6-μm-pore-size Nuclepore filters to remove larger particulate materials, and diluted for growth in 0.22-μm-pore-size Millipore filter-sterilized unenriched seawater. This provided cells comparable in size and morphology to those in natural seawater, but the cultures were free of the interfering particulate detritus naturally present. Cells were collected on glass-fiber GF/F filters, and biovolumes were corrected for cells passing these filters; C and N were measured with a CHN analyzer. Our criteria for size measurement by epifluorescence photomicrography were confirmed with fluorescent microspheres of known diameters. Surprisingly, in six cultures with average per-cell biovolumes ranging from 0.036 to 0.073 μm³, the average per-cell carbon biomass was relatively constant at 20 ± 0.08 fg of C (mean ± standard error of the mean). The biovolume-to-biomass conversion factor averaged 0.38 ± 0.05 g of C cm⁻³, which is about three times higher than the value previously estimated from Escherichia coli, and decreased with increasing cell volume. The C:N ratio was 3.7 ± 0.2. We conclude that natural marine bacterial biomass and production may be higher than was previously thought and that variations in bacterial size may not reflect variations in biomass per cell.     

Hydrodynamic focusing and electronic cell-sizing techniques

The technique of hydrodynamic focusing, used to improve the resolution of the Coulter counter for the sizing of bacteria, was examined. Latex particles of 0.26 μm³ to 6.7 μm³ volume were used to examine the characteristics of the system with and without hydrodynamic focusing. The system then was evaluated for sizing mixed bacterial populations as well as single populations. Possible applications are also discussed.     

Antimicrobial Action of Oleanolic Acid on Listeria monocytogenes,Enterococcus faecium,and Enterococcus faecalis

This study investigated the antimicrobial action of oleanolic acid against Listeria monocytogenes, Enterococcus faecium, and Enterococcus faecalis. To determine the cytotoxicity of oleanolic acid, HEp-2 cells were incubated with oleanolic acid at 37^oC. MICs (minimal inhibition concentrations) for L. monocytogenes, E. faecium, and E. faecalis were determined using two-fold microdilutions of oleanolic acid, and bacterial cell viability was then assessed by exposing the bacteria to oleanolic acid at 2 × MIC. To investigate the mode of antimicrobial action of oleanolic acid, we measured leakage of compounds absorbing at 280 nm, along with propidium iodide uptake. Scanning electron microscope (SEM) images were also analysed. The viability of HEp-2 cells decreased (P < 0.05) at oleanolic acid concentrations greater than 128 μg mL^-1. The MICs were 16-32 μg mL^-1 for L. monocytogenes and 32-64 μg mL^-1 for E. faecium and E. faecalis, and bacterial cell viability decreased (P < 0.05) about 3-4 log CFU mL^-1 after exposure to 2 × MIC of oleanolic acid. Leakage of 280 nm absorbing materials and propidium iodide uptake was higher in oleanolic acid –treated cells than in the control. The cell membrane was damaged in oleanolic acid-treated cells, but the control group had intact cell membrane in SEM images. The results indicate that oleanolic acid can kill L. monocytogenes, E. faecium, and E. faecalis by destroying the bacterial cell membrane.     

Biodegradation of Methyl tert-Butyl Ether by a Bacterial Pure Culture

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 × 10⁶ cells ml⁻¹ were 0.07, 1.17, and 3.56 μg ml⁻¹ h⁻¹ for initial concentrations of 5, 50, and 500 μg MTBE ml⁻¹, respectively. When incubated with 20 μg of uniformly labeled [¹⁴C]MTBE ml⁻¹, strain PM1 converted 46% to ¹⁴CO₂ and 19% to ¹⁴C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE⁻¹. Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 μg of MTBE ml⁻¹ added to the core material. The rate of MTBE removal increased with additional inputs of 20 μg of MTBE ml⁻¹. These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.