Acridine Orange Direct Counts Research Articles

Structure and activity of the bacterial community in the rhizosphere of different plant species and the effect of arbuscular mycorrhizal colonisation

The aim of this study was to determine if arbuscular mycorrhizal (AM) colonisation influences rhizosphere bacteria differently depending on plant species. Thus, the effect of AM colonisation (Glomus intraradices) on rhizosphere bacteria of subterranean clover, cucumber, leek and maize was studied. The bacterial activity was measured as thymidine or leucine incorporation and bacterial numbers as colony forming units and acridine orange direct counts. The phospholipid fatty acid (PLFA) pattern was used to characterise the bacterial community structure and was compared with the community substrate utilisation pattern using Biolog. The bacterial activity and bacterial densities differed between the rhizospheres of the plant species. AM colonisation had a low impact on bacterial activity, but affected bacterial numbers differently depending on the plant species. Only small effects of AM colonisation were detected with the PLFA technique, and no effects were seen with Biolog, while similar effects of the plant species were found for these techniques. Thus, the plant species had greater effects on the bacterial community in the rhizosphere than AM colonisation and the effect of AM differed between plant species.

Biofilm formation in drinking water affected by low concentrations of phosphorus.

Abstract: There are geographical regions where microbial growth in drinking waters is limited by phosphorus instead of organic carbon. In these drinking waters even a low amount of phosphorus can strongly enhance microbial growth. The formation of biofilm can be limited by low availability of phosphorus in drinking waters with low content of phosphorus. The formation of biofilms on polyvinyl chloride plates was studied in laboratory experiments with water containing 48 microg/L assimilable organic carbon and 0.19 microg/L microbially available phosphorus. We found that low additions of phosphate (1-5 microg/L PO4(3-)-P) to water increased microbial growth in the water and in the biofilm. The effect of phosphorus on microbial growth could be detected by determining either the microbial cell production or the content of ATP in biofilms. Also, in steady-state biofilms, microbial concentrations were higher with phosphorus addition as enumerated by heterotrophic plate counts on R2A-agar and acridine orange direct counting. This work confirms the earlier findings of the importance of phosphorus for microbial growth in humic-rich drinking waters.

Spatial and temporal variation in hexachlorocyclohexane isomers in a temperate estuary

Hexachlorocylcohexanes (HCHs) are pesticides that persist in air and water of the Northern hemisphere. To understand the spatial and temporal variability in HCH levels in estuarine surface waters we measured concentrations of two HCH isomers (α-HCH and γ-HCH) at six sites in the York River estuary at bimonthly intervals for a year. Bacterial abundance and activity were also monitored using acridine orange direct counts and uptake of tritiated substrates, respectively. α-HCH was consistently observed to be significantly higher in marine water compared to river water entering the estuary, suggesting that the Chesapeake Bay or Atlantic Ocean is a larger source of this compound to the York River estuary compared to riverine input. Moreover, following periods of high freshwater flow into the estuary during spring and early summer, both α- and γ-HCH mixing curves indicated an additional source of these pollutants to the estuary such as land-derived runoff or groundwater discharge. In contrast, during low freshwater flow (late summer and fall) the estuary was a sink for HCHs, with γ-HCH more rapidly removed from the estuary than α-HCH. During the period of low freshwater flow, concentrations of both α- and γ-HCH were negatively correlated with bacterial activity. Bacterial activity as opposed to abundance appears to control HCH degradation in estuarine surface waters.

Structure and activity of the bacterial community in the rhizosphere of different plant species and the effect of arbuscular mycorrhizal colonisation

The aim of this study was to determine if arbuscular mycorrhizal (AM) colonisation influences rhizosphere bacteria differently depending on plant species. Thus, the effect of AM colonisation ( Glomus intraradices) on rhizosphere bacteria of subterranean clover, cucumber, leek and maize was studied. The bacterial activity was measured as thymidine or leucine incorporation and bacterial numbers as colony forming units and acridine orange direct counts. The phospholipid fatty acid (PLFA) pattern was used to characterise the bacterial community structure and was compared with the community substrate utilisation pattern using Biolog. The bacterial activity and bacterial densities differed between the rhizospheres of the plant species. AM colonisation had a low impact on bacterial activity, but affected bacterial numbers differently depending on the plant species. Only small effects of AM colonisation were detected with the PLFA technique, and no effects were seen with Biolog, while similar effects of the plant species were found for these techniques. Thus, the plant species had greater effects on the bacterial community in the rhizosphere than AM colonisation and the effect of AM differed between plant species.

Culturability as an indicator of succession in microbial communities.

Successional theory predicts that opportunistic species with high investment of energy in reproduction and wide niche width will be replaced by equilibrium species with relatively higher investment of energy in maintenance and narrower niche width as communities develop. Since the ability to rapidly grow into a detectable colony on nonselective agar medium could be considered as characteristic of opportunistic types of bacteria, the percentage of culturable cells may be an indicator of successional state in microbial communities. The ratios of culturable cells (colony forming units on R2A agar) to total cells (acridine orange direct microscopic counts) and culturable cells to active cells (reduction of 5-cyano-2,3-ditolyl tetrazolium chloride) were measured over time in two types of laboratory microcosms (the rhizosphere of hydroponically grown wheat and aerobic, continuously stirred tank reactors containing plant biomass) to determine the effectiveness of culturabilty as an index of successional state. The culturable cell:total cell ratio in the rhizosphere decreased from approximately 0.25 to less than 0.05 during the first 30-50 days of plant growth, and from 0.65 to 0.14 during the first 7 days of operation of the bioreactor. The culturable cell:active cell ratio followed similar trends, but the values were consistently greater than the culturable cell:total cell ratio, and even exceeded I in early samples. Follow-up studies used a cultivation-independent method, terminal restriction fragment length polymorphisms (TRFLP) from whole community DNA, to assess community structure. The number of TRFLP peaks increased with time, while the number of culturable types did not, indicating that the general decrease in culturability is associated with a shift in community structure. The ratio of respired to assimilated C-14-labeled amino acids increased with the age of rhizosphere communities, supporting the hypothesis that a shift in resource allocation from growth to maintenance occurs with time. Results from this work indicate that the percentage of culturable cells may be a useful method for assessing the successional state of microbial communities.

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 106 or 109 total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/106-AODC) than in water samples (0.02 to 0.4 PRM/106-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/109-AODC) than from other sites (nondetectable to 0.77 PRM/109-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.

Impact of dilution on microbial community structure and functional potential: comparison of numerical simulations and batch culture experiments.

A series of microcosm experiments was performed using serial dilutions of a sewage microbial community to inoculate a set of batch cultures in sterile sewage. After inoculation, the dilution-defined communities were allowed to regrow for several days and a number of community attributes were measured in the regrown assemblages. Based upon a set of numerical simulations, community structure was expected to differ along the dilution gradient; the greatest differences in structure were anticipated between the undiluted-low-dilution communities and the communities regrown from the very dilute (more than 10(-4)) inocula. Furthermore, some differences were expected among the lower-dilution treatments (e.g., between undiluted and 10(-1)) depending upon the evenness of the original community. In general, each of the procedures used to examine the experimental community structures separated the communities into at least two, often three, distinct groups. The groupings were consistent with the simulated dilution of a mixture of organisms with a very uneven distribution. Significant differences in community structure were detected with genetic (amplified fragment length polymorphism and terminal restriction fragment length polymorphism), physiological (community level physiological profiling), and culture-based (colony morphology on R2A agar) measurements. Along with differences in community structure, differences in community size (acridine orange direct counting), composition (ratio of sewage medium counts to R2A counts, monitoring of each colony morphology across the treatments), and metabolic redundancy (i.e., generalist versus specialist) were also observed, suggesting that the differences in structure and diversity of communities maintained in the same environment can be manifested as differences in community organization and function.

Rapid determination of bacteria in drinking water using an ATP assay

AbstractThe presently used heterotrophic plate count (HPC) for the evaluation of the total number of bacteria in a sample of drinking water takes 7 days of incubation. When the results are known, the water has been consumed and is ineffective for the protection of the health of the consumers. Operators of water treatment systems need to know the bacterial water quality in near real time. Contamination of the system, whether it is intentional, accidental, or due to an inadequate disinfectant residual needs to be discovered much sooner because intervention can then take place in the form of flushing low quality water and/or raising the disinfectant residual. The purpose of this study was therefore to determine if a rapid ATP assay can estimate the HPC in minutes. Two additional methods were used for some samples. The first method was the acridine orange direct count (AODC) that enumerates both viable and nonviable bacteria. The second method was the direct viable count (DVC) that enumerates only viable bacteria. Water samples were obtained from local, national, and international locations. The sample selection criteria were based on proximity to the laboratory, cooperating water utilities, and the travel of the authors. The results of the study show that the rapid ATP assay is highly correlated with the conventional plate count method and the DVC method. The significance of the ATP assay is that it can determine the bacterial quality of the drinking water in less than 5 min. © 2001 John Wiley & Sons, Inc. Field Analyt Chem Technol 5: 185–189, 2001

Dynamics of thraustochytrid protists in the water column of the Arabian Sea

Thraustochytrids, a group of osmoheterotrophic chromistan protists are ubiquitous in the sea. However, little is known of their ecological role, particularly in oceanic waters. To obtain an insight into their dynamics in this realm, we investigated thraustochytrids and bacteria in the water column, up to 2000 m in the central Arabian Sea. Four seasons, namely the end of the southwest sum- mer monsoon (September/October 1993), the summer pre-monsoon (April/May 1994), the end of the northeast winter monsoon (February/March 1995) and the peak period of the southwest monsoon (July/August 1995) were studied. Thraustochytrids were estimated using the acriflavine direct detec- tion method (AfDD) and bacteria by the acridine orange direct counts (AODC). Thraustochytrids were present in substantial numbers throughout the 150 m water column in all the stations investi- gated during the end of the biologically productive summer and winter monsoons (0 to 1313 × 10 3 and 3.7 to 183 × 10 3 cells l -1 water respectively). Their populations in the upper 150 m were positively re- lated to bacteria and particulate organic matter during these 2 periods, the relationship being signifi- cant during the end of the winter monsoon. They were infrequent during the low productive summer pre-monsoon period (present in < 29% of the total 41 samples), a period of high bacterial abundance, as well as in the peak-productive southwest summer monsoon (present in 10 of 19 samples). Vertical distribution was seasonally variable. Thraustochytrids were regularly detected in the Arabian Sea oxygen minimum zone (OMZ) at 200 to 1000 m. Similar to the upper 150 m, thraustochytrids below 200 m were least frequent in the summer pre-monsoon (present in 49% of samples), although high numbers, up to 266 × 10 3 l -1 were found at 1000 m in 1 station. Their numbers in the OMZ ranged from 0 to 38 × 10 3 l -1 during the end of the summer and winter monsoons. Both thraustochytrids and bacteria showed a distinct peak at 250 to 500 m in the OMZ during these 2 seasons. Dense popula- tions of thraustochytrids were detected even at depths up to 2000 m. We hypothesise that seasonal in- creases of thraustochytrid populations are related to particles following phytoplankton degradation at the end of the biologically productive seasons. Our estimates suggest that during the 3 seasons, maximum thraustochytrid C biomass reached values ranging from 36 to 217% of bacterial C biomass in the upper 150 m and 34.5 to 56% of bacterial C biomass between 200 and 2000 m.

Double-stranded DNA measurement in lakes with the fluorescent stain PicoGreen and the application to bacterial bioassays

We used the double-stranded DNA (dsDNA) stain PicoGreen with a microplate fluo- rometer to measure bacterial abundance, biomass, and growth rates in lake water. PicoGreen fluo- rescence units (PFU) correlated closely with bacterial abundance measured with acridine orange direct counts (R 2 = 0.95 to 0.98) as well as bacterial biomass inferred from image analysis (R 2 = 0.95 to 0.98) in eutrophic waters. PicoGreen fluorescence increased proportionally with bacterial size, indi- cating that it was a good indicator of biomass as well as abundance. In oligotrophic Lake Superior, there was a weaker, but significant (p < 0.05) correlation between PFU and abundance (R 2 = 0.52) as well as PFU and biomass (R 2 = 0.54). Growth rate measurements in bottle cultures showed a similar relationship, with PFU, abundance, and biomass being more tightly correlated in productive waters than in oligotrophic waters. Parallel dilution cultures were performed in microplate wells (nanocosms) and 1 l bottles. The slope of nanocosm fluorescence to bottle fluorescence was ca 1, indicating that nanocosms mimicked bacterial abundance and growth in bottle cultures. Preservation of Escherichia coli with formaldehyde showed that there was an initial loss of dsDNA of about 10 to 15% with little subsequent loss for 2 wk, indicating that bacteria from growth experiments conducted in the field could be preserved for subsequent analysis in the laboratory. Bacterial cellular dsDNA content in 3 Minnesota lakes varied between 0.6 and 6.2 fg cell -1 and was highest in the most eutrophic and most rapidly growing bacterial community, i.e., the more eutrophic lakes. These results suggest that the PicoGreen method is effective for growth bioassays in systems with moderate to high levels of bacterial productivity. PicoGreen coupled with a microplate fluorescence reader is a promising method for determining bacterial biomass and growth rates, especially in meso- to eutrophic systems.

Evaluation of Bacterial Recovery Efficiency and Counting Precision from Decaying Leaf Litter in Little Rock Lake, Wisconsin, USA

We studied the effectiveness of sonication and filter-size-fractionation to efficiently remove and separate bacterial leaf-surface colonizers from other members of the microbial community. Leaf discs from red oak and paper birch leaves were colonized in situ for 8 and 22 days in Little Rock Lake, WI, and sonicated at 90 W power output for 60 seconds, followed by filter-fractionation (3.0 μm) to remove and isolate bacteria. This technique consistently yielded the highest bacterial activity (relative Electron Transport System activity) and resulted in bacterial recovery efficiencies from leaf surfaces ranging from 96.5% to 98.8%. In addition, comparisons among various methods currently used to estimate bacterial densities (e.g., malachite green-INT, acridine orange direct counts, scanning electron microscopy approaches) were completed to assess the utility of the MINT method for counting bacteria removed by sonication. Bacterial counts derived from MINT and AODC methods were never significantly different, but always were significantly higher and less variable than counts from SEM. Finally, an attempt was made to address sources of variability in counting precision using the MINT method for bacteria removed from leaf surfaces. For two leaf species, on two sampling dates, most of the variability in MINT cell counts was due to variability among microscopic fields counted, while little was due to either subsample or filter used in cell counts. Thus, the combination of sonication and filter-size-fractionation to remove bacterial leaf surface colonizers appears to be an effective approach, allowing reproducible and precise measurements of bacterial densities and activities.

Temperature effects on wastewater treatment under aerobic and anoxic conditions

Current evaluations find savings in operating costs of greater than 25% when denitrification is added to a system already achieving nitrification. The economic evaluations performed were based on a sludge reduction of 25% under anoxic conditions compared to aerobic conditions. Conflicting reports of biosolids production for reactors operating under aerobic, anoxic and alternating aerobic conditions exist. This study quantifies and compares biosolids production rates for SBRs achieving carbon removal under aerobic or anoxic conditions as a function of temperature (14°C and 20°C). Proteins were chosen as model substrates because they are a major contributor to the total COD of raw sewage and by virtue of their high molecular weight, require hydrolysis. This study is unique in that the bacterial population was also enumerated using acridine orange direct counts (AODC). Observed yields were 35–52% higher for the anoxic reactors than they were for the aerobic reactors. The observed yield for the aerobic reactor decreased by 4% at a lower temperature whereas the anoxic yield increased by 8%. Further analysis discounted that differences in observed yield were caused by an accumulation of influent proteins. A factorial analysis revealed that the anoxic reactor contained more bacteria than its aerobic counterpart. Microscopic examination of the mixed liquor showed the presence of protozoa in the aerobic reactor while none were noted in the anoxic reactor. Predation is thought to be responsible for differences in the observed sludge production between anoxic and aerobic conditions.

Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM

Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat. Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC). However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively. The total bacterial abundance ranged from 1.1 to 3.2×10 6 ml −1. As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria. Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria. Counts of filamentous bacteria made by AODC were only about half of those detected by TEM. In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates. Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4×10 5 ml −1, respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids. Besides, cocci might easily be mistaken for large viral particles when counted by AODC. Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters.

A RAPID METHOD FOR DETECTING BACTERIA IN DRINKING WATER

Abstract A rapid determination of the total bacterial count in drinking water is important to the operators of treatment plants and distribution systems. It will allow corrective measures in real‐time, such as increasing the disinfectant dose or removing water that has high bacterial numbers. The present heterotrophic plate count (HPC) analysis takes seven days and is not useful for operational intervention. The purpose of this study was to determine if a rapid adenosine triphosphate (ATP) assay would estimate the total number of bacteria in minutes. For quality control purposes and also to test the accuracy of both the ATP and HPC test, direct enumeration of the bacteria in a water sample was done using two epifluorescence methods. One was acridine orange direct count (AODC) method, which allows enumeration of both viable and nonviable bacteria. The other was direct viable count (DVC) method, which enumerates viable bacteria. Water samples originated from local, national, and international locations. The sample selection criteria were based on proximity to the laboratory, cooperating water utilities, and the travel of the authors. The results of the study show that the rapid ATP assay is highly correlated with the conventional plate count method and the DVC method, and estimates the bacterial quality of drinking water in minutes.

Distribution and Composition of Microbial Populations in a Landfill Leachate Contaminated Aquifer (Grindsted, Denmark).

> Abstract To investigate whether landfill leachates affected the microbial biomass and/or community composition of the extant microbiota, 37 samples were collected along a 305-m transect of a shallow landfill-leachate polluted aquifer. The samples were analyzed for total numbers of bacteria by use of the acridine orange direct count method (AODC). Numbers of dominant, specific groups of bacteria and total numbers of protozoa were measured by use of the most probable number method (MPN). Viable biomass estimates were obtained from measures of ATP and ester-linked phospholipid fatty acid (PLFA) concentrations. The estimated numbers of total bacteria by direct counts were relatively constant throughout the aquifer, ranging from a low of 4.8 x 10(6) cells/g dry weight (dw) to a high of 5.3 x 10(7) cells/g dw. Viable biomass estimates based on PLFA concentrations were one to three orders of magnitude lower with the greatest concentrations (up to 4 x 10(5) cells/g dw) occurring at the border of the landfill and in samples collected from thin lenses of clay and silt with sand streaks. Cell number estimates based on ATP concentrations were also found to be lower than the direct count measurements (<2.2 x 10(6) cells/g dw), and with the greatest concentrations close to the landfill. Methanogens (Archaea) and reducers of sulfate, iron, manganese, and nitrate were all observed in the aquifer. Methanogens were found to be restricted to the most polluted and reduced part of the aquifer at a maximum cell number of 5.4 x 10(4) cells/g dw. Populations of sulfate reducers decreased with an increase in horizontal distance from the landfill ranging from a high of 9.0 x 10(3) cells/g dw to a low of 6 cells/g dw. Iron, manganese, and nitrate reducers were detected throughout the leachate plume all at maximum cell numbers of 10(6) cells/g dw. Changes in PLFA profiles indicated that a shift in microbial community composition occurred with increasing horizontal distance from the landfill. The types and patterns of lipid biomarkers suggested that increased proportions of sulfate- and iron-reducing bacteria as well as certain microeukaryotes existed at the border of the landfill. The presence of these lipid biomarkers correlated with the MPN results. There was, however, no significant correlation between the abundances of the specific PLFA biomarkers and quantitative measurements of redox processes. The application of AODC, MPN, PLFA, and ATP analyses in the characterization of the extant microbiota within the Grindsted aquifer revealed that as distance increased from the leachate source, viable biomass decreased and community composition shifted. These results led to the conclusion that the landfill leachate induced an increase in microbial cell numbers by altering the subsurface aquifer so that it was conducive to the growth of methanogens and of iron-and sulfate-reducing bacteria and fungi.

Microbial biomass and numbers of denitrifiers related to macropore channels in agricultural and forest soils

In clay and loamy soils the vertical flow of dissolved and particulate matter primarily takes place through macropore channels supplying the attached microorganisms with nutrients and substrates for their growth. Consequently the walls of the macropore channels may have a higher microbial biomass and a higher rate potential for microbial processes, e.g. denitrification, than the soil matrix not affected by macropores. This was tested by determining the content of NO 3 −-N, water-soluble carbon (WSC) and the size of the microbial biomass in bulk soil, in soil adjacent to macropore channels (macropore soil) and in soil not affected by macropores (matrix soil) from one agricultural site and one forest site located in Jutland, Denmark. Determinations of the bacterial biomass in the agricultural bulk soil profile (0 to 250 cm) generally showed the following sequence of bacterial abundance: acridine orange direct counts>most probable numbers>total plate counts>nitrate reducers>denitrifiers. The numbers of culturable bacteria were 1 to 2 orders of magnitude lower in the forest profile than in agricultural profile. There was a tendency for a slight decrease in the numbers of bacteria with increasing depth. The comparison of macropore soil and matrix soil clearly showed that macropore soil contained larger concentrations NO 3 −-N and WSC and a larger bacterial biomass. In the agricultural site the average contents of NO 3 −-N were 6.7 and 3.7 μg g −1 dry soil the macropore soil and the matrix soil, respectively, whereas the very low contents of NO 3 −-N in the forest soil (<1 μg N g −1 dry soil) resulted in smaller differences between the macropore and the matrix soils. The average contents of WSC in the macropore soil and the matrix soil were 51 and 40 μg C g −1 dry soil at the agricultural site and 33 and 19 μg C g −1 dry soil at the forest site, respectively. With respect to the bacterial biomass the results showed that with only four exceptions, the numbers of bacteria were higher in the macropore soil samples than in the matrix soil samples and in 19 out of 44 analyses this difference was statistically significant. These results of direct measurements in macropore soil and matrix soil substantiate the role of macropore channels as the preferential pathways for transportation of solutes such as nitrate and organic compounds. Consequently, the higher numbers of bacterial cells in the macropore environments may be attributed to a better substrate supply and the transportation of bacterial cells through the macropore channels.

Assessment of heavy metals associated with bacteria in soil

The heavy metals associated with bacteria in soil are dicult to quantify due to the problem of extracting sucient soil microbial biomass for analysis. The e€ects of soil minerals, organic matter and dissolved ions on the solubility and the reactivity of metals would all complicate the quanti®cation of heavy metals associated with bacteria. One approach to overcome this problem would be to separate the bacterial cells from soil in adequate quantities followed by analysis of heavy metals in the bacterial fraction. The essential steps are dispersal of the soil aggregates, followed by centrifugation in which cells and soil particles are separated according to size, density or both (Bakken and Lindhal, 1994). We describe a new method to quantify bacterial heavy metals that involve cell extraction and analysis of heavy metals in and absorbed onto cells. The method was tested in soil with or without manure addition. Samples were collected from two soil treatments (with or without addition of cow manure at the rate of 4% w/w) of a cultivated soil (Typic Cryoboroll). The soil samples (two replicates) were moistened with a tracer solution (a mixed solution of Cd and Zn) to bring the moisture content to 35% (w/w) and the total activity of the two tracers to 2000 Bq gy1 soil. After 1 week of soil incubation at 228C, the soils were used for cell dispersion and cell separation as follows. The soils were suspended in distilled water (soil:water=1:10) and homogenized in a Waring blender (3 1 min homogenization at full speed). The homogenates were placed in 250 ml centrifuge tubes and a cushion of Nycodenz (Nycomed Pharma AS, Torshov, Oslo, Norway) with a density of 1.3 g mly1 (24 g Nycodenz to 30 ml ®ltered distilled water) was placed underneath each homogenate. The cushions were placed with care to reduce mixing of the soil homogenate with the Nycodenz. The cushion of Nycodenz was large enough to extend above the rounded end of the centrifuge tubes. In a 250 ml centrifugation tube, we used a 50 ml Nycodenz cushion underneath 200 ml of soil homogenate. The samples were placed in a swing-out rotor to centrifuge at 10,000g for 60 min. After centrifugation, the bacterial cells had oated to the top of the Nycodenz cushion and soil material formed a soil pellet at the bottom of the tube. The supernatant above the bacterial layer was sampled separately (20 ml aliquots removed consecutively) to investigate the distribution of heavy metal in the washed portion. The bacterial layer (with or without a cushion) was harvested with a pipette. This bacterial fraction was diluted (1:20) and was subjected to prolonged centrifugation (2 h). This is because in the ®rst centrifugation (60 min at 10,000g), a true isopycnic equilibrium was often not reached. This is required for higher purity, since the cells lost are those with adhering soil colloids. After the prolonged centrifugation, the supernatents were discarded. The pure bacterial pellet was diluted (1:50) in distilled water and one portion analyzed for a bacterial count and the other for the activity of Cd and Zn. The soil pellet was also sampled for bacterial counts and for activity of the Cd and Zn. The percentage distribution of Cd and Zn between the fractions (supernatant, bacterial fractions and soil pellets) were estimated after counting the gamma emissions by Cd and Zn in the ®nal extracts and residues, by a NaI-detector. The counts were corrected for background emission and the energy spectra overlapping between the two nuclides, whereas the energy maxima are 88 and 1115 keV for Cd and Zn, respectively. The extraction eciency and the total bacterial count was investigated by acridine orange direct counting (AODC). The activity of Cd and Zn associated with bacteria (Table 1) are estimated by taking account of the bacterial count in both the bacterial fraction and soil pellet (Table 2). The Cd activity associated with bacteria was less than 1.3% of the total activity. Similarly, the Zn activities in the bacterial fraction were less than 3.6% of the total activities (Table 1). This shows that soil bacSoil Biology and Biochemistry 31 (1999) 315±316

Differences of Image between Epifluorescence Microscope with Universal Infinity System BX60 and Epifluorescence Microscope BHS-RFK on the Study of the River Microbial Community stained by Acridine Orange.

Acridine orange direct count (AODC) of bacterial community was examined with two types of epifluorescence microscope, BX-60 (OLYMPUS OPTICAL CO., LTD.) and BHS-RFK (OLYMPUS OPTICAL CO., LTD.). When observed under the naked eye, AO-stained particles appeared clearer under the BX60 than under the BHS-RFK. This difference in the image was considered to influence the AODC containing particles of various size and shape. AODC of a bacterial pure culture did not provide significant difference when determined under the naked eye with these two epifluorescence microscopes. However, the AODC of complex communities from river water and epilithon showed difference in counting among 4 investigators. To clarify the reason for these differences, the particle image of each scope was compared with an image analyzer. Mean AO-stained particle area of BHS-RFK was larger than that of BX60, probably caused by the existence of a dim margin surrounding particles for the former system. As a result, it is not necessary to correct AODC when determined under the naked eye. However, when an image analyzer is employed to determine AODC of natural bacterial community, procedure for application should be checked carefully for newly developed microscope.

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages.

Potential digestive function of bacteria in krill Euphausia superba stomachs

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 14:129-136 (1998) - doi:10.3354/ame014129 Potential digestive function of bacteria in krill Euphausia superba stomach Stuart P. Donachie*, Marek K. Zdanowski Department of Antarctic Biology, Polish Academy of Sciences, ul. Ustrzycka 10, PL-02-141 Warsaw, Poland *Present address: Department of Oceanography, SOEST, University of Hawai'i, Honolulu, Hawai'i 96822, USA. E-mail: donachie@soest.hawaii.edu Evidence is presented that bacteria in the stomach of live Antarctic krill Euphausia superba participate in the digestion of host dietary components. Total (AODC: Acridine Orange Direct Count) and culturable (CFU: Colony Forming Units) bacteria in this organ in fresh adult krill were compared in numerical and physiological terms with those in contemporaneously collected seawater samples in Admiralty Bay, King George Island, South Shetland Islands. On a per unit volume basis, AODC and CFU numbers in the stomach were greater than in the seawater, and higher than could be accounted for through selective prey filtration alone. CFU in the stomach and seawater averaged ~4% and <0.01% of the respective AODC, and in the stomach ~6% of AODC were dividing, compared to less than 1% in seawater, observations suggesting that bacterial growth occurs in the stomach. Biovolume of cells in the stomach exceeded that of cells in seawater (t2854 = -6.262, p < 0.001), but the former were too small to have been selectively filtered. Maltose and gluconate assimilation, and lipase (C14), 'trypsin'-like, and acid phosphatase activities were significantly more prominent in culturable bacteria from the stomach than seawater, further indicating selection. Conversely, a-fucosidase production appears host derived. Yeasts isolated from the stomach were identified as Leucosporidium antarcticum and Metschnikowia australis. Antarctic krill · Euphausia superba · Bacteria · Digestive enzyme · Yeast Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 14, No. 2. Publication date: February 13, 1998 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1998 Inter-Research.