Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.     

Direct estimate of active bacteria: CTC use and limitations

During the last 10 years, the dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used to determine the in situ number of "active" bacteria in different ecosystems. A part of this success is due to a simple protocol, which does not require sophisticated equipment. However, it has not been established whether the method determines viable cells, e.g. those capable of growth and cell division, as opposed to cells that are active in the sense of having some detectable metabolic activity. In this study, the number of CTC-positive cells through the growth stages of Escherichia coli was estimated and compared to counts of the total number of bacteria, the culturability (CFU counts) and respiratory activity (CO(2) evolution). There was a good correlation between the number of CTC-positive cells and the CFU count, regardless of the growth phase. However, CTC could still be reduced by a large part of the population during the first hours of stationary phase even if the bacteria were no longer releasing CO(2). Thus, the reduction of CTC is a good estimator for cell viability, rather than cell activity. Additionally, a review of the literature showed that there is presently no standardized protocol for using CTC, which makes difficult at present the comparison of active bacterial numbers in different samples from different sites.     

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [³H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.     

Selachohemecus benzi n. sp. (Digenea: Sanguinicolidae) from the blacktip shark Carcharhinus limbatus (Carcharhinidae) in the northern Gulf of Mexico

Selachohemecus benzi Bullard & Overstreet n. sp. infects the heart and kidney of the blacktip shark Carcharhinus limbatus in the northern Gulf of Mexico off Florida and Mississippi, USA. Specimens of S. olsoni Short, 1954, the only congener and only other named blood fluke reported from a chondrichthyan in the Gulf of Mexico, were collected from the heart of the Atlantic sharpnose shark Rhizoprionodon terraenovae from two new localities, Apalachicola Bay, Florida, and Mississippi Sound, Mississippi, USA. The new species differs from S. olsoni by having a larger body (1.4-3.8 mm long), robust tegumental body spines numbering 51-63 along each lateral body margin, a testis extending from the posterior caeca to the ovary, and a medial ovary with lobes. We amend the diagnosis of Selachohemecus Short, 1954 to accommodate it and provide a diagnostic key for all named chondrichthyan blood flukes.     

Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures.

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

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

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

Selachohemecus benzi n. sp. (Digenea: Sanguinicolidae) from the blacktip shark Carcharhinus limbatus (Carcharhinidae) in the northern Gulf of Mexico

Selachohemecus benzi Bullard & Overstreet n. sp. infects the heart and kidney of the blacktip shark Carcharhinus limbatus in the northern Gulf of Mexico off Florida and Mississippi, USA. Specimens of S.␣olsoni Short, 1954, the only congener and only other named blood fluke reported from a chondrichthyan in the Gulf of Mexico, were collected from the heart of the Atlantic sharpnose shark Rhizoprionodon terraenovae from two new localities, Apalachicola Bay, Florida, and Mississippi Sound, Mississippi, USA. The new species differs from S. olsoni by having a larger body (1.4–3.8 mm long), robust tegumental body spines numbering 51–63 along each lateral body margin, a testis extending from the posterior caeca to the ovary, and a medial ovary with lobes. We amend the diagnosis of Selachohemecus Short, 1954 to accommodate it and provide a diagnostic key for all named chondrichthyan blood flukes.     

Analysis of the microbial functional diversity within water-stressed soil communities by flow cytometric analysis and CTC+ cell sorting

Total and active cell counts within soil samples were determined by culture-independent methods using flow cytometry and preparative Nycodenz gradient centrifugation. Whole cells were purified from soil cores and total extractable cell counts assessed by SYBR Green II fluorescence, while active cell counts were determined by 5-cyano-2,3-ditolyl tetrazolium chloride reduction (CTC+ cells). Parallel microcosms, maintained at either field water capacity or subjected to drying, indicated that the total extractable cell count remained between 10(8) and 10(9) g(-1) (dry weight). In contrast, the CTC+ active count fell threefold in dried microcosms (6% of total cell count) when compared to wetted microcosms (18% of total cell count). Specifically, these data highlighted an overall deactivation of microbial biomass during water stress, with 16S rDNA analyses of flow-sorted CTC+ cells demonstrating shifts within the active diversity. Flow cytometry coupled with cell purification techniques represents a significant tool for operationally defining an active and redundant microbial component within soil communities and is demonstrated during water stress.     

Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates.

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.     

Phages Infecting Vibrio vulnificus Are Abundant and Diverse in Oysters (Crassostrea virginica) Collected from the Gulf of Mexico

Phages infecting Vibrio vulnificus were abundant (>10⁴ phages g of oyster tissue⁻¹) throughout the year in oysters (Crassostrea virginica) collected from estuaries adjacent to the Gulf of Mexico (Apalachicola Bay, Fla.; Mobile Bay, Ala.; and Black Bay, La.). Estimates of abundance ranged from 10¹ to 10⁵ phages g of oyster tissue⁻¹ and were dependent on the bacterial strain used to assay the sample. V. vulnificus was near or below detection limits (<0.3 cell g⁻¹) from January through March and was most abundant (10³ to 10⁴ cells g⁻¹) during the summer and fall, when phage abundances also tended to be greatest. The phages isolated were specific to strains of V. vulnificus, except for one isolate that caused lysis in a few strains of V. parahaemolyticus. Based on morphological evidence obtained by transmission electron microscopy, the isolates belonged to the Podoviridae, Styloviridae, and Myoviridae, three families of double-stranded DNA phages. One newly described morphotype belonging to the Podoviridae appears to be ubiquitous in Gulf Coast oysters. Isolates of this morphotype have an elongated capsid (mean, 258 nm; standard deviation, 4 nm; n = 35), with some isolates having a relatively broad host range among strains of V. vulnificus. Results from this study indicate that a morphologically diverse group of phages which infect V. vulnificus is abundant and widely distributed in oysters from estuaries bordering the northeastern Gulf of Mexico.     

Inhibition of Listeria in dry fermented sausages by the bacteriocinogenic Lactobacillus sake CTC494

Lactobacillus sake CTC494 isolated from a naturally fermented sausage, produced an antibacterial agent active against selected strains of Listeria monocytogenes and L. innocua. The agent was bacteriolytic against L. monocytogenes and sensitive to proteolytic enzymes; it was identified as a bacteriocin and was designated as sakacin K. The ability of Lact. sake CTC494 to inhibit the growth of listeria, compared to a bacteriocinogenic negative control strain, was examined in a model sausage system and in dry fermented sausages. In dry fermented sausages Lact. sake CTC494 was able not only to suppress the growth of listeria but to diminish their number by 1.25 log compared to the non-bacteriocinogenic control strain. Thus, Lact. sake CTC494 has proved to be a good starter culture providing good organoleptical and sensorial qualities to the product and can be employed as a bioprotective starter culture in fermented meat products.     

Molecular analyses of the diversity in marine bacterioplankton assemblages along the coastline of the northeastern Gulf of Mexico

Bacterial community diversity in marine bacterioplankton assemblages were examined in 3 coastal locations along the northeastern Gulf of Mexico (GOM) using 16S rRNA gene libraries and fluorescence in situ hybridization approaches. The majority of the sequences (30%-60%) were similar to the 16S rRNA gene sequences of unknown bacteria; however, the operational taxonomic units from members of the Cyanobacteria, Proteobacteria, and Bacteroidetes were also present at the 3 GOM sites. Overall, sequence diversity was more similar between the Gulf sites of Carrabelle and Ochlockonee than between either of the Gulf sites and Apalachicola Bay. Fluorescence in situ hybridization analyses revealed the quantitative predominance of members of the Alphaproteobacteria subclass and the Cytophaga-Flavobacterium cluster within the bacterioplankton assemblages. In general, the study further reveals the presence of many bacterial taxa that have been previously found to be dominant in coastal marine environments. Differences observed in the representation of the various bacterial phylogenetic groups among the GOM coastal sites could be partly attributed to dynamic variations in several site-specific conditions, including intermittent tidal events, nutrient availability, and anthropogenic influences.     

Large differences in the fraction of active bacteria in plankton,sediments, and biofilm

Generally, only a small fraction of free-living pelagic bacteria are metabolically active, while particle-associated bacteria usually exhibit a larger proportion of active bacteria. Most previous studies on the active fraction of bacteria focus on planktonic communities, and there are only a few studies on sediment and epiphytic biofilm bacteria. We compared the active fraction of the total number of bacteria in three different habitats of the littoral zone of Lake Erken, Sweden, including the sediments, the epiphytic biofilm on the submerged macrophyte Ranunculus circinatus, and the water column. Active bacteria were detected as those with an active electron transport system, identified by the capacity to reduce the tetrazolium salt CTC (5-cyano-2,3-ditolyltetrazolium chloride) into its fluorescent, water insoluble state. There were large differences between habitats. The active fraction of the total number of bacteria detected by fluorescence microscopy (annual mean +/- SD) in the sediments was 46 +/- 10%, on R. circinatus 37 +/- 18%, and in the water column 4 +/- 4%. The abundance of CTC-reducing cells was correlated with total bacterial abundance, and the fraction of CTC-reducing bacteria generally increased with total bacterial abundance, for all the habitats. Consequently, the difference in the fraction of CTC-reducing bacteria between the habitats could be attributed to different densities of bacteria, with a larger proportion of active bacteria at higher bacterial densities.     

Comparison of an inactive submarine spring with an active nearshore anchialine spring in Florida

Jewfish Sink is a former anchialine karst feature located in the Gulf of Mexico off the coast of West Central Florida. Freshwater flowed from the feature until 1962 and it is now an anoxic marine basin. The current biodiversity within Jewfish Sink was examined in terms of Bacteria, Archaea, and Eukaryota using a combination of 16S and 18S ribosomal RNA analysis from environmental samples. Analysis of 16S rRNA sequences from microbial mats in the anoxic zones revealed a broad diversity of bacteria (265 clones) and archaea (392 clones), many of which had previously been identified in anoxic environmental samples and are likely to be involved with sulfur, nitrogen, and methane metabolism. Sequence analysis of 785 18S clones revealed that fungi and dinoflagellate sequences dominate the eukaryote sequences. Because Jewfish Sink water is anoxic and high in sulfide, we investigated the effect of Jewfish Sink on the nearby shallow benthic environment. We compared the shallow benthic macrofauna near Jewfish Sink with that near Crystal Beach Spring, an active submarine spring. We found significantly greater species richness, abundance, and diversity of benthic fauna near the Jewfish Sink site than near Crystal Beach Spring. This comparison suggests that greater submarine groundwater discharge in an area with active submarine springs is a significant factor reducing the richness and diversity of the benthic community structure in the nearshore, shallow marine environment.     

Characterization of depth-related bacterial communities and their relationships with the environmental factors in the river sediments

To better understand the bacterial processes in river sediments, it is necessary to investigate the depth-related bacterial communities in the whole sediment profile. Sediment samples were collected to a depth of 25 cm from the Pearl River. Bacterial abundance, activity, cell-specific respiration rate, and diversity were measured, respectively, by 4′, 6-diamidino-2-phenylindole direct count, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining, electron transport system by CTC reduction, and denaturing gradient gel electrophoresis analysis of 16S rRNA amplification fragments. Results showed that the bacterial metabolism activities decreased with the sediment depth. The total bacterial abundance was highest in the surface sediment with 65.1 × 10⁷ cells g⁻¹, and decreased to 11.1 × 10⁷ cells g⁻¹ below 20 cm in the sample location that suffered from heavy sewage inputs. The active bacteria accounted for 7.50–46.7% of the total bacterial number and decreased with the sediment depth. Electron transport system by the CTC reduction showed that bacterial respiration rate declined from 1.093 μmol CTC-formazan h⁻¹ g⁻¹ in the surface sediment to a half in the bottom sediment, while the cell-specific respiration increased significantly with the depth from 3.56 to 93.75 fmol CTC-formazan cell⁻¹. The bacterial diversity also changed with the depth. Beta-Proteobacteria were the dominant species in the surface sediment, whereas Delta-Proteobacteria were the main species below 10 cm. Results of canonical correspondence analysis (CCA) indicated that the distribution of bacteria was affected by the combined effect of various dissolved inorganic matter, while the respiration rate was independent of the nutrient conditions. The specific bacterial distribution contributed to not only the nutrient cycle but also enhanced pollutant decomposition in sediment of the Pearl River. The results showed that some specific bacterial species had a strong activity in the deeper layers. Therefore, the metabolic functions of the deeper bacterial species should not be neglected.     

Active bacteria (CTC+) in temperate lakes: temporal and cross-system variations

The temporal variation in the abundance and proportion of highlyrespiration-active bacteria in the eutrophic lakes Esrum andFrederiksborg Slotssø was determined with the redox dye5-cyano-2,3-ditolyl tetrazolium chloride (CTC). In addition,a comparative late summer study was undertaken across a gradientof nutrient enrichment in Danish lakes. The purpose was to investigatethe importance of substrate (chlorophyll) and temperature forthe control of CTC-active cells (CTC+). The abundance of CTC+cells was much lower and more variable than the total numberof cells counted after 4',6-diamidino-2-phenylindole (DAPI)staining. The proportion of CTC+ cells in Lake Esrum and FrederiksborgSlotssø was normally <5%, and between 2.5 and 20%in 14 other lakes. The abundance as well as the proportion ofCTC+ cells increased with chlorophyll in Lake Esrum and FrederiksborgSlotssø, and chlorophyll explained 43% of the variabilityin CTC+ abundance. In the comparative study, the abundance ofCTC+ cells increased along the chlorophyll gradient, which explained49% of the variability. The results showed that the abundanceand, to a lesser degree, the proportion of CTC+ bacteria werecontrolled by substrate supply. One consequence of the low abundanceof active bacteria is that in situ growth rates scaled to CTC+cells are 3- to 7-fold higher than those scaled to DAPI counts.It is suggested that studies on factors controlling bacterioplanktonactivity at the single-cell level should be investigated scaledto active cells.     

Daily variations of highly active bacteria in the Northern Adriatic Sea

Nowadays, it is recognized that only a fraction of aquatic bacteriaare actively growing, but there is little information aboutthe factors constraining their metabolism. Marine bacterioplanktoncan rapidly modify their metabolic activity level in responseto environmental changes. In this study, we focused on the dailychanges in abundance and activity of active bacterial fractionover a 20-day period preceded by intense rainfalls which slightlymodified water column conditions. Cells capable of reducingthe membrane-penetrable dye 5-cyano-2,3-ditolyl tetrazoliumchloride (CTC), estimated by epifluorescence microscopy, areconsidered very active (CTC+ bacteria). Total bacterial abundance(TBA) ranged from 0.8 to 2.4 x 10⁹ cells L^–1, whereasCTC+ bacteria were more variable (1.6–9.2 x 10⁷ cellsL^–1), accounting for 1.2–4.4% of TBA. Bacterialactivity (BA) quantified as the incorporation of [3H]-leucinevaried by more than one order of magnitude over the period (25.0–662.5pmol L^–1 h^–1). BA was strongly related to CTC+ bacteria,suggesting that they were mainly responsible for the bacterialcommunity metabolism. Nevertheless, cell-specific activity,scaled to only CTC+ cells, was very high, suggesting that afraction of cells not detectably CTC+ may be able to assimilate[3H]-leucine. The correlation between salinity and TBA, CTC+bacteria and BA supported the hypothesis of the active roleof freshwater input in enhancing cell activity. Our resultssuggest that freshwater inputs rather than phytoplanktonic bloomsare able to induce shifts in bacterial metabolism over a timescale of days in the area studied.     

A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water.

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.