Effects of Phosphorus Limitation on the Bioavailability of DOM Released by Marine Heterotrophic Prokaryotes.

Heterotrophic prokaryotes (HP) contribute largely to dissolved organic matter (DOM) processing in the ocean, but they also release diverse organic substances. The bioavailability of DOM released by HP under varying environmental conditions has not been fully elucidated. In this study, we investigated the bioavailability of DOM released by a single bacterial strain (Sphingopyxis alaskensis) and 2 natural HP communities grown under P-replete and P-limited conditions. The released DOM (HP-DOM) was used as a substrate for natural HP communities at a coastal site in the Northwestern Mediterranean Sea. We followed changes in HP growth, enzymatic activity, diversity, and community composition together with the consumption of HP-DOM fluorescence (FDOM). HP-DOM produced under P-replete and P-limited conditions promoted significant growth in all incubations. No clear differences in HP-DOM lability released under P-repletion and P-limitation were evidenced based on the HP growth, and P-limitation was not demonstrated to decrease HP-DOM lability. However, HP-DOM supported the growth of diverse HP communities, and P-driven differences in HP-DOM quality were selected for different indicator taxa in the degrading communities. The humic-like fluorescence, commonly considered recalcitrant, was consumed during the incubations when this peak was initially dominating the FDOM pool, and this consumption coincided with higher alkaline phosphatase activity. Taken together, our findings emphasize that HP-DOM lability is dependent on both DOM quality, which is shaped by P availability, and the composition of the consumer community.

Dissolved organic matter released by two marine heterotrophic bacterial strains and its bioavailability for natural prokaryotic communities.

Marine heterotrophic prokaryotes (HP) play a key role in organic matter processing in the ocean; however, the view of HP as dissolved organic matter (DOM) sources remains underexplored. In this study, we quantified and optically characterized the DOM produced by two single marine bacterial strains. We then tested the availability of these DOM sources to in situ Mediterranean Sea HP communities. Two bacterial strains were used: Photobacterium angustum (a copiotrophic gammaproteobacterium) and Sphingopyxis alaskensis (an oligotrophic alphaproteobacterium). When cultivated on glucose as the sole carbon source, the two strains released from 7% to 23% of initial glucose as bacterial derived DOM (B-DOM), the quality of which (as enrichment in humic or protein-like substances) differed between strains. B-DOM induced significant growth and carbon consumption of natural HP communities, suggesting that it was partly labile. However, B-DOM consistently promoted lower prokaryotic growth efficiencies than in situ DOM. In addition, B-DOM changed HP exoenzymatic activities, enhancing aminopeptidase activity when degrading P. angustum DOM, and alkaline phosphatase activity when using S. alaskensis DOM, and promoted differences in HP diversity and composition. DOM produced by HP affects in situ prokaryotic metabolism and diversity, thus changing the pathways for DOM cycling (e.g. respiration over biomass production) in the ocean.

Use of organic exudates from two polar diatoms by bacterial isolates from the Arctic Ocean.

Global warming affects primary producers in the Arctic, with potential consequences for the bacterial community composition through the consumption of microalgae-derived dissolved organic matter (DOM). To determine the degree of specificity in the use of an exudate by bacterial taxa, we used simple microalgae-bacteria model systems. We isolated 92 bacterial strains from the sea ice bottom and the water column in spring-summer in the Baffin Bay (Arctic Ocean). The isolates were grouped into 42 species belonging to Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Forty strains were tested for their capacity to grow on the exudate from two Arctic diatoms. Most of the strains tested (78%) were able to grow on the exudate from the pelagic diatom Chaetoceros neogracilis, and 33% were able to use the exudate from the sea ice diatom Fragilariopsis cylindrus. 17.5% of the strains were not able to grow with any exudate, while 27.5% of the strains were able to use both types of exudates. All strains belonging to Flavobacteriia (n = 10) were able to use the DOM provided by C. neogracilis, and this exudate sustained a growth capacity of up to 100 times higher than diluted Marine Broth medium, of two Pseudomonas sp. strains and one Sulfitobacter strain. The variable bioavailability of exudates to bacterial strains highlights the potential role of microalgae in shaping the bacterial community composition. This article is part of the theme issue 'The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'.

Distinct Spatial Patterns of SAR11, SAR86, and Actinobacteria Diversity along a Transect in the Ultra-oligotrophic South Pacific Ocean.

Distinct distribution patterns of members of the major bacterial clades SAR11, SAR86, and Actinobacteria were observed across a transect from the Marquesas islands through the ultra-oligotrophic South Pacific Gyre into the Chilean upwelling using 16S rRNA gene sequencing and RNA-DNA fingerprinting. Three different Actinobacteria sequence clusters belonging to "Candidatus Actinomarinidae" were localized in the western half of the transect, one was limited to the gyre deep chlorophyll maximum (DCM) and sequences affiliated to the OCS155 clade were unique to the upwelling. The structure of the surface bacterial community was highly correlated with water mass and remained similar across the whole central gyre (1300 nautical miles). The surface hyperoligotrophic gyre was dominated (>70% of all sequences) by highly diverse SAR11 and SAR86 operational taxonomic units and these communities were significantly different from those in the DCM. Analysis of 16S rRNA fingerprints generated from RNA allowed insights into the potential activity of assigned bacterial groups. SAR11 and Prochlorococcus showed the highest potential activity in all water masses except for the upwelling, accounting together for 65% of the total bacterial 16S rRNA in the gyre surface waters in equal proportions whereas the contribution of SAR11 decreased significantly at the DCM.

Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions.

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 10(6) cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 10(4) cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 10(6) bacteria per ml per day) and bacterial production (from 3 to 379 µg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.

Changes in bacterial community metabolism and composition during the degradation of dissolved organic matter from the jellyfish Aurelia aurita in a Mediterranean coastal lagoon.

Spatial increases and temporal shifts in outbreaks of gelatinous plankton have been observed over the past several decades in many estuarine and coastal ecosystems. The effects of these blooms on marine ecosystem functioning and particularly on the dynamics of the heterotrophic bacteria are still unclear. The response of the bacterial community from a Mediterranean coastal lagoon to the addition of dissolved organic matter (DOM) from the jellyfish Aurelia aurita, corresponding to an enrichment of dissolved organic carbon (DOC) by 1.4, was assessed for 22 days in microcosms (8 l). The high bioavailability of this material led to (i) a rapid mineralization of the DOC and dissolved organic nitrogen from the jellyfish and (ii) the accumulation of high concentrations of ammonium and orthophosphate in the water column. DOM from jellyfish greatly stimulated heterotrophic prokaryotic production and respiration rates during the first 2 days; then, these activities showed a continuous decay until reaching those measured in the control microcosms (lagoon water only) at the end of the experiment. Bacterial growth efficiency remained below 20%, indicating that most of the DOM was respired and a minor part was channeled to biomass production. Changes in bacterial diversity were assessed by tag pyrosequencing of partial bacterial 16S rRNA genes, DNA fingerprints, and a cultivation approach. While bacterial diversity in control microcosms showed little changes during the experiment, the addition of DOM from the jellyfish induced a rapid growth of Pseudoalteromonas and Vibrio species that were isolated. After 9 days, the bacterial community was dominated by Bacteroidetes, which appeared more adapted to metabolize high-molecular-weight DOM. At the end of the experiment, the bacterial community shifted toward a higher proportion of Alphaproteobacteria. Resilience of the bacterial community after the addition of DOM from the jellyfish was higher for metabolic functions than diversity, suggesting that jellyfish blooms can induce durable changes in the bacterial community structure in coastal lagoons.

High contribution of SAR11 to microbial activity in the north west Mediterranean Sea.

We investigated the abundance and activity of SAR11 on a monthly time scale between January 2008 and October 2008 in the oligotrophic NW Mediterranean Sea. Applying MICRO-CARD-FISH, we observed that SAR11 had a large contribution to bulk abundance (37 ± 6% of DAPI-stained cells) and to bulk bacterial heterotrophic production (BHP), as estimated from leucine incorporation (55 ± 15% of DAPI-cells assimilating leucine) in surface waters (5 m) throughout the study period. SAR11 contributed also substantially to the assimilation of glucose, ATP, and a combination of amino acids (44 ± 17%, 37 ± 14%, and 43 ± 12% of DAPI cells assimilating these compounds, respectively), organic compounds that provide either single or combined sources of C, P, and N. Temporal changes in the abundance of SAR11 cells that assimilated leucine, glucose, amino acids, and ATP revealed a pattern consistent with that of substrate-active DAPI cells, suggesting that the activity of SAR11 can explain to a large extent the variability in total cells contributing to the utilization of these compounds. Short-term nutrient enrichment experiments performed on each sampling date revealed a strong co-limitation of at least two of the three elements analyzed (C, N, P), in particular, during summer and early autumn. The in situ abundance of SAR11 cells assimilating leucine appeared to increase with P limitation as determined in the nutrient enrichment experiments (r = 0.81, p = 0.015). Our results demonstrate that SAR11 is an important component of the active bacterial community in the NW Mediterranean Sea. Our observations further indicate that the activity of the bulk bacterial community is linked to the activity of SAR11, possibly due to its adaptation to nutrient limitation.

Phylogenetic and functional diversity of Bacteria and Archaea in a unique stratified lagoon, the Clipperton atoll (N Pacific).

The Clipperton lagoon in the North Pacific Ocean has been isolated from the surrounding sea for c. 160 years. It has a stratified water column that comprises an oxic and brackish upper water layer (mixolimnion) and a deep sulfuric anoxic saline layer (monimolimnion), separated by a steep pycnocline. Here, we test whether the Clipperton lagoon with its distinctive physico-chemical features, geographic isolation, recent water column stratification, and large nutrient input harbors original microbial communities. The combination of capillary electrophoresis single-strand polymorphism (CE-SSCP) fingerprinting and sequencing of cloned bacterial and archaeal 16S rRNA genes, and functional genes for methanogenesis (mcrA), methanotrophy (pmoA), and sulfate reduction (dsrAB), revealed that microbial communities and pathways were highly stratified down the water column. The mixolimnion contained ubiquitous freshwater clades of Alpha- and Betaproteobacteria, while the pycnocline contained mostly green sulfur bacteria (phylum Chlorobi). Sequences of the upper layers were closely related to sequences found in other aquatic ecosystems, suggesting that they have a strong potential for dispersal and colonization. In contrast, the monimolimnion contained new deeply branching bacterial divisions within the OP11 cluster and the Bacteroidetes, and was the most diverse of the layers. The unique environmental conditions characterizing the deep layers of the lagoon may explain the novelty of the microbial communities found at the Clipperton atoll.

Pelagic food web patterns: do they modulate virus and nanoflagellate effects on picoplankton during the phytoplankton spring bloom?

As agents of mortality, viruses and nanoflagellates impact on picoplankton populations. We examined the differences in interactions between these compartments in two French Atlantic bays. Microbes, considered here as central actors of the planktonic food web, were first monitored seasonally in Arcachon (2005) and Marennes-Oléron (2006) bays. Their dynamics were evaluated to categorize trophic periods using the models of Legendre and Rassoulzadegan as a reference framework. Microbial interactions were then compared through 48 h batch culture experiments performed during the phytoplankton spring bloom, identified as herbivorous in Marennes and multivorous in Arcachon. Marennes was spatially homogeneous compared with Arcachon. The former was potentially more productive, featuring a large number of heterotrophic pathways, while autotrophic mechanisms dominated in Arcachon. A link was found between viruses and phytoplankton in Marennes, suggesting a role of virus in the regulation of autotroph biomass. Moreover, the virus-bacteria relation was weaker in Marennes, with a bacterial lysis potential of 2.6% compared with 39% in Arcachon. The batch experiments (based on size-fractionation and viral enrichment) revealed different microbial interactions that corresponded to the spring-bloom trophic interactions in each bay. In Arcachon, where there is a multivorous web, flagellate predation and viral lysis acted in an opposite way on picophytoplankton. When together they both reduced viral production. Conversely, in Marennes (herbivorous web), flagellates and viruses together increased viral production. Differences in the composition of the bacterial community composition explained the combined flagellate-virus effects on viral production in the two bays.

Ekhidna lutea gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from the South East Pacific Ocean.

A novel aerobic, heterotrophic bacterium, designated BiosLi39(T), was isolated from the South East Pacific Ocean. Cells were Gram-negative gliding rods forming yellow colonies on marine agar. The isolate was oxidase-, catalase- and alkaline phosphatase-positive and ß-galactosidase-negative. Strain BiosLi39(T) grew at 20-37°C (optimum 30°C), at pH7.0-9.0 (optimum pH8.0) and with 20-60 g NaCl l(-1) (optimum 30-50 g NaCl l(-1)). The fatty acids (>1 %) comprised iso-C(14 : 0), iso-C(15 : 1) G, iso-C(15 : 0), anteiso-C(15 : 0), C(15 : 1) G, C(15 : 0), iso-C(15 : 0) 2-OH, iso-C(16 : 1) G, iso-C(16 : 0), iso-C(16 : 0) 3-OH, iso-C(16 : 0) 2-OH, iso-C(17 : 0) 3-OH, C(17 : 0) 2-OH and three unidentified components with equivalent chain lengths of 17.87, 18.10 and 18.71. A significant proportion of the hydroxylated fatty acids are amide-linked. The lipid pattern indicated the presence of phosphatidylethanolamine, two unidentified aminolipids and three unidentified polar lipids. The strain contained menaquinone 7 as the sole respiratory lipoquinone and did not produce flexirubin-type pigments. The G+C content of the genomic DNA was 37.2 mol%. Comparative 16S rRNA gene sequence analysis indicated that strain BiosLi39(T) was distantly related to all of the representatives of the phylum Bacteroidetes. Its closest relative was Marinoscillum furvescens IFO 15994(T), with which it shared 92.5 % 16S rRNA gene sequence similarity. On the basis of genotypic, phenotypic and chemotaxonomic characteristics, we propose a novel genus and species, Ekhidna gen. nov., sp. nov., with type strain BiosLi39(T) (=DSM 19307(T) =CIP 109600(T) =OOB 398(T)).

Linkage between bacterial carbon processing and the structure of the active bacterial community at a coastal site in the NW Mediterranean Sea.

The temporal dynamics in bulk bacterial parameters and in the richness of the total and active bacterial community, determined from CE-SSCP fingerprints of 16S rRNA genes and 16S rRNA transcripts, respectively, were followed weekly to bimonthly at an oligotrophic coastal site in the NW Mediterranean Sea. Bacterial abundance, bacterial heterotrophic production, and bacterial and community respiration determined over two seasonal cycles displayed large short-term variability and no pronounced temporal pattern was detectable for these parameters. Concentrations in inorganic nutrients, salinity, or concentrations of chlorophyll a could not significantly explain the temporal variability of the bacterial parameters determined. By contrast, bacterial respiration and the bacterial carbon demand were both negatively correlated with the richness of the active bacterial community, while the bacterial parameters determined herein were not related to the richness of the total bacterial community present. Our results indicate that a reduced number of ribotypes is active when rates of bacteria-mediated carbon processes are high. Our approach, based on fingerprints of 16S rRNA transcripts, could represent an interesting tool to investigate the relationship between the structure and function of marine bacteria, in particular, on short temporal and spatial scales.

Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea.

We investigated the impact of water masses originating from freshwater input on bacterial heterotrophic metabolism and community structure at an offshore site in the oligotrophic NW Mediterranean Sea in 2007 and 2008. By combining 16S rRNA gene clone libraries and MICRO-CARD-FISH we determined the dominant operational taxonomic units (OTU) and their contribution to bulk abundance and activity in the presence of buoyant water masses characterized by lower salinity (LSW, < 37.9) and compared these with the winter and spring phytoplankton blooms. We demonstrate that organic matter associated with LSW markedly stimulated bacterial heterotrophic production as determined by [(3) H]-leucine incorporation. The OTUs SAR11-IA, SAR11-IIB, SAR86-I and SAR86-III were dominant in all clone libraries, while the Roseobacter clade and the Bacteroidetes OTU NorSea72 were more specific to the spring phytoplankton bloom. The relative contribution of these OTUs to leucine incorporation varied between 23% and 69% for SAR11, 2% and 17% for Roseobacter and was up to 4% for NorSea72. Together, they accounted for roughly 50% of bulk abundance and leucine incorporation during the four situations investigated. Our results suggest that a few cosmopolitan OTUs respond to different DOM sources in the NW Mediterranean Sea.

Spatial comparison of total vs. active bacterial populations by coupling genetic fingerprinting and clone library analyses in the NW Mediterranean Sea.

Spatial distributions of both total (i.e. 16S rDNA-based fingerprints) and active (i.e. 16S rRNA-based fingerprints) bacterial populations, together with total bacterial activity measured by 3H-leucine incorporation, were studied along a 98 km transect in the NW Mediterranean Sea. Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) fingerprinting was coupled to a clone library, allowing CE-SSCP peaks identification and the monitoring of the spatial variation of bacterial phylotypes. Up to 80% of the community peaks matched those obtained from clone library sequences, accounting for 86.7% of the total fingerprinting area. A good agreement was found between the relative abundance of Prochlorococcus in the CE-SSCP fingerprints and flow cytometry counts (r2=0.66, P<0.05). The largest differences between total and active bacterial populations distribution were found at depths with higher bacterial activity (i.e. surface and deep chlorophyll maximum, DCM). SAR11 at the surface and Gammaproteobacteria at the DCM were the most abundant groups on the 16S rDNA-based fingerprints. However, their ratio of relative importance between rRNA : rDNA was <1 in most cases. Conversely, ratios observed for Prochlorococcus, were consistently >1 both at the surface and at the DCM. These results emphasize the need for combining rDNA- and rRNA-based analyses to better understand the functional role of individual bacterial populations in situ.

Hellea balneolensis gen. nov., sp. nov., a prosthecate alphaproteobacterium from the Mediterranean Sea.

A novel aerobic, heterotrophic, prosthecate bacterium, designated 26III/A02/215(T), was isolated from surface water of the north-western Mediterranean Sea. Cells stained Gram-negative and were straight to slightly curved rods, forming red colonies on agar plates. The strain grew at 15-37 degrees C inclusive (optimum 30 degrees C) and grew optimally at seawater salinity. Growth was observed on organic acids, amino acids and complex organic substrates. The fatty acids (>5 %) detected in strain 26III/A02/215(T) were C(17 : 1)omega6c, C(18 : 1)omega7c and C(17 : 0). The lipid pattern indicated the presence of phosphatidylglycerol, glucuronopyranosyldiglyceride, monoglycosyldiglyceride, an unidentified glycolipid and three unidentified phospholipids. Phosphatidylethanolamine and diphosphatidylglycerol were absent. Ubiquinone Q-10 was the only respiratory lipoquinone. The G+C content of the genomic DNA was 46.8 mol%. Comparative 16S rRNA gene sequence analysis indicated that strain 26III/A02/215(T) belonged to the Hyphomonas-Hirschia-Robiginitomaculum branch of the order Caulobacterales. This affiliation was consistent with the results of polar lipid analyses. Among this group, the novel isolate was most closely related to Robiginitomaculum antarcticum (93.9 % 16S rRNA gene sequence similarity to the type strain). On the basis of genotypic, chemotaxonomic and phenotypic distinctness, we propose a novel genus, Hellea gen. nov., with Hellea balneolensis sp. nov. as the type species. The type strain of Hellea balneolensis is 26III/A02/215(T) (=DSM 19091(T) =CIP 109500(T) =OOB 269(T)).

Eudoraea adriatica gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae.

A novel aerobic, non-motile, Gram-negative bacterium, designated AS06/20a(T), was isolated from coastal waters of the Adriatic Sea and subjected to polyphasic taxonomic analysis. Cells were rod-shaped and formed non-pigmented punctiform colonies on agar plates. The novel isolate grew heterotrophically on a variety of carbon compounds, including organic acids, carbohydrates, polyols, amino acids and complex organic substrates. Cells grew at 15-33 degrees C (optimum 30 degrees C), pH 6.5-8.5 (optimum pH 7.5-8.0) and between 2 and 6 % (w/v) NaCl (optimum 2 %). The dominant fatty acids (>5 %) detected in strain AS06/20a(T) were iso-C(15 : 1) G, iso-C(15 : 0), iso-C(15 : 0) 3-OH, iso-C(17 : 1)omega9c and iso-C(17 : 0) 3-OH. The major respiratory quinone was MK-6. The G+C content of the genomic DNA was 38.9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain AS06/20a(T) fell within the family Flavobacteriaceae in the phylum Bacteroidetes. Comparative 16S rRNA gene sequence analysis indicated that strain AS06/20a(T) was most closely related to members of the genera Zeaxanthinibacter and Robiginitalea, sharing 92-93 % 16S rRNA gene sequence similarity with representatives of these genera. It is proposed, from the results of the polyphasic taxonomic analysis, that the strain should be placed in a new genus, Eudoraea gen. nov., with Eudoraea adriatica sp. nov. as the type species. The type strain is AS06/20a(T) (=DSM 19308(T)=CIP 109577(T)=OOB 358(T)).

Fountain Flow cytometry, a new technique for the rapid detection and enumeration of microorganisms in aqueous samples.

BACKGROUND: Pathogenic microorganisms are known to cause widespread waterborne disease worldwide. There is an urgent need to develop a technique for the real-time detection of pathogens in environmental samples at low concentrations, <10 microorganisms/ml, in large sample volumes, > or =100 ml. METHODS: A novel method, Fountain Flowtrade mark cytometry, for the rapid and sensitive detection of individual microorganisms in aqueous samples is presented. Each sample is first incubated with a fluorescent label and then passed as a stream in front of a laser, which excites the label. The fluorescence is detected with a CCD imager as the sample flows toward the imager along its optical axis. The feasibility of Fountain Flow cytometry (FFC) is demonstrated by the detection of Escherichia coli labeled with ChemChrome CV6 and SYBR Gold in buffer and natural river water. RESULTS: Detections of labeled E. coli were made in aqueous suspensions with an efficiency of 96% +/- 14% down to a concentration approximately 200 bacteria/ml. CONCLUSIONS: The feasibility of FFC is demonstrated by the detection of E. coli in buffer and natural river water. FFC should apply to the detection of a wide range of pathogenic microorganisms including amoebae.

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry.

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.