Development and Application of a Monoclonal-Antibody Technique for Counting Aureococcus anophagefferens, an Alga Causing Recurrent Brown Tides in the Mid-Atlantic United States

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called “brown tides” in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.     

Immunofluorescence Flow Cytometry Technique for Enumeration of the Brown-Tide Alga, Aureococcus anophagefferens

A new immunologically based flow cytometry (IFCM) technique was developed to enumerate Aureococcus anophagefferens, a small pelagophyte alga that is the cause of “brown tides” in bays and estuaries of the mid-Atlantic states along the U.S. coast. The method utilizes a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) to label the surface of A. anophagefferens cells which are then detected and enumerated by using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations, were determined. The FITC-MAb method was tested for cross-reactivity with nontarget, similarly sized, photoautotrophic protists, and the method was compared to an enzyme-linked immunosorbent assay (ELISA) using the same MAb. Comparisons of the IFCM technique to traditional microscopy enumeration of cultures and spiked environmental samples showed consistent agreement over several orders of magnitude (r² > 0.99). Comparisons of the IFCM and ELISA techniques for enumerating cells from a predation experiment showed a substantial overestimation (up to 10 times higher) of the ELISA in the presence of consumers of A. anophagefferens, presumably due to egested cell fragments that retained antigenicity, using the ELISA method, but were not characterized as whole algal cells by the IFCM method. Application of the IFCM method to environmental “brown-tide” samples taken from the coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundance levels throughout the course of a bloom and over a large range of abundance values. IFCM counts of the brown-tide alga from natural samples were consistently lower than those obtained using the ELISA method and were equivalent to those of the polyclonal immunofluorescence microscopy technique, since both methods discriminate intact cells. Overall, the IFCM approach was an accurate and relatively simple technique for the rapid enumeration of A. anophagefferens in natural samples over a wide range of abundance values (10³ to 10⁶ cells ml⁻¹).     

PHYLOGENY OF AUREOCOCCUS ANOPHAGEFFERENS AND A MORPHOLOGICALLY SIMILAR BLOOM-FORMING ALGA FROM TEXAS AS DETERMINED BY 18S RIBOSOMAL RNA SEQUENCE ANALYSIS1

The 18S ribosomal RNA genes from isolates of the east coast “brown tide” alga Aureococcus anophagefferens (strain Pt-1) and the Texas “brown tide” alga (strain TBA-2) were sequenced and compared to the gene sequences of Pelagomonas calceolata, a member of the Pelagophyceae, and 10 other organisms. The genes of A. anophagefferens and strain TBA-2 consisted of 1814 and 2236 bases, respectively. The difference in length was largely due to a 423-base insert occurring in the gene of strain TBA-2. Excluding the insert, 93% of the bases of the aligned sequences were identical. Phylogenetic analyses were performed based on two different alignment refinement methods (eye refinement and Gatesy refinement). Trees inferred from the Jukes-Cantor distance matrices using the neighbor-joining method were similar for both alignment methods. In both trees, A. anophagefferens, strain TBA-2, and P. calceolata formed a monophyletic group with A. anophagefferens and P. calceolata being sister taxa and strain TBA-2 occurring on a deeper-rooted branch. Bootstrap data sets for both alignment methods gave strong support for the Pelagophyceae group and the branches within that group. Parsimony analyses using the two alignments gave one tree for the eye-refined alignment and two trees for the Gatesy method. All three trees had nearly the same topology as the trees inferred from the distance method. In addition, the Pelagophyceae group and the branches within that group were supported by bootstrap analyses and decay indices. Based on the available data, A. anophagefferens and strain TBA-2 should be placed in the Pelagophyceae but not in the same order and family as P. calceolata.     

ULTRASTRUCTURE AND ECOLOGY OF AUREOCOCCUS ANOPHAGEFERENS GEN. ET SP. NOV. (CHRYSOPHYCEAE): THE DOMINANT PICOPLANKTER DURING A BLOOM IN NARRAGANSETT BAY,RHODE ISLAND,SUMMER 19851

Observations of a marked cessation of feeding in filter feeding animals maintained in flowing Narragansett Bay seawater in June 1985 drew our attention to a bloom of a golden alga 2 μm in diameter at unprecedented populations of 10⁹ cells. L^?1. This picoplankter lacked morphological features useful in discriminating it from other similar sized forms with either phase contrast or epifluorescence light microscopy. Natural populations of picoplankton, obtained from the height of the bloom until its decline, were examined in thin section with transmission electron microscopy. A cell with a single chloroplast, nucleus, and mitochondrion and an unusual exocellular polysaccharide-like layer was apparently the bloom alga. The ultrastructure of this alga is consistent with that of the Chrysophyceae, and a new genus and species, Aureococcus anophagefferens is described. Attempts to grow this previously unrecognized picoplanktonic alga as an obligate phototroph failed and only yielded cultures of other previously described picoalgae. Facultative and obligate phagotrophic protists with ingested cells of Aureococcus were only observed as the bloom waned and minute diatoms became common. Cells of A. anophagefferens with virus particles typical for picoalgae occurred throughout the bloom. Populations of the usually dominant photosynthetic picoplankter, the cyanobacterium Synechococcus Nägeli, were depressed during the bloom. This could be due in part to selective grazing on Synechococcus rather than Aureococcus by elevated populations of Calycomonas ovalis Wulff which accompanied the algal bloom.     

CHARACTERIZATION OF A LYTIC VIRUS INFECTIOUS TO THE BLOOM-FORMING MICROALGA AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE)

Aureococcus anophagefferens Hargraves and Sieburth has caused recurring monospecific blooms in Long Island embayments since it was first described in 1985. It was termed the "brown tide," due to the resulting water color, and has had a devastating effect on Long Island's (New York) marine ecosystem. In 1992, a virus that was capable of causing lysis of A. anophagefferens was isolated and maintained in culture. We report on the further characterization of this virus, Aureococcus anophagefferens virus-1 (AaV-1), indicated by a buoyant density of 1.2776 g·mL⁻¹ in a CsCl equilibrium gradient. Electron microscopy revealed a phage with a hexagonal head and tail similar to previously described phages. By using adenovirus for calibration, the virus was found to have a head 50—55 nm wide and a tail 70–75 nm long. The viral band was infectious to A. anophagefferens after dialysis. The virus was composed of at least 16 distinct polypeptides ranging in molecular weight from 20 to 230 kDa. The adsorption coefficient for the virus was 7.2 × 10⁻⁹ mL·min⁻¹, and the burst size was calculated to be 9.4 viruses per A. anophagefferens cell at 20° C. Complete lysis of A. anophagefferens occurred with a titer as low as 893 viruses·mL⁻¹, and the lower limit of infectivity was 93 viruses·mL⁻¹. The virus lost its infectivity between 30° and 40° C. These results suggest that AaV-1 is highly infectious and that the role of the virus in preventing or ending A. anophagefferens blooms needs further investigation.     

A TRANSIENT BLOOM OF OSTREOCOCCUS (CHLOROPHYTA, PRASINOPHYCEAE) IN WEST NECK BAY, LONG ISLAND, NEW YORK

The smallest known eukaryote, Ostreococcus tauri Courties et Chrétiennot-Dinet, was first reported as the dominant picoplankter in a French lagoon known for its diverse phytoplankton community and high oyster productivity. Long-term seasonal blooms of this picoeukaryote were observed in association with stable plankton communities. On 5 June 2001, a distinctive monotypic picoplankton bloom was detected by flow cytometry as part of an ongoing study of "brown tide" ( Aureococcus anophagefferens ) bloom initiation in Long Island bays. The bloom reached a concentration of 5 × 10⁵ cells·mL⁻¹ in West Neck Bay and lasted less than 2 weeks. Epifluorescence microscopy and TEM indicated that the bloom organism was an Ostreococcus -like picoalga, the first ever observed in a Long Island bay. Many cells of this alga contained numerous virus-like particles. The Ostreococcus -like picoalga, which resembles O. tauri , was rare in samples collected the following week. Instead, a substantial increase in the Synechococcus population was observed. Such rapid population changes have not previously been reported for Ostreococcus . Viral lysis and grazing by heterotrophic nanoflagellates may have contributed to the rapid decline of the Ostreococcus -like cells in West Neck Bay.     

Flora and fauna associated with the introduced red alga Gracilaria vermiculophylla

This paper presents the first detailed study of the spread of the introduced marine red alga Gracilaria vermiculophylla on the west coast of Sweden, and of the fauna and flora associated with this alga in Scandinavia and the western mid-Atlantic. G. vermiculophylla was discovered in the archipelago of Göteborg, Sweden, in the summer of 2003, and in 2005 its distribution range covered at least 150 km. The species is typically found as loose-lying thalli or attached to small stones and mollusc shells within low-energy bays and estuaries. Both gametophytic and tetrasporophytic specimens were found, as well as specimens with mixed reproductive stages. In order to assess the importance of this introduced alga as a habitat for native benthic organisms, attached and loose-lying individuals of G. vermiculophylla were sampled from invaded locations in Sweden, Denmark and Virginia (United States). In total we found 92 taxa associated with G. vermiculophylla. The dominant classes were Malacostraca, Gastropoda and Florideophyceae. The diversity of the associated taxa was not affected by attachment status, or G. vermiculophylla biomass. In Virginia and Sweden animal abundances were positively correlated with the biomass of algae and plants associated with G. vermiculophylla. If G. vermiculophylla primarily invades non-vegetated soft-sediment estuaries, the invasion may lead to an increase in abundances of small native invertebrates (e.g. gastropods and crustaceans) and epiphytic algae, with likely cascading effects on higher trophic levels.     

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.     

Distinct ecological niches of marine symbiotic N2‐fixing cyanobacterium Candidatus Atelocyanobacterium thalassa sublineages

A recently described symbiosis between the metabolically streamlined nitrogen‐fixing cyanobacterium UCYN‐A and a single‐celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN‐A sublineages have been defined based on UCYN‐A nitrogenase (nifH) sequences. Due to the low abundances of UCYN‐A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN‐A sublineages based on high throughput sequencing of UCYN‐A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN‐A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN‐A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN‐A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN‐A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN‐A distributions and the ecological importance of the different sublineages.     

The fish community of a newly restored southern California estuary: ecological perspective 3 years after restoration

Bays and estuaries are considered essential fish habitat, yet in many parts of the world, these areas have been degraded or destroyed. In southern California, habitat restoration has become a widely used approach for protecting coastal ecosystems; however, there is little information available on the success of these efforts. Monthly abundance surveys were employed to examine spatial and temporal trends in the fish assemblages 3 years after the restoration of the Bolsa Chica Full Tidal Basin (BCFTB). This was used as a short-term success assessment of the BCFTB restoration, as well as an important baseline against which future studies can determine the long-term trajectory of the restoration. Forty-four species of fish were caught inside the BCFTB, at an average density of 116.8 fish 100 m^?2 and an average biomass of 4.2 kg 100 m^?2. There was a seasonal pattern in fish abundances but no overall increase or decrease in abundances during the entire study period. Marine, estuarine and migrant fish species were found in the BCFTB, each showing different seasonal patterns in abundance, similar to nearby estuaries, with 14 species driving these patterns. Water temperature and season were the most influential factors on the species composition of the fish community in the BCFTB. Therefore, 3 years after restoration the BCFTB is providing habitat for coastal fish species where none existed previously, and shows a community structure similar to natural estuaries in southern California. The BCFTB restoration has been initially successful but needs to be monitored periodically to assess its long-term success.     

Use of Quantitative Real-Time PCR to Investigate the Dynamics of the Red Tide Dinoflagellate Lingulodinium polyedrum

A new method based on quantitative real-time polymerase chain reaction (qPCR) was developed and applied to quantify the red tide dinoflagellate Lingulodinium polyedrum in natural seawater samples and in laboratory cultures. The method uses a Molecular Beacon™ approach to target a species-specific region of the small subunit ribosomal RNA gene. The accuracy of the method was verified by microscopical counts using cultures of the dinoflagellate isolated from coastal waters near Los Angeles, CA, and with natural water samples spiked with cultured L. polyedrum. The method was applied to document the pattern and timing of vertical migration by the dinoflagellate in a 2-m water column on an 11:13 h light/dark photoperiod established in the laboratory. Positive phototaxis of L. polyedrum resulted in dense aggregations of the dinoflagellate within the top few centimeters of the water column during the light period. This pattern of distribution was readily established by both methods, although abundances of L. polyedrum determined using qPCR were higher than abundances determined by microscopy in the morning and lower in the afternoon and evening. These differences may have been a consequence of variability in the DNA content per cell because of synchrony of cell division. Counts using both methods to analyze natural samples collected from coastal waters in the Long Beach–Los Angeles area and adjacent San Pedro Channel were in close agreement. However, the qPCR method exhibited greater sensitivity than the microscopical method when L. polyedrum was present at low abundances, and qPCR had a much higher rate of sample throughput than microscopy. The development of this new approach for enumerating L. polyedrum provides a useful tool for studying the ecology of this important red tide species.     

DESCRIPTION AND CHARACTERIZATION OF THE ALGAL SPECIES AUREOUMBRA LAGUNENSIS GEN. ET SP. NOV. AND REFERRAL OF AUREOUMBRA AND AUREOCOCCUS TO THE PELAGOPHYCEAE1

The Texas brown tide alga (strain TBA-2) is described as Aureoumbra lagunensis Stockwell, DeYoe, Hargraves, et Johnson, gen. et sp. nov. Pigment composition, chloroplast structure, and 18s ribosomal RNA gene sequence data indicate that A. lagunensis and the east coast brown tide alga Aureococcus anophagefferens (originally placed in the Chrysophyceae) belong in the class Pelagophyceae. The new genus Aureoumbra with A. lagunensis as the type species differs from Aureococcus in 18s ribosomal RNA gene sequence, pyrenoid form, nitrogen physiology, and possession of basal bodies. The genus Aureococcus is placed in the order Pelagomonadates and family Pelagomonadaceae while ordinal placement of Aureoumbra is deferred.     

Molecular Cloning and Antiserum Development of Cyclin Box in the Brown Tide Alga Aureococcus anophagefferens

Cyclins can be useful cell cycle markers for growth rate studies on harmful algal blooms. In this study, a gene fragment corresponding to cyclin box was cloned for the brown tide alga Aureococcus anophagefferens. This algal gene fragment, designated as Btcycl1, was most similar to cyclin B. Oligopeptides based on the deduced amino acid sequence were synthesized and used to raise an antiserum that reacted on Western blots with a protein of about 63 kDa, the same size as cyclin B in other organisms. The cyclin B–like protein recognized by this antiserum, and the messenger RNA amplified using the primers, were more abundant in exponential cultures and decreased markedly in stationary cultures. This protein also appeared to be cell cycle dependent. Immunofluorescence labeling showed that this antiserum specifically stained a protein in Aureococcus cells and had no cross-reaction with bacteria that were present in the algal culture. The Btcycl1 sequence and the antiserum will provide a useful tool for studies on regulation of in situ growth rate for this brown tide alga. Received May 22, 2000; accepted July 13, 2000.     

Identifying ‘hot spots’ of biological and anthropogenic activity in two Irish estuaries using means and frequencies

The available data for two important Irish estuaries, Cork and Wexford harbours, were analysed to identify hot spots: locations where water quality variables are likely to differ from background levels. The approach taken reflects the limitations imposed by restricted spatial and temporal replication in the available datasets. Information for many estuaries may exist in such fragmented datasets. Averages drawn from small sample sizes are susceptible to extreme values. To lessen this problem, a novel approach was used: identifying locations where high measurements of a variable were relatively more frequent. The locations of relatively high chlorophyll measurements in Cork and Wexford harbours indicated estuarine origins for the majority of algal blooms. Nutrient cycling in Wexford Harbour appeared to be coupled with phytoplankton growth. The estuary acted as a source for dissolved inorganic nutrients during periods with low chlorophyll levels and as a sink during plankton blooms. High chlorophyll levels in Cork Harbour were generally associated with sub-surface samples in stratified water. Sources of ammonia and phosphate in Cork harbour appeared to result from direct anthropogenic input. Residual variation in biological oxygen demand reflected point pollution sources in both Cork and Wexford Harbours. Algal blooms were common in both estuaries, with 20% of chlorophyll a measurements exceeding 20 g l^–1 in each system. However, despite the presence of blooms and influences of point sources, there is currently little evidence for environmental impacts such as extensive deoxygenation. This conclusion is tentative, given the fragmented nature of the datasets. The locations of hot spots can be used to inform future research on potential impacts and estuarine function.     

AN INVESTIGATION OF DISTRIBUTIONAL PATTERNS IN THE DIATOM FLORA OF NETARTS BAY,OREGON, BY CORRESPONDENCE ANALYSIS1

Distributional patterns in assemblages of epiphytic and sediment-associated diatoms were investigated in Netarts Bay, Oregon. The method of reciprocal averaging revealed a floristic discontinuity between the epiphytic and sediment samples in ordination space. The basis for this discontinuity was the presence of a large number of sediment-associated taxa that were either very rare or not observed in the epiphytic samples. Within the sediment samples, the diatom flora formed a distributional continuum which had relatively high correlations with mean grain size, a sediment sorting coefficient, and the organic matter content of the sediment. A comparison of the flora in Netarts Bay with floras in other Oregon estuaries indicates that epiphytic, epilithic, and sediment-associated diatom assemblages do not exhibit conspicuous latitudinal changes along the coast of Oregon, and that many of the same taxa can be expected to occur in samples from comparable habitats in estuaries throughout the temperate regions of the world.     

Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains

AIMS: Production of a monoclonal antibody (MAb) to Escherichia coli O157 to develop a rapid test using a sandwich ELISA (sELISA) format. METHODS AND RESULTS: A MAb (7A6) was developed to the long-chain lipopolysaccharide of E. coli O157. A sELISA developed with the MAb reacted with 28 bovine and seven human enterohaemorrhagic E. coli (EHEC) O157 strains and also with two enterotoxigenic E. coli O157 strains. Cross-reaction to a rabbit diarrhoeal E.coli O15, Citrobacter freundii, Salmonella urbana and Vibrio cholerae O1 Inaba was detected. CONCLUSION: A MAb-based sELISA to detect E. coli O157 was produced. Its application to field samples is required to fully determine its prospective use for the detection of EHEC O157, to evaluate the non-specific interference of the cross-reacting strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay produced is not wholly specific to EHEC O157, but has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157.     

An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains.

A monoclonal antibody (MAb), MAb 900, which detects a 43-kDa protein present on Escherichia coli was found. Subsequently, more than 90 organisms, belonging to either the bacterial, archaeal, or eucaryal domain, were tested for reactivity to this MAb. Of the bacterial and archaeal domains, almost all species proved to be positive, whereas all organisms from the eucaryal domain gave negative results. The 43-kDa protein was purified by affinity chromatography and subsequently analyzed by microsequencing methods. Two peptide sequences which showed a high degree of homology (> 99%) to the prokaryotic elongation factor Tu (EF-Tu) were obtained. Western blot (immunoblot) analysis using both purified EF-Tu and EF-Tu domains confirmed that the unknown protein was EF-Tu. The panbacterial distribution of EF-Tu, which is present in large amounts in every prokaryotic cell, renders this protein a good candidate for a diagnostic approach. In consequence, we have used the anti-EF-Tu MAb 900 to design both a dot blot assay and an enzyme-linked immunosorbent assay. From either blood culture, urine, or gall-bladder fluid, bacterial contamination could be detected. The sensitivity of these tests is currently 10(4) bacteria per ml.