An indirect fluoresent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples

Xu, Huai Shu; Roberts, N C; Adams, Linda B.; West, Paul; Siebeling, R. J.; Huq, Anwar; Huq, M. I.; Rahman, Rosliza Abdul; Colwell, Rita R.

doi:10.1016/0167-7012(84)90017-4

Cited by 60 publications

(28 citation statements)

References 20 publications

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“…In recent years, a concomitant effort has been made to reduce the time required for detection of V. cholerae O1 in clinical and environmental sampies. To this end, several investigators have employed the fluorescent antibody (FA) technique for detection of V. cholerae [1,3,4]. Polyclonal anti-01 serum was originally used in direct fluorescent antibody (DFA) procedures [6] to detect V. cholerae O1 cells in smears prepared directly from specimens.…”

Section: Introductionmentioning

confidence: 99%

“…Unfortunately, problems were encountered, arising from cross-reactions and non-specific fluorescence. Nevertheless, Xu et al [4] were able to detect V. cholerae O1 in environmental samples, using enrichment broths followed by indirect fluorescent antibody (IFA) staining and employing a commercially prepared polyclonal anti-O1 sera which had to be absorbed seriatim with a set of Gram-negative organisms to eliminate non-specific staining.…”

Section: Introductionmentioning

confidence: 99%

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Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

Hasan

1994

FEMS Microbiology Letters

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An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well as improved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

Hasan

1994

FEMS Microbiology Letters

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“…The size of the bacterial population producing thiocyanate hydrolase was estimated by fluorescent immunostaining (24) by using antisera raised against the ␣ and ␥ subunits of the enzyme and fluorescein isothiocyanate-labeled secondary antibody. To stain the cytoplasmic enzyme, the cell wall was permeabilized by treatment for 30 to 60 min at 37°C with a lysozyme solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl 2 , lysozyme (40 g/ml), DNase I (1 mg/ml), and gelatin (10 g/ml).…”

mentioning

confidence: 99%

Genetic and Immunochemical Characterization of Thiocyanate-Degrading Bacteria in Lake Water

Yamasaki¹,

Matsushita

Namura³

et al. 2002

Appl Environ Microbiol

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Natural aquatic and soil samples were screened for the presence of thiocyanate-degrading bacteria. Using thiocyanate supplementation, we established an enrichment culture containing such bacteria from lake water. The dominant bacteria had the scnC-LS5 gene encoding thiocyanate hydrolase, which was closely related to the enzyme found previously in Thiobacillus thioparus THI115 isolated from activated sludge.Carbonyl sulfide (COS) is a major sulfur compound in the troposphere contributing to sulfate aerosol, which accumulates in the stratosphere and influences the climate (3). About onehalf of the COS originates from marine and soil environments (11). Biological processes have been considered to be responsible for the unique sulfur flow (14). However, little is known about the diversity and composition of the bacterial communities that are involved in the production and degradation of COS in the environment. It has been observed that production of COS in soil is stimulated by addition of thiocyanate (2,14,15). Katayama et al. (6,7) isolated Thiobacillus thioparus THI112 and THI115 from activated sludge and showed that these bacteria are obligate chemolithotrophs that utilize thiocyanate as a sole energy source and produce ammonia and COS as the reaction products (8,12). A unique enzyme responsible for degradation of thiocyanate was purified from this bacterium and designated thiocyanate hydrolase (7). The scnA, scnB, and scnC genes encoding the three subunits (␣, ␤, and ␥) of this enzyme have been cloned and sequenced. The deduced amino acid sequences exhibit significant homology to the sequences of nitrile hydratases from various bacteria (10). Furthermore, thiocyanate-degrading activity has been found in the facultative chemolithotroph Paracoccus thiocyanatus (9), in which a thiocyanate hydrolase-like enzyme has been detected (K. Hatayama and Y. Katayama, unpublished data), indicating that microbes carrying this enzyme may be distributed widely in water and soil environments.In the present study, we detected thiocyanate-degrading and COS-producing bacteria in lake water by using an enrichment culture supplemented with thiocyanate. To quantify such bacteria in a natural aquatic environment, we used a fluorescent immunostaining technique with thiocyanate hydrolase-specific antibodies. Furthermore, scnC-specific primers were used to characterize the microbes harboring the related genes. To determine phylogenetic relationships among thiocyanate-degrading bacteria, we analyzed 16S ribosomal DNA (rDNA) fragments by a PCR-denaturing gradient gel electrophoresis (DGGE) method.Thiocyanate degradation and COS emission in environmental bacteria. Water and soil samples were collected from various locations in Japan (Table 1). The microorganisms in the samples were grown at 30°C in TC medium (7) containing 0.1 g of potassium thiocyanate per liter (TC1 medium). Consumption of thiocyanate in the medium was measured spectrophotometrically (7). To measure the amount of COS, a gas sample was collected from the headspace of a flas...

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“…1997). Total V. cholerae counts as immunofluorescence positive V. cholerae O1 (FA‐DC) were evaluated using an indirect fluorescent antibody staining method (Xu et al. 1984; Macek et al.…”

Section: Micro‐organism Countsmentioning

confidence: 99%

“…During the last decades, V. cholerae has been reported from brackish or saline waters, mostly in a viable but non‐culturable (VBNC) form (Xu et al. 1984; Colwell et al.…”

Section: Introductionmentioning

confidence: 99%

In situ measured elimination of Vibrio cholerae from brackish water

Pérez

Macek

Galván

2004

Tropical Med Int Health

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In situ elimination of fluorescently labelled Vibrio cholerae (FLB) was measured in two saline water bodies in Mexico: in a brackish water lagoon, Mecoacán (Gulf of Mexico; State of Tabasco) and an athalassohaline lake, Alchichica (State of Puebla). Disappearance rates of fluorescently labelled V. cholera O1 showed that they were eliminated from the environment at an average rate of 32% and 63%/day, respectively (based on the bacterial standing stocks). The indirect immunofluorescence method confirmed the presence of V. cholerae O1 in the lagoon. However, the elimination of FLB was not directly related either to the presence or absence of the bacterium in the water body or to the phytoplankton concentration.

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An indirect fluoresent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples

Cited by 60 publications

References 20 publications

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

Genetic and Immunochemical Characterization of Thiocyanate-Degrading Bacteria in Lake Water

In situ measured elimination of Vibrio cholerae from brackish water

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