a dormant state, and are not enumerated in the dormant state because they are incapable
of growth in conventional culture media. The cells are viable, but non-culturable. Therefore, waters, the quality of which are judged acceptable, may indeed be hazardous,
placing the public health safety of those waters in question.
By adapting indirect immunofluorescent-epifluorescent techniques developed in our
laboratory for environmental studies, a more realistic estimate of population size has
been obtained (Xu, et al., 1983 and 1984). An evaluation of this new approach, compared
with conventional enumeration methods was conducted for waters of Chesapeake Bay,
Louisiana, Puerto Rico, Zimbabwe (Africa) and Bangladesh for the presence of V.
cholerae serovar 01 (Colwell, et ai, 1984, Xu, el al., 1984). Determinations were performed in sewage and water samples using the three-tube, three-dilution, most probable
number (MPN) index. Loopfuls from each tube of alkaline peptone enrichment broth
(peptone, 1 % w v; NaCl ; 1 % w/v ox-bile; 0.5 % w/v, pH 8.6, were transferred to
thiosulfate-citrate-bile salts agar (TCBS, Oxoid, Columbia, MD USA) and suspect
colonies were examined by slide agglutination, employing commercial antiserum (Difco
Laboratory, Detroit, MI) for the presence of the 01 antigen. A loopful was also spread on
a clean glass slide for fluorescent antibody staining (FAB) and an MPN enumeration by
microscopic detection was made (Xu, el al., 1984).
Slides were air-dried, fixed with alcohol, and air dried once more. A drop of RITC for
background staining (Rhodamine isothiocyanate-conjugated bovine serum albumin,
BBL Microbiology Systems, Cockeysville, MD) was placed on each slide and incubated
in a moist chamber for 30 minutes at 35°C. Phosphate buffered saline (PBS) was then
used to wash each slide. Next, a drop of specific antiserum was placed on the slides,
followed by incubation. After another washing with PBS, one drop of FITC (fluoresceinisothiocyanate conjugate of goat anti-rabbit globulin antiserum, Difco) was delivered to
the slides. A final incubation period with subsequent washing followed. After air drying, a
cover-glass was mounted on each slide using FA mounting fluid (Difco, pH 9.O.). Slides
were viewed using a Zeiss standard 18 microscope equipped with a 12.5 x eyepiece, 100 x
neofluor objective, IVFL epifluorescence condenser, 100 W halogen lamp, BP 450-490
band pass filter, FT 510 beam splitter, and LP 520 barrier filter (Carl Zeiss Inc.,
New-York, NY) or equivalent.
Upon examination stained preparations of V. cholerae revealed rod-shaped cells, with
distinct curvature, in some cases, and a peripheral green band just below the cell wall was
visible.
Results of MPN enumerations of sample collections in Louisiana are presented in
Table 1. Only one sewage sample in June, 1981 proved positive for V. cholerae 01, using
standard culture methods, i.e. media and slide agglutinations. In contrast, all water,
sewage and sediment samples from June and September were positive by fluorescent
staining, yielding a significantly higher bacterial count per 100 ml volume of water.
Canals and village water sources in Bangladesh were the sites of sampling in April and
May, 1982. To determine the presence or absence of V. cholerae Ol, 100 ml volumes of
water were filtered through 0.45 µm filters (Gelman, GN-6) which were placed in enrichment broths. Broths were incubated at 30°C for 12 h, after which 1.0 ml aliquots were
removed and dispensed to fresh enrichment broth. Incubation at 37°C followed for an
additional 22 h. Loopfuls of inoculum were transferred, after each incubation stage, to
taurocholate-tellurite-gelatin agar and slides. Slide agglutinations were performed on
portions of bacterial colonies and fluorescent antibody staining was done to determine
the presence of the V. cholerae 0l antigen. From a total of 52 water samples collected,
508
of growth in conventional culture media. The cells are viable, but non-culturable. Therefore, waters, the quality of which are judged acceptable, may indeed be hazardous,
placing the public health safety of those waters in question.
By adapting indirect immunofluorescent-epifluorescent techniques developed in our
laboratory for environmental studies, a more realistic estimate of population size has
been obtained (Xu, et al., 1983 and 1984). An evaluation of this new approach, compared
with conventional enumeration methods was conducted for waters of Chesapeake Bay,
Louisiana, Puerto Rico, Zimbabwe (Africa) and Bangladesh for the presence of V.
cholerae serovar 01 (Colwell, et ai, 1984, Xu, el al., 1984). Determinations were performed in sewage and water samples using the three-tube, three-dilution, most probable
number (MPN) index. Loopfuls from each tube of alkaline peptone enrichment broth
(peptone, 1 % w v; NaCl ; 1 % w/v ox-bile; 0.5 % w/v, pH 8.6, were transferred to
thiosulfate-citrate-bile salts agar (TCBS, Oxoid, Columbia, MD USA) and suspect
colonies were examined by slide agglutination, employing commercial antiserum (Difco
Laboratory, Detroit, MI) for the presence of the 01 antigen. A loopful was also spread on
a clean glass slide for fluorescent antibody staining (FAB) and an MPN enumeration by
microscopic detection was made (Xu, el al., 1984).
Slides were air-dried, fixed with alcohol, and air dried once more. A drop of RITC for
background staining (Rhodamine isothiocyanate-conjugated bovine serum albumin,
BBL Microbiology Systems, Cockeysville, MD) was placed on each slide and incubated
in a moist chamber for 30 minutes at 35°C. Phosphate buffered saline (PBS) was then
used to wash each slide. Next, a drop of specific antiserum was placed on the slides,
followed by incubation. After another washing with PBS, one drop of FITC (fluoresceinisothiocyanate conjugate of goat anti-rabbit globulin antiserum, Difco) was delivered to
the slides. A final incubation period with subsequent washing followed. After air drying, a
cover-glass was mounted on each slide using FA mounting fluid (Difco, pH 9.O.). Slides
were viewed using a Zeiss standard 18 microscope equipped with a 12.5 x eyepiece, 100 x
neofluor objective, IVFL epifluorescence condenser, 100 W halogen lamp, BP 450-490
band pass filter, FT 510 beam splitter, and LP 520 barrier filter (Carl Zeiss Inc.,
New-York, NY) or equivalent.
Upon examination stained preparations of V. cholerae revealed rod-shaped cells, with
distinct curvature, in some cases, and a peripheral green band just below the cell wall was
visible.
Results of MPN enumerations of sample collections in Louisiana are presented in
Table 1. Only one sewage sample in June, 1981 proved positive for V. cholerae 01, using
standard culture methods, i.e. media and slide agglutinations. In contrast, all water,
sewage and sediment samples from June and September were positive by fluorescent
staining, yielding a significantly higher bacterial count per 100 ml volume of water.
Canals and village water sources in Bangladesh were the sites of sampling in April and
May, 1982. To determine the presence or absence of V. cholerae Ol, 100 ml volumes of
water were filtered through 0.45 µm filters (Gelman, GN-6) which were placed in enrichment broths. Broths were incubated at 30°C for 12 h, after which 1.0 ml aliquots were
removed and dispensed to fresh enrichment broth. Incubation at 37°C followed for an
additional 22 h. Loopfuls of inoculum were transferred, after each incubation stage, to
taurocholate-tellurite-gelatin agar and slides. Slide agglutinations were performed on
portions of bacterial colonies and fluorescent antibody staining was done to determine
the presence of the V. cholerae 0l antigen. From a total of 52 water samples collected,
508