M I C R O B I O L O G Y A N D
V I R O L O G Y
O F
W A S T E W A T E R
RAPID ASSESSMENT OF WATER QUALITY
BY FLUORESCENT ANTIBODY IDENTIFICATION
OF FECAL STREPTOCOCCI
MARIA T. PAVLOVA, E. BEAUVAIS, F.T. BREZENSKI and WARREN LITSKY*
♦Marshall Hall, University of Massachusetts, Amherst,
Massachusetts 01002, U.S.A.
INTRODUCTION
Studies conducted in our laboratory indicated that all species of group D streptococci
(fecal streptococci) yielded positive fluorescent antibody (FA) reactions with laboratory
prepared labeled antibodies.
The specificity of these reactions was also demonstrated in that no cross-reactions with
non-group D streptococci occurred after trypsinization of the smears prior to staining.
These findings suggested that FA techniques may be employed in the determination of
the presence and source of fecal pollution in water employing the fecal streptococci as
indicator organisms. This report summarizes our efforts in this endeavor.
MATERIALS AND METHODS
Collection and plating of samples.
Water samples from the Connecticut and Mill rivers and raw sewage from the Amherst
treatment plant were examined within 15-40 minutes. One, 10, 20, 50 and 100 ml of
water were filtered and duplicate membranes (millipore HA 0.45 μ) were transferred to
Pfeizer selective enterococcus (PSE) and KF streptococcus agars. One tenth ml of the
appropriate dilutions from sewage was spread with a glass rod on each of the above media
in triplicate.
The inverted PSE plates were incubated aerobically at 37°C for 24 hr whereas the KF
plates were incubated for 48 hr. All membrane filter plates were incubated in rn
atmosphere saturated with water vapor. The densities of fecal streptococci (group D)
were reported as colony counts per 100 ml of water and sewage.
Identification of isolates.
The isolates from the selective media were identified on the basis of the results from
the following physiological, biochemical and serological tests: growth at 10° and 45°C;
growth in brain heart infusion (BHI) with 6.5% NaCl and pH 9.6; survival at 60°C for 30
minutes; reduction of 0.1%methylene blue, potassium tellurite and triphenyl tetrazolium
chloride (TTC); hemolysis on blood agar; hydrolysis of gelatin and 0.2% starch; catalase
production; fermentation of lactose, d-sorbitol and 1-arabinose; precipitin and FA
reactions. Specific streptococcal D antisera for the performing of precipitin and FA tests
were prepared in 15 rabbits by conventional methods. The precipitin tests were
performed according to the Lancefield method (1933) employing our laboratory
prepared and commercial streptococcal group D antisera. Globulin fractions of individual
and pooled antisera were prepared by the method of Moody et al (1958) and labeled witn
fluorescein isothiocyanate (FITC) according to the procedures described by Hebert et al
(1967).
Smears were made directly from colonies on PSE agar which were suspended in a drop
63
V I R O L O G Y
O F
W A S T E W A T E R
RAPID ASSESSMENT OF WATER QUALITY
BY FLUORESCENT ANTIBODY IDENTIFICATION
OF FECAL STREPTOCOCCI
MARIA T. PAVLOVA, E. BEAUVAIS, F.T. BREZENSKI and WARREN LITSKY*
♦Marshall Hall, University of Massachusetts, Amherst,
Massachusetts 01002, U.S.A.
INTRODUCTION
Studies conducted in our laboratory indicated that all species of group D streptococci
(fecal streptococci) yielded positive fluorescent antibody (FA) reactions with laboratory
prepared labeled antibodies.
The specificity of these reactions was also demonstrated in that no cross-reactions with
non-group D streptococci occurred after trypsinization of the smears prior to staining.
These findings suggested that FA techniques may be employed in the determination of
the presence and source of fecal pollution in water employing the fecal streptococci as
indicator organisms. This report summarizes our efforts in this endeavor.
MATERIALS AND METHODS
Collection and plating of samples.
Water samples from the Connecticut and Mill rivers and raw sewage from the Amherst
treatment plant were examined within 15-40 minutes. One, 10, 20, 50 and 100 ml of
water were filtered and duplicate membranes (millipore HA 0.45 μ) were transferred to
Pfeizer selective enterococcus (PSE) and KF streptococcus agars. One tenth ml of the
appropriate dilutions from sewage was spread with a glass rod on each of the above media
in triplicate.
The inverted PSE plates were incubated aerobically at 37°C for 24 hr whereas the KF
plates were incubated for 48 hr. All membrane filter plates were incubated in rn
atmosphere saturated with water vapor. The densities of fecal streptococci (group D)
were reported as colony counts per 100 ml of water and sewage.
Identification of isolates.
The isolates from the selective media were identified on the basis of the results from
the following physiological, biochemical and serological tests: growth at 10° and 45°C;
growth in brain heart infusion (BHI) with 6.5% NaCl and pH 9.6; survival at 60°C for 30
minutes; reduction of 0.1%methylene blue, potassium tellurite and triphenyl tetrazolium
chloride (TTC); hemolysis on blood agar; hydrolysis of gelatin and 0.2% starch; catalase
production; fermentation of lactose, d-sorbitol and 1-arabinose; precipitin and FA
reactions. Specific streptococcal D antisera for the performing of precipitin and FA tests
were prepared in 15 rabbits by conventional methods. The precipitin tests were
performed according to the Lancefield method (1933) employing our laboratory
prepared and commercial streptococcal group D antisera. Globulin fractions of individual
and pooled antisera were prepared by the method of Moody et al (1958) and labeled witn
fluorescein isothiocyanate (FITC) according to the procedures described by Hebert et al
(1967).
Smears were made directly from colonies on PSE agar which were suspended in a drop
63