64
Maria T. Pavlova, Elizabeth Beauvais, Francis T. Brezenski and Warren Litsky
of phosphate buffered saline (PBS) on a microscope slide and from washed sediments of
6-18 hr glucose-Lemco broth cultures. Direct fluorescent antibody staining technique was
applied as described by Pavlova et al (1971). Pooled conjugate from individual labeled
antisera was utilized for staining.
Fluorescent microscopy and photography.
A Leitz-Ortholux microscope, fitted with dark-field condenser and oil immersion lens,
a lamp housing with 150 watts high pressure xenon bulb and appropriate ultraviolet
filters, was used for examining the smears. Photographs were originally taken on 35 mm
high speed Ektachrome B color reversal film at an exposure of 3 to 6 minutes.
Fluorescent intensity was measured subjectively and recorded as follows: 4+ brilliant
yellow-green fluorescence, cells sharply outlined; 3+ bright yellow-green fluorescence,
cells sharply outlined; 2+ dull yellow-green fluorescence, cells not sharply outlined; 1 +
faint green discernible in dense areas, cells not outlined; 0 no fluorescence. Fluorescence
with intensity of 4+, 3+ and 2+ was considered positive.
RESULTS AND DISCUSSION
The fecal streptococci densities varied between 2.7 x 10
1 to 1.4 x 10
3 per 100 ml
sample from the Connecticut and Mill rivers respectively. In sewage these organisms were
in order of 2.4 x 10
6 . Streptococcal counts on PSE agar were generally higher than those
on KF streptococcus agar which confirmed our previous findings (Pavlova et al, 1970).
The fecal streptococci distribution from 15 river samples and 7 sewage samples is shown
in Table 1. Streptococcus faecalis and its variant S. liquefaciens were found in all the
samples and were the predominant species. Streptococcus faecium was found in 9 water
and 3 sewage samples in low numbers. Likewise, S. durans was found in low numbers in
water, however it could not be demonstrated in sewage. S. zymogenes was not found.
While the rivers flowed through pasture land above the sampling station S. bovis could
not be isolated throughout this study even though a conscientious effort was made to do
so.
Of the 166 isolates which were classified biochemically, only 67 were found to be
typical, using the classical reactions for identification. Of this number 7.5% were S.
faecalis, 62.7% S. liquefaciens, 6.Wo S. faecium, and 23.8% S. durans. The remaining
59.6% of strains, classified as atypical fecal streptococci, failed to satisfy one or more of
the Sherman criteria, to produce typical growth on potassium tellurite or TTC agars, or to
ferment the appropriate carbohydrates.
Prior to the serological identification of the isolates a comparison of commercial and
laboratory prepared D antisera was carried out in order to determine the effectiveness of
the latter. For this 191 fecal streptococcal strains from our culture collection were used
in the precipitin reactions with the above antisera. A higher proportion of negative
precipitin reactions for S. faecium, S. durans and S. bovis was observed with the
commercial antiserum than with that prepared in the laboratory. Only 18.3% of 191
strains tested were negative with laboratory prepared antiserum as opposed to 53.9% of
commercial D antiserum. This may be due to the fact that the agglutination titers of the
laboratory prepared D antiserum against the group D strains employed, with the
exception of S. zymogenes, were twice that of the commercial antiserum. Most probably
the higher titers of the laboratory prepared antiserum resulted from the pooling effect of
the high titer sera prepared with the freshly isolated strains in our collection.
Maria T. Pavlova, Elizabeth Beauvais, Francis T. Brezenski and Warren Litsky
of phosphate buffered saline (PBS) on a microscope slide and from washed sediments of
6-18 hr glucose-Lemco broth cultures. Direct fluorescent antibody staining technique was
applied as described by Pavlova et al (1971). Pooled conjugate from individual labeled
antisera was utilized for staining.
Fluorescent microscopy and photography.
A Leitz-Ortholux microscope, fitted with dark-field condenser and oil immersion lens,
a lamp housing with 150 watts high pressure xenon bulb and appropriate ultraviolet
filters, was used for examining the smears. Photographs were originally taken on 35 mm
high speed Ektachrome B color reversal film at an exposure of 3 to 6 minutes.
Fluorescent intensity was measured subjectively and recorded as follows: 4+ brilliant
yellow-green fluorescence, cells sharply outlined; 3+ bright yellow-green fluorescence,
cells sharply outlined; 2+ dull yellow-green fluorescence, cells not sharply outlined; 1 +
faint green discernible in dense areas, cells not outlined; 0 no fluorescence. Fluorescence
with intensity of 4+, 3+ and 2+ was considered positive.
RESULTS AND DISCUSSION
The fecal streptococci densities varied between 2.7 x 10
1 to 1.4 x 10
3 per 100 ml
sample from the Connecticut and Mill rivers respectively. In sewage these organisms were
in order of 2.4 x 10
6 . Streptococcal counts on PSE agar were generally higher than those
on KF streptococcus agar which confirmed our previous findings (Pavlova et al, 1970).
The fecal streptococci distribution from 15 river samples and 7 sewage samples is shown
in Table 1. Streptococcus faecalis and its variant S. liquefaciens were found in all the
samples and were the predominant species. Streptococcus faecium was found in 9 water
and 3 sewage samples in low numbers. Likewise, S. durans was found in low numbers in
water, however it could not be demonstrated in sewage. S. zymogenes was not found.
While the rivers flowed through pasture land above the sampling station S. bovis could
not be isolated throughout this study even though a conscientious effort was made to do
so.
Of the 166 isolates which were classified biochemically, only 67 were found to be
typical, using the classical reactions for identification. Of this number 7.5% were S.
faecalis, 62.7% S. liquefaciens, 6.Wo S. faecium, and 23.8% S. durans. The remaining
59.6% of strains, classified as atypical fecal streptococci, failed to satisfy one or more of
the Sherman criteria, to produce typical growth on potassium tellurite or TTC agars, or to
ferment the appropriate carbohydrates.
Prior to the serological identification of the isolates a comparison of commercial and
laboratory prepared D antisera was carried out in order to determine the effectiveness of
the latter. For this 191 fecal streptococcal strains from our culture collection were used
in the precipitin reactions with the above antisera. A higher proportion of negative
precipitin reactions for S. faecium, S. durans and S. bovis was observed with the
commercial antiserum than with that prepared in the laboratory. Only 18.3% of 191
strains tested were negative with laboratory prepared antiserum as opposed to 53.9% of
commercial D antiserum. This may be due to the fact that the agglutination titers of the
laboratory prepared D antiserum against the group D strains employed, with the
exception of S. zymogenes, were twice that of the commercial antiserum. Most probably
the higher titers of the laboratory prepared antiserum resulted from the pooling effect of
the high titer sera prepared with the freshly isolated strains in our collection.