74
N. Buras and Y. Kott
EXPERIMENTAL PROCEDURES
/. Water samples
Samples were collected from various sources including reservoirs, pools, springs and
effluent from the local wastewater treatment plant.
//. Media used
(a) M-Endo (Difco).
(b) Modified mFC medium (MmFC) (Buras & Kott, 1969).
(c) mFC medium (Difco).
(d) Phage assay base agar (Fischer Scientific) and phage broth (Kott, 1966) were used for
the preparation of bacteriophage stocks and for phage assays.
///. Bacteriophages used
T t — T 7 E. coli B bacteriophages.
70 E. coli bacteriophages isolated from wastewater on E. coli strains of bacteria
isolated from water and wastewater as well.
Stocks were prepared from every one of the bacteriophages isolated. For the
convenience of performance of the test, equal aliquots of the bacteriophage stocks were
collected into a sterile bottle and the mixture obtained used in the test described below.
IV. Test for the quantitative estimation ofcoliforms and identification ofE. coli bacteria
by specific bacteriophages in water
Two equal portions of a water sample (usually 100 ml) were filtered separately
through membrane filters grade HA, 47 mm in diameter, 0.45 micron pore size (Millipore
filter HAWP04700). Following filtration both filters were placed separately, on MmFC
soaked pads in small MF plastic Petri dishes (Millipore). The first filter was incubated
immediately at 37°C. The second filter was exposed to E. coli bacteriophages by
pipetting one ml of the bacteriophage mixture onto the filter. After 10 minutes, the Petri
dish was inverted and incubated overnight at 37° C after which the results were read.
For comparison of performance with other tests two additional filtrations of the same
water sample were carried out. One of the filters was placed on a M-Endo soaked pad and
incubated at 37°C; the other one was placed on a mFC (Difco) soaked pad and incubated
in a water bath at 44.5°C as recommended by Geldreich (1965) for the fecal coliform test.
All the results were recorded after 22-24 hours incubation and the number of
coliforms was calculated per 100 ml of water sample tested.
Occasionally random blue and pink colonies picked from the membrane filters were
tested for their IMViC reaction. These tests were performed as described in Standard
Methods (1965).
RESULTS
As already published (Buras & Kott, 1969), E. coli bacteria grown on MmFC medium
37°C, produce dark blue colonies, A. aerogenes produce pink colonies, Klebsiella olive
green colonies, Salmonella red colonies. Intermediate coliforms grew into light green or
pink colonies.
The results of a typical field sample are seen in Scheme 1, where different coliform
colonies are represented: E. coli — blue colonies, A. aerogenes — pink and light green
N. Buras and Y. Kott
EXPERIMENTAL PROCEDURES
/. Water samples
Samples were collected from various sources including reservoirs, pools, springs and
effluent from the local wastewater treatment plant.
//. Media used
(a) M-Endo (Difco).
(b) Modified mFC medium (MmFC) (Buras & Kott, 1969).
(c) mFC medium (Difco).
(d) Phage assay base agar (Fischer Scientific) and phage broth (Kott, 1966) were used for
the preparation of bacteriophage stocks and for phage assays.
///. Bacteriophages used
T t — T 7 E. coli B bacteriophages.
70 E. coli bacteriophages isolated from wastewater on E. coli strains of bacteria
isolated from water and wastewater as well.
Stocks were prepared from every one of the bacteriophages isolated. For the
convenience of performance of the test, equal aliquots of the bacteriophage stocks were
collected into a sterile bottle and the mixture obtained used in the test described below.
IV. Test for the quantitative estimation ofcoliforms and identification ofE. coli bacteria
by specific bacteriophages in water
Two equal portions of a water sample (usually 100 ml) were filtered separately
through membrane filters grade HA, 47 mm in diameter, 0.45 micron pore size (Millipore
filter HAWP04700). Following filtration both filters were placed separately, on MmFC
soaked pads in small MF plastic Petri dishes (Millipore). The first filter was incubated
immediately at 37°C. The second filter was exposed to E. coli bacteriophages by
pipetting one ml of the bacteriophage mixture onto the filter. After 10 minutes, the Petri
dish was inverted and incubated overnight at 37° C after which the results were read.
For comparison of performance with other tests two additional filtrations of the same
water sample were carried out. One of the filters was placed on a M-Endo soaked pad and
incubated at 37°C; the other one was placed on a mFC (Difco) soaked pad and incubated
in a water bath at 44.5°C as recommended by Geldreich (1965) for the fecal coliform test.
All the results were recorded after 22-24 hours incubation and the number of
coliforms was calculated per 100 ml of water sample tested.
Occasionally random blue and pink colonies picked from the membrane filters were
tested for their IMViC reaction. These tests were performed as described in Standard
Methods (1965).
RESULTS
As already published (Buras & Kott, 1969), E. coli bacteria grown on MmFC medium
37°C, produce dark blue colonies, A. aerogenes produce pink colonies, Klebsiella olive
green colonies, Salmonella red colonies. Intermediate coliforms grew into light green or
pink colonies.
The results of a typical field sample are seen in Scheme 1, where different coliform
colonies are represented: E. coli — blue colonies, A. aerogenes — pink and light green