16 4 7 Pollution of Aquatic Systems: Pollution Through Eutrophication, Fecal Materials, and Oil Spills
volumes are suitable. If it is of doubtful quality,
one 50 ml, five 10 ml, and five 1-ml quantities
could be used. When the water is heavily pol-
luted the original water may have to be diluted
by a factor of 100 or more in order to obtain
some negative results in the series put up, and
thus obtain a finite figure for the MPN. Whatever
the series used, the volumes of water in the
individual tubes and the number of tubes con-
taining each volume of water should be such
that an estimate of the MPN number of orga-
nisms present in 100 ml of the original water
can be obtained from the table.
In the procedure outlined in Standard
Methods for carrying out the presumptive test,
water is inoculated into multiple tubes (chosen
as indicated above) of lactose or lauryl tryptose
broth and incubated at 35°C. The production of
acid and gas within 48 h indicates a positive
presumptive test.
In the confirmatory test, one to three loop-
fuls of broth are transferred from positive tubes
to brilliant green lactose broth (BGLB) or
streaked on endo or EMB agar (although the
latter is not recommended). Gas formation after
48 h at 35°C indicates a positive Confirmed
Test using the liquid medium.
On EMB agar, a typical positive colony of
E. coli is nucleated with or without a metallic
sheen. An atypical colony is opaque unnucleated,
mucoid, and pink after 24 h in the incubator.
In the Completed Test, a positive growth
from BGLB broth is streaked on endo or EMB
agar. If plates were used in the confirmatory test
typical and atypical colonies are transferred to
lactose or lauryl tryptose broth, as well as to
nutrient agar. Formation of gas in secondary
fermentation and the occurrence of Gram-
negative rods on staining is a positive Completed
Test and indicates that E. coli is present. It ought
to be pointed out that the media and some
aspects of the techniques set out in Table 7.4
and which are available in Standard Methods
are not universally adopted. On the European
continent, the media used are different.
(b) Membrane filtration method
After filtration the pad is picked up with sterile
forceps and placed on an absorbent pad soaked
in lauryl tryptose broth (or agar) if enrichment
is desired or directly on to M-Endo soaked pad
or M-Endo agar. The result is expressed as
coliform density (total coliform colonies per
100 ml). This is calculated as follows:- Fecal coliforms
The above tests are for general coliforms; they do
not distinguish coli forms of animal origin from
others. They are used for the examination of potable
water since no coliform of any kind should be toler-
ated in treated water. For investigations of stream
pollution, raw water sources, sewage treatment sys-
tems, bathing water, and general water-quality
monitoring, it is important to know whether or not
the coliform is of fecal origin, that is, whether or
not from the intestine of warm-blooded animals.
The procedure for detecting fecal coliform is simi-
lar to the above in both the multiple tube or the mem-
brane technique. In each the confirmatory test is
carried out at an elevated temperature of 44.5°C for 24
h (i.e., the Eijkmann test). Gas production indicates
coliform of warm-blooded animals. Prior to the con-
firmatory test, however, the coliform must be enriched
in the lactose broth used in the presumptive test. A
loopful is then transferred from all positive tubes to the
confirmatory broth (Brilliant Green Lactose Bile Broth
at 44.5°C for 24 h or Boric Acid Lactose Broth at
45°C for 48 h). When the membrane filter method is
used, the incubation of the filters at 44.5°C may be
done directly after the filtration of water.
Fecal Streptococci
(a) Multiple-tube method
Multiple portions of water are inoculated into
tubes of glucose azide broth and incubated at
37°C for 72 h. When acidity is observed a heavy
inoculation is introduced into further tubes of
glucose azide broth and incubated at 44.5°C for
48 h. Tubes showing acidity at this temperature
contain fecal streptococci.
Multiple portions of water may also be inoc-
ulated into tubes of buffered azide-glucose-
glycerol broth (BAGG) and incubated at 44.5°C
for 48 h. Acid production is indicative of fecal
streptococcal presence, but the broth should be
checked with Gram stain to confirm the presence
of Gram-negative rods.
Coliform colonies counted 100
ml sample filtered