7.2 Pollution of Water with Reference to Human Health: Bacterial Indicators of Fecal Pollution 159
Among these the most commonly used indicators
are E. coli and the coliform group as a whole. E. coli is
of fecal origin and E. coli Type I (Eijkmann positive),
which is of human (and warm-blooded animal) origin
grows at 44.5°C. Hence, the presence of fecal E. coli is
a definite indication of fecal pollution. All coliforms in
general are not necessarily of fecal origin; nevertheless,
since they are not indigenous to water their presence in
drinking water should cast suspicion on the water and
should indicate pollution in the widest sense.
Coliform bacteria are defined as Gram-negative,
nonspore-forming rods capable of fermenting lactose,
aerobically or facultatively, with production of acid
and gas at 35°C in less than 48 h.
S. fecalis occurs regularly in feces in numbers that
usually probably disappear at the same rate as E. coli,
but quicker than other coliforms. When, therefore, it is
found in water in which E. coli cannot be detected, this
is an important confirmatory evidence of fecal pollution.
C. welchii is also regularly present in human feces
but fewer than E. coli. Spores survive longer in water
and usually resist chlorination. Its presence suggests
fecal contamination and the absence of E. coli suggests
contamination in the distant past.
7.2.1.2 Procedure for the Determination of
Fecal Contamination
- Collection of samples
Samples of water for bacteriological examination
should be collected in clean sterile bottles, and
should not be less than 100 ml. If the water to be
examined is chlorinated, then a dechlorination agent
should be added to the bottle before sterilizing.
Sodium thiosulfate is usually used for the purpose,
the amount employed depending on the amount of
residual chlorine in the water (0.1 ml of 10% solu-
tion of sodium thiosulfate will neutralize a 250-ml
sample of water containing 15 mg of residual chlo-
rine per liter). For samples containing metals such
as tin or copper, a chorinating agent such as EDTA
should be added. If the water is collected from a tap
it should be allowed to run for 2–3 min before the
bottle is filled. If from a river, stream, lake, reser-
voir, etc. the aim should be to collect the water in
the same way as for consumers. In samples sus-
pected to be highly contaminated, dilutions of up to
104 may be required. The colonies should, as in
routine plate counting, lie between 30 and 300
(Anonymous 2006 ).
2. Media used for enumerating indicator organisms of
fecal pollution
Various media are used as indicator organisms. In
effect they are selective media, which eliminate
other organisms while encouraging the develop-
ment of the indicators. The active components of the
more common media are discussed briefly.
(a) Coliform media
Among the short Gram-negative rods the ability
to ferment lactose appears to be limited to the
family Enterobacteriaceae within which the
coliforms are found. Anaerobic spore formers
notably C. welchii, which are also of fecal origin
also ferment lactose, as do some aerobic spore
formers. In order to inhibit these Gram-positive
organisms during the initial (presumptive) tests
for coliforms, a number of inhibitors are
employed; some of the Gram-positive inhibitors’
activities promote the growth of coliforms. The
inhibitors act by lowering the surface tension of
the medium thereby making the medium more
favorable for the growth of intestinal organisms,
which are already adapted to the low surface ten-
sion (due to bile salts) of the lower alimentary
canal. Sometimes dyes and other chemicals,
which selectively inhibit Gram-positive bacteria
are used. Some surface tension-reducing salt
compounds appear to act by both methods.
The surface tension-reducing agents, which
have been used include the following: Ox bile,
bile salt (or sodium taurocholate), lauryl sul-
fate, and sodium ricinoleate (i.e., soap made
from castor oil). The dyes, which have been
used are the triphenyl methane dyes which in
low concentrations inhibit Gram-positive orga-
nisms. In much higher concentrations they
inhibit even Gram-positives. Thus, brilliant
green inhibits C. welchii at 1:30,000, but coli-
forms grow at 1:350. Gentian (crystal) violet
inhibits the anaerobe at 1:9,000, but at 1:100,000
begins to inhibit the coliforms. Rosolic acid, a
dye in this family, has also been used.
After the initial (presumptive and confirma-
tory) tests, which are described latter in this
chapter, the media used in the subsequent com-
pleted tests do not need to contain Gram-positive
inhibitors. The media used on both sides of the
Atlantic vary slightly, but the principles dictat-
ing which medium is used at which stage are