160 7 Pollution of Aquatic Systems: Pollution Through Eutrophication, Fecal Materials, and Oil Spills
Table 7.4 Some bacteriological media used for the presumptive and confirmed tests in water examination (Compiled from Standard
Methods for the Examination of Water and Wastewater; Anonymous 2006 )
Medium
Dye for inhibiting
Gram-positive bacteriaReducing
agent BufferNitrogen
source pH indicator Remarks
Media for presumptive test
Brilliant green
lactose bile (BGLB)
Brilliant green Ox bile Peptone Peptone Brilliant green –Lauryl sulfate
tryptose broth (LST)
- Sodium lauryl
sulfate/bile
salts
Na/K 2 HPO 4 ,
KH 2 PO 4Tryptose – –McConkey broth – Bile salts Peptone Peptone Neutral red
Media for confirmed test
Eosin methylene
blue (EMB) agar
- – Peptone,
K 2 HPO 4
Peptone Eosin Metallic sheen
produced by
Escherichia coli
Endo agar – – Peptone Peptone Basic fuchsin
the same. Table 7.4 lists some of the media,
which have been used. They all contain lactose,
a buffer, a nitrogen source, while those used for
the presumptive test contain besides the above, a
pH indicator, Gram-positive inhibitor, and/or a
surface-tension reducer.
One of these media, E.M.B. agar, characteristically
shows E. coli colonies as having a metallic sheen,
whereas other coliforms, particularly Enterobacter and
Klebsiella, are not only larger on this agar, but do not
show this metallic sheen The metallic sheen typical of
E. coli depends on the action of the eosin and methyl-
ene blue to form a methylene blue eosinate, which acts
as an acidic dye. Absorption of the eosinate by the E.
coli cells is facilitated by the lowering of the pH by the
acid produced by the fermentation of the lactose.
(b) Streptococcus medium
Several media are available for selectively
growing Streptococcus, including azide dextrose
broth, Ethyl violet azide broth, and KF strepto-
coccus, the compositions of which are found in
Standard Methods. They all have in common
the presence of sodium azide, which inhibits
Gram-negative bacteria. They also contain
buffers, nitrogen source, and other requirements
of a bacteriological medium.
(c) C. perfringens (welchii) medium
C. perfringens produces a large number of gas
bubbles in litmus milk. The clot of protein pro-
duced by the acid from the fermentation of the
milk lactose is broken up by the gas bubbles to
produce the so-called stormy clot.
7.2.1.3 Methods Used in the Enumeration
of Indicator Organisms in Water
Two general methods are used for enumerating fecal
contaminants in water (and indeed any liquid): the
multiple tube method and the membrane-filter method.
With either method it is customary to relate the num-
bers to 100 ml of water. Both methods may be used
with all three indicator organisms.- The multiple-tube fermentation method
The multiple tube method is basically the dilution
count (or dilution to extinction), which is made more
accurate by the use of several tubes. In the ordinary
dilution count, a serial tenfold dilution of the liquid
whose bacterial load is to be counted is made. A loop-
ful is introduced from each dilution to a suitable liquid
medium in a tube, which contains a Durham tube to
trap any gas produced. This method can only be used
if no growth occurs in one of the dilutions introduced
into the broth (hence the alternate name of “dilution to
extinction” (see Fig. 2.2). In the ordinary dilution, the
number of organisms in the original liquid is assumed
to be the reciprocal of the dilution just before the nega-
tive one, that is, the highest dilution. Thus, if of a series
of ten tubes in serial tenfold dilution, the highest dilu-
tion in which growth occurred is 10^3 , the number of
organisms in the original tube is taken as 10^3 or 100 (or
more accurately more than 10^3 but less than 10^4. The
rationale behind this is illustrated in Fig. 2.2.
The basic reason for the development of the most
probable number (MPN) method can be seen from
Fig. 2.2. Suppose several replicates were taken of
the 10^3 dilution, then growth might occur in one at