Microbial Analysis of water

Indicator organism:
The indicator bacterium should be suitable for the analysis of all types of
water: tap, river, ground, impounded, recreational, estuary, sea, and waste.
It should be present whenever enteric pathogens are present.
It should survive longer than the hardiest enteric pathogen.
It should not reproduce in the contaminated water, as this would produce an
inflated value.
It should be harmless to humans.
Its level in contaminated water should have some direct
relationship to the degree of fecal pollution.
The assay procedure for the indicator should have great specificity; in other
words, other bacteria should not give positive results. In addition, the
procedure should have high sensitivity and detect low levels of the indicator.
The testing method should be easy to perform.

 Coliforms are defined as facultatively aerobic, gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas
formation within 48 hours at 35C.
The coliforms include a wide range of bacteria whose primary
source may not be the intestinal tract. To address this, tests have
been developed that assay for the presence of fecal coliforms.
These are coliforms derived from the intestine of warm blooded
animals, which grow at the more restrictive temperature of 44.5C.
Enterobacter; Escherichia coli, a common intestinal organism and
occasional pathogen; and Klebsiella pneumoniae

 The original test for coliforms involved the

presumptive, confirmed, and completed tests
The presumptive step is carried out by means of tubes
inoculated with three different sample volumes to give an
estimate of the most probable number (MPN) of coliforms
in the water.
The complete process, including the confirmed and
completed tests, requires at least 4 days of incubations
and transfers.

 The confirmation test:

The confirmed test is done by selective and differential media such
as eosinmethylene blue (EMB) or Endo agar.
The medium will be streaked from a positive lactose broth tube
obtained from the presumptive test.
Eosinmethylene blue contains the dye methylene blue, which
inhibits the growth of gram-positive organisms.
In the presence of an acid environment, EMB forms a complex that
precipitates out onto the coliform colonies, producing dark centers
and a green metallic sheen. The reaction is characteristic for
Escherichia coli,

 The membrane filter technique:

The water sample is passed through a membrane filter.
The filter with its trapped bacteria is transferred to the
surface of a solid medium or to an absorptive pad
containing the desired liquid medium.
A resuscitation step often is needed with chlorinated
samples, where the microorganisms are especially
stressed. Membrane filters have been widely used with
water that does not contain high levels of background
organisms, sediment, or heavy metals.

 The presence-absence (P-A) test:

This is a modification of the most probable number
procedure in which a larger water sample (100 milliliters) is
incubated in a single culture bottle with a triple-strength
broth containing lactose broth, lauryl tryptose broth, and
bromocresol purple indicator.
The P-A test is based on the assumption that no coliforms
should be present in 100 milliliters of drinking water. A
positive test results in the production of acid (a yellow
color), which requires confirmation.

 Colilert defined substrate test:

A water sample of 100 milliliters is added to
a specialized medium containing onitrophenyl-13-ogalactopyranoside (ONPG)
and 4-methylumbelliferyl-13-oglucuronide
(MUG) as the only nutrients.
If coliforms are present, the medium will
turn yellow within 24 hours at 35C due to
the hydrolysis of ONPG, which releases onitrophenol (figure 43.3).
To check for E. coli, the medium is observed
under long-wavelength ultraviolet (UV) light
for fluorescence.
When E. coli is present, MUG is modified to
yield a fluorescent product.