Bacteriological water analysis
E. coli
Bacteriological water analysis is a method of analysing water to
estimate the numbers of bacteria present and, if needed, to find out what
sort of bacteria they are. It represents one aspect of water quality. It is
a microbiological analytical procedure which uses samples of water and
from these samples determines the concentration of bacteria. It is then
possible to draw inferences about the suitability of the water for use
from these concentrations. This process is used, for example, to
routinely confirm that water is safe for human consumption or that
bathing and recreational waters are safe to use.
The interpretation and the action trigger levels for different waters vary
depending on the use made of the water. Whilst very stringent levels
apply to drinking water, more relaxed levels apply to marine bathing
waters, where much lower volumes of water are expected to be ingested
by users.
Approach[edit]
The common feature of all these routine screening procedures is that the
primary analysis is for indicator organisms rather than the pathogens that
might cause concern. Indicator organisms are bacteria such as non-
specific coliforms, Escherichia coli and Pseudomonas aeruginosa that
are very commonly found in the human or animal gut and which, if
detected, may suggest the presence of sewage. Indicator organisms are
used because even when a person is infected with a more pathogenic
bacteria, they will still be excreting many millions times more indicator
organisms than pathogens. It is therefore reasonable to surmise that if
indicator organism levels are low, then pathogen levels will be very
much lower or absent. Judgements as to suitability of water for use are
based on very extensive precedents and relate to the probability of any
sample population of bacteria being able to be infective at a reasonable
statistical level of confidence.
Analysis is usually performed using culture, biochemical and sometimes
optical methods. When indicator organisms levels exceed pre-set
triggers, specific analysis for pathogens may then be undertaken and
these can be quickly detected (where suspected) using specific culture
methods or molecular biology.[1]
Methodologies
The most reliable methods are direct plate count method and membrane
filtration method. mEndo Agar is used in the membrane filtration while
VRBA Agar is used in the direct plate count method. VRBA stands for
violet red bile agar. A media that contains bile salts which promotes the
growth of gram negative and has inhibitory characteristic to gram
positive although not complete inhibitory. These media contain lactose
which is usually fermented by lactose fermenting bacteria producing
colonies that can be identified and characterised. lactose fermenting
produce colored colonies while non lactose fermenting produce colorless
ones. Because the analysis is always based on a very small sample taken
from a very large volume of water, all methods rely on statistical
principles.[2]
Multiple tube method[edit]
One of the oldest methods is called the multiple tube method.[3] In this
method a measured sub-sample (perhaps 10 ml) is diluted with 100 ml
of sterile growth medium and an aliquot of 10 ml is then decanted into
each of ten tubes. The remaining 10 ml is then diluted again and the
process repeated. At the end of 5 dilutions this produces 50 tubes
covering the dilution range of 1:10 through to 1:10000.
The tubes are then incubated at a pre-set temperature for a specified time
and at the end of the process the number of tubes with growth in is
counted for each dilution. Statistical tables are then used to derive the
concentration of organisms in the original sample. This method can be
enhanced by using indicator medium which changes colour when acid
forming species are present and by including a tiny inverted tube called
a Durham tube in each sample tube. The Durham inverted tube catches
any gas produced. The production of gas at 37 degrees Celsius is a
strong indication of the presence of Escherichia coli.
ATP Testing[edit]
An ATP test is the process of rapidly measuring active microorganisms
in water through detection adenosine triphosphate (ATP). ATP is a
molecule found only in and around living cells, and as such it gives a
direct measure of biological concentration and health. ATP is quantified
by measuring the light produced through its reaction with the naturally
occurring enzyme firefly luciferase using a luminometer. The amount of
light produced is directly proportional to the amount of biological
energy present in the sample.
Second generation ATP tests are specifically designed for
water, wastewater and industrial applications where, for the most part,
samples contain a variety of components that can interfere with the ATP
assay.
Plate count[edit]
The plate count method relies on bacteria growing a colony on a nutrient
medium so that the colony becomes visible to the naked eye and the
number of colonies on a plate can be counted. To be effective, the
dilution of the original sample must be arranged so that on average
between 30 and 300 colonies of the target bacterium are grown. Fewer
than 30 colonies makes the interpretation statistically unsound whilst
greater than 300 colonies often results in overlapping colonies and
imprecision in the count. To ensure that an appropriate number of
colonies will be generated several dilutions are normally cultured. This
approach is widely utilised for the evaluation of the effectiveness of
water treatment by the inactivation of representative microbial
contaminants such as E. coli following ASTM D5465.[4][5]
The laboratory procedure involves making serial dilutions of the sample
(1:10, 1:100, 1:1000, etc.) in sterile water and cultivating these
on nutrient agar in a dish that is sealed and incubated. Typical media
include plate count agar for a general count or MacConkey agar to
count Gram-negative bacteria such as E. coli. Typically one set of plates
is incubated at 22 °C and for 24 hours and a second set at 37 °C for 24
hours. The composition of the nutrient usually includes reagents that
resist the growth of non-target organisms and make the target organism
easily identified, often by a colour change in the medium. Some recent
methods include a fluorescent agent so that counting of the colonies can
be automated. At the end of the incubation period the colonies are
counted by eye, a procedure that takes a few moments and does not
require a microscope as the colonies are typically a few millimetres
across.
Membrane filtration[edit]
Most modern laboratories use a refinement of total plate count in which
serial dilutions of the sample are vacuum filtered through purpose
made membrane filters and these filters are themselves laid on nutrient
medium within sealed plates.[6] The methodology is otherwise similar to
conventional total plate counts. Membranes have a printed millimetre
grid printed on and can be reliably used to count the number of colonies
under a binocular microscope.
Pour plate method[edit]
When the analysis is looking for bacterial species that grow poorly in
air, the initial analysis is done by mixing serial dilutions of the sample in
liquid nutrient agar which is then poured into bottles which are then
sealed and laid on their sides to produce a sloping agar surface. Colonies
that develop in the body of the medium can be counted by eye after
incubation.
The total number of colonies is referred to as the Total Viable
Count (TVC). The unit of measurement is cfu/ml (or colony forming
units per millilitre) and relates to the original sample. Calculation of this
is a multiple of the counted number of colonies multiplied by the
dilution used.
Pathogen analysis[edit]
When samples show elevated levels of indicator bacteria, further
analysis is often undertaken to look for specific pathogenic bacteria.
Species commonly investigated in the temperate zone
include Salmonella typhi and Salmonella Typhimurium. Depending on
the likely source of contamination investigation may also extend to
organisms such as Cryptosporidium spp. In tropical areas analysis
of Vibrio cholerae is also routinely undertaken.
Types of nutrient media used in analysis[edit]
MacConkey agar is culture medium designed to grow Gram-negative
bacteria and stain them for lactose fermentation. It contains bile salts (to
inhibit most Gram-positive bacteria), crystal violet dye (which also
inhibits certain Gram-positive bacteria), neutral red dye (which stains
microbes fermenting lactose), lactose and peptone. Alfred Theodore
MacConkey developed it while working as a bacteriologist for the Royal
Commission on Sewage Disposal in the United Kingdom.
Endo agar contains peptone, lactose, dipotassium phosphate, agar,
sodium sulfite, basic fuchsin and was originally developed for the
isolation of Salmonella typhi, but is now commonly used in water
analysis. As in MacConkey agar, coliform organisms ferment the
lactose, and the colonies become red. Non-lactose-fermenting organisms
produce clear, colourless colonies against the faint pink background of
the medium.[7]
mFC medium is used in membrane filtration and contains selective and
differential agents. These include rosolic acid to inhibit bacterial growth
in general, except for faecal coliforms, bile salts inhibit non-enteric
bacteria and aniline blue indicates the ability of faecal coliforms to
ferment lactose to acid that causes a pH change in the medium.[8]
TYEA medium contains tryptone, yeast extract, common salt and L-
arabinose per liter of glass distilled water and is a non selective medium
usually cultivated at two temperatures (22 and 36 °C) to determine a
general level of contamination (a.k.a. colony count).
