MIC09102 Environmental Microbiology
Faecal Indicator Organisms: techniques for enumeration
Faecal indicator microorganisms can be useful in assessment of faecal
contamination of waters and the concomitant risk from infection from pathogens.
Levels of indicator organisms have been used to set limits for the acceptable
quality of drinking water, water for abstraction for treatment, bathing water, and
water used for shellfish rearing. It is therefore necessary to be able to easily and
rapidly enumerate such indicator organisms, and it is the techniques for doing
this that we will examine next. You will also conduct some of these in the
laboratory practical classes.
Elective-, selective- and differential media
Techniques using elective-, selective- and differential media in growth and
enumeration of particular microorganisms in environments such as foods, soils
and waters are well-established in microbiology. An elective medium is designed
to specifically stimulate the growth of target organisms, a selective medium is
designed to specifically inhibit the growth of non-target organisms, and a
differential medium is one that contains factors that visually demonstrate the
presence of target organisms. These types of media for enumeration of faecal
indicator bacteria are described below.
For growth and enumeration of coliforms, an elective medium contains factors
such as glutamic acid and ammonia that coliform organisms can use as nitrogen
sources, and lactose that coliform organisms can use as carbon and energy
sources. Minerals Modified Glutamate Medium (MMGM) is an example.
A selective medium for coliforms uses bile salts or a synthetic detergent to
suppress growth of non-enteric organisms (detergents make the medium
selective for enteric organisms). MacConkey medium and Teepol are examples.
To make such media differential for coliforms, lactose is added along with a pH
indicator, which demonstrates an organisms’ ability to ferment lactose (resultant
production of organic acids is indicated by change in colour of colonies on agar
or colour of liquid medium brought about as the pH indicator changes colour).
Alternatively, ONPG can be added to medium which coliforms convert to yellow
ONP (which demonstrates activity of galactopyranosidase enzyme) or MUG can
be added which E. coli converts to fluorescent MU (which demonstrates activity
of glucuronidase enzyme) .
For growth and enumeration of enterococci, growth media can be made selective
by addition of sodium azide at levels that inhibit virtually all other bacteria but to
which enterococci are resistant. Glucose Azide medium is an example of this.
For growth and enumeration of Cl. perfringens, growth media can be made
selective by addition of mixture of antibiotics that Cl. perfringens is resistant to.
Alternatively, before enumeration tests are performed water samples can be
pasteurised at 75⁰C for 10 minutes to select for endospores (pasteurisation kills
vegetative cells and only the endospores survive the treatment as these are heat
resistant). Conditions are made elective for this organism by running tests in
anaerobic conditions and incorporating factors such as cysteine into the medium.
Medium is made differential by addition of iron salts and sodium sulphite, so that
black iron sulphide is formed by reduction of sulphite (which leads to formation of
sulphide). Differential Reinforced Clostridial medium is an example of this.
Enumeration methods
Routine enumeration of faecal indicator bacteria in waters is carried out by two
methods.
• Membrane filtration (MF)
MF involves filtering a given volume of water through
a filter (0.22 or 0.45µm) diameter pore size) to
capture bacteria on the surface which is then
incubated on selective and differential medium. After
incubation individual bacterial cells grow into
colonies that are visible with the naked eye and
target organisms can be recognised by the
differential reaction with the medium. Colonies of
target organisms are then counted. The number of cells in the original volume of
water filtered is then calculated.
• Most Probable Number (MPN)
The MPN or multiple tube or multiple tube
fermentation test involves distributing known
volumes of the test water (for example 50, 10,
5, 1 and 0.1ml) as replicates (typically 5 per
volume) into liquid medium in a series of
vessels e.g. test tubes or bottles. After
incubation growth and differential reaction in
each vessel is recorded, and the pattern of
growth/no-growth in each replicate and for
each water volume is recorded and compared with a standard table. Alternatively,
the data is entered into a computer programme to give the most probable number
of organisms.
These two techniques have relative advantages and disadvantages – some are
summarised below.
Membrane filtration (MF)
Advantages Disadvantages
More simple technically than MPN Only detect <250 organisms in the
volume sampled as at greater numbers
the colonies are uncountable. Not a
problem with treated potable waters
which usually have none-very low
numbers.
Results may be obtained within 16h Cannot be used on turbid waters as the
filters become blocked by suspended
solids. Not a problem with treated potable
waters.
Sampling errors are less than MPN
Large volumes of water can be sampled,
if this is technically possible.
.
Most Probable Number MPN
Advantages Disadvantages
Can in a single test detect between 1 Sampling error can be large (up to
and 1900 organisms per 100ml water. three times the MPN value).
Can be used with turbid waters. Results may take 48h or longer
Not so reliable where organisms occur
in very low numbers.
A disadvantage inherent in both techniques is that false positive or false
negatives can arise. To minimise this a great deal of developmental work goes
into improving selective and differential media, and you will find in the literature
that differential media may be used in different laboratories.
**Please take some time to note the types of selective and differential media
reported in recent articles for the enumeration of coliforms and
enterococci.**
** Please develop the list of advantages and disadvantages for MF and MPN
through reading and your practical experience in the laboratory classes.
This will be very useful when it comes to critically appraising your own
laboratory data.**
In testing of potable waters for coliforms a negative result is expected (EU
standards state that coliforms should be absent from any 100ml sample).
Incidentally this is why concentrations of organisms in waters are often expressed
as number/100ml - as 100ml is the standard sample size in testing potable
waters. It seems rather pointless to perform an MPN test (using several
tubes/bottles) on water which should produce a negative result; it would seem
more sensible simply to put all the water sample in one vessel - the expected
negative result can be obtained more simply. This technique is known as the
presence/absence method (P/A).
In contrast to membrane filtration and MPN, which are quantitative, the
presence/absence method is essentially qualitative (it reveals simply that
organisms are present or absent from the volume of water sampled - if present it
gives no indication of numbers), and so may not be as useful in monitoring natural
waters. However, if sufficient samples are taken the proportion of samples
positive over a given period can be used to estimate the frequency of coliforms
in that water and thus give an indication of water quality and any changes in it
over time. Studies in the USA have shown the P/A testing to be as useful as
membrane filtration and MPN in a variety of waters and this system is used in
parts of the USA where standards stipulate that over a set time not more than
5% of P/A tests should be positive.
