To find the Most Probable Number (MPN) of bacterial density by E.coli test.
Coliform group comprises of all the aerobic, facultative and anaerobic gram-negative non-spore forming rod
shaped bacteria that ferment lactose with gas formation within 48 hours at 35°C. The standard test for this group
may be carried out either by multiple tube fermentation technique or by membrane filter technique. The E.coli test
by multiple tube fermentation technique consists of 3 phases – presumptive, confirmed and completed.
Escherichia coli (E.coli) for the purpose of sanitary examination of water, is defined as a gram-negative, nonspore
forming rod which is capable of fermenting lactose with the production of acid and gas at 35°C in less than
48 hours, which produces indole peptone water containing tryptophan, which is incapable of utilising sodium
citrate as its sole source of carbon, which is incapable of producing acetyl methyl carbinol, and which gives a
positive methyl red test. The results are expressed in terms of MPN (Most Probable Number), which is based on
certain probability formulae. The estimate may give a value greater than the actual number of coliform present. The
accuracy of any single test depends on the number of tubes fermented. This method helps in describing the sanitary
quality of water.
The safety of the water is generally judged from the knowledge of sanitary condition and mentioned by the
number of samples yielding positive or negative results. If more than 95% should yield negative results, the safety
is usually assured. The scheme of the MPN test is given as follows:
Reagents (» click to check the preparation of reagents)
- Fermentation tubes
- Petri dishes
- Test tubes
- Measuring jars
- Inoculating equipments
- Media preparation utensils etc.
- Lactose broth
- Lauryl tryptose broth
- Brilliant green lactose bile broth
- Endo agar
- Eosin methylene blue agar etc.
|Schematic outline for presumptive and confirmed test for coliform detection.
|Schematic outline of presumptive, confirmed and completed test for
total coliform detection.
Clean and sterilise all the glasswares.
Inoculate a series of fermentation tubes with appropriate graduated quantities (multiples and sub-multiples
of 10) of the water to be tested. The concentration of nutritive ingredients in the mixture of the medium
should conform to the specifications. The partitions of the water sample used for inoculating lactose or
lauryl tryptose broth fermentation tubes will vary in size and number with the character of the water
under examination. Usually, decimal multiples and sub-multiples of 1mL of the sample are selected.
Inoculate 10 mL portion of each water sample provided into different one of the three large tubes
containing 10 mL of lactose or lauryl tryptose broth which has been prepared with twice the normal
concentration of constituent to allow for dilution. Inoculate 1.0 mL and 0.1 mL of water into small tubes
(two sets of three each) of single strength lactose or lauryl tryptose broth.
Incubate the inoculated fermentation tubes at 35±0.5°C. At the end of 24±2 hrs shake each tube gently
and examine and if no gas is formed, repeat this test at the end of 48±3 hrs.
Record the presence or absence of gas formation at each examination of the tubes. Formation within
48±3 hrs of gas in any amount in the inverted fermentation tubes constitutes a positive presumptive test.
Active fermentation may be shown by the continued appearance of small bubbles of gas throughout
the medium outside the inner vial in the fermentation tubes. Presumptive test without confirmation should
not be used routinely except in the analysis of heavily polluted water, sewage or other waste, which
are not suitable for drinking purpose.
Lactose or lauryl tryptose broth may be used for primary fermentation in presumptive test to avoid false
Brilliant green lactose bile broth fermentation tubes are used in confirmed test.
Submit all primary fermentation tubes showing any amount of gas at the end of 24 hrs incubation to
the confirmed test.
Gently shake primary fermentation tube showing gas formation and with a sterile metal loop, transfer
one loop full of medium to a fermentation tube containing brilliant green lactose bile broth.
Incubate the inoculated brilliant green lactose bile broth tube for 48±3 hrs at 35±0.5°C.
The formation of gas in any amount in the inverted vial of the brilliant green lactose bile broth fermentation
tube at any time within 48±3 hrs constitutes a positive confirmed test.
- If no gas is formed, it is a negative confirmed test and E.coli is absent.
Completed test is the next step following the confirmed test. It is applied to the brilliant green lactose bile broth
fermentation tubes showing gas in the confirmed test.
Differentiation of E.coli and A. aerogenes on eosin or EMB agar
- Streak one or more endo or Eosin Methylene Blue (EMB) agar plates (taken in Petri dishes) from each
tube of brilliant green lactose bile broth showing gas.
- While streaking it is essential to ensure the presence of some discrete colonies separated by at least
0.5 cm from one another.
- Insert the end of the streaking needle into the liquid in the tube to a depth of 5mm.
- Streak the plate by bringing only the curved section of the needle in contact with the agar surface so
that the latter will not be scratched or torn.
- Incubate the Petri dishes (inverted) at 35 ± 0.5°C for 24 ± 2 hrs.
- The colonies developing on endo or eosin methylene blue agar may be typical (unnucleated, with or
without metallic sheen) atypical (opaque, unnucleated, mucoid, pink after incubation for 24 hrs) or
negative (all others).
- From each of these plates fish out one or two colonies and transfer to lauryl tryptose broth fermentation
tubes and to nutrient agar slants.
- Incubate the secondary broth tubes and agar slants at 35 ± 0.5°C for 24 ± 2 hrs or 48 ± 3 hrs and
if gas is not produced in 24 hrs gram stained preparation from these agar slant cultures are made.
- The gas formation in the secondary lauryl tryptose broth tubes and the demonstration of gram-negative
non-spore forming rod shaped bacteria in agar culture may be considered a satisfactory positive
- If after 48 ± 3 hrs gas is produced in the secondary fermentation tubes and no spore of gram positive
rod are found on the slant, the test may be considered as positive completed test and this demonstrates
the presence of coliform organisms.
Differentiation of E. coli and A. aerogenes on eosin or EMB agar can be done by referring the following table.
Reagents (» click to check the preparation of reagents)
- Ammonium oxalate-crystal violet (Hucker's)
- Lugol's solution
- Counter stain
- Acetone alcohol.
Steps in the gram staining is shown in the following table.
- Prepare a light emulsion of the bacterial growth on an agar slant in a drop of distilled water on a glass
- Air-dry or fix by passing the slide through a flame and stain for 1minute with ammonium oxalate-crystal
- Rinse the slide in tap water and then apply Lugol's solution for 1minute.
- Rinse the stained slide in tap water.
- Decolorise with acetone alcohol till the stain is just removed.
- Counter-stain with safranin for 15 seconds and then rinse with tap water.
- Blot dry with blotting paper and view through the microscope.
- Cells that decolorise and accept the safranin stain are pink and are defined as gram negative. Cells that
do not decolorise but retain the crystal violet stain (deep blue) are defined as gram positive.
Computation of MPN
The number of positive finding of coliform group organisms resulting from the multiple portion decimal dilution
planting should be computed as the combination of positives and recorded in terms of the Most Probable Number
(MPN). The MPN for the variety of planting series are presented in table in Appendix III. The values are at the
95% confidence limit for each of the MPN determined. These values are prepared for 10,1 and 0.1mL combination.
If the combination is 100, 10, 1mL, the MPN is 0.1 times the value in the table. If on the other hand a combination
corresponding to 1, 0.1, and 0.01 mL is planted, record MPN as 10 times the value shown in the table.
The MPN for combination not appearing on the table or for other combinations of tubes and dilutions, may be
estimated by Thomas' simple formula:
- Case (i)
For three each of 10 mL, 1 mL and 0.1 mL sample concentration combinations
MPN from the MPN table (Appendix-III) = .........
- Case (ii)
For other combinations and dilutions
MPN/100 mL = .........