MPN Test
Principle: The three basic tests to detect coliform bacteria in water are presumptive, confirmed,
and completed. The tests are performed sequentially on each sample under analysis. They
detect the presence of coliform bacteria (indicators of fecal contamination), through the
fermentation of lactose that will produce acid and gas that is detectable following a 24-hour
incubation period at 37°C.
The Presumptive Test: The presumptive test is specific for detection of coliform bacteria.
Measured aliquots of the water to be tested are added to a lactose fermentation broth containing
an inverted gas vial. Because these bacteria are capable of using lactose as a carbon source (the
other enteric organisms are not), their detection is facilitated by the use of this medium. In this
experiment, you will use lactose fermentation broth containing an inverted Durham tube for
gas collection. Tubes of this lactose medium are inoculated with 10-ml, 1-ml, and 0.1-ml
aliquots of the water sample. The series consists of at least three groups, each composed of five
tubes of the specified medium. The tubes in each group are then inoculated with the designated
volume of the water sample. The greater the number of tubes per group, the greater the
sensitivity of the test. Development of gas in any of the tubes is presumptive evidence of the
presence of coliform bacteria in the sample. The presumptive test also enables the
microbiologist to obtain some idea of the number of coliform organisms present by means of
the most probable number (MPN) test. The MPN is estimated by determining the number of
tubes in each group that show gas following the incubation period.
The Confirmed Test: The presence of a positive or doubtful presumptive test immediately
suggests that the water sample is nonpotable. Confirmation of these results is necessary because
positive presumptive tests may be the result of organisms of noncoliform origin that are not
recognized as indicators of fecal pollution. The confirmed test requires that selective and
differential media (e.g., eosin–methylene blue (EMB) or Endo agar) be streaked from a positive
lactose broth tube obtained from the presumptive test. Eosin–methylene 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 major indicator of fecal pollution. Endo agar is a nutrient medium
containing the dye fuchsin, which is present in the decolorized state. In the presence of acid
produced by the coliform bacteria, fuchsin forms a dark pink complex that turns the E. coli
colonies and the surrounding medium pink.
The Completed Test: The completed test is the final analysis of the water sample. It is used
to examine the coliform colonies that appeared on the EMB or Endo agar plates used in the
confirmed test. An isolated colony is picked up from the confirmatory test plate, inoculated
into a tube of lactose broth, and streaked on a nutrient agar slant to perform a Gram stain.
Following inoculation and incubation, tubes showing acid and gas in the lactose broth and
presence of gram-negative bacilli on microscopic examination is further confirmation of the
presence of E. coli, and they are indicative of a positive completed test.
Materials: Lactose broth, MacConkey agar, nutrient agar, test tubes, Petridish, slide, crystal
violet, safranin, gram’s iodine, 95% ethanol.
Procedure:
Presumptive Test
1. Examine the tubes from your presumptive test after 24 and 48 hours of incubation. Your
results are positive if the Durham tube fills 10% or more with gas in 24 hours, doubtful if gas
develops in the tube after 48 hours, and negative if there is no gas in the tube after 48 hours.
2. Determine the MPN using standard table, and record your results in the Lab Report.
Confirmed Test
1. Using a positive 24-hour lactose broth culture from the sewage water series from the
presumptive test, streak the surface of one EMB or one Endo agar plate, to obtain discrete
colonies.
2. Incubate all plate cultures in an inverted position for 24 hours at 37°C.
3. Examine all the plates from your confirmed test for the presence or absence of E. coli
colonies.
4. Based on your results, determine whether each of the samples is potable or nonpotable. The
presence of E. coli is a positive confirmed test, indicating that the water is nonpotable. The
absence of E. coli is a negative test, indicating that the water is not contaminated with fecal
wastes and is therefore potable.
Completed Test
1. Label each tube of nutrient agar slants and lactose fermentation broths with the source of its
water sample.
2. Inoculate one lactose broth and one nutrient agar slant with a positive isolated E. coli colony
obtained from each of the experimental water samples during the confirmed test.
3. Incubate all tubes for 24 hours at 37°C.
4. Prepare a Gram stain, using the nutrient agar slant cultures of the organisms that showed a
positive result in the lactose fermentation broth.
5. Examine the slides microscopically for the presence of gram-negative short bacilli, which
are indicative of E. coli and thus nonpotable water.
Observation and Result: