Water Quality Analysis by Most Probable Number (MPN)


The presumptive test is the first step in the most probable number (MPN) analysis, which is a statistical method to estimate the concentration of viable microorganisms in a water sample. The presumptive test is used to screen the water sample for the presence of coliform bacteria, which are indicators of fecal contamination and potential pathogens. Coliform bacteria are Gram-negative, non-spore-forming rods that can ferment lactose with acid and gas production.

The presumptive test involves inoculating measured volumes of water into a series of tubes containing a selective growth medium, such as MacConkey lactose broth, Lauryl tryptose broth, or brilliant green lactose bile broth. The medium contains lactose as the sole carbohydrate source, a surfactant such as sodium lauryl sulfate or bile salt to inhibit non-coliform bacteria, and a pH indicator dye such as bromcresol purple or brilliant green to detect acid production. The tubes also contain inverted Durham tubes to collect gas bubbles.

The inoculated tubes are incubated at 35°C for 24 to 48 hours and observed for color change and gas production. A positive presumptive test is indicated by the formation of at least 10% gas in the Durham tube and a change in the color of the medium from purple or green to yellow, indicating acid production. A negative presumptive test is indicated by no gas formation and no color change. A doubtful presumptive test is indicated by gas formation without color change or vice versa.

The number and distribution of positive tubes in each set are recorded and compared with a standard MPN table to estimate the number of coliform bacteria per 100 ml of water sample. The MPN table gives a range of possible values with a 95% confidence interval. The presumptive test is only an estimation and not a definitive identification of coliform bacteria. Therefore, if any tube shows a positive or doubtful result, a confirmatory test is performed to verify the presence of coliform bacteria.