Rapid Estimation of the Most Probable Number (MPN) of Viable Bacteria In Water Samples

Tuesday, October 18, 2011
Exhibit Hall B (Minneapolis Convention Center)
Sachidevi Puttaswamy, Byung-Doo Lee, Ashley Jurgensmeyer and Shramik Sengupta, Biological Engineering, University of Missouri, Columbia, MO

Various regulatory agencies such as the USDA, FDA and EPA require that the number of viable bacteria present in certain samples of food or water be less than a certain specified number. For instance the US Pasteurized Milk Ordinance requires “Grade A” pasteurized milk to have a total viable bacterial count of ≤ 20,000 CFU/ml, and the EPA requires that the concentration of coliforms in primary recreational water bodies not exceed 500 CFU/100ml (5 CFU/ml)

The enumeration of VIABLE bacteria is typically done using a statistical technique known as the Most Probable Number (MPN) method. The method involves progressively diluting an aliquot from the sample of interest into microbial growth media (repeated in triplicate) and keeping a track of which dilutions show evidence of the presence of even a single VIABLE bacterium. Currently, the latter (whether a vial contains any viable bacteria, or not) is determined by culturing the vials for 1-2 days, and looking for the development of turbidity in the sample (either manually, or using a spectrometer). It is known that if there is even a single viable bacterium present, it will give rise to hundreds of millions, which will turn the solution turbid.   

 Since the concentrations of bacteria in the sample have to rise to millions of CFU/ml before they can be detected via turbidity measurements, it takes a long time (typically 1-2 days) before the MPN can be determined. This delay has many adverse economic (and even, health) consequences.

In contrast, our method promises to deliver MPN results in less than 1 day. The principle underlying our method of detection is the polarizability of viable bacterial cells. In the presence of an alternating electric field, there occurs a build-up of charge at the membrane, causing the cells to act like capacitors. As the bacteria multiply in number, there will be a corresponding increase in the charge stored in the interior of the suspension (its “bulk capacitance”), and this increase in bulk capacitance over time serves as a “signature” for the presence of viable bacteria.

We were able to quantify viable E. coli in Tryptic Soy Broth (TSB) in <6 hours as opposed to 1-2 days taken by the current methods and also have a cut off time of ~10 hours which significantly reduces the wait time for industries.

This method can thus have a significant impact on many modern water and food quality applications.

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