Method to partition between freely suspended Escherichia coli and Escherichia coli attached to clay particles
Currently, about 29% of waters across the United States are impaired because of elevated bacterial levels (USEPA, 2009). While attachment of bacteria to particulates is one likely mode of transport through the environment, understanding of environmental transport mechanisms is lacking. Previous studies have shown that some bacteria preferentially attach to sediment but a standard procedure does not exist to separate attached and unattached bacteria. In this project, we are developing a practical and accurate method to distinguish and quantify between E. coli attached to clay particles and E. coli freely suspended in solution. Two methods to detect differences between unattached and attached E. coli were compared, settling (or centrifugation followed by settling) and flow cytometry. Each method was tested using three environmental strains collected from swine facilities and one research strain of E. coli (ATCC 43888, E. coli O157:H7 with Shiga-like toxin I and II removed); four clay particles: Hectorite (diameter: 1 μm, surface area: 63 m2/g), Kaolinite (diameter: 1.25 μm, surface area: 11.2 m2/g), Ca-Montmorillonite (diameter: 3 μm, surface area: 84 m2/g), Montmorillonite K-10 (diameter: 6 μm, surface area: 240 m2/g); and a range of surface area ratios (clay particle surface area to E. coli surface area).
From the results, E. coli were more likely to attach to clay particles with smaller sizes. As the surface area ratio increased from 1 to 1,000, the attachment ratio increased with greatest attachment occurring at a clay particle surface area to E. coli surface area ratio of 1,000, where and an average of 59% of cells were attached. Moreover, the attachment ratio reached a maximum value of 99.8% for E. coli attachment to Kaolinite. When comparing the results of the two methods, the detected attachment ratios were always lower when using the flow cytometry method, especially for Hectorite, the smallest particle size tested in this project. The main limitation of the settling method is its inability to detect viable but non-culturable cells while the inability to discriminate live and dead cells in the main reason for the underestimated attachment fractions by flow cytometry method. Nevertheless, the increasing trend in attachment ratio from flow cytometry was similar to the results from the settling method. Our results indicate that flow cytometry is a rapid and accurate method to test the attachment ratio of E. coli to clay particles, but the method is still in need of further development.