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 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; three clay particles: Kaolinite, Ca-Montmorillonite, Montmorillonite K-10; and a range of surface area ratios (clay particle surface area to E. coli surface area).

E. coli were more likely to attach to clay particles with smaller sizes. As the surface area ratio increased from 1 to 500, the percent attached increased with greatest attachment (an average of 47%) occurring at surface area ratio of 500. Moreover, the percent attached reached a maximum value of 99.8% for E. coli attachment to Kaolinite. When comparing the two methods, the detected attachment ratios were always lower when using the flow cytometry method. 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 is the main reason for the underestimated attachment fractions by flow cytometry 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.