AimA standard procedure does not exist to distinguish between attached and unattached micro-organisms. In this study, we compared two methods to quantify between Escherichia coli attached to clay particles and E.coli freely suspended in solution: flow cytometry (attachment assay and viability assay) and settling (or centrifugation followed by settling). Methods and ResultsMethods were tested using three environmental strains collected from swine facilities (A, B and C) and one purchased modified pathogenic strain (ATCC 43888); four clay particles: Hectorite, Kaolinite, Ca-Montmorillonite, Montmorillonite K-10; and a range of surface area ratios (particle surface area to E.coli surface area). When comparing the two methods, the per cent attached obtained from the flow cytometry was lower, but not significantly different from the per cent attached obtained from the settling method for all conditions except when the particle was Hectorite or Montmorillonite K-10; when the strain was C; and when the surface area ratio was below 100. Differences between the methods are likely because traditional culture-based methods cannot detect the viable but nonculturable (VBNC) population, whereas flow cytometry can detect the fraction of VBNC with intact membranes. ConclusionOur results indicate that flow cytometry is a rapid and culture-independent method for differentiating between attached and unattached micro-organisms. Significance and Impact of the StudyFlow cytometry is useful for laboratory-based studies of micro-organism-particle interactions.