Feasibility of using a particle counter or flow-cytometer for bacterial enumeration in the assimilable organic carbon (AOC) analysis method

Abstract

Assimilable organic carbon (AOC) is one of the major determinants of microbial growth and stability in drinking water distribution systems. Nevertheless, AOC measurements are rarely conducted in practice owing, in part, to the tedious and time-consuming nature of the bioassay. Herein, we compared three alternative cell count approaches [flow cytometry with staining (FC-S), flow cytometry without staining (FC-NS), and particle counting (Coulter counter; CC)] for bacterial enumeration as a means to expedite the AOC bioassay. Our results suggest that of the three methods only FC-S provides a suitable alternative to plate counting for rapid and accurate enumeration of both P17 and NOX in the AOC bioassay. While the cell counts obtained by FC-NS were linearly correlated with those obtained using the traditional heterotrophic plate count (HPC) method (FC-NS: R² = 0.89–0.96), the AOC values obtained by FC-NS were overestimated by 18–57 %. The CC approach was unsuccessful in enumerating Spirillum strain NOX cells because of the relatively small size of that organism. The CC counts were linearly correlated with HPC for Pseudomonas fluorescens strain P-17 (P17) cells (R² = 0.83) but like FC-NS, the CC approach also overestimated the AOC values (for P-17). The advantage of the FC-S method over the other two is improved sensitivity and the ability to specifically enumerate whole cells (and likely viable) as opposed to non-viable cells, cell debris, and other contaminating particles introduced by the test water itself or sample handling.