Absolute bacterial cell enumeration using flow cytometry

To evaluate a flow cytometry protocol that uses reference beads for the enumeration of live and dead bacteria present in a mixture. Mixtures of live and dead Escherichia coli with live:dead concentration ratios varying from 0 to 100% were prepared. These samples were stained using SYTO 9 and propidium iodide and 6‐μm reference beads were added. Bacteria present in live samples were enumerated by agar plate counting. Bacteria present in dead samples were enumerated by agar plate counting before treatment with isopropanol. There is a linear relationship between the presented flow cytometry method and agar plate counts for live (R2 = 0·99) and dead E. coli (R2 = 0·93) concentrations of c. 104 to 108 bacteria per ml within mixtures of live and dead bacteria. Reliable enumeration of live E. coli within a mixture of both live and dead was possible for concentration ratios of above 2·5% live and for the enumeration of dead E. coli the lower limit was c. 20% dead. The ability to obtain absolute cell concentrations is only available for selected flow cytometers, this study describes a method for accurate enumeration that is applicable to basic flow cytometers without specialized counting features. By demonstrating the application of the method to count E. coli, we raised points of consideration for using this FCM counting method and aim to lay the foundation for future work that uses similar methods for different bacterial strains.