Aim: To evaluate a flow cytometry protocol that uses reference beads for the
enumeration of live and dead bacteria present in a mixture. Methods and
Results: 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 {\mu}m 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 ca. 104 to 108 bacteria ml-1 within
mixtures of live and dead bacteria. Conclusions: 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 ca. 20% dead. Significance and Impact of the Study: 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 specialised 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.Comment: 31 pages, 14 figure
