Absolute bacterial cell enumeration using flow cytometry

TL;DR

This study presents a flow cytometry protocol using reference beads for accurate enumeration of live and dead bacteria, demonstrating high correlation with traditional plate counts across a range of concentrations.

Contribution

The paper introduces a reliable flow cytometry method for absolute bacterial cell counting applicable to basic cytometers without specialized features.

Findings

High correlation (R2 > 0.9) with plate counts for live and dead bacteria.

Effective for bacteria concentrations from 10^4 to 10^8 per mL.

Reliable for live bacteria ratios above 2.5% and dead bacteria above 20%.

Abstract

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.