Flow Cytometric Enumeration of Bacteria Using TO-PRO®-3 Iodide as a Single-Stain Viability Dye. (December 2014)

• Record Type: Journal Article
• Authors: Kerstens, Monique; Boulet, Gaëlle; Tritsmans, Christian; Horemans, Tessa; Hellings, Mario; Delputte, Peter; Maes, Louis; Cos, Paul
• Is Part Of: Journal of laboratory automation. Volume 19:Number 6(2014:Dec.)
• Page Start: 555
• Page End: 561
• Publication Date: 2014-12
• Subjects: flow cytometry -- quantification -- Bacillus subtilis -- Staphylococcus aureus -- Escherichia coli; Medical laboratory technology -- Periodicals; Laboratories -- Equipment and supplies -- Periodicals; Diagnosis, Laboratory -- Periodicals; 616.075
• DOI: 10.1177/2211068214546745
• Languages: English
• ISSNs: 2211-0682

Abstract

Quantification of bacteria using conventional viable plate counting (VPC) is labor-intensive and time-consuming. Flow cytometry (FCM) can be proposed as a faster alternative. This study aimed to develop a flow cytometric, single-stain approach using TO-PRO®-3 iodide (TP3) for the quantification of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis cells. Live or dead bacterial suspensions were stained with TP3 and analyzed using a FACSCalibur flow cytometer. After optimization of staining parameters and instrument settings, an excellent separation of viable and dead cells was achieved for all species. The quantitative performance of the technique was assessed by analyzing serial dilutions of bacterial suspensions using FCM and VPC. A highly linear correlation (r2 > 0.99) was observed between the colony forming units (CFU)/mL as determined by FCM and by VPC over a concentration range of about 104 to 108 CFU/mL. As such, FCM quantification of viable bacteria using TP3 can be considered as an accurate and reliable alternative for VPC. The monostain procedure is easy to apply and cost-effective, and it allows bacterial enumeration in a broad variety of samples.