title: Flow cytometric enumeration of bacteria using TO-PRO®-3 iodide as a single-stain viability dye.
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.
authors.affiliation: University of Antwerp, Antwerp, Belgium.
authors.forename: Monique
authors.initials: M
authors.lastname: Kerstens
authors.affiliation: University of Antwerp, Antwerp, Belgium.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Boulet
authors.affiliation: University of Antwerp, Antwerp, Belgium.
authors.forename: Christian
authors.initials: C
authors.lastname: Tritsmans
authors.affiliation: University of Antwerp, Antwerp, Belgium.
authors.forename: Tessa
authors.initials: T
authors.lastname: Horemans
authors.affiliation: Janssen Research & Development, Division of Janssen Pharmaceutica N.V., Beerse, Belgium.
authors.forename: Mario
authors.initials: M
authors.lastname: Hellings
authors.affiliation: University of Antwerp, Antwerp, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Delputte
authors.affiliation: University of Antwerp, Antwerp, Belgium.
authors.forename: Louis
authors.initials: L
authors.lastname: Maes
authors.affiliation: University of Antwerp, Antwerp, Belgium paul.cos@uantwerpen.be.
authors.forename: Paul
authors.initials: P
authors.lastname: Cos
mesh_terms: D001412:Bacillus subtilis / Q000302:isolation & purification; D058491:Bacterial Load / Q000379:methods*; D002232:Carbocyanines / Q000378:metabolism*; D015169:Colony Count, Microbial; D003362:Cost-Benefit Analysis; D004926:Escherichia coli / Q000302:isolation & purification; D005434:Flow Cytometry / Q000379:methods*; D007454:Iodides / Q000378:metabolism; D050296:Microbial Viability; D013194:Staining and Labeling / Q000379:methods*; D013211:Staphylococcus aureus / Q000302:isolation & purification
chemical_list: D002232:Carbocyanines; D007454:Iodides; C098830:TO-PRO-3
keywords: Bacillus subtilis; Escherichia coli; Staphylococcus aureus; flow cytometry; quantification