Table 1.

Examples of individual uses of dyes to perform a multi-parameter flow cytometry analysis in order to characterize antimicrobial mechanisms.

Reference	Objective	Dyes and their function	Main result
Antolinos et al., 2014	Effect of acid shock on cell viability of Bacillus cereus and Bacillus weihenstephanensis vegetative cells	• PI (membrane permeability) • cFDA (esterase activity)	pH 3.4, 3.8, and 4.2 caused membrane disruption and subsequent bacterial cell death in the first 24 h exposition to these acidic environments.
Boda et al., 2015	Investigate the generated reactive oxygen species (ROS) causing peroxidation of the membrane lipids and ion channel proteins, leading to greater permeabilization of the bacterial membranes	• DCFH-DA [intracellular indicator of reactive oxygen species (ROS)]	~60 and ~70% reduction was recorded in the survival of staphylococcal species and Escherichia coli, respectively at pulsed magnetic field as evaluated by colony forming unit (CFU) analysis and flow cytometry. A 2–5 fold increase in intracellular ROS (reactive oxygen species) levels suggests oxidative stress as the key mediator in PMF induced bacterial death/injury.
Caldeira et al., 2013	Assessment of antibacterial properties of L-cysteine and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniae	• PI (membrane permeability) • DiBAC4 (membrane potential) • CTC (respiratory activity)	The main mechanism of action of L-Cys on both bacteria, K. pneumonia, and S. aureus, was the reduction in metabolic activity, consistent with the results that confirmed the bacteriostatic effect of L-Cys on both these bacteria.
Kramer and Muranyi, 2014	Investigate the effects of a pulsed light treatment on the physiological properties of Listeria innocua and Escherichia coli	• PI (membrane permeability) • DiBAC4 (membrane potential) • EB (efflux pump activity) • cFDA (esterase activity)	Oxidative stress with concomitant damage to the DNA molecule was shown to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes.
Kramer and Thielmann, 2016	Monitoring the live to dead transition of bacteria during thermal stress	• PI (membrane permeability) • DiBAC4 (membrane potential) • cFDA (esterase activity) • 2-NBGD (glucose uptake)	Exposure to moderate heat first of all compromised the function of the respiration chain and other heat sensitive proteins of the cell membrane such as efflux pumps. Membrane rupture and intracellular esterase activity were less affected and strong differences depending on the type of bacteria regarding their Gram-staining behavior were observed.
Lee et al., 2015	Mechanism of action of scopolendin 2 against E. coli O157 and Candida albicans	• Sytox Green • (membrane permeability) • DiBAC4 (membrane potential) • DiSC3 (membrane potential)	Scopolendin 2 led to the formation of pores in microbial plasma membrane, subsequent leakage of cytoplasmic matrix components and consequent membrane depolarization, ultimately resulting in microbial cell death.
Morishige et al., 2015	Analysis of the metabolic response of H2O2-treated Salmonella cells	• CTC (respiratory activity) • 2-NBGD (glucose uptake) • EdU-Alexa 488 • (DNA synthesis)	H2O2-treated Salmonella cells did not lose their biological activities of living cells all together. Different subpopulations of Viable But Non-Culturable bacteria were detected. Cells lost their DNA-synthesis activity first, then CTC-reducing activity, and finally glucose-uptake activity to a lesser extent.
Silva et al., 2011	Coriandrum sativum essential oil mode of action against Candida species	• PI (membrane permeability) • DiBAC4 (membrane potential) • DRAQ5 (DNA staining)	Coriander essential oil kills Candida spp. by damaging the cytoplasmic membrane, leading to an impairment of cellular functions.
Teng et al., 2014	Elucidate further the antimicrobial mechanism of AvBD103b, an avian defensin, on the Salmonella enteritidis CVCC3377 cell membrane and intracellular DNA	• PI (membrane permeability) • FITC-labeled AvBD103b • (AvBD103b permeation of the membrane)	Antimicrobial target of AvBD103b was the cell membrane.

PI, Propidium Iodide; cFDA, carboxyFluorescein DiAcetate; DiBAC₄, Bis-(1,3-DibutylBarbituric ACid) Trimethine Oxonol = bis-oxonol = BOX; CTC, 5-Cyano-2,3-ditolyl Tetrazolium Chloride; EB, Ethidium Bromide; 2-NBGD, 2-[N-(7-nitrobenz-2-oxa-1,3-Díazol-4-yl) amino]-2-deoxy-Dglucose; EdU: 5-ethynyl-2-deoxyuridine; FITC, fluorescein isothiocyanate; DRAQ5, 1,5-bis{[2-(di-methylamino) ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione; DiSC₃, 3,3′-Dipropylthiadicarbocyanine Iodide, DCFH-DA, 2′,7′-dichlorofluorescein-diacetate.