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Flow cytometry is a rapid and quantitative method to determine bacterial viability. Although different stains can be used to establish viability, staining protocols are inconsistent and lack a general optimization approach. Very few "true" multicolor protocols, where dyes are combined in one sample, have been developed for microbiological applications. In this mini-review, the discrepancy between protocols for cell-permeant nucleic acid and functional stains are discussed as well as their use as viability dyes. Furthermore, optimization of staining protocols for a specific setup are described. Original data using the red-excitable SYTO dyes SYTO 59 to 64 and SYTO 17, combined with functional stains, for double and triple staining applications is also included. As each dye and dye combination behaves differently within a certain combination of medium matrix, microorganism, and instrument, protocols need to be tuned to obtain reproducible results. Therefore, single, double, and triple stains are reviewed, including the different parameters that influence staining such as stain kinetics, optimal stain concentration, and the effect of the chelator EDTA as membrane permeabilizer. In the last section, we highlight the need to investigate the stability of multicolor assays to ensure correct results as multiwell autoloaders are now commonly used.