Abstract
SYTO 9 is a fluorescent nucleic acid stain that is widely used in microbiology, particularly for fluorescence microscopy and flow cytometry analyzes. Fluorimetry-based analysis, i.e., analysis of fluorescence intensity from a bulk sample measurement, is more cost effective, rapid and accessible than microscopy or flow cytometry but requires application-specific calibration. Here we show the relevance of SYTO 9 for food safety analysis. We stained four bacterial species of relevance to food safety (Bacillus cereus, Escherichia coli, Salmonella enterica subspecies enterica ser. Typhimurium, Staphylococcus aureus) with different concentrations of SYTO 9, with and without the presence of ethylenediaminetetraacetic acid (EDTA), for varying amounts of time, to investigate the effect of these treatment parameters on fluorescence intensity. The addition of EDTA and an increased staining duration did not significantly affect fluorescence intensity, and over the bacterial cell concentration range investigated (∼105–108 CFU/ml) there was no significant difference in using 0.5 or 1 μM SYTO 9. The effect of bacterial cell concentration on fluorescence intensity was species specific. At different bacterial cell concentrations, the effect of species on fluorescence intensity is different. This interaction complicates the development of a general fluorimetry-based protocol for the determination of bacterial cell concentration in a mixed bacterial suspension, as would be expected from samples taken from food safety settings.
Highlights
Applications involving fluorescence are widespread throughout microbiology as alternatives to culture-based methods
Only a small increase in fluorescence was observed with the addition of SYTO 9 to peptone water (Figure 1)
Species A significant interaction was observed between species and bacterial cell concentration
Summary
Applications involving fluorescence are widespread throughout microbiology as alternatives to culture-based methods. A variety of measurement techniques are used to collect fluorescence data, predominantly fluorescence microscopy (Donoso et al, 2017; Jackson et al, 2018), flow cytometry (De Sousa Guedes and De Souza, 2018; Zahavy et al, 2018), and fluorimetry (Bianchi et al, 2004). Many bacterial species are not natively fluorescent at visible wavelengths of excitation and a dye is added that will bind to a component of the target bacteria to generate a fluorescent signal (Sohn et al, 2009). Most importantly is that SYTO 9 exhibits a significant enhancement in quantum yield upon binding to nucleic acids. SYTO 9 has been used in fluorescence microscopy (Larrosa et al, 2012; Zotta et al, 2012; Tawakoli et al, 2013; Bogachev et al, 2018), flow cytometry
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