Abstract
BackgroundViability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. However, data generated by such routinely used method are often not critically evaluated for their accuracy. In this study we aim to investigate the critical aspects of this staining method using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms for high throughput studies in microtiter plates. SYTO9 or PI was added alone or consecutively together to cells and the fluorescence intensities were measured using microplate reader and confocal laser scanning microscope.ResultsWe found that staining of S. aureus cells with SYTO9 alone resulted in equal signal intensity for both live and dead cells, whereas staining of P. aeruginosa cells led to 18-fold stronger signal strength for dead cells than for live ones. After counterstaining with PI, the dead P. aeruginosa cells still exhibited stronger SYTO9 signal than the live cells. We also observed that SYTO9 signal showed strong bleaching effect and decreased dramatically over time. PI intensity of the culture increased linearly with the increase of dead cell numbers, however, the maximum intensities were rather weak compared to SYTO9 and background values. Thus, slight inaccuracy in measurement of PI signal could have significant effect on the outcome.ConclusionsWhen viability staining with SYTO9 and PI is performed, several factors need to be considered such as the bleaching effect of SYTO9, different binding affinity of SYTO9 to live and dead cells and background fluorescence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0376-x) contains supplementary material, which is available to authorized users.
Highlights
Viability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies
We identified and studied the critical aspects of the SYTO9/PI staining using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms and based on the data acquired from microplate reader
Isopropanol treated cells led to no colonies in the agar plating experiments, whereas expected number of colonies was obtained from the Sodium chloride (NaCl) treated cells
Summary
Viability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. Bacterial viability tests are often performed with premixed, ready for use, dual staining kits, such as the BacLightTM (Live/Dead Bacterial Viability Kit, L-7007, Molecular Probes, [7,8]), composed of two fluorophores SYTO9 and propidium iodide (PI) based on the detection of membrane integrity. Advantages of using such a kit are a rapid procedure, quantitative analyses, as well as the possibility to measure using various instruments such as flow cytometer [8,9,10], microplate reader [11,12], and microscope [13,14]. For the 645 nm signal it is highly recommended to subtract background of unbound PI and cross-signal of SYTO9
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