Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide

Abstract

Background: Viability 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.

Results: We 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.

Conclusions: When 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.

Figure 1

SYTO9 staining analyzed with microplate reader. Relative fluorescence intensity (RFU) at 528 nm is shown for different live/dead proportions of S. aureus (a) and P. aeruginosa (b). Values were measured after staining with SYTO9 for 15 minutes (green diamonds) and after additional 15 minutes counterstaining with PI (red circles). As a control 0.9% NaCl solution (NS) was added (green circles) instead of PI to consider dilution and bleaching effects of SYTO9. Cell optical densities (OD) of 0.25 for S. aureus and 0.12 for P. aeruginosa were used. Error bars represent 3 individual repeats with 3 replicas for P. aeruginosa and S. aureus, respectively. The error bars for some data points are too small to be seen (hidden behind the symbols).

Figure 2

Bleaching of SYTO9 over time. Different amounts of live or dead cells of S. aureus (S.a.) and P. aeruginosa (P.a.) were stained with SYTO9, respectively. After 15 min incubation fluorescence intensity at 528 nm was automatically measured every 5 minutes with the microplate reader. Starting RFU value was set to 100% which was used to normalize other values.

Figure 3

Propidium iodide (PI) staining measured with microplate reader. Relative fluorescence intensity at 645 nm is shown for different live/dead proportions of S. aureus (a) and P. aeruginosa (b). Values were measured using the microplate reader after 15 minutes staining with PI of SYTO9 pre-stained cells. OD values of 0.25 for S. aureus and 0.12 for P. aeruginosa were used. Error bars represent 3 individual repeats with 3 replicas. The error bars for some data points are too small to be seen (hidden behind the symbols). Blue diamonds: mean values of the raw data; green diamonds: calculated values after subtraction SYTO9 cross-signal at 645 nm; red diamonds: values after additionally subtracting background of unbound PI.

Figure 4

SYTO9 staining analyzed with confocal microscopy. Different live/dead proportions of S. aureus (right) and P. aeruginosa (left) cells were stained with SYTO9 and examined with CLSM. The live/dead ratios of 10:90 and 90:10 are shown for illustration and comparison. For P. aeruginosa, a small proportion of approximately 10% of the total stained cells exhibits weaker (in 10:90 ratio) or stronger (in 90:10 ratio) fluorescence compared with the rest of the cells. The cells having weaker or stronger fluorescence are indicated by arrowheads. For S. aureus no difference in SYTO9 signal intensity can be observed for live and dead cells.

Figure 5

SYTO9/PI staining analyzed with confocal microscopy. Fluorescence images of the same samples at 528 nm (green) for SYTO9 signal, 645 nm (red) for PI signal and merged images are shown. 50:50 ratio of live and dead cells of P. aeruginosa (left) and S. aureus (right) was used. The cells were stained with SYTO9 and PI. Bacteria exhibiting red or yellow fluorescence are considered as dead cells.

Figure 6

Standard curves for determination of live/dead ratio. Standard curves were generated according to the manufacturers’ instructions for the determination of live/dead ratios through dividing fluorescence intensity at 528 nm (G: green) by that at 645 nm (R: red), referred as G/R ratio. Values for different live/dead proportions of S. aureus and P. aeruginosa were plotted either with (a) or without (b) background subtraction.