Hypothesis
SYTO9 and propidium iodide staining, read in microtiter plates, gives an accurate high-throughput measure of live versus dead bacteria.
What failed
With SYTO9 alone, live and dead Staphylococcus aureus gave equal signal, and dead Pseudomonas aeruginosa gave an 18-fold stronger signal than live cells — still stronger after PI counterstaining. SYTO9 bleached strongly over time, and PI's maximum signal was weak relative to SYTO9 and background.
Broken assumption
SYTO9 stains live and dead cells equally, so the SYTO9/PI ratio reads out viability.
Why
The authors attribute the errors to SYTO9 bleaching, its different binding to live and dead cells, and background fluorescence, which make small inaccuracies in the weak PI signal change the outcome.
From the published paper: Stiefel et al. BMC Microbiology, 2015. ↗CC BY