SYTO-9

Operating Instructions (The following is a recommended experimental protocol for guidance only and should be adjusted according to specific requirements)

1. Working Solution Preparation
- Diluent: Phosphate-buffered saline (PBS) or HBSS is optional.
- Working concentration: 0.5-5 µM for single staining; 33.4 µM for co-staining with propidium iodide (PI).
- Notes: The concentration of the working solution can be adjusted as needed, and it must be prepared and used immediately.

3. Staining Procedures
3.1 Suspended bacterial samples[1][2][3]:
3.1.1 Sample preparation: Adjust the bacterial suspension to a concentration of approximately 10⁵-10⁸ CFU/mL in the selected diluent; if removal of culture medium or fixative (if applicable) is required, centrifuge at 4000-8000 × g for 2-10 min before resuspension; centrifugation is not required for direct staining of live or formaldehyde-fixed aquatic bacteria.
3.1.2 Incubation conditions: Use a working concentration of 0.5-33.4 µM; incubate at room temperature in the dark for 5-45 min; for pure bacterial cultures, perform staining in amber tubes to avoid light exposure. For PI co-staining, use a combined system of 33.4 µM SYTO 9 and 0.4 mM PI, and incubate at room temperature in the dark for 15 min.
3.2 Adherent bacterial biofilms^[4]:
3.2.1 Sample preparation: Use biofilms grown on silicon wafers, coverslips or glass-bottom dishes; trypsin digestion is not required.
3.2.2 Washing before staining: Wash the samples 3 times with PBS.
3.2.3 Incubation conditions: Direct co-staining with SYTO 9-PI: Use the prepared co-staining working solution and incubate at room temperature for 15 min. Sequential staining: First incubate with SYTO 9 working solution at room temperature for 15 min, then directly add PI and image immediately; or after staining with DAPI and PI, directly add SYTO 9 and image immediately.
- Washing after staining: Wash the samples 3 times with PBS to remove excess dye.

4. Control Setup
4.1 Bacterial samples without dye.
4.2 Blank control containing only peptone water.
4.3 Peptone water solution containing 0.5 µM SYTO 9.
4.4 Peptone water solution containing 1.0 µM SYTO 9.
4.5 Untreated *Staphylococcus aureus* biofilm control.
4.6 *Staphylococcus aureus* biofilm samples treated with 70% EtOH.

5. Detection and Analysis
5.1 Instrument types: Flow cytometer, fluorescence microscope, etc.
5.2 Excitation/emission wavelengths: RNA: Ex/Em = 486/501 nm; DNA: Ex/Em = 485/498 nm.
5.3 Result analysis:
After SYTO 9 binds to bacterial nucleic acids, the fluorescence intensity increases significantly, and the intensity varies with bacterial species, cell concentration, and the state of live/fixed bacteria.
5.3.1 Suspended bacteria: Staining duration (5-45 min) has minimal effect on fluorescence intensity, but gram-negative bacteria may show low signals at 5 min; the fluorescence intensity of live bacteria may be higher than that of fixed bacteria; double staining with PI allows differentiation between live and dead bacteria via intensity ratio or spectral analysis.
5.3.2 Biofilms: SYTO 9 can stain intracellular nucleic acids (intact cells appear solid green) or substances around cells (damaged cells appear hollow green); co-staining with PI shows overlapping green and magenta fluorescence, and SYTO 9 signals often distribute around PI signals; fluorescence intensity has no direct correlation with cell size.