May consider using Permai fluorescence dye.

This protocol provides a reliable method for evaluating antibacterial activity using Hoechst 33342 and propidium iodide (PI) as a cost-effective alternative to commercial viability kits. The method is particularly suitable for Gram-negative bacteria like E. coli and Klebsiella, though it can be adapted for other species.

Begin by preparing bacterial cultures in mid-log phase growth (OD600 ≈ 0.4-0.6), then treat with your test antimicrobial compound. After treatment, wash cells twice with phosphate-buffered saline (PBS, pH 7.4) to remove residual compounds and resuspend in fresh PBS to an OD600 of approximately 0.1 for optimal staining. For staining, first add Hoechst 33342 to a final concentration of 1-5 μg/mL and incubate at 37°C for 15-20 minutes in the dark. This cell-permeant dye will stain DNA in all bacterial cells, serving as a total cell counterstain.

Following the Hoechst incubation, add propidium iodide to a final concentration of 5-10 μg/mL and continue incubation for an additional 5-10 minutes. The restricted incubation time with PI is crucial as this membrane-impermeant dye will only stain cells with compromised membranes (dead/damaged cells), and prolonged exposure may lead to false positives. For Gram-negative bacteria, consider including 0.5 mM EDTA in the staining solution to enhance PI penetration in damaged cells while maintaining specificity for membrane-compromised cells.

After staining, cells may be washed once with PBS (5,000-8,000 ×g for 5 minutes) to reduce background fluorescence, though this step can be omitted for rapid assessments. For microscopy analysis, visualize using standard fluorescence filters: Hoechst 33342 (excitation/emission ~350/461 nm) through a DAPI filter set, and PI (excitation/emission ~535/617 nm) through a TRITC filter set. In live cells, only blue Hoechst staining will be visible, while dead cells will show both blue (Hoechst) and red (PI) fluorescence.

For flow cytometry, detect Hoechst 33342 using a UV or violet laser (405 nm excitation) with emission collected in the blue channel, while PI can be detected using a green laser (532 or 561 nm) with emission collected in the red or PE channel. Always include appropriate controls: untreated bacteria (live control, should be Hoechst-positive/PI-negative) and ethanol-treated bacteria (dead control, 70% ethanol for 30 minutes, should be double-positive).

Key optimization considerations include dye concentration titration (excess PI increases background, insufficient Hoechst reduces sensitivity) and strict timing of PI exposure. For biofilm samples, extend staining times to 30 minutes and include gentle washing steps. This protocol provides a robust, reproducible method for antimicrobial susceptibility testing that correlates well with commercial viability assays while being significantly more cost-effective. The dual-stain approach allows simultaneous quantification of total cell counts (Hoechst) and viability ratios (PI), making it particularly valuable for dose-response studies of novel antimicrobial compounds.