Glass bottomed cell culture dish

Strains were cultured in TSB in a glass-bottomed cell culture dish (NEST Biotechnology, Wuxi, China) without shaking for 24 h (Kannappan et al., 2017 (link)), in the presence of 4 μg/ml ZY-214-4; untreated samples served as a positive control. After rinsing with 0.85% saline, the dish was air-dried and 300 μl of SYTO-9 [0.02%; component A of the LIVE/DEAD BacLight Bacterial Viability kit (Thermo Fisher Scientific, Waltham, MA, United States)] and propidium iodide (0.067%) were added for 30 min in the dark at room temperature. Optical sections were scanned by CLSM (Nikon, Tokyo, Japan) using a 60 × oil-immersion objective lens. 3D reconstruction of the images was performed using NIS-Elements software (Nikon). The assay was performed in triplicate.

SYTO9 green fluorescent nucleic acid staining was used to assess the effect of TPCP on the viability of MSSA and MRSA biofilm (Jefferson et al., 2005 (link)). Bacterial suspension (2 mL, 1.5 × 10⁸ CFU/mL) was added to a glass-bottomed cell culture dish (NEST, Wuxi, China) and incubated for at 35°C 72 h. The planktonic bacteria were then removed, and 500 μL of fresh MHB containing different final concentrations of TPCP (2×, 1×, and 1/2 × MIC) were added. A control group was set up separately. After 8 h of incubation, the supernatant was discarded and the cells were washed with 5 mM 4-(2-hydroxyethy)-1-piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 7.2). STYO9 (1 μL, Thermo Fisher, USA) was added to each dish and incubated for 20 min, and observed under a CLSM (Olympus FV1200, Japan) after the dye was removed. This experiment was repeated three times.