Ultraviolet spectrophotometer

Vibrio splendidus strains, including WTVs, MTVs and fur^C were coated to 2216E agar at 28°C for 24 h. Single colonies were transferred into 10 ml of fresh 2216E broth and allowed to reach the optical density at 600 nm (OD₆₀₀) of 1.0. The culture was diluted proportionally to the same concentration. One hundred microlitre aliquots of WTVs, MTVs, and fur^C were reinoculated into flasks with 100 ml of fresh 2216E broth, 2216E broth supplemented with 100 μM iron chelator 2,2′-dipyridyl (DP), and 2216E broth supplemented with 50 μM FeCl₃ and grown at 28°C with agitation at 180 rpm. The OD₆₀₀ was recorded at different time points with an ultraviolet spectrophotometer (Mapada Instruments Co. Ltd., Shanghai, China). The experiment was repeated three times for each sample.

The cytotoxicity of the hemostatic materials (BACG, BACG-1B, BACG-3B, BACG-6B, and CMPHP) was tested by MTT assay using L6 cells (mouse myoblast cell line). Three different concentrations of the experimental materials were set for testing: 1 mg/L, 10 mg/L, and 100 mg/L. Besides, the cytotoxicity of berberine was also assessed at the same three concentrations. Briefly, all materials were diluted into a suitable test concentration using PBS and then dissolved at 37 °C. The cells were cultured (90 μL/well) in 96-well microtiter plates and incubated at 37 °C in a humidified atmosphere of 5% CO₂ for 24 h. Then, the lysate of the material or the drug (10 μL) was added to the 96-well plates for a further 72 h of incubation. After that, the cells were treated with MTT solution (5 mg/mL, 10 μL/well) for another 4 h at 37 °C. Finally, the absorbance was measured at 490 nm through an ultraviolet spectrophotometer (Shanghai Mapada Instruments Co., Ltd., Shanghai, China), which could indirectly reflect the number of living cells.
The cell viability (CV) was calculated according to Equation (5):
(A_s: absorbance of the sample; A_c: absorbance of the control group; A_b: absorbance of the blank group)