Culture media Bacterial growth and biofilm formation were quantified in nine different media: Marine Broth (MB) (Conda); Mueller-Hinton Broth (Scharlau) supplemented with NaCl to give a final concentration of 2% (MH2); cation-adjusted
MH2 (CAMH2), that consisted in MH2 supplemented with 55 mg/l CaCl2 and 40 mg/l MgCl2; Brain Heart Infusion (Scharlau) supplemented with NaCl to a final concentration of 2% (BHI2); Tryptic Soy Broth (BD) supplemented with NaCl to a final concentration of 2% (TSB2); Luria Marine Broth (LMB); Supplemented Artificial Seawater (SASW); Väätänen Nine-Salt Solution (VNSS); and Marine Minimal Medium (MMM). LMB and SASW were prepared according to Lang et al. , NSS and VNSS followed the recipe described by Mårdén et al. ; and MMM was prepared as described by Östling et al. . A summary of the composition of each medium is provided as additional https://www.selleckchem.com/products/jph203.html information (Additional file 1: Table S1). Assessment of growth and biofilm production Each well of the microtiter plate contained 100 μl of bacterial inoculum and 100 μl the appropriate culture medium. Growth at two temperatures (26 and 32°C) was quantified after an incubation period of 24 h by measuring the optical density at 625 nm (OD 625) with an automatic plate reader (Perkin-Elmer EnSpire). MK5108 supplier Eight replicates were used for
each medium. Once the growth was measured, biomass was quantified by the crystal violet (CV) staining method . Briefly, wells were thoroughly washed three times with water to remove the culture medium and planktonic cells as well as loosely adhered bacteria. Firmly attached bacteria were heat fixed (65°C) for 30–45 min and then 200 μL of a 0.2% CV solution (Sigma-Aldrich) were added to each well. After 15 min wells were emptied and washed carefully with water. Plates were air-dried and then the dye was solubilised by addition of 200 μl of absolute ethanol. Absorbance was recorded at 590 nm. When OD590 readings were above
2.5, the sample was tenfold diluted and OD was measured again . Three classic antifouling agents: TBTO, tralopyril and zinc pyrithione were purchased from Sigma-Aldrich. Stock solutions of the products 4��8C (40 mM) dissolved in dimethylsulfoxide (DMSO) were diluted in the culture medium to give a final test concentration of 100 μM. Serial dilutions (100, 50, 10, 5, 1, 0.5, 0.1 and 0.05 μM) were selleck screening library performed for the determination of the IC50 in MB, MH2, LMB and SASW. OD readings were normalised with respect to the absorbance of the blank wells and then the growth inhibition percentage respect to a control with the proportional amount of DMSO was calculated. Experiments were run by triplicate. Preparation of inocula Bacterial inocula were prepared in 0.22 μm filtered seawater (FSW). Isolated colonies were suspended until they matched a McFarland turbidity of 0.5 (bioMérieux Vitek Densichek). One hundred microliters were transferred to test tubes containing 9.