U shaped 96 well plates

This experiment was performed according to the microdilution protocol of CLSI (CLSI, 2015 ) using Mueller-Hinton broth (MHB; Carl Roth, Karlsruhe, Germany) and U-shaped 96-well plates (Greiner Bio One, Kremsmünster, Austria). The compounds were diluted in MHB in 2 mL tubes (Eppendorf, Wesseling-Berzdorf, Germany) to a concentration of 1.6 mM. Twelve twofold serial dilutions of the compounds were prepared in a 96-well plate to a final concentration ranging from 800 to 0.39 μM. The highest concentration was in well 12 of each row and the lowest concentration in well 1 of each row. Bacteria were grown overnight on Columbia blood agar plates and on the day of the experiment suspended in 0.9% saline solution with turbidity adjusted to a 0.5 McFarland standard. The bacterial solution was diluted in MHB to reach a bacterial concentration of 1.5 × 10⁸ CFU/mL and then added to the compounds in the wells to a final concentration of 5 × 10⁵ CFU/mL. After overnight incubation at 37°C the minimum inhibitory concentration (MIC) was determined by visual inspection to detect the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in the tubes.

Confluent VeroB4 cells were cultured in Medium199 including 5% FCS in T75 tissue culture flasks (Sarstedt, Germany) and transferred into 96-well tissue culture plates (Sarstedt, Germany). Passage 1 isolates of SARS-CoV-2 were thawed from −80 °C freezer and titrated from 1:10 to 1:10⁻¹² in U-shaped 96-well plates (Greiner, Germany) and pipetted into each corresponding well of the 96-well tissue culture plate. Plates were incubated at 36 °C. Three days post infection, incubation was stopped by gently removing the supernatant, washing the cells three times with PBS and fixing cells in 1:1 ice-cold acetone–methanol. For easier optical evaluation, cells were dyed by crystal violet staining and tissue culture infectious dose of 70% (TCID₇₀) and PFU were calculated^{57 (link)}.

Confluent VeroB4 cells were cultured in Medium199 including 5% FCS in T75 tissue culture flasks (Sarstedt, Germany) and transferred into 96-well tissue culture plates (Sarstedt, Germany). Passage 1 isolates of SARS-CoV-2 were thawed from − 80 °C freezer and titrated from 1:10 to 1:10⁻¹² in U-shaped 96-well plates (Greiner, Germany) and pipetted into each corresponding well of the 96-well tissue culture plate. Plates were incubated at 36 °C. Three days post infection, incubation was stopped by gently removing the supernatant, washing the cells three times with PBS and fixing cells in 1:1 ice-cold acetone-methanol. For easier optical evaluation, cells were dyed by crystal violet staining and tissue culture infectious dose of 70% (TCID₇₀) and plaque-forming units (PFU) were calculated [15 (link)]. Titration was performed twice with our isolates no. I1 and I2.