Gram s crystal violet solution

Phosphate buffered saline (PBS), alginate, sulfuric acid, absolute ethanol (99.7%), trichloroacetic acid, sulforhodamine B, acetic acid and tris(hydroxymethyl)aminomethane (TRIS base) were purchased from Sigma-Aldrich^® (Darmstadt, Germany). Gram’s crystal violet solution, dimethyl sulfoxide (DMSO), deuterated dimethyl sulfoxide (DMSO-d6), anhydrous chloroform, deuterated chloroform (CDCl₃), methanol, ethyl acetate (AcOEt) and calcium chloride were bought from Merck^® (Lisboa, Portugal). Dehydroabietic acid was obtained from Wako^® (Neuss, Germany) (FUJIFILM Wako Pure Chemical Corporation; CAS RN^®: 1740-19-8, Molecular Formula: C20H28O2; Molecular Weight: 300.44). Mueller–Hinton Broth (MHB), Mueller–Hinton agar (MHA), Sabouraud dextrose broth (SDB), Sabouraud dextrose agar (SDA) and tryptic soy broth (TSB) were purchased from Biokar^® Diagnostics (Allonne, France).

To quantify the biofilm biomass, the washed biofilms were stained with 200 μl of a 0.1% crystal violet solution (1:4 dilution in DDW of the Gram's crystal violet solution, Merck) for 20 min at room temperature^{57 (link)}. The stained biofilms were washed twice with DDW, and after drying the wells, the stain was dissolved in 200 μl of a 33% acetic acid solution, and quantified by reading the OD at 595 nm in the M200 Tecan microplate reader. Different dilutions of the test compounds in the absence of bacteria were used to measure background signals. The percentage biofilm formation was calculated by dividing the OD of treated samples on OD of control samples, multiplied with 100%.

To quantify the resulting biofilm biomass, the washed biofilms were stained with 200 μl of a 0.1% crystal violet solution (1:4 dilution in DDW of the Gram's crystal violet solution, Merck) for 20 min at room temperature [24 (link)]. Thereafter the biofilms were washed twice with DDW and the stain dissolved in 200 μl of a 33% acetic acid solution. The OD at 595 nm was measured spectrophotometrically using the M200 Tecan microplate reader. Different dilutions of the test compounds in the absence of bacteria were used to measure background signals.

Ten μL of control and AEA (50 μg/mL, 2 h)-treated MDRSA CI-M cultures at an OD_600nm of 0.3 were inoculated on tryptic soy agar plates (Acumedia, Neogen, Lansing, MI, USA) containing 1.5% gelatin followed by a 24 h incubation at 37 °C. Alternatively, 10 μL of their supernatants were inoculated on these plates. The clearance zone around the inoculation site was detected after staining the plates with 0.1% crystal violet solution prepared from a 0.4% Gram’s crystal violet solution (Merck, EMD Millipore Corporation, Billerica, MA, USA) diluted with DDW. After a 15 min incubation, the plates were washed with DDW, and images were captured using the Bio-Rad ChemiDoc XRS+ Imager system and the Image Lab software.

SARS-CoV-2 strain G614 and VOCs (Delta and Omicron) were isolated from patients in Pavia and used for microneutralization assay [24 (link), 25 ]. Briefly, 50 μL of the sample, starting from 1:10 in a serial twofold dilution series (up to 1:640), was added to two wells of a flat-bottom tissue-culture microtiter plate (COSTAR, Corning Incorporated), mixed with an equal volume of 100 Tissue Culture Infection Dose 50 (TCID50) of a SARS-CoV-2 strain, previously titrated and incubated at 33°C in 5% CO₂. All dilutions were made in Eagle’s minimum essential medium with addition of 1% penicillin, streptomycin, and glutamine and 5 μg/mL of trypsin. After 1 h of incubation at 33 °C in 5% CO₂, VERO E6 cells (VERO C1008 (Vero 76, cloneE6, Vero E6); ATCC® CRL-1586™) were added to each well. After 48 h of incubation at 33°C in 5% CO₂, wells were stained with Gram’s crystal violet solution (Merck) plus 5% formaldehyde 40% m/v (Carlo ErbaSpA) for 30 min. Microtiter plates were then washed in running water. Wells were scored to evaluate the degree of cytopathic effect (CPE) compared with the virus control. Blue staining of wells indicated the presence of neutralizing antibodies. The neutralizing titer was defined as the maximum dilution giving a reduction of 90% of the CPE. The cut-off for positivity was ≥1:10. Positive and negative controls were included in all test runs.