S. aureus biofilm removal multi-assay

Culture bacteria strains in TSB at 35°C for approximately 4h 0m 0s in order to reach mid-log phase (OD₆₀₀ 0.5-0.6).

Pellet the cells by centrifugation (4500rpm,20°C) then wash twice with potassium phosphate buffer ( PPB ; 100millimolar (mM), 7).

For washing, redisperse and repeat the centrifugation. First two washes with 3mL of PPB , then redisperse in 3mL of PPB

Adjust bacterial suspensions to 10⁷ CFU/mL in PPB (OD₆₀₀= 0.01, ref: PPB ).

Initiate biofilm formation in 24-well plate by covering surface with 1mL of the adjusted cell suspension, and incubate the plate at Room temperature for 2h 0m 0s to allow the bacterial adhesion. Remove the content of the wells after this time.

Cover the wells with 1mL of TSB , and put a protective film before putting the lid to minimize the evaporation. Incubate the bacteria for at least 18h 0m 0s

Prepare your substance in either PBS or NaCl ( ) 9Mass Percent) by serial dilution ranging from C to C/(X²) where X is the number of concentrations tested. A 24-well plate can hold 7 concentrations + negative control.

After the biofilm matured, remove the 24-well plate from the incubator.

Gently remove the TSB using either pipette or vacuum aspirator. After removing medium from a cell, immediately wash it with PPB , PBS or NaCl two times. After last washing add 1 mL of your sample. Repeat for concentration in triplicate. Same as before, apply protective film before incubating the plate.

Treat the biofilms at suitable temperature and time. This two factors have to be estimated prior to the treatment, for example by means of crystal violet staining (see below).

After the time has passed, remove the plate, and start removing the content of the well. After each removal, wash the remaining biofilm three times with either PBS , NaCl or PPB . Remove remaining liquid after and perform on of the techniques described below.

Add 0.5mL of TSB to each well and indubate the plate at 37°C for 6h 0m 0s 8h 0m 0s

Transfer the supernatant to 96-well plate and measure OD₆₀₀, dilute with NaCl if necessary.

Add 1mL of either PBS , NaCl or PPB to the well with a pipette, directly onto the bioflm, so the thrust of the liquid damage the biofilm.

Scratch the bottom of the well with a pipette tip and transfer the content to eppendorf tubes. Vortex-shake eppendorfs for couple of seconds or until reaching homogeneity.

Serially dilute the content of the eppendorf tubes in 96-well plate.

Plate 20µL of the content of the wells on the BHI medium prepared in the square Petri dish.

Incubate Petri dish 4h 0m 0s

Next day, count the colonies and asses the CFU/mL or CFU/cm².

Dry the biofilms for 0h 30m 0s in Room temperature

After that time, fix the biofilms by adding 200µL of methanol to each well. Wait 0h 15m 0s and remove any remaining methanol.

Add250µL of 0.06Mass Percent solution of crystal violet to each well and wait 0h 15m 0s.

Remove any non-absorbed dye by washing gently stained biofilms with either PBS , NaCl or PPB .

At this moment you can perform brightfield microscopy of the biofilm to image better its structure.

Add 1mL of acetic acid to the wells and gently shake 120rpm them horizontally for 0h 30m 0s.

Remove 200µL od the solubilised pigment with a pipette and transfer it to 96-well plate and dilute serially with water.

Record the absorption of the wells at 570 nm . The percent of the remaining biomass is calculated by dividing the A₅₇₀ of the sample to A₅₇₀ of the control.