TITLE: S. aureus biofilm removal multi-assay
AUTHORS: Tomasz Swebocki

[DESCRIPTION]
The following protocol describes culture and treatment of the S.aureus-based biofilm and techniques that can be used to asses its eradication/removal. The protocol covers basic preparation and three techniques, namely: growth control, colony forming assay, and crystal violet staining.

[STEPS]
SECTION: Biofilm culture
1. Culture bacteria strains in TSB at 35 °C for approximately 240 min in order to reach mid-log phase (OD600 0.5-0.6).

SECTION: Biofilm culture
2. Pellet the cells by centrifugation (4500 rpm, 5 min, 20 °C) then wash twice with potassium phosphate buffer (PPB; 100 millimolar (mM), pH 7).

For washing, redisperse and repeat the centrifugation. First two washes with 3 mL of PPB, then redisperse in 3 mL ofPPB
SECTION: Biofilm culture
3. Adjust bacterial suspensions to 107 CFU/mL in PPB (OD600= 0.01, ref: PPB).
SECTION: Biofilm culture
4. Initiate biofilm formation in 24-well plate by covering surface with 1 mL of the adjusted cell suspension, and incubate the plate at Room temperature for 120 min to allow the bacterial adhesion. Remove the content of the wells after this time.

SECTION: Biofilm culture
5. Cover the wells with 1 mL of TSB, and put a protective film before putting the lid to minimize the evaporation. Incubate the bacteria for at least 1080 min
SECTION: Treatment of the biofilm
6. Prepare your substance in either PBS or NaCl (9 Mass 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.

SECTION: Treatment of the biofilm
8. 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.
 

SECTION: Treatment of the biofilm
9. 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).

SECTION: Treatment of the biofilm
7. After the biofilm matured, remove the 24-well plate from the incubator.
SECTION: Treatment of the biofilm
10. 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.
SECTION: Colony forming assay
13. Add 1 mL 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. 
SECTION: Colony forming assay
14. 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.

SECTION: Colony forming assay
15. Serially dilute the content of the eppendorf tubes in 96-well plate.

SECTION: Colony forming assay
16. Plate 20µL of the content of the wells on the BHI medium prepared in the square Petri dish.
SECTION: Colony forming assay
17. Incubate Petri dish 240 min 

SECTION: Crystal violet staining + brightfield microscopy
19. Dry the biofilms for 30 min in Room temperature

SECTION: Crystal violet staining + brightfield microscopy
20. After that time, fix the biofilms by adding 200 µL of methanol to each well. Wait 15 min and remove any remaining methanol.

SECTION: Colony forming assay
18. Next day, count the colonies and asses the CFU/mL or CFU/cm2.
SECTION: Growth control
11. Add 0.5 mL of TSB to each well and indubate the plate at 37 °C for 360 min  480 min
SECTION: Growth control
12. Transfer the supernatant to 96-well plate and measure OD600, dilute with NaCl if necessary.
SECTION: Crystal violet staining + brightfield microscopy
21. Add250 µL of 0.06 Mass Percent solution of crystal violet to each well and wait 15 min.

SECTION: Crystal violet staining + brightfield microscopy
22. Remove any non-absorbed dye by washing gently stained biofilms with either PBS, NaCl or PPB.
SECTION: Crystal violet staining + brightfield microscopy
22.1. At this moment you can perform brightfield microscopy of the biofilm to image better its structure.
SECTION: Crystal violet staining + brightfield microscopy
23. Add 1 mL of acetic acid to the wells and gently shake    them horizontally for 30 min.

SECTION: Crystal violet staining + brightfield microscopy
24. Remove 200 µL od the solubilised pigment with a pipette and transfer it to 96-well plate and dilute serially with water.

SECTION: Crystal violet staining + brightfield microscopy
25. 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.