Jun 14, 2023
Version 1
S. aureus biofilm removal multi-assay V.1
- 1Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520-IEMN-Institut d’Electronique de Microélectronique et de Nanotechnologie, 59000 Lille, France
Protocol Citation: Tomasz Swebocki 2023. S. aureus biofilm removal multi-assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xk6klqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 14, 2023
Last Modified: June 16, 2023
Protocol Integer ID: 83400
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Abstract
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.
Biofilm culture
Biofilm culture
1d 0h 5m
1d 0h 5m
Culture bacteria strains in TSB at 35 °C for approximately 04:00:00 in order to reach mid-log phase (OD600 0.5-0.6).
4h
Pellet the cells by centrifugation (4500 rpm, 20°C, 00:05:00 ) then wash twice with potassium phosphate buffer (PPB; 100 millimolar (mM) , 7 ).
For washing, redisperse and repeat the centrifugation. First two washes with 3 mL of PPB, then redisperse in 3 mL ofPPB
5m
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 1 mL of the adjusted cell suspension, and incubate the plate at Room temperature for 02:00:00 to allow the bacterial adhesion. Remove the content of the wells after this time.
2h
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 18:00:00
18h
Treatment of the biofilm
Treatment of the biofilm
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.
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.
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | C | C | C | C/16 | C/16 | C/16 |
| B | C/2 | C/2 | C/2 | C/32 | C/32 | C/32 |
| C | C/4 | C/4 | C/4C | C/64 | C/64 | C/64 |
| D | C/8 | C/8 | C/8 | NEGATIVE CONTROL | NEGATIVE CONTROL | NEGATIVE CONTROL |
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.
Growth control
Growth control
Add 0.5 mL of TSB to each well and indubate the plate at 37 °C for 06:00:00 08:00:00
14h
Transfer the supernatant to 96-well plate and measure OD600, dilute with NaCl if necessary.
Colony forming assay
Colony forming assay
4h
4h
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.
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 Overnight
4h
Next day, count the colonies and asses the CFU/mL or CFU/cm2.
Crystal violet staining + brightfield microscopy
Crystal violet staining + brightfield microscopy
1h 30m
1h 30m
Dry the biofilms for 00:30:00 in Room temperature
30m
After that time, fix the biofilms by adding 200 µL of methanol to each well. Wait 00:15:00 and remove any remaining methanol.
15m
Add250 µL of 0.06 Mass Percent solution of crystal violet to each well and wait 00:15:00 .
15m
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 1 mL of acetic acid to the wells and gently shake 120 rpm, Room temperature them horizontally for 00:30:00 .
30m
Remove 200 µL 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.