3.2 Crystal Violet
Biofilm Staining
All these steps are completed at room temperature.
1. Following desired incubation time, remove planktonic cells by
gently pipetting the supernatant and add 150 μL of fixative
buffer (4% PFA) to increase biofilm stability and incubate
30 min.
2. Gently remove the fixative buffer, and rinse two times with
200 μL of PBS by gentle pipetting.
3. Tilt the plate and remove as much PBS as possible.
4. Pipette 175 μL of 0.1% crystal violet solution into wells. This
volume ensures that the stain will cover the biofilm (see Note 4).
5. Incubate for 10 min.
6. Gently remove the crystal violet solution and rinse two times
with 200 μL PBS.
7. Tilt the plate and remove as much PBS as possible and dry for a
few hours or overnight.
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
H 2 O
Condition 1
Condition 2
Condition 3
TFig. 1 Classical working plate layout including 8 blank wells containing sterile EMJH medium and 18 technical
replicates for each conditions tested (three conditions maximum per plate in this configuration). Also, note the
presence of sterile distilled water in the peripheral wells of the plate to ensure correct moisture level and
reduce border effect
210
Roman Thibeaux et al.