Crystal violet assay

Protocol biofilm culture and CV staining 

Materials

Plasma coating

Growing biofilm

Crystal violet staining 

Protocol

Coating

• Grow overnight cultures in 3 ml TSB, shaking 600 rpm at 37°C. 
• Dissolve plasma to 20% in filter sterilized carbonate-bicarbonate buffer
• Coat plates with 50 uL coating solution
• Incubate overnight at 4 °C.

Grow biofilm

• Gently aspirate coating solution, take care to not touch the bottom of the well
• Start biofilm with a 1:1000 inoculation in biofilm medium 
• Add 200 uL to each well of a 96-wells plate. Don’t use the outer ring of the plate and fill all empty wells in the plate with 200 uL Milli Q. Include control well with biofilm medium alone.
• Grow biofilms for 24h at 37°C, static. 

During growth or after growth you can add a treatment that you want to test by adding it to the growth medium. For example, for treatment during biofilm formation add 100 uL 2x bacteria and 100 uL 2x treatment, incubate 24 h and perform CV staining.

For treatment after biofilm formation, remove medium, wash 1 time and add treatment.

CV staining

• 1x wash biofilms carefully with 200 µL PBS
• Fix biofilm for 30 minutes to 1 hour at 60°C 
  • Now the material is heat fixed to the well. After this step biofilm can’t be washed away.
• Stain with 160 µL 0.1 % crystalviolet for exactly 5 min. Discard CV and wash 2x with tap water. 
• Dry overnight / at 60 degrees
• Elute CV by adding 160 uL 33% acetic acid
• Measure the eluted crystal violet at 595nm 
  • If the signal is too high, dilute 2x or more in 33% acetic acid