Biofilm formation test

Reagents:

1. Staphylococcus aureus solution (37℃, 200rpm, overnight culture)
2. Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China Peptides, Suzhou)
3. LB Broth
4. 96-well transparent plate

Procedure:

1. Dilute 200μl Staphylococcus aureus of stationary phase to 2ml volume using LB.
2. Mix 100 μl diluted S. aureus (in step 1) and 100 μl peptide solutions of different concentrations and transfer the mixture in 96-well plate.
3. Add 200μl LB to each well in column 1 of both plates.
4. Add 20μl S. aureus (from step 2) and 180μl LB to each well in column 2 of both plates.
5. Add 200μl S. aureus+AMP solution (from step 3) to wells 1-4, 5-8 in column 3,4,5,6,7,8,9,10,11,12 (4 repeats for each concentration of AMP).
6. Seal plates with tape and incubate at 37℃ in plate reader for 24h.

Our setting for LL-37 and Grammistin-Pp1:

Sample plate for GF-17 and Mixed anti-microbial peptides:

Biofilm staining

1. Pipette LB out of the 96-well plate.
2. Add 200ul ddH₂O in each well.
3. Pipette H₂O out.
4. Add 200μl ddH₂O in each well.
5. Pipette H₂O out.
6. Incubate for 15min in 37 ℃.
7. Air dry (for around 15min).
8. Add 200μl crystal violet (0.1%) in each well.
9. Wash with ddH₂O three times.
10. Add 200μl 95% ethanol.