Biofilm Formation of S.maltophilia with Vancomycin

Today I am repeating the crystal violet assay with several modifications to the staining procedure. In attempt to reduce the variability between trials, I will be using a different method to wash the plates after 24 hours of incubation. I will only be using three of our isolates of S.maltophilia (BAA2423, BAA84, and 13270) with vanc25 and vanc100, with no vanc as a control.  I have updated the protocol below to accommodate for the new wash method.

Materials

• Trypticase soy broth
• Cultures of bacteria on plates
• 15ml conical tubes
• Pipet aid with 5ml sterile glass pipet
• Shaker, 37°C
• 96 well plate, sterile
• Vacomycin stock
• Sterile distilled water
• 0.1% crystal violet
• p200 pipet and tips
• 95% ethanol
• 96 well plate reader

Procedure

1. Create an overnight culture of selected bacteria strains by adding 3ml TBS into each 15ml conical tube, add a loop of bacteria, and vortex well until bacteria is thoroughly suspended.
2. Place tubes in 37°C shaking incubator overnight.
3. Add 3mls of 2xTSB into new 15ml conical tubes labeled appropriately. From overnight cultures, pipet 30µl from culture into new media to create a 1:100 dilution.
4. Create a microtiter plate with different concentrations of vancomycin so that each strain will have a total of 18 wells (3 concentrations with 6 replicated wells).
• Create a 50μg/mL and 200μg/mL working stock
• Add 100μL to the wells that half of that vanc concentration
• Add 100μL of sterile water to all other wells
• Concentrations of vancomycin should be 0, 50, and 200 μg/ml.
5. Add 100µl of inoculated media into each designated well, final volume of all wells should be 200μL and final vanco concentrations will be 0, 25, and 100 μg/ml
6. Incubate plates at 37°C for 24 hours.
7. Carefully transfer culture from each inoculated well into a new 96-well plate and measure OD600 by using a plate reader set to 600nm.
8. Gently submerge the original plate in a 2 inch deep pan containing 0.1% crystal violet.
9. Let plate sit in the crystal violet for 15 minutes at room temp.
10. Remove the plate and gently submerge in a new pan containing distilled water.
11. Repeat in clean water 3 times to remove excess crystal violet and any unattached cells.
12. Let the plate dry for 15 minutes in the hood.
13. Add 200µl 95% Ethanol to each well, let sit for at least 5 minutes; vortex gently if necessary.
14. Read plate at 600 nm with plate reader.

//BEE