Biofilm formation of S.maltophilia with delayed addition of vancomycin

Today I will be testing the impact on biofilm formation by S. maltophilia when vancomycin is added 6 hours after inoculation of a 96-well plate. I prepared liquid cultures yesterday in 3mLs of TSB and followed the procedure outlined below.

Materials

• Trypticase soy broth
• Cultures of 8 ATCC S. maltophilia bacteria isolates
• 15ml conical tubes
• Pipet aid with sterile pipets
• 37°C shaking incubator
• 37°C shaking incubator
• 96 well plate, sterile
• Vancomycin stock
• PBS
• 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 selected media into new 15ml conical tubes labeled appropriately. From overnight cultures, pipet 30µl from culture into new media to create a 1:100 dilution.
4. Add 200µl of inoculated media into each designated well
• For each isolate fill a 4x4 area for a total of 16 wells
5. Incubate plates at 37°C for 6 hours.
6. Remove liquid media from wells very carefully
7. Add 200µl of fresh TSB with to each row of 4 wells, with each column having a different concentration of vancomycin (0, 25, 50, 100µg/ml) and incubate at 37°C for 18 hours.
8. Remove liquid culture media from wells and transfer to a new 96 well plate--read OD600.
9. Wash overnight plate 3 times with 200µl PBS, let dry for 30 minutes in the hood.
10. Stain with 200µl 0.1% crystal violet for 15 minutes.
11. Remove crystal violet and dispose of appropriately.
12. Wash wells 3 times with 200µl PBS, let dry for 15 minutes in the hood.
13. Add 200µl 95% Ethanol to each well, let sit for 5 minutes.
14. Read plate at 600 nm with plate reader.

Tomorrow I will continue the procedure at step 8.

//BEE