Biofilm Formation of S.maltophilia with Vancomycin

Today I am repeating an experiment I have done in the past that observers the effects the antibiotic vancomycin has on formation of biofilm on a polystyrene 96-well plate by the bacterium S.maltophilia. I started overnight liquid cultures yesterday of 8 ATCC strains that we have in the lab (BAA84, BAA2423, 700, 51, 49, 13270, 17666, 13637).  I followed the following procedure and will finish after 24 hours of incubation.

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
• 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 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 2 microtiter plates with 4 different concentrations of vancomycin so that each strain will have a total of 16 wells (4 concentrations with 4 replicated wells).
• Create 200μg/mL working stock
• Add 200μL to the wells that will have the highest vanco concentration
• Add 100μL of sterile water to all other wells.
• Serial dilute 1:2 subsequent wells by transferring 100μL and discard final 100μL so all wells have a final volume of 100μL
• Concentrations of vancomycin should be 0, 50, 100, 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, 50, and 100 μg/ml
6. Incubate plates at 37°C for 24 hours.
7. Transfer culture from each inoculated well into a new 96-well plate and measure OD600 by using a plate reader set to 600nm.
8. Wash overnight plate 3 times with 200µl PBS per well, let dry for 30 minutes in the hood.
9. Stain wells with 200µl 0.1% crystal violet for 15 minutes.
10. Remove crystal violet and dispose of appropriately.
11. Wash wells 3 times with 200µl PBS, let dry for 15 minutes in the hood.
12. Add 200µl 95% Ethanol to each well, let sit for at least 5 minutes; vortex gently if necessary.
13. Read plate at 600 nm with plate reader.

//BEE