M465:Biofilms

Biofilm Assay

The crystal violet assay is great to quantify biofilm formation quickly in a high-throughput way. We will be growing our cultures in polystyrene dishes (12 well) on PVC coverslips.

"Biofilm disruption solution"

• 33% acetic acid

You will perform the protocol below for each of your isolates
"Protocol" 1. The day before, create an overnight culture of your isolates to be tested by inoculating a single colony into a tube of broth (the appropriate medium).
2. In the morning, measure tho OD of the culture and dilute it to 0.05. You can use excel to calculate your dilutions here. You want at least 15 mL of media at a 0.05 OD600 so you can use the C1V1 = C2V2 equation to figure out how much you need to put into the 15 mL medium
. 3. In each well of a 12-well polystyrene dish, place a single PVC coverslip, such that it is standing upright.
4. Use this dilution to inoculate 3 wells in your polystyrene dish (add ~5 ml of media to each well). Include a medium only control in one of the wells.
5. Incubate for 2 days at 37°C, at the appropriate atmosphere. At the completion of incubation, aspirate the liquid from the wells using a pasteur pipette.
6. Wash the wells and coverslips GENTLY once with deionized, distilled water (you can use the squeeze bottles on your bench).
7. Stain the sample with crystal violet by adding 1-2 drops from your droppers into each coverslip in each well and waiting 2 minutes at room temperature.
8. Wash the sample again with water twice. At this point, you can take a picture of the stained biofilm on the coverslip for each of your isolates (sacrificing one of the three replicates).
9. In the next few steps we will quantify the biofilm by first disrupting it with acetic acid and then measuring the OD600. Fill each well with 33% acetic acid, 5 mL each
10. Shake for 30 mins at room temp using the platform shaker in the room
11. At the end of this final incubation, measure the OD600 of each well with the Synergy H1 plate reader