M465:Biofilms

Biofilm Assay

Bacterial communities in the environment will often exist in biofilms, adhered to a surface. That surface may be a grain of soil, the basalt on the bottom of the ocean near a hydrothermal vent, or a medical catheter. One way to identify whether or not your isolates can form a biofilm is to provide them with a surface to which they can adhere (in our case, chitin), allow them time to adhere and grow the biofilm, and then stain the biofilm with crystal violet to visualize and quantify the amount of biomass on the surface. The crystal violet assay is great to quantify biofilm formation quickly in a high-throughput way. We will be growing our cultures in round bottom plates with chitin. The protocol will be conducted over 48 hours (today and our next meeting).

Take a look at the image to the right for an example of what published crystal violet assays look like (this one from Chaves et al., BMC Research Notes 2009 2:50)

You will perform the protocol below for each of your isolates

On Sunday/Monday

1. The day before our lab (Sunday), create an overnight broth culture of your isolate to be tested by inoculating a 16mm tube of broth (of the appropriate medium).

2. In the morning (Monday), measure the OD600 of the culture and dilute it to 0.05. You can use excel to calculate your dilutions here (using the OD600 calculation spreadsheet). You want at least 5 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 5 mL medium

3. You will perform the assay in triplicate for each of your samples as well as sterile media controls. Use a 96-well plate map to mark out where your samples will go.

4. Obtain a sterile, round bottom, 96-well plate. To each of the wells that you are going to inoculate, aseptically transfer sterile chitin into the bottom of the plate, just enough to cover the bottom. Try to make the amount even in all of your wells.

5. Add 200 μL of your diluted culture or sterile media to the plate.

6. Incubate your dish for 2 days at 30°C.

On Wednesday

7. At the completion of incubation, aspirate the liquid from all the wells using a pasteur pipette.

8. Wash the wells GENTLY once with deionized, distilled water (you can use the squeeze bottles on your bench).

9. Stain the sample with crystal violet by adding 1-2 drops from your droppers into each well and waiting 2 minutes at room temperature.

10. Wash the sample, gently, with water, twice.

Note: In the next few steps we will quantify the biofilm by first disrupting it with acetic acid and then measuring the OD600.

11. Fill each well with 33% acetic acid.

12. Shake your dish for 30 mins at room temp using the platform shaker in the room.

13. At the end of this final incubation, transfer the liquid to a clear bottom, 96-well plate. Be careful to avoid transferring any chitin over. Measure the OD600 of each well with the Synergy H1 plate reader