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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, PVC coverslips), 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 polystyrene dishes (12 well) on PVC coverslips. 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 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 four wells of a sterile, 12-well polystyrene dish, place a single PVC coverslip, such that it is standing upright.

4. Use your 15 mL 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. You will only need one control well for the entire polystyrene dish.

5. Incubate your dish for 2 days at 37°C, at the appropriate atmosphere.

On Wednesday

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

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

8. 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.

9. Wash the sample, gently, 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).

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

10. Fill each well with 33% acetic acid, 5 mL each

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

12. At the end of this final incubation, measure the OD600 of each well with the Synergy H1 plate reader

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