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 bacterial community can form a biofilm is to provide them with a surface to which they can adhere, 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 performing this assay in round bottom plates and 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)
1. You will perform the assay in triplicate for each of your fly genotypes. Use a 96-well plate map to mark out where your samples will go.
2. Obtain a sterile, round bottom, 96-well plate.
3. Obtain a 1.5 mL tube of Drosophila flies from your instructor. Each of these is wild-caught, collected by us on our field trip. You will be asking if the microbes associated with the fly can form biofilms as a community by first making a lysate from your flies.
note: move all of your materials to a laminar flow hood now to limit contamination!
4. Add 500 uL of sterile LB to the flies and grind using a sterile pestle - 20 strokes should do it.
5. Add another 500 μL of sterile LB medium to the test tube - you will have 1 mL of lysate total now. Add 100 uL of your lysate to each of three wells in your 96-well plate.
6. As a negative control, add 100 ul of sterile LB medium to three wells in your 96-well plate.
7. Repeat your experiment above, steps 3-6, this time using MRS medium.
8. Incubate your dish at 30°C in the incubators in the back.
1. At the completion of incubation, aspirate the liquid from all the wells using a sterile pasteur pipette. You can place the liquid into a "liquid waste" container on your bench.
2. Wash the wells GENTLY once with deionized, distilled water (you can use the squeeze bottles on your bench). To wash, fill the well 1/2 way with your ddH2O, then gently aspirate this liquid from the well using a sterile pipette, placing it in the liquid waste container.
3. Stain the sample with crystal violet by adding 1-2 drops from your droppers containing crystal violet into each well and waiting 2 minutes at room temperature.
4. Wash the sample, gently, with water, twice, following the same protocol above for washing.
Note: In the next few steps we will quantify the biofilm by first disrupting it with acetic acid and then measuring the OD600.
5. Fill each well with 33% acetic acid.
6. Shake your dish for 30 mins at room temp using the platform shaker in the room.
7. At the end of this final incubation, transfer the liquid to a clear, flat-bottom, 96-well plate.Measure the OD600 of each well with the Synergy H1 plate reader