M465:Communityanalyses

Interaction Assays

Antibiotic Resistance

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

"Protocol"

• The day before, create an overnight culture of your isolates to be tested by inoculating a single colony into a tube of nutrient agar (or other appropriate medium).
• In the morning, measure tho OD of the culture and dilute to 0.05. Use this dilution to inoculate at least 3 wells in your polystyrene dish (add ~5 ml of media to each well). Include a medium only control.
• Incubate for 24-48 hours at 37C, monitoring turbidity and growth daily.
• At the completion of incubation, aspirate the liquid from the wells using a pasteur pipette.
• Wash the wells once with deionized, distilled water.
• Stain the sample with crystal violet by adding 1-2 drops from your droppers into each well and waiting 2 minutes at room temperature.
• Wash the sample again with water twice.
• Fill each well with acetic acid
• Shake for 30 mins at room temp
• Measure OD600

Quorum Sensing