M465:Communityanalyses: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
| Line 14: | Line 14: | ||
"Protocol" | "Protocol" | ||
* The day before, create an overnight culture of your isolates to be tested by inoculating a single colony into a tube of nutrient | * The day before, create an overnight culture of your isolates to be tested by inoculating a single colony into a tube of nutrient broth (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. | * 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 | * Incubate for 24-48 hours at 37°C, monitoring turbidity and growth daily. | ||
* At the completion of incubation, aspirate the liquid from the wells using a pasteur pipette. | * At the completion of incubation, aspirate the liquid from the wells using a pasteur pipette. | ||
* Wash the wells once with deionized, distilled water. | * Wash the wells once with deionized, distilled water. | ||
Revision as of 15:58, 8 January 2013
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 broth (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 37°C, 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
