M465:Communityanalyses

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(Biofilm Assay)
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"Protocol"
"Protocol"
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* 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).   
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* 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.
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* Incubate for 24-48 hours at 37C, monitoring turbidity and growth daily.
+
* 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 18:58, 8 January 2013

M465

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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


==Quorum Sensing==
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