IGEM:Virginia 2012/Protocols/Passaging Pertussis

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Contents

Overview

Detailed below is the procedure to passage B. pertussis cells.

Materials

  • Plate of B. Pertussis
  • SSM Buffer with Proline and SSM Supplements
  • Sterile Polyester-Tipped Swabs
  • Sterile Hood
  • 25 mL sterile plastic pipette
  • 37°C Water Bath
  • 50 mL Erlenmeyer Flasks

Note: Media is stored in the refrigerator, while supplements are stored in the freezer. Note: Supplements are stored in the freezer, but once they are used they can be stored in the refrigerater, but must be used within a week.

Procedure

  • Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.

Move all materials to a sterile hood.

  • Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to (each) flask
  • Next, pipette 150 μL of each supplement (Proline & SSM) into each flask.
  • Swab the Pertussis plate with a sterile polyester-tipped swab and mix it into the flask. Do this again to ensure you have transferred bacteria into the flask. Try to get a visible amount of bacteria into the flask, so it is a little cloudy.
  • Re-cover the flask with foil.
  • Replace used swabs into their original covering and discard them into the biohazard waste.
  • Place the flasks in the INFORS Minitron shaker/incubator (35.5°C and 150 RPM). Press Start.
  • Incubate for 1 day. -- Later, measure at 650 nm wavelength.
  • Wipe down the bench and hood with Cavicide and then Ethanol.


Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!


References

Contact

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