IGEM:Virginia 2012/Protocols/Passaging Pertussis
Detailed below is the procedure to make CaCl2-competent stock cells. This is adapted from The Department of Biology at Indiana University.
- 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.
- 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.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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