IGEM:Virginia 2012/Protocols/Preparing a Pertussis Culture

Overview

Below is the procedure for preparing a Bordetella pertussis culture. First, the frozen stock is streaked onto a sheep blood agar plate. This plate is then incubated for ~3 days, until it produces pertussis colonies which are gray/white and dull rather than shiny. These colonies are then used to inoculate a liquid culture. Since pertussis is aerobic, the liquid culture must have a high amount of surface area. This liquid culture can then be diluted so that it is in the logarithmic growth phase in time for manipulation. Pertussis can't be stored in the fridge, so the liquid culture can be used to start new cultures for a limited period of time, so that mutations do not accumulate.

Materials

• Pertussis frozen stock (keep on ice)
• BG Agar plates
• Sterilized sticks
• 50 mL flask
• SSM media

Procedure

Note: This procedure should be done in a hood to prevent aerosolization.

1. Using a sterilized stick, streak the entire surface of the plate. Discard the stick in the biohazard container.
2. Put the plates in a plastic bag to prevent drying out and incubate at 37°C for three days.
3. Using 10 mL of SSM media in a 50 mL flask, inoculate the liquid culture.
4. Incubate at 35.5°C for a day.
5. Dilute the liquid culture.

Notes

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

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