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

Materials

• Pertussis frozen stock (keep on ice)
• Sheep blood agar plates
• Sterilized sticks

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. Inoculate the liquid culture. Incubate at 35.5°C.
4. The next day, 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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