IGEM:Virginia 2012/Protocols/Preparing a Pertussis Culture

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*50 mL flask
*50 mL flask
*[http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/SSM_Media_Preparation SSM media]
*[http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/SSM_Media_Preparation SSM media]
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*Proline and SSM Supplements
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*Sterile Polyester-Tipped Swabs
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* 25 mL sterile plastic pipette
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* 37°C Water Bath
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* 3 50 mL Erlenmeyer Flasks
==Procedure==
==Procedure==
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# Using a sterilized stick, streak the entire surface of the [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Bordet_Gengou_%28BG%29_Agar_Plate_Preparation BG Agar plate]. Discard the stick in the biohazard container.
# Using a sterilized stick, streak the entire surface of the [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Bordet_Gengou_%28BG%29_Agar_Plate_Preparation BG Agar plate]. Discard the stick in the biohazard container.
# Put the plates in a plastic bag to prevent drying out and incubate at 37°C for three days.
# Put the plates in a plastic bag to prevent drying out and incubate at 37°C for three days.
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# Using 10 mL of [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/SSM_Media_Preparation SSM media] in a 50 mL flask, inoculate the liquid culture.
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# Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.
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# Incubate at 35.5°C for a day.
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# Using a sterile 25 mL plastic pipette,  add 15 mL of warm SSM media to each 50 mL flask.
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# Dilute the liquid culture.
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# Pipette 150 μL of '''each''' supplement (Proline & SSM) into each flask.
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# Swab the ''Pertussis'' plate with a sterile polyester-tipped swab and mix it into the flask. 'Try to get a visible amount of bacteria into the flask, so it is a little cloudy.'
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# Re-cover the flask with foil.
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# Replace used swabs into their original covering and discard them into the biohazard waste.
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# Place the flasks in the INFORS Minitron shaker/incubator (35.5°C and 150 RPM). Press Start.
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# Incubate for 1 day. -- Later, measure at 650 nm wavelength.
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# Wipe down the bench and hood with Cavicide and then Ethanol.
==Notes==
==Notes==

Current revision

Contents

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
  • Proline and SSM Supplements
  • Sterile Polyester-Tipped Swabs
  • 25 mL sterile plastic pipette
  • 37°C Water Bath
  • 3 50 mL Erlenmeyer Flasks

Procedure

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

  1. Using a sterilized stick, streak the entire surface of the BG Agar 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. Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.
  4. Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to each 50 mL flask.
  5. Pipette 150 μL of each supplement (Proline & SSM) into each flask.
  6. Swab the Pertussis plate with a sterile polyester-tipped swab and mix it into the flask. 'Try to get a visible amount of bacteria into the flask, so it is a little cloudy.'
  7. Re-cover the flask with foil.
  8. Replace used swabs into their original covering and discard them into the biohazard waste.
  9. Place the flasks in the INFORS Minitron shaker/incubator (35.5°C and 150 RPM). Press Start.
  10. Incubate for 1 day. -- Later, measure at 650 nm wavelength.
  11. 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

  • Who has experience with this protocol?

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