IGEM:Virginia 2012/Protocols/Preparing a Pertussis Culture - Revision history

Jacqueline K. Grimm at 18:22, 4 June 2012

Jacqueline K. Grimm — Mon, 04 Jun 2012 18:22:55 GMT

←Older revision		Revision as of 18:22, 4 June 2012
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	# Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.		# Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.
	# Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to each 50 mL flask.		# Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to each 50 mL flask.
-	# ~~Next, pipette~~ 150 μL of '''each''' supplement (Proline & SSM) into each flask.	+	# 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. 'Try to get a visible amount of bacteria into the flask, so it is a little cloudy.'		# 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.'
	# Re-cover the flask with foil.		# Re-cover the flask with foil.

Jacqueline K. Grimm at 18:22, 4 June 2012

Jacqueline K. Grimm — Mon, 04 Jun 2012 18:22:09 GMT

←Older revision		Revision as of 18:22, 4 June 2012
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	# 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.
	# Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.		# Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.
-	# Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to (each) flask	+	# Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to each 50 mL flask.
	# Next, pipette 150 μL of '''each''' supplement (Proline & SSM) into 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.''	+	# 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.'
	# Re-cover the flask with foil.		# Re-cover the flask with foil.
	# Replace used swabs into their original covering and discard them into the biohazard waste.		# Replace used swabs into their original covering and discard them into the biohazard waste.

Jacqueline K. Grimm at 18:21, 4 June 2012

Jacqueline K. Grimm — Mon, 04 Jun 2012 18:21:13 GMT

←Older revision		Revision as of 18:21, 4 June 2012
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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]
		+	*Proline and SSM Supplements
		+	*Sterile Polyester-Tipped Swabs
		+	* 25 mL sterile plastic pipette
		+	* 37°C Water Bath
		+	* 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.
-	# 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~~.	+	# Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.
-	# ~~Incubate at~~ 35.5°C for a day.	+	# Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to (each) flask
-	# ~~Dilute~~ the ~~liquid culture~~.	+	# 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==		==Notes==

Alex Zorychta at 18:56, 31 May 2012

Alex Zorychta — Thu, 31 May 2012 18:56:51 GMT

←Older revision		Revision as of 18:56, 31 May 2012
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	*Pertussis frozen stock (keep on ice)		*Pertussis frozen stock (keep on ice)
-	*[http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Bordet_Gengou_%28BG%29_Agar_Plate_Preparation~~#Materials~~ BG Agar plates]	+	*[http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Bordet_Gengou_%28BG%29_Agar_Plate_Preparation BG Agar plates]
	*Sterilized sticks		*Sterilized sticks
	*50 mL flask		*50 mL flask
Line 15:		Line 15:
	'''Note: This procedure should be done in a hood to prevent aerosolization.'''		'''Note: This procedure should be done in a hood to prevent aerosolization.'''

-	# 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~~#Materials~~ 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.
-	# Using 10 mL of SSM media in a 50 mL flask, inoculate the liquid culture.	+	# 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.
	# Incubate at 35.5°C for a day.		# Incubate at 35.5°C for a day.
	# Dilute the liquid culture.		# Dilute the liquid culture.

Alex Zorychta: /* Procedure */

Alex Zorychta — Thu, 31 May 2012 18:56:16 GMT

Procedure

←Older revision		Revision as of 18:56, 31 May 2012
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	'''Note: This procedure should be done in a hood to prevent aerosolization.'''		'''Note: This procedure should be done in a hood to prevent aerosolization.'''

-	# Using a sterilized stick, streak the entire surface of the 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#Materials 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.
	# Using 10 mL of SSM media in a 50 mL flask, inoculate the liquid culture.		# Using 10 mL of SSM media in a 50 mL flask, inoculate the liquid culture.

Alex Zorychta at 18:55, 31 May 2012

Alex Zorychta — Thu, 31 May 2012 18:55:42 GMT

←Older revision		Revision as of 18:55, 31 May 2012
Line 6:		Line 6:

	*Pertussis frozen stock (keep on ice)		*Pertussis frozen stock (keep on ice)
-	*~~Sheep blood agar~~ plates	+	*[http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Bordet_Gengou_%28BG%29_Agar_Plate_Preparation#Materials BG Agar plates]
	*Sterilized sticks		*Sterilized sticks
	*50 mL flask		*50 mL flask

Jacqueline K. Grimm at 18:32, 31 May 2012

Jacqueline K. Grimm — Thu, 31 May 2012 18:32:41 GMT

←Older revision		Revision as of 18:32, 31 May 2012
Line 13:		Line 13:
	==Procedure==		==Procedure==

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

	# Using a sterilized stick, streak the entire surface of the plate. Discard the stick in the biohazard container.		# Using a sterilized stick, streak the entire surface of the 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.
-	# Using 10 mL of SSM media in a 50 mL flask, inoculate the culture.	+	# Using 10 mL of SSM media in a 50 mL flask, inoculate the liquid culture.
	# Incubate at 35.5°C for a day.		# Incubate at 35.5°C for a day.
	# Dilute the liquid culture.		# Dilute the liquid culture.

Jacqueline K. Grimm at 18:31, 31 May 2012

Jacqueline K. Grimm — Thu, 31 May 2012 18:31:31 GMT

←Older revision		Revision as of 18:31, 31 May 2012
Line 9:		Line 9:
	*Sterilized sticks		*Sterilized sticks
	*50 mL flask		*50 mL flask
-	*~~Liquid~~ media	+	*[http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/SSM_Media_Preparation SSM media]

	==Procedure==		==Procedure==
Line 17:		Line 17:
	# Using a sterilized stick, streak the entire surface of the plate. Discard the stick in the biohazard container.		# Using a sterilized stick, streak the entire surface of the 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.
-	# Using 10 mL of media in a 50 mL flask, inoculate the culture.	+	# Using 10 mL of SSM media in a 50 mL flask, inoculate the culture.
	# Incubate at 35.5°C for a day.		# Incubate at 35.5°C for a day.
	# Dilute the liquid culture.		# Dilute the liquid culture.

Jacqueline K. Grimm at 18:30, 31 May 2012

Jacqueline K. Grimm — Thu, 31 May 2012 18:30:28 GMT

←Older revision		Revision as of 18:30, 31 May 2012
Line 18:		Line 18:
	# 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.
	# Using 10 mL of media in a 50 mL flask, inoculate the culture.		# Using 10 mL of media in a 50 mL flask, inoculate the culture.
-	# Incubate at 35.5°C for a day	+	# Incubate at 35.5°C for a day.
	# Dilute the liquid culture.		# Dilute the liquid culture.

Jacqueline K. Grimm at 18:29, 31 May 2012

Jacqueline K. Grimm — Thu, 31 May 2012 18:29:12 GMT

←Older revision		Revision as of 18:29, 31 May 2012
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	==Overview==		==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	+	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==		==Materials==
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	*Sheep blood agar plates		*Sheep blood agar plates
	*Sterilized sticks		*Sterilized sticks
		+	*50 mL flask
		+	*Liquid media

	==Procedure==		==Procedure==
Line 15:		Line 17:
	# Using a sterilized stick, streak the entire surface of the plate. Discard the stick in the biohazard container.		# Using a sterilized stick, streak the entire surface of the 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.
-	# ~~Inoculate~~ the ~~liquid~~ culture. Incubate at 35.5°C.	+	# Using 10 mL of media in a 50 mL flask, inoculate the culture.
-	# ~~The next day, dilute~~ the liquid culture.	+	# Incubate at 35.5°C for a day
		+	# Dilute the liquid culture.

	==Notes==		==Notes==