IGEM:Virginia 2012/Protocols/SSM Media Preparation - Revision history

Jacqueline K. Grimm at 17:43, 1 June 2012

Jacqueline K. Grimm — Fri, 01 Jun 2012 17:43:09 GMT

←Older revision		Revision as of 17:43, 1 June 2012
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	==Procedure==		==Procedure==

		+	* Put a 1 L bottle on a magnetic mixer, and mix while adding all components. The last three are available as stock solutions-- add 1 mL of each.

	===Basal Medium===		===Basal Medium===

Jacqueline K. Grimm at 17:41, 1 June 2012

Jacqueline K. Grimm — Fri, 01 Jun 2012 17:41:00 GMT

←Older revision		Revision as of 17:41, 1 June 2012
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	==Overview==		==Overview==
-	This is the procedure for making SSM medium (Stainer and Scholte Modified Medium), which ~~can be~~ used for growing ''Bordetella pertussis''.	+	This is the procedure for making SSM medium (Stainer and Scholte Modified Medium), which is used for growing ''Bordetella pertussis''.
	==Materials==		==Materials==
	===Basal Medium===		===Basal Medium===
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	==Procedure==		==Procedure==
		+
		+
	===Basal Medium===		===Basal Medium===
-	* Adjust to pH 7.60 with concentrated HCl (about 0.5-0.7 mL/L).	+	* '''Adjust to pH 7.60''' with concentrated HCl (about 0.5-0.7 mL/L). Rinse the bulb of the pH meter with DI H2O and check the pH meter using one of the standards. Rinse the bulb again and hold it in the medium while slowly adding HCl using a pipette. Press "Read" to get the new pH. When the medium is at pH 7.60, rinse off the pH meter again. Make sure to adjust the pH, or the salt will come out of solution.
-	* Autoclave for 20 min. ''(May store at this stage at 4°C as "SSM Incomplete.")'''	+	* Add the rest of the DI H2O.
		+	* Autoclave for 25 min (select "Liquid 25", then enter, okay, and start). ''(May store at this stage at 4°C as "SSM Incomplete.")'''
	* After cooling and before use, add 10 mL/L of both proline and SSM supplements (see below).		* After cooling and before use, add 10 mL/L of both proline and SSM supplements (see below).
	*Note: for SSM-S, also add 1 mL/L SSM-S supplement. May store basal medium at 4°C for short periods of time and at -20°C for somewhat longer.		*Note: for SSM-S, also add 1 mL/L SSM-S supplement. May store basal medium at 4°C for short periods of time and at -20°C for somewhat longer.

Jacqueline K. Grimm at 16:44, 1 June 2012

Jacqueline K. Grimm — Fri, 01 Jun 2012 16:44:07 GMT

←Older revision		Revision as of 16:44, 1 June 2012
Line 1:		Line 1:
		+	==Overview==
		+	This is the procedure for making SSM medium (Stainer and Scholte Modified Medium), which can be used for growing ''Bordetella pertussis''.
	==Materials==		==Materials==
	===Basal Medium===		===Basal Medium===

Alex Zorychta: /* Procedure */

Alex Zorychta — Thu, 31 May 2012 18:53:06 GMT

Procedure

←Older revision		Revision as of 18:53, 31 May 2012
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	** 80mg of nicotinic acid (niacin)		** 80mg of nicotinic acid (niacin)
	** 2.0g of glutathione; reduced form		** 2.0g of glutathione; reduced form
		+	*Filter sterilize, and make 10 mL aliquots. Store at -20°C.
		+
		+	===Proline Supplement===
		+
		+	*Mix 200 mL of water with 4.8g of L-proline.
	*Filter sterilize, and make 10 mL aliquots. Store at -20°C.		*Filter sterilize, and make 10 mL aliquots. Store at -20°C.

Alex Zorychta: /* Materials */

Alex Zorychta — Thu, 31 May 2012 18:52:19 GMT

Materials

←Older revision		Revision as of 18:52, 31 May 2012
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	==Materials==		==Materials==
		+	===Basal Medium===
	{\| border="0" cellpadding="0"		{\| border="0" cellpadding="0"
	\|-		\|-
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	* 80mg of nicotinic acid (niacin)		* 80mg of nicotinic acid (niacin)
	* 2.0g of glutathione; reduced form		* 2.0g of glutathione; reduced form
		+
		+	===Proline Supplement===
		+	*200 mL H<sub>2</sub>O
		+	* 4.8g L-proline

	==Procedure==		==Procedure==

Alex Zorychta: /* SSM Supplement */

Alex Zorychta — Thu, 31 May 2012 18:50:50 GMT

SSM Supplement

←Older revision		Revision as of 18:50, 31 May 2012
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	* Bring to volume 200 mL with deionized H<sub>2</sub>O. ''May need to add a little more HCl at this point if cystine comes out of solution.''		* Bring to volume 200 mL with deionized H<sub>2</sub>O. ''May need to add a little more HCl at this point if cystine comes out of solution.''
	* Then add the following:		* Then add the following:
-	**	+	** 0.2g FeSO<sub>4</sub>
		+	** 0.4g of ascorbic acid
		+	** 80mg of nicotinic acid (niacin)
		+	** 2.0g of glutathione; reduced form
		+	*Filter sterilize, and make 10 mL aliquots. Store at -20°C.

	==Notes==		==Notes==

Alex Zorychta: /* Procedure */

Alex Zorychta — Thu, 31 May 2012 18:49:55 GMT

Procedure

←Older revision		Revision as of 18:49, 31 May 2012
Line 40:		Line 40:

	==Procedure==		==Procedure==
		+	===Basal Medium===
	* Adjust to pH 7.60 with concentrated HCl (about 0.5-0.7 mL/L).		* Adjust to pH 7.60 with concentrated HCl (about 0.5-0.7 mL/L).
	* Autoclave for 20 min. ''(May store at this stage at 4°C as "SSM Incomplete.")'''		* Autoclave for 20 min. ''(May store at this stage at 4°C as "SSM Incomplete.")'''
	* After cooling and before use, add 10 mL/L of both proline and SSM supplements (see below).		* After cooling and before use, add 10 mL/L of both proline and SSM supplements (see below).
	*Note: for SSM-S, also add 1 mL/L SSM-S supplement. May store basal medium at 4°C for short periods of time and at -20°C for somewhat longer.		*Note: for SSM-S, also add 1 mL/L SSM-S supplement. May store basal medium at 4°C for short periods of time and at -20°C for somewhat longer.
		+
		+	===SSM Supplement===
		+	*Mix 2 mL conc. HCl and 6 mL distilled H<sub>2</sub>O.
		+	* Add 0.80g L-cystine.
		+	* Vortex to dissolve.
		+	* Bring to volume 200 mL with deionized H<sub>2</sub>O. ''May need to add a little more HCl at this point if cystine comes out of solution.''
		+	* Then add the following:
		+	**

	==Notes==		==Notes==

Alex Zorychta: /* SSM Supplement */

Alex Zorychta — Thu, 31 May 2012 18:48:07 GMT

SSM Supplement

←Older revision		Revision as of 18:48, 31 May 2012
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	*A Vortex		*A Vortex
	*200 mL deionized H<sub>2</sub>O		*200 mL deionized H<sub>2</sub>O
-	~~{\| border="~~0~~" cellpadding="0"~~	+	* 0.2g FeSO<sub>4</sub>
-	\|-	+	* 0.4g of ascorbic acid
-	~~\| width="50 pt" \|~~ FeSO<sub>4</sub> ~~\|\| width="50 pt" \|~~ 0.2g	+	* 80mg of nicotinic acid (niacin)
-	\|-	+	* 2.0g of glutathione; reduced form
-	\| ascorbic acid ~~\|\| 0.4g~~	+
-	\|-	+
-	\| nicotinic acid (niacin) ~~\|\| 80 mg~~	+
-	\|-	+
-	\| glutathione; reduced form ~~\|\| 2.0g~~	+
-	\|}	+

	==Procedure==		==Procedure==

Alex Zorychta: /* Materials */

Alex Zorychta — Thu, 31 May 2012 18:47:02 GMT

Materials

←Older revision		Revision as of 18:47, 31 May 2012
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	\| CaCl<sub>2</sub> 2H<sub>2</sub>O ''*20 mg/mL'' \|\| 20mg \|\| 40mg \|\| 80mg \|\| 100mg		\| CaCl<sub>2</sub> 2H<sub>2</sub>O ''*20 mg/mL'' \|\| 20mg \|\| 40mg \|\| 80mg \|\| 100mg
	\|-		\|-
-	\| ~~Q.S.~~ with deionized water to** \|\| 980mL \|\| 1960mL \|\| 3920mL \|\| 4900mL	+	\| Bring to Volume with deionized water to** \|\| 980mL \|\| 1960mL \|\| 3920mL \|\| 4900mL
	\|}		\|}

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	*Note: if pouring blood plates, bring volume up to 100 mL/L '''LESS''' than above.		*Note: if pouring blood plates, bring volume up to 100 mL/L '''LESS''' than above.
	*Note: it is convenient to make up the last three salts in a concentrated stock solution, such that you can use 1mL/liter of SSM.		*Note: it is convenient to make up the last three salts in a concentrated stock solution, such that you can use 1mL/liter of SSM.
		+
		+	===SSM Supplement===
		+
		+	*2 mL conc. HCL
		+	*6 mL distilled H<sub>2</sub>O
		+	*0.80g L-cystine
		+	*A Vortex
		+	*200 mL deionized H<sub>2</sub>O
		+	{\| border="0" cellpadding="0"
		+	\|-
		+	\| width="50 pt" \| FeSO<sub>4</sub> \|\| width="50 pt" \| 0.2g
		+	\|-
		+	\| ascorbic acid \|\| 0.4g
		+	\|-
		+	\| nicotinic acid (niacin) \|\| 80 mg
		+	\|-
		+	\| glutathione; reduced form \|\| 2.0g
		+	\|}

	==Procedure==		==Procedure==

Alex Zorychta: /* Materials */

Alex Zorychta — Thu, 31 May 2012 18:35:41 GMT

Materials

←Older revision		Revision as of 18:35, 31 May 2012
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	Note: Make stock solutions and add this amount.		Note: Make stock solutions and add this amount.
	Note: *These Volumes are to allow for 20 mL/L worth of supplements to be added later.		Note: *These Volumes are to allow for 20 mL/L worth of supplements to be added later.
-	*Note: For SSM-S, add 2.0 g/L of 2-ketoglutarate and pyruvate before pH'ing.	+	*Note: For SSM-S, add 2.0 g/L of 2-ketoglutarate and pyruvate before pH'ing.
-	*Note: if pouring blood plates, bring volume up to 100 mL/L '''LESS''' than above.	+	*Note: if pouring blood plates, bring volume up to 100 mL/L '''LESS''' than above.
-	*Note: it is convenient to make up the last three salts in a concentrated stock solution, such that you can use 1mL/liter of SSM.	+	*Note: it is convenient to make up the last three salts in a concentrated stock solution, such that you can use 1mL/liter of SSM.

	==Procedure==		==Procedure==