IGEM:Virginia 2012/Protocols/SSM Media Preparation

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==Overview==
 +
This is the procedure for making SSM medium (Stainer and Scholte Modified Medium), which is used for growing ''Bordetella pertussis''.
==Materials==
==Materials==
 +
===Basal Medium===
{| border="0" cellpadding="0"
{| border="0" cellpadding="0"
|-
|-
Line 12: Line 15:
| KH<sub>2</sub>PO<sub>4</sub> || 0.50g || 1.00g || 2.00g || 2.50g
| KH<sub>2</sub>PO<sub>4</sub> || 0.50g || 1.00g || 2.00g || 2.50g
|-
|-
-
| KCl || 0.20g || 0.40g || 0.80g || 1.00g
+
| KCl ''*200 mg/mL'' || 0.20g || 0.40g || 0.80g || 1.00g
|-
|-
-
| MgCl<sub>2</sub> 6H<sub>2</sub>O || 100mg || 200mg || 400mg || 500mg
+
| MgCl<sub>2</sub> 6H<sub>2</sub>O   ''*100mg/mL (0.5 mM)'' || 100mg || 200mg || 400mg || 500mg
|-
|-
-
| CaCl<sub>2</sub>  2H<sub>2</sub>O || 20mg || 40mg || 80mg || 100mg
+
| CaCl<sub>2</sub>  2H<sub>2</sub>O   ''*20 mg/mL'' || 20mg || 40mg || 80mg || 100mg
 +
|-
 +
| Bring to Volume with deionized water to** || 980mL || 1960mL || 3920mL || 4900mL
|}
|}
 +
 +
*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: 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: 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===
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 +
*2 mL conc. HCL
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*6 mL distilled H<sub>2</sub>O
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*0.80g L-cystine
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*A Vortex
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*200 mL deionized H<sub>2</sub>O
 +
* 0.2g FeSO<sub>4</sub>
 +
* 0.4g of ascorbic acid
 +
* 80mg of nicotinic acid (niacin)
 +
* 2.0g of glutathione; reduced form
 +
 +
===Proline Supplement===
 +
*200 mL H<sub>2</sub>O
 +
* 4.8g L-proline
==Procedure==
==Procedure==
-
* Add 2.9g of NaCl to a 1 L bottle.
+
 
-
* Add in 25 mL of 7.5 pH Tris-Cl
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* 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.
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* Bring this to a volume of 500 mL by adding distilled water.
+
 
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* Autoclave (large liquid).
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===Basal Medium===
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* '''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.
 +
* 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).
 +
*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:
 +
** 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.
 +
 
 +
===Proline Supplement===
 +
 
 +
*Mix 200 mL of water with 4.8g of L-proline.
 +
*Filter sterilize, and make 10 mL aliquots. Store at -20°C.
==Notes==
==Notes==

Current revision

Contents

Overview

This is the procedure for making SSM medium (Stainer and Scholte Modified Medium), which is used for growing Bordetella pertussis.

Materials

Basal Medium

BASAL MEDIUM to make 1 Liter 2 Liters 4 Liters 5 Liters
Glutamic acid, monosodium salt, 1H2O 10.72g 21.44g 42.88g 53.60g
Trisma Base 1.525g 3.05g 6.10g 7.63g
NaCl 2.50g 5.00g 10.00g 12.50g
KH2PO4 0.50g 1.00g 2.00g 2.50g
KCl *200 mg/mL 0.20g 0.40g 0.80g 1.00g
MgCl2 6H2O *100mg/mL (0.5 mM) 100mg 200mg 400mg 500mg
CaCl2 2H2O *20 mg/mL 20mg 40mg 80mg 100mg
Bring to Volume with deionized water to** 980mL 1960mL 3920mL 4900mL
  • 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: 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: 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 H2O
  • 0.80g L-cystine
  • A Vortex
  • 200 mL deionized H2O
  • 0.2g FeSO4
  • 0.4g of ascorbic acid
  • 80mg of nicotinic acid (niacin)
  • 2.0g of glutathione; reduced form

Proline Supplement

  • 200 mL H2O
  • 4.8g L-proline

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

  • 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.
  • 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).
*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 H2O.
  • Add 0.80g L-cystine.
  • Vortex to dissolve.
  • Bring to volume 200 mL with deionized H2O. May need to add a little more HCl at this point if cystine comes out of solution.
  • Then add the following:
    • 0.2g FeSO4
    • 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.

Proline Supplement

  • Mix 200 mL of water with 4.8g of L-proline.
  • Filter sterilize, and make 10 mL aliquots. Store at -20°C.

Notes

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


References

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