IGEM:Virginia 2012/Protocols/SSM Media Preparation
From OpenWetWare
Jump to navigationJump to search
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
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.