M2GSC Medium
From Global Methane Hub Wikipedia
Description
M2GSC medium is based on a modified M2 medium [1] supplemented with glucose, soluble starch, cellobiose, and rumen fluid in an anaerobic broth.
Recipe
M2GSC medium composition
| Bacto casitone | 10 g |
| Bacto yeast extract | 2.5 g |
| NaHCO3 | 4 g |
| Glucose | 2 g |
| Soluble starch | 2 g |
| Cellobiose | 2 g |
| Rumen fluid (spun) | 300 ml |
| Mineral solution 1 | 150 ml |
| Mineral solution 2 | 150 ml |
| Resazurin (0.1%) | 1 ml |
| Cysteine HCl | 1 g |
| Add distilled H2O to 1 L | |
Mineral solution 1
| K2HPO4 | 3 g |
| Add distilled H2O to 1 L | |
Mineral solution 2
| NaCl | 6 g |
| KH2PO4 | 3 g |
| (NH4)2SO4 | 6 g |
| CaCl2.2H2O | 0.6 g |
| MgSO4.7H2O | 0.6 g |
| Add distilled H2O to 1 L | |
Method
- Weigh all ingredients as listed into a Pyrex conical glass flask, with the exception of cysteine.
- Add mineral solutions, rumen fluid, resazurin and water.
- Microwave flask on high until rapidly boiling.
- Remove flask from microwave and bubble with a gentle stream of CO2 until cool. Slowly add cysteine and then flush headspace only as bubble can change pH.
- Dispense M2GSC medium in 9.5 ml volumes into CO2 flushed Hungate tubes.
- Butyl rubber stoppers should be placed in the neck of tubes while withdrawing the CO2 sparger and the tubes sealed with screw caps.
- Autoclave tubes at 121ºC for 15 min. on same day as prepared.
Notes
Note: for rumen fluid
- Thaw once-spun (10,000 g for 20 min) rumen fluid and bubble with N2 (100%) for 20 minutes.
- Autoclave under N2 at 121⁰C for 20 minutes in a high pressure Schott bottle to inactivate viruses, etc. The anoxic conditions are to limit the formation of unwanted oxidation products. Allow to cool.
- Freeze at -80⁰C for 2 hours or -20⁰C overnight.
- Spin at 10,000 G for 20 minutes.
Sources
[1] Hobson PN. (1969). Rumen bacteria. In: Methods in Microbiology, vol 3B. Academic Press, pp. 133–149.
