M2GSC Medium
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.
