M10 Medium
Description
M10 is a medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria [1].
Recipe
| Component | Percentage in mediuma |
|---|---|
| Glucose | 0.05 |
| Cellobiose | 0.05 |
| Soluble Starch | 0.05 |
| Minerals (v/v)b | 3.75 |
| Na2S·9H2O | 0.025 |
| Cystein·HCl·H2O | 0.025 |
| Resazurin | 0.001 |
| Na2CO3 | 0.4 |
| Trypticase | 0.2 |
| Yeast extract | 0.05 |
| Volatile fatty acids (v/v)c | 0.31 |
| Hemin | 0.0001 |
a Weight/ volume unless otherwise indicated.
b Except for K2HPO4, which was prepared separately, the minerals were prepared as a common stock solution. The final mineral concentrations in the medium were as follows: KH2PO4, 1.3 X 10-3 M; NaCl, 7.6 X 10-4 M; (NH)2SO4, 3.4 X 10-3 M; K2HPO4, 1.7 X 10-3 M; Ca(Cl)2, 4.1 X 10-4 M; MgSO4c·7H20, 3.8 X 10-4 M.
c The volatile fatty acid mixture used was the following: acetic, 17 ml (2.9 X 10-2 M); propionic, 6ml (8.0 X 10-3 M); butyric, 4 ml (4.3 X 10-3 M); isobutyric, 1 ml (1.1 X 10-3 M); n-valeric, isovaleric, and DL-α-methylbutyric, 1 ml each (9 X 10-4 M). The figures in parentheses indicate the final concentration of each acid when 0.31 ml of mixture was placed in a final medium volume of 100 ml.
The final pH of media is 6.7 to 6.8.
Directions
Suspend 38 g of RCM in 1 liter of distilled water. Boil to dissolve solids completely, aliquot, and sterilize by autoclaving at 121 °C for 15 minutes.
Notes
Store bought RCM contains 0.5 g/L agar to mitigate oxygen permeability into the medium. The addition of this agar can have a compounding effect on the formation of extracellular polymeric substances (EPS) in some species like Sharpea azubuensis and Megasphaera elsdeni. This EPS generation makes the formation of cell pellets difficult and can affect the procedure of making competent cells. It's recommended to make your RCM "in house" without agar to avoid this complication.
Sources
- ThermoFisher CM0149 (Used by Guss Lab)
