For plates, see MRS Agar Plates from MRS Broth
Base Recipe (per 1 L)
Use pre-made powder
We typically use pre-made powder for this - check bottle for amount (typically 55 g MRS powder / L)
MRS typically contains:
- 1.0% peptone
- 1.0% beef extract
- 0.4% yeast extract
- 2.0% glucose
- 0.5% sodium acetate trihydrate
- 0.1% polysorbate 80 (also known as Tween 80)
- 0.2% dipotassium hydrogen phosphate
- 0.2% triammonium citrate
- 0.02% magnesium sulfate heptahydrate
- 0.005% manganese sulfate tetrahydrate
- pH adjusted to 6.2 at 25 °C
Materials
- MRS medium (Criterion Lactobacilli MRS Broth, Hardy Diagnostics)
- Deionized water
- NaCl (analytical grade) — as required
- 100 mM cysteine-HCl solution
- 1 M NaOH (wear protective eyewear)
- Anaerobic culture tubes and bottles
- N₂ gas supply
For a larger batch, the protocol also uses 1300 mL water + 71.5 g MRS powder.
NaCl Concentrations (per 100 mL bottle)
| Concentration (M) | NaCl per 100 mL (g) |
|---|---|
| 0 | 0 |
| 0.05 | 0.292 |
| 0.1 | 0.584 |
| 0.2 | 1.17 |
| 0.25 | 1.46 |
| 0.3 | 1.75 |
| 0.325 | 1.899 |
| 0.375 | 2.19 |
| 0.4 | 2.33 |
| 0.45 | 2.63 |
| 0.5 | 2.92 |
Procedure
Step 1: Prepare MRS Base
- Combine water with MRS powder at the ratio above.
- Stir with heating until fully dissolved.
Step 2: Add NaCl
- Weigh NaCl in a 25 mL graduated cylinder.
- Use a 60 mL syringe with needle to add MRS (4 × 25 mL).
- Hold the needle against the cylinder wall to minimize bubbles.
- Gently stir after each addition.
- Verify volume remains at the 25 mL mark.
- Proceed in order of increasing salt concentration.
- Wash and dry the graduated cylinder between concentrations.
Step 3: Dispense and Sterilize
- Dispense into anaerobic bottles (100 mL each).
- Seal bottles with rubber stoppers and metal crimp caps.
- Flush with N₂ gas for 20 minutes.
- Autoclave on liquid cycle (121 °C, 15 min).
Step 4: Post-Autoclave Additions and pH Adjustment
- Add 0.3 mL of 100 mM cysteine-HCl per 100 mL.
- Measure the pH of each bottle.
- Adjust to pH 6.4–6.6 with sterile 1 M NaOH:
- Starting pH ~6.15 → add ~0.7 mL NaOH
- Starting pH ~6.0 → add ~0.9 mL NaOH
Storage
Use within 2 weeks; store at room temperature in the dark.