M2 medium

NaCl, 5.53 g

KCl, 0.36 g

CaCl₂•2H₂O, 0.25 g

KH₂PO₄, 0.16 g

MgSO₄•7H₂O, 0.29 g

NaHCO₃, 0.35 g

HEPES, 4.97 g

Sodium lactate, 2.6 g (or 4.35 g of 60% syrup)

Sodium pyruvate, 0.04 g

Glucose, 1 g

Bovine serum albumin (BSA), 4 g

Penicillin G (potassium salt), 0.06 g

Streptomycin sulfate, 0.05 g

Phenol red, 0.01 g

H₂O, 2x glass-distilled, to 1 liter

1. Dissolve HEPES in 50-100 ml of 2x distilled H₂O.

2. Adjust the pH to 7.4 with 0.2 M NaOH.

3. Weigh the penicillin and streptomycin and dissolve them in a small volume of 2x distilled H₂O.

4. Weigh the CaCl₂ and dissolve it in 2x distilled H₂O.

5. Weigh the remaining components (except BSA and lactate) into a designated 1-liter volumetric flask and add approximately 500 ml of 2x distilled H₂O. Allow to dissolve.

6. Add the penicillin, streptomycin, HEPES, and CaCl₂ to a volumetric flask.

7. Weigh the lactate syrup into a designated 10-ml beaker and add it to the volumetric flask. Rinse the beaker several times with 2x distilled H₂O, add the washings to the volumetric flask, and adjust the volume to 1 liter.

8. Sprinkle BSA on top of the medium and allow it to dissolve slowly. Mix gently. Do not shake the medium, because it will froth and denature the protein.

9. If necessary, readjust the pH of the medium to 7.2-7.4 with 0.2 M NaOH.

10. Filter through a Millipore filter with postive pressure to reduce foaming. Discard the first few millitliters and aliquot into sterile containers.

11. Store at 4°C for up to 2 weeks. The osmolarity should be 285-287 mosmoles.