M16 medium

NaCl, 5.53 g

KCl, 0.36 g

CaCl₂•2H₂O, 0.25 g

KH₂PO₄, 0.16 g

MgSO₄•7H₂O, 0.29 g

NaHCO₃, 2.10 g

Sodium lactate, 2.61 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. Weigh the penicillin and streptomycin and dissolve them in a small volume of 2x distilled H₂O.

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

3. 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.

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

5. 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.

6. Determine the pH of the medium; it should be 7.2-7.4. If it is less, it can be adjusted using 0.2 M NaOH. Bring the volume to 1 liter. Gas the medium by bubbling with 5% CO₂, 95% air for 5 minutes to adjust the pH (this step can be omitted if the pH is 7.4).

7. 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.

8. Filter the medium through a Millipore filter into small sterile containers, gas the air space with 5% CO₂, 95% air, and cap tightly to maintain a pH of 7.2-7.4. Use positive pressure to filter to reduce foaming. Discard the first few milliliters that come through the filter.

9. Store at 4°C for up to 2 weeks. The osmolarity should be 288-292 mosmoles.