Solution A: Dissolve 19.84 g anhydrous magnesium chloride (MgCl2) or an equivalent of hydrated magnesium chloride and 46.24 g anhydrous calcium chloride (CaCl2) or an equivalent of hydrated calcium chloride in distilled water and dilute to 1,000 mL. Sterilize in an autoclave. Store the solution at 2°C to 8°C for no longer than one month.
Solution B: Dissolve 35.02 g sodium hydrogencarbonate (NaHCO3) in distilled water and dilute to 1,000 mL. Sterilize by membrane filtration. Store the solution at 2°C to 8°C for no longer than one week.
Hard water: For the preparation of 1 L, place 600 mL to 700 mL distilled water in a 1,000-mL volumetric flask and add 6.0 mL of Solution A, then 8.0 mL of Solution B. Mix and dilute to 1,000 mL with water. The pH of the hard water should be 7.0 + 0.2. If necessary, adjust the pH by using a solution of approximately 40 g/L (about 1 mol/L) of sodium hydroxide (NaOH) or approximately 36.5 g/L (about 1 mol/L) of hydrochloric acid (HCl). The hard water should be freshly prepared under aseptic conditions and used within 12 hr.