Media Preparation from Powder and Concentrates

1. To a mixing container that is as close to the final volume as possible, add 950 mL of distilled water.
2. Add powdered medium to room temperature (15°C to 30°C) water with gentle stirring.
3. Rinse inside of package to remove all traces of powder.
4. When completely dissolved, adjust the pH to 4.1 to 4.2 with 1 N HCl.
5. Dispense 95 mL of the liquid into 100-mL bottles. Do not cap tightly.
6. Autoclave the medium for at least 15 minutes at 121°C on a slow exhaust cycle. However, due to equipment variability, we recommend that you qualify your autoclave sterilization cycle.
7. Allow the medium to cool to room temperature.
8. Aseptically add 3 mL of 7.5% NaHCO₃ solution to each bottle. Mix well.
9. Aseptically add L-glutamine and antibiotic solutions, serum, or other supplements, as desired. Mix well.
10. Aseptically adjust the medium to final pH 7.2 to 7.4 by slowly adding, with stirring, 1 N NaOH or 1 N HCl, if necessary. 

Procedure B: Prepare Powdered Leibovitz’s L-15 Medium (1X) (Cat. no. 41300*)

1. To a mixing container that is as close to the final volume as possible, add 950 mL of distilled water.
2. Add powdered medium to room temperature (15°C to 30°C) water with gentle stirring. Do not heat the water.
3. Rinse the inside of package to remove all traces of powder.
4. Adjust pH to 7.6 by slowly adding, with stirring, 1 N NaOH or 1 N HCl.
5. Adjust the final volume with distilled water.
6. Process the medium immediately into sterile containers by membrane filtration with a 0.2-μm filter using a positive pressure system. 

Procedure C: Prepare Powdered Iscove’s Modified Dulbecco’s Medium (1X) (Cat. nos. 42200*, 12200*)

1. To a mixing container that is as close to the final volume as possible, add 950 mL of distilled water.
2. Add powdered medium to room temperature (15°C to 30°C) water with gentle stirring. Do not heat the water.
3. Rinse the inside of package to remove all traces of powder.
4. Add 3.024 g of sodium bicarbonate/L of medium.
5. Adjust the final volume with distilled water and stir until dissolved.
6. Do not adjust the pH. Keep the container closed until the medium is filtered.
7. Process the medium immediately into sterile containers by membrane filtration with a 0.2-μm filter using a positive pressure system.

Procedure D: Prepare Powdered Medium

1. To a mixing container that is as close to the final volume as possible, add 950 mL of distilled water.
2. Add powdered medium to room temperature (15°C to 30°C) water with gentle stirring. Do not heat the water.
3. Rinse the inside of package to remove all traces of powder.
4. Add sodium bicarbonate (see Tables 1, 2 for quantity) to medium.
5. Adjust the pH to 0.2 and 0.3 units below the desired final working pH by slowly adding, with stirring, 1 N NaOH or
6. 1 N HCl. The pH may rise 0.1 to 0.3 units upon filtration.
7. Adjust the final volume with distilled water.
8. Process the medium immediately into sterile containers by membrane filtration with a 0.2-μm filter using a positive-pressure system.

Table 1: Powdered Media Preparation (Procedure D)