Media Preparation from Powder and Concentrates

Liquid Media:   Preparing 1X Solutions from 10X Concentrates

To prepare an acceptable, final single-strength solution, perform the following procedure under aseptic conditions.  We recommend Distilled Water (Cat. No. 15230) and 7.5% Sodium Bicarbonate Solution (Cat. No. 25080) for use in this protocol.

Use sterile solutions of 1 N sodium hydroxide or hydrochloric acid prepared by membrane filtration using appropriate filters.  Also note, because we adjust the pH of all 10X media and 10X balanced salt solution concentrates for solubility, you may need to adjust the pH after the dilution and to add sodium bicarbonate, as appropriate.

1. Aseptically dilute 100 ml of 10X concentrate with approximately 850 ml of distilled water.
2. Aseptically add the correct amount of 7.5% sodium bicarbonate solution.
3. Adjust the pH as necessary with 1 N hydrochloric acid or 1 N sodium hydroxide.
4. QS to volume with distilled water.
5. Dispense the solution into sterile containers.  Cap the bottles tightly with sterile closures and store them at the recommended temperature

Procedure 1:  Preparing Powdered Media

1. To a mixing container that is as close to the final volume as possible, add 5% less distilled water than the desired total volume of medium.
2. Add powdered medium to room temperature (20°C to 30°C) water with gentle stirring.  Do not heat water.
3. Rinse inside of package to remove all traces of powder.
4. Add NaHCO₃ to 1 L of medium.
5. Dilute the medium to the desired volume with distilled water and stir until dissolved.  Do not over-mix.
6. Adjust the pH to between 0.2 and 0.3 below the desired final working pH by slowly adding, with stirring, either 1 N NaOH or 1 N HCl.  The pH usually will rise 0.1 to 0.3 units upon filtration.  Keep the container closed until the medium is filtered.
7. Process the medium immediately into sterile containers by membrane filtration using a 0.2-μm filter.  We recommend using a positive-pressure system.

Procedure 2:  Preparing Autoclavable Powdered Media

1. To a mixing container that is as close to the final volume as possible, add 5% less distilled water than the desired total volume of medium.
2. Add powdered medium to room temperature (20°C to 30°C) water with gentle stirring.  Do not heat water.
3. Rinse inside of package to remove all traces of powder.
4. When completely dissolved, adjust 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. It is recommended that the medium be autoclaved for at least 15 min. 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 liquid 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.