Cell Culture Media Preparation from Powder

Save these protocols on how to mix media and reagents from powders or concentrates.

In addition to the materials listed below, you will also need:

• Media or reagent of choice (see protocols below for catalog numbers)
• Digital pH meter
• 0.2-μm membrane filtration system, such as Nalgene Rapid-Flow Filters
• PPE as designated by your lab's rules

While powdered media takes additional time to prepare, cost savings and extended shelf life make it a good choice for many labs. Media concentrates are also a great alternative to pre-diluted formulas because they are easier to store and handle compared to powdered media, while offering convenience and flexibility for precise dilution to desired concentrations.

To make cell culture media from powder, refer to the generic protocol below. Precise quantities and volumes of the pH adjustors for your chosen cell culture media can be found on Table 1.

The following protocol is a generic powdered cell culture media preparation protocol. To prepare DMEM from powder—or any additional media formulations, tailor this protocol with the values provided in Table 1.

1. To a mixing container that is as close to the final volume as possible, add 950 mL of distilled water.
2. Add cell culture media powder 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 to medium in either powder or 7.5% solution form (see Table 1 for quantity/volume).
5. Adjust the pH to 0.2 and 0.3 units below the desired final working pH (see Table 1 for quantity/volume) by slowly adding, with stirring, 1 N sodium hydroxide or 1 N hydrochloric acid. The pH may rise 0.1 to 0.3 units upon filtration.
6. Adjust the final volume with distilled water.
7. Process the medium immediately into sterile containers by membrane filtration with a 0.2-μm filter using a positive-pressure system.

The following is a general cell culture media preparation protocol for 1X solutions from common 10X liquid media concentrates. Please 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 (see Table 2 for concentrations).
3. Adjust the pH as necessary with 1 N hydrochloric acid or 1 N sodium hydroxide (see Table 2 for 1X pH values).
4. Adjust the final volume with distilled water.
5. Dispense the solution into sterile containers. Cap the bottles tightly with sterile closures and store at the recommended temperature (see product label).

* pH range varies depending on the pH of water used in dilution.

Powdered reagents and liquid concentrates, such as DPBS (Dulbecco’s Phosphate Buffered Saline), are a good option for labs that want to save money and storage space because they are typically less expensive than traditional liquid reagents and take up less room. Powdered reagents also have a longer shelf life, and allow personnel to prepare only the amount needed, minimizing the risk of reagent waste due to expiration.

The following protocol prepares a 1x concentration of powdered Dulbecco’s Phosphate Buffered Saline with calcium and magnesium.

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 the inside of package to remove all traces of powder.
4. If calcium chloride is included separately with the medium, add 0.1 g calcium chloride/L of medium.
5. Adjust the final volume with distilled water. Process the medium immediately into sterile containers by membrane filtration with a 0.2-μm filter using a positive pressure system.

To prepare an acceptable, final 1X solution from 10X concentrates of cell culture reagents, perform the following procedure under aseptic conditions. Use distilled water and 7.5% sodium bicarbonate solution for use in this protocol. 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. See Table 3 for concentrations.
3. Adjust the pH as necessary with 1 N HCl or 1 N NaOH.
4. Adjust the final volume with distilled water.
5. Dispense the solution into sterile containers. Cap the bottles tightly with sterile closures and store at the recommended temperature (see product label).

* pH range varies depending on the pH of water used in dilution.

Media sterilization reduces the risk of contamination by eliminating potential contaminants through autoclaving or manual filtration. In an autoclave, the media is subjected to high pressure and temperature, after which it must cool to room temperature before any supplements are added to prevent the degradation of heat-sensitive components (such as vitamins and amino acids). When media cannot be sterilized in an autoclave, such as when the media already contains heat-sensitive components, filtration is recommended. In the protocols above, a 0.2 μm filter system is used. This size is considered sterilizing grade and removes all bacteria and larger microbes.

Media supplementation involves adding essential nutrients and growth factors to the sterilized media to help provide an adequate environment for the cells. Supplements can introduce the proper pH balance, osmotic pressure, and essential nutrients to basal mediums. Common supplements include 7.5% sodium bicarbonate solution, L-glutamine, antibiotics, and serum.

Following proper storage and preparation procedures can help ensure success in routine cell culture.

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