Chapter 3
Preparation of Media
Subprotocol 1
Direct Method of Preparing Media Consisting of a Few
Chemicals
Media consisting of a few chemicals may be prepared by dissolving these
chemicals in solution one after the other. Following is the preparation of
MM medium (Mitsuhashi and Maramorosch, 1964) as an example of such
media. (For the composition of the medium, see Appendix 1.)
• • Materials
PROTOCOL
- Chemical balance (capacity 200 g, readability 0.1 mg)
Equipment
- Magnetic stirrer (one with an installed heating system is preferable)
- pH meter (readability 0.01)
The use of tissue culture-grade chemicals is recommended.
Chemicals
Lactalbumin hydrolysate: either acid hydrolyzed or enzyme hydrolyzed lactalbumin can be used. Although the contents vary among products from different manufacturers or even among different lots from the
same manufacturer, the differences do not usually create any problems.
Any brand products can be used.
TC-yeastolate (Difco Laboratories): this is a water-soluble extract of
yeast. Difco Laboratories is the sole producer.
FBS: any brand and any lot can be used. However, it is recommended
that the compatibility of the FBS be checked before use by incorporating
it into cell cultures. For primary culture, two or more brands may be
used for comparison.
Procedure
In order to prepare 1,000 ml medium, the following process is recommended.
1. Place approximately 800 ml distilled water (double-distilled water, distilled and deionized water, or water passed through a Milli-Q system
is preferable) into a 1,000-ml beaker.
2. Stir the water with a magnetic stirrer.
3. Weigh each ingredient and dissolve in water, one after the other. The
order of dissolution makes no difference.
4. Add FBS if necessary (sterile FBS can be added after sterilization of
the mixed solution).
Preparation of Media
Subprotocol 1
Direct Method of Preparing Media Consisting of a Few
Chemicals
Media consisting of a few chemicals may be prepared by dissolving these
chemicals in solution one after the other. Following is the preparation of
MM medium (Mitsuhashi and Maramorosch, 1964) as an example of such
media. (For the composition of the medium, see Appendix 1.)
• • Materials
PROTOCOL
- Chemical balance (capacity 200 g, readability 0.1 mg)
Equipment
- Magnetic stirrer (one with an installed heating system is preferable)
- pH meter (readability 0.01)
The use of tissue culture-grade chemicals is recommended.
Chemicals
Lactalbumin hydrolysate: either acid hydrolyzed or enzyme hydrolyzed lactalbumin can be used. Although the contents vary among products from different manufacturers or even among different lots from the
same manufacturer, the differences do not usually create any problems.
Any brand products can be used.
TC-yeastolate (Difco Laboratories): this is a water-soluble extract of
yeast. Difco Laboratories is the sole producer.
FBS: any brand and any lot can be used. However, it is recommended
that the compatibility of the FBS be checked before use by incorporating
it into cell cultures. For primary culture, two or more brands may be
used for comparison.
Procedure
In order to prepare 1,000 ml medium, the following process is recommended.
1. Place approximately 800 ml distilled water (double-distilled water, distilled and deionized water, or water passed through a Milli-Q system
is preferable) into a 1,000-ml beaker.
2. Stir the water with a magnetic stirrer.
3. Weigh each ingredient and dissolve in water, one after the other. The
order of dissolution makes no difference.
4. Add FBS if necessary (sterile FBS can be added after sterilization of
the mixed solution).