26
General Methods
5. Make the solution up to 1,000 ml by the addition of distilled water. If
adding sterile FBS after sterilization of the medium, the final volume
of the solution should be 1,000 ml minus the volume of FBS to be
added.
6. Adjust the pH to 6.3-6.5 with concentrated KOH solution (higher than
10 N), and 1 N HCi. Without adjustment, the pH of the solution is usually approximately 6.5.
Subprotocol 2
Indirect Method of Preparing Media Consisting of a Few
Chemicals
For frequent preparation of the medium, the preparation of stock solution is recommended, even if the medium consists of only a few chemicals. Following is the preparation of the MM medium (Mitsuhashi and
Maramorosch, 1964) as an example of such media. (For the composition
of the medium, see Appendix 1.) Stock solutions consist of lO-fold concentrated NaHC0 3 and 10-fold concentrated other inorganic salts. If all
inorganic salts are mixed at a 10-fold concentration of that in the medium, precipitates will appear.
Materials
Equipment and chemicals are the same as for subprotocol1.
Solutions - Stock solution A
70 g NaCl, 2 g NaHzP04, 2 g KCl, 2 g CaClz·2Hp, and 19 MgClz·6HzO
per 1,000 ml distilled water.
- Stock solution B
1.2 g NaHC0 3 per 1,000 ml distilled water.
Procedure
1. In order to make 1,000 ml MM medium, mix 100 ml of both stock
solutions and approximately 750 ml distilled water in a 1,000-ml beaker.
2. Stir the solution with a magnetic stirrer.
3. Dissolve 6.5 g lactalbumin hydrolysate, 5.0 g TC-yeastolate, and 4.0 g
o-glucose in the solution.
4. Add FBS if necessary.
5. Make the solution up to 1,000 ml by adding distilled water.
6. Adjust the pH to 6.3-6.5 with concentrated KOH solution and 1 N
HCl, if necessary.
General Methods
5. Make the solution up to 1,000 ml by the addition of distilled water. If
adding sterile FBS after sterilization of the medium, the final volume
of the solution should be 1,000 ml minus the volume of FBS to be
added.
6. Adjust the pH to 6.3-6.5 with concentrated KOH solution (higher than
10 N), and 1 N HCi. Without adjustment, the pH of the solution is usually approximately 6.5.
Subprotocol 2
Indirect Method of Preparing Media Consisting of a Few
Chemicals
For frequent preparation of the medium, the preparation of stock solution is recommended, even if the medium consists of only a few chemicals. Following is the preparation of the MM medium (Mitsuhashi and
Maramorosch, 1964) as an example of such media. (For the composition
of the medium, see Appendix 1.) Stock solutions consist of lO-fold concentrated NaHC0 3 and 10-fold concentrated other inorganic salts. If all
inorganic salts are mixed at a 10-fold concentration of that in the medium, precipitates will appear.
Materials
Equipment and chemicals are the same as for subprotocol1.
Solutions - Stock solution A
70 g NaCl, 2 g NaHzP04, 2 g KCl, 2 g CaClz·2Hp, and 19 MgClz·6HzO
per 1,000 ml distilled water.
- Stock solution B
1.2 g NaHC0 3 per 1,000 ml distilled water.
Procedure
1. In order to make 1,000 ml MM medium, mix 100 ml of both stock
solutions and approximately 750 ml distilled water in a 1,000-ml beaker.
2. Stir the solution with a magnetic stirrer.
3. Dissolve 6.5 g lactalbumin hydrolysate, 5.0 g TC-yeastolate, and 4.0 g
o-glucose in the solution.
4. Add FBS if necessary.
5. Make the solution up to 1,000 ml by adding distilled water.
6. Adjust the pH to 6.3-6.5 with concentrated KOH solution and 1 N
HCl, if necessary.