3 Preparation of Media
27
Comments
TC-yeastolate may be replaced with an extract of yeast sold by several
manufacturers. However, some yeast extracts may not be replaceable. It
is better to check the compatibility of the yeast extract before use. Some
yeast extracts may not dissolve completely in water.
When all chemicals are dissolved, the pH of the solution is usually
approximately 6.5. Because insect cells are generally tolerant to a wide
range of pH (Mitsuhashi, 1989), the solution may be used as the culture
medium without adjustment of the pH.
Osmolarity is not usually adjusted, because insect cells are tolerant
of a wide range of osmotic pressures (Mitsuhashi, 1989). If adjustment
of osmolarity is necessary, this can be achieved by changing the amount
of NaCI in the solution.
Stock solutions of inorganic salts can be stored in a refrigerator for
several months.
The stock solutions A and B of the MM medium are the same as those
of Carlson's solution (see Appendix 1), and may be called stock solutions
A and B of Carlson's solution, respectively.
Subprotocol 3
Preparation of Complex Media
Many insect cell culture media consist of mixtures of chemically defined
and natural substances, such as sera, insect hemolymph, and so on. The
well-known Grace's medium (GMA; Grace, 1962) is an example of this
type of medium. The method for preparation of GMA is shown below.
Other media with a similar composition will be prepared in a similar
manner. The composition of the GMA is given in Appendix 1.
Materials
The equipment necessary is the same as given for subprotocol1.
Equipment
- GMA salt stock solution A (lO-fold concentrated)
Solutions
Stir 900 ml distilled water on a magnetic stirrer.
Dissolve 3.5 g NaHC03 and 27.8 g MgS04·7HzO in the water.
Make the solution up to 1,000 ml by adding distilled water.
Store the solution at 5°C (can be kept for several months).
- GMA salt stock solution B (lO-fold concentrated)
Stir 900 ml distilled water on a magnetic stirrer.
Dissolve 11.4 g NaHzP04·2HzO, 22.4 g KCI, 22.8 g MgCl z ·6H z O and
10.0 g CaCl z in the water.
Make the solution up to 1,000 ml by adding distilled water.
Store the solution at 5°C (can be kept for several months).
- GMA stock solution OSA (organic acids, sugars and amino acids) (5fold concentrated)
27
Comments
TC-yeastolate may be replaced with an extract of yeast sold by several
manufacturers. However, some yeast extracts may not be replaceable. It
is better to check the compatibility of the yeast extract before use. Some
yeast extracts may not dissolve completely in water.
When all chemicals are dissolved, the pH of the solution is usually
approximately 6.5. Because insect cells are generally tolerant to a wide
range of pH (Mitsuhashi, 1989), the solution may be used as the culture
medium without adjustment of the pH.
Osmolarity is not usually adjusted, because insect cells are tolerant
of a wide range of osmotic pressures (Mitsuhashi, 1989). If adjustment
of osmolarity is necessary, this can be achieved by changing the amount
of NaCI in the solution.
Stock solutions of inorganic salts can be stored in a refrigerator for
several months.
The stock solutions A and B of the MM medium are the same as those
of Carlson's solution (see Appendix 1), and may be called stock solutions
A and B of Carlson's solution, respectively.
Subprotocol 3
Preparation of Complex Media
Many insect cell culture media consist of mixtures of chemically defined
and natural substances, such as sera, insect hemolymph, and so on. The
well-known Grace's medium (GMA; Grace, 1962) is an example of this
type of medium. The method for preparation of GMA is shown below.
Other media with a similar composition will be prepared in a similar
manner. The composition of the GMA is given in Appendix 1.
Materials
The equipment necessary is the same as given for subprotocol1.
Equipment
- GMA salt stock solution A (lO-fold concentrated)
Solutions
Stir 900 ml distilled water on a magnetic stirrer.
Dissolve 3.5 g NaHC03 and 27.8 g MgS04·7HzO in the water.
Make the solution up to 1,000 ml by adding distilled water.
Store the solution at 5°C (can be kept for several months).
- GMA salt stock solution B (lO-fold concentrated)
Stir 900 ml distilled water on a magnetic stirrer.
Dissolve 11.4 g NaHzP04·2HzO, 22.4 g KCI, 22.8 g MgCl z ·6H z O and
10.0 g CaCl z in the water.
Make the solution up to 1,000 ml by adding distilled water.
Store the solution at 5°C (can be kept for several months).
- GMA stock solution OSA (organic acids, sugars and amino acids) (5fold concentrated)