30
General Methods
Procedure
1. Water-insoluble components are prepared as follows. Dissolve 10 mg
cod liver oil, 4.5 mg cholesterol, 2 mg (X-tocopherol acetate, and 25 mg
Tween-80 in 1.0 ml ethyl alcohol. Sterilize the mixture by passing it
through a membrane filter (0.2-j.lm pore size). Add 10 ml filter-sterilized 10% Pluronic polyol F-68 in water to this mixture by slow, stepwise
addition at 37°C while vortexing. The mixture will initially turn cloudy,
but will clear when the addition of Pluronic polyol is completed.
2. Add all the emulsified lipid supplement and 40 ml ultrafiltered TCyeastolate solution to 1,000 ml IPL-41 medium.
Comments
An alternative method for incorporating water-insoluble substances,such
as lipids, into culture media is as follows.
l. Prepare 2% (v/v) Tween-80 solution in ethyl alcohol.
2. Dissolve water-insoluble substances in 2% Tween-80 in ethyl alcohol
at a concentration 1000-fold that of the final concentration.
3. Pass the mixture through a 0.2-j.lm membrane filter (cellulose nitrate).
4. Add 1.0 ml filtered alcoholic solution to 1,000 ml sterilized culture
media. This makes a very fine emulsion and no turbidity is seen. The
water-insoluble substances do not separate from the water, even after
long preservation.
Subprotocol 5
Sterilization of Culture Media
Prepared media should be sterilized, and tested for their sterility before
use. There are several methods for sterilization. Elimination of microorganisms by passing the media through a filter is common. There are vari0us types of filters available. Several decades ago, the Seitz-type filter,
consisting of a pressed asbestos filter, was used widely. This filter liberates some toxic substances and changes the pH of the medium passed
through it, and has rarely been used after the appearance of the membrane filter.
Materials
Equipment - Disposable filter sets or sterilized filter and filter holder
- Autoclave
- A small pump (181Jmin) that can be used to both apply pressure and
create a vacuum
- Peristaltic pump
- Syringe with appropriate capacity
- Medium bottle
General Methods
Procedure
1. Water-insoluble components are prepared as follows. Dissolve 10 mg
cod liver oil, 4.5 mg cholesterol, 2 mg (X-tocopherol acetate, and 25 mg
Tween-80 in 1.0 ml ethyl alcohol. Sterilize the mixture by passing it
through a membrane filter (0.2-j.lm pore size). Add 10 ml filter-sterilized 10% Pluronic polyol F-68 in water to this mixture by slow, stepwise
addition at 37°C while vortexing. The mixture will initially turn cloudy,
but will clear when the addition of Pluronic polyol is completed.
2. Add all the emulsified lipid supplement and 40 ml ultrafiltered TCyeastolate solution to 1,000 ml IPL-41 medium.
Comments
An alternative method for incorporating water-insoluble substances,such
as lipids, into culture media is as follows.
l. Prepare 2% (v/v) Tween-80 solution in ethyl alcohol.
2. Dissolve water-insoluble substances in 2% Tween-80 in ethyl alcohol
at a concentration 1000-fold that of the final concentration.
3. Pass the mixture through a 0.2-j.lm membrane filter (cellulose nitrate).
4. Add 1.0 ml filtered alcoholic solution to 1,000 ml sterilized culture
media. This makes a very fine emulsion and no turbidity is seen. The
water-insoluble substances do not separate from the water, even after
long preservation.
Subprotocol 5
Sterilization of Culture Media
Prepared media should be sterilized, and tested for their sterility before
use. There are several methods for sterilization. Elimination of microorganisms by passing the media through a filter is common. There are vari0us types of filters available. Several decades ago, the Seitz-type filter,
consisting of a pressed asbestos filter, was used widely. This filter liberates some toxic substances and changes the pH of the medium passed
through it, and has rarely been used after the appearance of the membrane filter.
Materials
Equipment - Disposable filter sets or sterilized filter and filter holder
- Autoclave
- A small pump (181Jmin) that can be used to both apply pressure and
create a vacuum
- Peristaltic pump
- Syringe with appropriate capacity
- Medium bottle