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Methods for Setting Up Primary Cultures Specific to Animal Groups
- Culture vessel
- Pasteur pipette
Solutions - Physiological solution
- Culture medium
- 70% Ethanol
Animals - Eggs of dipteran insects
Procedure
Sterilization 1. Collect as many eggs as possible.
2. Wash eggs in city water.
3. Sterilize the surface of the eggs with 70% ethanol for 3-5 min.
4. Wash the surface-sterilized eggs in sterile distilled water.
Culture 1. Transfer the eggs into a glass homogenizer with a small amount of a
set-up
physiological solution or culture medium.
2. Crush the eggs with several gentle strokes of a pestle (the degree of
crushing is determined empirically).
3. Wash the crushed eggs out of the homogenizer with physiological solution or culture medium onto a stainless steel mesh to remove chorions and uncrushed eggs.
4. Centrifuge the cell suspension passed through the stainless steel mesh
at 150 g for 5 min.
5. Suspend the cells in the culture medium.
6. Transfer the cell suspension to a culture vessel.
Alternative Procedure
Dissection 1. Follow the instructions for sterilization as given for the preceding procedure.
2. Transfer the eggs into a physiological solution placed on a Maximov
slide.
3. Cut the tips of the eggs with a knife or with needles.
4. Squeeze the embryos and yolk from the eggs.
5. Remove the yolk that envelops the embryos.
6. Wash the embryos by transferring them into fresh physiological solution.
Culture 1. Transfer the embryos into culture medium.
set-up 2. Disintegrate the embryos by vigorous pipetting.
3. Transfer the suspension of embryonic fragments into a culture vessel.
Methods for Setting Up Primary Cultures Specific to Animal Groups
- Culture vessel
- Pasteur pipette
Solutions - Physiological solution
- Culture medium
- 70% Ethanol
Animals - Eggs of dipteran insects
Procedure
Sterilization 1. Collect as many eggs as possible.
2. Wash eggs in city water.
3. Sterilize the surface of the eggs with 70% ethanol for 3-5 min.
4. Wash the surface-sterilized eggs in sterile distilled water.
Culture 1. Transfer the eggs into a glass homogenizer with a small amount of a
set-up
physiological solution or culture medium.
2. Crush the eggs with several gentle strokes of a pestle (the degree of
crushing is determined empirically).
3. Wash the crushed eggs out of the homogenizer with physiological solution or culture medium onto a stainless steel mesh to remove chorions and uncrushed eggs.
4. Centrifuge the cell suspension passed through the stainless steel mesh
at 150 g for 5 min.
5. Suspend the cells in the culture medium.
6. Transfer the cell suspension to a culture vessel.
Alternative Procedure
Dissection 1. Follow the instructions for sterilization as given for the preceding procedure.
2. Transfer the eggs into a physiological solution placed on a Maximov
slide.
3. Cut the tips of the eggs with a knife or with needles.
4. Squeeze the embryos and yolk from the eggs.
5. Remove the yolk that envelops the embryos.
6. Wash the embryos by transferring them into fresh physiological solution.
Culture 1. Transfer the embryos into culture medium.
set-up 2. Disintegrate the embryos by vigorous pipetting.
3. Transfer the suspension of embryonic fragments into a culture vessel.