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Methods for Setting Up Primary Cultures Specific to Animal Groups
Collection
of eggs
Sterilization
Dissection
Culture
set-up
Usually leafhoppers and planthoppers lay their eggs in the tissue of their
host plants.
1. Dissect the plant tissue and remove single eggs or egg masses.
2. Accumulate the eggs freed of plant tissue in water placed in a centrifuge tube.
1. When sufficient eggs have been accumulated, decant the water, and
introduce 70% ethanol into the centrifuge tube.
2. Repeatedly turn the centrifuge tube for 1 min.
3. Stand the tube upright. When all the eggs sink, decant the ethanol.
4. Add sterile distilled water to the centrifuge tube.
5. Decant the water and add physiological solution.
6. Transfer the eggs into physiological solution placed in the depression
of a Maximov slide.
1. Cut the tip of an egg with a sharp knife and forceps (Fig. 1).
2. Squeeze the contents of the egg out by pressing the egg with forceps
under a dissecting microscope.
3. With needles, remove the yolk that wraps the embryos. If the embryos
are at stages later than blastokinesis, pull the yolk out from the embryos that have enclosed the yolk in their abdomen by dorsal closure.
4. Collect embryonic tissue, and transfer it into fresh physiological solution.
1. If the embryos are large, cut the embryos into several pieces.
2. Replace the physiological solution with culture medium.
3. Transfer embryo fragments into culture vessels with the medium.
Comments
After the first step of culture set-up, partial digestion of the tissue surface may be undertaken. In this case, replace the physiological solution
or culture medium with trypsin solution or dispase solution. After incubating the tissues in this solution for 10-15 min at room temperature,
replace the enzyme solution with culture medium, and transfer into a
culture vessel.
Results
Three types of cells usually migrate from explants of primary cultures of
embryonic tissues ofleafhoppers. These cells are epithelial cell-like cells,
which form a monolayer cell sheet, fibroblastic cells, which form cellular
networks, and free cells, which do not form any structure and actively
move around (Fig. 2; Mitsuhashi and Maramorosch, 1964; Mitsuhashi,
1965). Several continuous cell lines have been established from leafhop-
Methods for Setting Up Primary Cultures Specific to Animal Groups
Collection
of eggs
Sterilization
Dissection
Culture
set-up
Usually leafhoppers and planthoppers lay their eggs in the tissue of their
host plants.
1. Dissect the plant tissue and remove single eggs or egg masses.
2. Accumulate the eggs freed of plant tissue in water placed in a centrifuge tube.
1. When sufficient eggs have been accumulated, decant the water, and
introduce 70% ethanol into the centrifuge tube.
2. Repeatedly turn the centrifuge tube for 1 min.
3. Stand the tube upright. When all the eggs sink, decant the ethanol.
4. Add sterile distilled water to the centrifuge tube.
5. Decant the water and add physiological solution.
6. Transfer the eggs into physiological solution placed in the depression
of a Maximov slide.
1. Cut the tip of an egg with a sharp knife and forceps (Fig. 1).
2. Squeeze the contents of the egg out by pressing the egg with forceps
under a dissecting microscope.
3. With needles, remove the yolk that wraps the embryos. If the embryos
are at stages later than blastokinesis, pull the yolk out from the embryos that have enclosed the yolk in their abdomen by dorsal closure.
4. Collect embryonic tissue, and transfer it into fresh physiological solution.
1. If the embryos are large, cut the embryos into several pieces.
2. Replace the physiological solution with culture medium.
3. Transfer embryo fragments into culture vessels with the medium.
Comments
After the first step of culture set-up, partial digestion of the tissue surface may be undertaken. In this case, replace the physiological solution
or culture medium with trypsin solution or dispase solution. After incubating the tissues in this solution for 10-15 min at room temperature,
replace the enzyme solution with culture medium, and transfer into a
culture vessel.
Results
Three types of cells usually migrate from explants of primary cultures of
embryonic tissues ofleafhoppers. These cells are epithelial cell-like cells,
which form a monolayer cell sheet, fibroblastic cells, which form cellular
networks, and free cells, which do not form any structure and actively
move around (Fig. 2; Mitsuhashi and Maramorosch, 1964; Mitsuhashi,
1965). Several continuous cell lines have been established from leafhop-