18 Platyhelminthes
215
Materials
- Incubator
Equipment
- Centrifuge and centrifuge tubes
- Petri dishes
- Razor blades
- Culture vessel (culture dish, 7 cm diameter)
- Pasteur pipettes
- TTP medium (see Appendix 1)
Solutions
- Germ-free freshwater Planarians (rear planarians in autoclaved rear- Animals
ing water. Wash the animals in antibiotic solution (200 j.1g/ ml streptomycin and 200 IU/ml penicillin in autoclaved rearing water) every 2
or 3 days)
- Sterilized filter paper
Miscellaneous
Procedure
l. Place a planarian in an open petri dish and irradiate it with a UV ray Sterilization
at a distance of30 cm above the animal for 5 min. Murray (1927,1931)
recommends reducing the water level so that there is sufficient water
only to keep the animal immersed, and continuous shaking of the dish
when the irradiation is made.
l. Stimulate the animal by touching it with sterilized filter paper in or- Demucusing
der to remove as much secreted mucus as possible.
1. Cut the animal as small as possible using two crossed razor blades. It Dissection
is recommended that the pharynx be removed so as to avoid parasitic
contamination.
2. Transfer the tissue fragments into a centrifuge tube containing Ca 2 +free water.
3. Leave the mixture to stand for 5 min at room temperature.
4. Apply vigorous pipetting with a Pasteur pipette.
5. Leave the mixture to stand for 1 min, keeping the tube in an upright
position.
6. Remove the supernatant, and centrifuge the supernatant at 150 g for
5 min.
1. Suspend the cell pellet in 4 ml TTP medium.
2. Transfer the cell suspension into a culture vessel.
3. Culture at 17°-18°C.
Culture
set-up
215
Materials
- Incubator
Equipment
- Centrifuge and centrifuge tubes
- Petri dishes
- Razor blades
- Culture vessel (culture dish, 7 cm diameter)
- Pasteur pipettes
- TTP medium (see Appendix 1)
Solutions
- Germ-free freshwater Planarians (rear planarians in autoclaved rear- Animals
ing water. Wash the animals in antibiotic solution (200 j.1g/ ml streptomycin and 200 IU/ml penicillin in autoclaved rearing water) every 2
or 3 days)
- Sterilized filter paper
Miscellaneous
Procedure
l. Place a planarian in an open petri dish and irradiate it with a UV ray Sterilization
at a distance of30 cm above the animal for 5 min. Murray (1927,1931)
recommends reducing the water level so that there is sufficient water
only to keep the animal immersed, and continuous shaking of the dish
when the irradiation is made.
l. Stimulate the animal by touching it with sterilized filter paper in or- Demucusing
der to remove as much secreted mucus as possible.
1. Cut the animal as small as possible using two crossed razor blades. It Dissection
is recommended that the pharynx be removed so as to avoid parasitic
contamination.
2. Transfer the tissue fragments into a centrifuge tube containing Ca 2 +free water.
3. Leave the mixture to stand for 5 min at room temperature.
4. Apply vigorous pipetting with a Pasteur pipette.
5. Leave the mixture to stand for 1 min, keeping the tube in an upright
position.
6. Remove the supernatant, and centrifuge the supernatant at 150 g for
5 min.
1. Suspend the cell pellet in 4 ml TTP medium.
2. Transfer the cell suspension into a culture vessel.
3. Culture at 17°-18°C.
Culture
set-up