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Organ Culture
Subprotocol 3
Integuments of Lepidopteran Insects (Lepidoptera)
Materials
Equipment - Dissecting microscope and dissecting tray (see chapter 1, section 3
"Tools")
- Scissors
- Forceps
- Needles
- Insect pins
- Petri dishes
- Pasteur pipettes
- Maximov slide
- Culture vessels
Solutions - Carlson's solution (see Appendix 1)
- Grace's medium (see Appendix 1)
- CSM-2F medium (see Appendix 1)
Animals - Larvae of the rice stem borer Chilo suppressalis or the tobacco horn
worm Manduca sexta
Procedure
Sterilization 1. Surface sterilize the animals by submersion in 70% ethanol for 10 min.
2. Wash the animals with sterilized distilled water.
Dissection l. Fix the animal, dorsal side up, on a dissecting tray using insect pins.
2. Excise the integument from the dorsal part of the animal.
3. Cut the integuments into pieces (3 x 5 mm) in Carlson's solution placed
in the depression of a Maximov slide.
4. Remove the fat bodies, trachea, muscles and oenocytes attached to
the integuments.
Culture 1. Transfer the integuments into culture vessels.
set-up 2. Add culture medium so as to soak the epidermis, leaving the outer
surface of the integuments in contact with air.
3. Keep the culture at 25°C.
Results
Agui et al. (1969, 1972) cultured the integuments of the rice stem borer
in CSM -2F medium or Grace's medium, and could keep the integuments
in good condition for more than 10 days. The cultured integuments could
molt in response to ecdysteroids added to the culture media (Fig. 2).
Organ Culture
Subprotocol 3
Integuments of Lepidopteran Insects (Lepidoptera)
Materials
Equipment - Dissecting microscope and dissecting tray (see chapter 1, section 3
"Tools")
- Scissors
- Forceps
- Needles
- Insect pins
- Petri dishes
- Pasteur pipettes
- Maximov slide
- Culture vessels
Solutions - Carlson's solution (see Appendix 1)
- Grace's medium (see Appendix 1)
- CSM-2F medium (see Appendix 1)
Animals - Larvae of the rice stem borer Chilo suppressalis or the tobacco horn
worm Manduca sexta
Procedure
Sterilization 1. Surface sterilize the animals by submersion in 70% ethanol for 10 min.
2. Wash the animals with sterilized distilled water.
Dissection l. Fix the animal, dorsal side up, on a dissecting tray using insect pins.
2. Excise the integument from the dorsal part of the animal.
3. Cut the integuments into pieces (3 x 5 mm) in Carlson's solution placed
in the depression of a Maximov slide.
4. Remove the fat bodies, trachea, muscles and oenocytes attached to
the integuments.
Culture 1. Transfer the integuments into culture vessels.
set-up 2. Add culture medium so as to soak the epidermis, leaving the outer
surface of the integuments in contact with air.
3. Keep the culture at 25°C.
Results
Agui et al. (1969, 1972) cultured the integuments of the rice stem borer
in CSM -2F medium or Grace's medium, and could keep the integuments
in good condition for more than 10 days. The cultured integuments could
molt in response to ecdysteroids added to the culture media (Fig. 2).