254
Organ Culture
Chemicals - Vaseline (silicone)
Animals - Third instar larvae of D. melanogaster
Procedure
Sterilization 1. Wash the larvae with running water to remove any adhering food.
2. Sterilize the larvae by submersion in 70% ethanol for 5 min.
3. Rinse the larvae with sterile distilled water.
Dissection 1. Transfer one larva into a drop of culture medium placed on a slide
glass.
2. Dissect out the salivary gland.
Culture 1. Transfer the gland into culture medium placed in the depression of a
set-up
Maximov slide, and remove as many fat bodies attached to the gland
as possible without damaging the gland.
2. Transfer the gland into a 50-I.ll drop medium in a depression slide,
and cover with a 35-mm 2 cover glass.
3. Seal the cover glasses with vaseline (silicone), and culture at 20°-22°C.
Results
Using this method,Ashburner (1972) maintained the salivary glands from
D. melanogaster in modified Grace's medium (Grace's medium (see Appendix 1) without hemolymph and antibiotics, and diluted 5 : 1 v/v with
10% ethanol in distilled water) for at least 48h, and observed puffing
activity of the chromosomes following stimulation with 20hydroxyecdysone. Later, using the same method, Farkas and Suicikovci
(1998) confirmed that the change in the fine structure of cultured glands
reflected progression of the puffing pattern, and these changes paralleled those seen in vivo. Using modified Shields and Sang's medium (see
Appendix 1), Poels (1972) cultured salivary glands from Drosophila hydei,
and Sinha and Lakhotia (1980) cultured the salivary glands of Drosophila nasuta; similar results were obtained in all studies. Using dipteran
insects other than Drosophila, Firling (1980) obtained similar results in
the culture of salivary glands from Chironomus tentans.
Subprotocol 12
Silk Glands of Lepidopteran Insects (Lepidoptera)
Materials
Equipment - Dissecting microscope and dissecting tray (see chapter 1, section 3
"Tools")
- Scissors
Organ Culture
Chemicals - Vaseline (silicone)
Animals - Third instar larvae of D. melanogaster
Procedure
Sterilization 1. Wash the larvae with running water to remove any adhering food.
2. Sterilize the larvae by submersion in 70% ethanol for 5 min.
3. Rinse the larvae with sterile distilled water.
Dissection 1. Transfer one larva into a drop of culture medium placed on a slide
glass.
2. Dissect out the salivary gland.
Culture 1. Transfer the gland into culture medium placed in the depression of a
set-up
Maximov slide, and remove as many fat bodies attached to the gland
as possible without damaging the gland.
2. Transfer the gland into a 50-I.ll drop medium in a depression slide,
and cover with a 35-mm 2 cover glass.
3. Seal the cover glasses with vaseline (silicone), and culture at 20°-22°C.
Results
Using this method,Ashburner (1972) maintained the salivary glands from
D. melanogaster in modified Grace's medium (Grace's medium (see Appendix 1) without hemolymph and antibiotics, and diluted 5 : 1 v/v with
10% ethanol in distilled water) for at least 48h, and observed puffing
activity of the chromosomes following stimulation with 20hydroxyecdysone. Later, using the same method, Farkas and Suicikovci
(1998) confirmed that the change in the fine structure of cultured glands
reflected progression of the puffing pattern, and these changes paralleled those seen in vivo. Using modified Shields and Sang's medium (see
Appendix 1), Poels (1972) cultured salivary glands from Drosophila hydei,
and Sinha and Lakhotia (1980) cultured the salivary glands of Drosophila nasuta; similar results were obtained in all studies. Using dipteran
insects other than Drosophila, Firling (1980) obtained similar results in
the culture of salivary glands from Chironomus tentans.
Subprotocol 12
Silk Glands of Lepidopteran Insects (Lepidoptera)
Materials
Equipment - Dissecting microscope and dissecting tray (see chapter 1, section 3
"Tools")
- Scissors