264
Organ Culture
Animals - Silkworm eggs (chilled to terminate diapause; elongated germ band
stage)
Miscellaneous - Parafilm (American National Can)
- Gauze
Sterilization
)echorionation
Culture
set-up
Hatching
Rearing
larvae
- Glass wool
- Mulberry leaves
- Artificial diet (i.e., Silkmate 2S; Nihon Nosan Kogyo)
Procedure
The following procedure is based on the methods of Takami et al. (1966)
and Kiguchi et al. (1990).
1. Wash the eggs in 5% formalin for 5 min.
2. Rinse the eggs in distilled water.
3. Surface sterilize the eggs by submersion in 94% ethanol (two changes
of 2-3 min each).
4. Wash the eggs in sterile distilled water.
5. Transfer the eggs into physiological solution.
1. Carefully cut the chorion using a sharpened needle, care being taken
to avoid injury to the serosal membrane.
2. Insert a needle between the chorion and the serosa, and peel the
chorion away.
1. Transfer each dechorionated egg into a drop of Grace's culture medium placed on the inner surface of the lid of a petri dish.
2. Turn the lid of the petri dish over the dish to make a hanging drop,
care being taken not to fuse the drops. Several hanging drops may be
made on a single petri dish lid.
3. Seal the petri dish with stretched Parafilm.
4. Maintain the culture at 25°C.
1. Rinse the embryos twice with sterilized distilled water approximately
12-24 h after the embryos finish swallowing serosa.
2. Transfer the embryos to a petri dish in which they are placed on a fine
gauze patch with a small amount glass wool underneath.
3. Add a few drops of water to the glass wool to maintain moderate moisture of 70-80% relative humidity. Excess humidity should be avoided.
1. After 24 h, supply the larvae with chopped mulberry leaves or artificial diet for silkworms.
2. Follow the regular rearing method for silkworms until the hatched
larvae become adults.
Organ Culture
Animals - Silkworm eggs (chilled to terminate diapause; elongated germ band
stage)
Miscellaneous - Parafilm (American National Can)
- Gauze
Sterilization
)echorionation
Culture
set-up
Hatching
Rearing
larvae
- Glass wool
- Mulberry leaves
- Artificial diet (i.e., Silkmate 2S; Nihon Nosan Kogyo)
Procedure
The following procedure is based on the methods of Takami et al. (1966)
and Kiguchi et al. (1990).
1. Wash the eggs in 5% formalin for 5 min.
2. Rinse the eggs in distilled water.
3. Surface sterilize the eggs by submersion in 94% ethanol (two changes
of 2-3 min each).
4. Wash the eggs in sterile distilled water.
5. Transfer the eggs into physiological solution.
1. Carefully cut the chorion using a sharpened needle, care being taken
to avoid injury to the serosal membrane.
2. Insert a needle between the chorion and the serosa, and peel the
chorion away.
1. Transfer each dechorionated egg into a drop of Grace's culture medium placed on the inner surface of the lid of a petri dish.
2. Turn the lid of the petri dish over the dish to make a hanging drop,
care being taken not to fuse the drops. Several hanging drops may be
made on a single petri dish lid.
3. Seal the petri dish with stretched Parafilm.
4. Maintain the culture at 25°C.
1. Rinse the embryos twice with sterilized distilled water approximately
12-24 h after the embryos finish swallowing serosa.
2. Transfer the embryos to a petri dish in which they are placed on a fine
gauze patch with a small amount glass wool underneath.
3. Add a few drops of water to the glass wool to maintain moderate moisture of 70-80% relative humidity. Excess humidity should be avoided.
1. After 24 h, supply the larvae with chopped mulberry leaves or artificial diet for silkworms.
2. Follow the regular rearing method for silkworms until the hatched
larvae become adults.