22 Arthropoda Other than Insecta
275
Subprotocol 6
Ovaries of Cirripeds (Crustacea)
Materials
- Aerated aquariums
Equipment
- Dissecting microscope and dissecting tray
- CO 2 incubator
- Scissors
- Forceps
- Pasteur pipettes
- Culture flasks (25 cm 2 ; Falcon; Becton-Dickinson)
- Seawater
Solutions
- Barnacle organ culture medium (see Appendix 1)
- Adults of the acorn barnacle Balanus amphitrite (10-15 mm basal di- Animals
ameter)
- Newly hatched Artemia nauplii
Procedure
1. Brush the animals clean and keep them in aerated aquariums at approximately 23°C and seawater of 3% salinity for at least 3 weeks. During this period, feed the animals with newly hatched Artemia nauplii
every 2nd day. Change the seawater weekly.
1. Carefully clean the animals and place them in a laminar flow hood for
16 to 20 h with the basal membrane pointing toward a UV lamp (30 W)
at a distance of 15 cm.
2. Dip the basal one-third of the animal into 5% sodium hypochlorite
for 2 s.
3. Rinse the animals in absolute ethanol for 5 s and wipe with sterile
cotton.
Preparation
of barnacles
Sterilization
1. Mount the animal upside down under a dissecting microscope.
Dissection
2. Remove the calcareous basis carefully.
3. Cut 1-2-mm 3 pieces of ovarioles with mantle tissue and cement glands
(cf. chapter. 12, Fig. 14).
1. Place four to six explants in a culture flask.
Culture
2. Allow the explants to adhere to the bottom surface of the flask for 30- set-up
60 s.
3. Add 2 ml culture medium to the flask.
4. Gas the flasks immediately with a mixture of 5% CO 2 in air, and cap
and incubate the flasks at 26°C in darkness.
275
Subprotocol 6
Ovaries of Cirripeds (Crustacea)
Materials
- Aerated aquariums
Equipment
- Dissecting microscope and dissecting tray
- CO 2 incubator
- Scissors
- Forceps
- Pasteur pipettes
- Culture flasks (25 cm 2 ; Falcon; Becton-Dickinson)
- Seawater
Solutions
- Barnacle organ culture medium (see Appendix 1)
- Adults of the acorn barnacle Balanus amphitrite (10-15 mm basal di- Animals
ameter)
- Newly hatched Artemia nauplii
Procedure
1. Brush the animals clean and keep them in aerated aquariums at approximately 23°C and seawater of 3% salinity for at least 3 weeks. During this period, feed the animals with newly hatched Artemia nauplii
every 2nd day. Change the seawater weekly.
1. Carefully clean the animals and place them in a laminar flow hood for
16 to 20 h with the basal membrane pointing toward a UV lamp (30 W)
at a distance of 15 cm.
2. Dip the basal one-third of the animal into 5% sodium hypochlorite
for 2 s.
3. Rinse the animals in absolute ethanol for 5 s and wipe with sterile
cotton.
Preparation
of barnacles
Sterilization
1. Mount the animal upside down under a dissecting microscope.
Dissection
2. Remove the calcareous basis carefully.
3. Cut 1-2-mm 3 pieces of ovarioles with mantle tissue and cement glands
(cf. chapter. 12, Fig. 14).
1. Place four to six explants in a culture flask.
Culture
2. Allow the explants to adhere to the bottom surface of the flask for 30- set-up
60 s.
3. Add 2 ml culture medium to the flask.
4. Gas the flasks immediately with a mixture of 5% CO 2 in air, and cap
and incubate the flasks at 26°C in darkness.