168
Methods for Setting Up Primary Cultures Specific to Animal Groups
Digestive system
Fig. 7. Internal structure of a slug. (Figure courtesy of Dr. E. Furuta, Institute of
Comparative Immunology, Saitama, Japan)
• • Materials
Equipment - CO2 incubator
- Surgical knife
- Forceps
- Plastic petri dish (no. 3002; Falcon, Becton-Dickinson)
- Pasteur pipette
Solutions - Salt solution containing antibiotics (SA; see Appendix 1)
- SH-7 medium (see Appendix 1)
Miscellaneous - Filter paper
Animals - Land slug (1. frukstorferi) reared on a piece of sweet potato on wet
filter paper
• • Procedure
Cleaning 1. Wipe the mucus off the skin of slug with tissue paper.
2. Anesthetize the slug by placing it on ice.
3. Wipe the body surface of the slug with sterile paper.
Dissection 1. Cut out the ventral foot and dorsal skin (Fig. 7).
2. Keep the excised tissues in saline containing antibiotics for 30 min at
room temperature.
Dissociation 1. Mince the tissue into small fragments.
2. Incubate the minced tissue fragments in the saline containing antibiotics for 10 min.
Culture 1. Transfer 10 fragments into a plastic petri dish, and add 2 ml culture
set-up
medium (SH-7).
2. Culture at 25°C in air containing 5% CO 2 •
Methods for Setting Up Primary Cultures Specific to Animal Groups
Digestive system
Fig. 7. Internal structure of a slug. (Figure courtesy of Dr. E. Furuta, Institute of
Comparative Immunology, Saitama, Japan)
• • Materials
Equipment - CO2 incubator
- Surgical knife
- Forceps
- Plastic petri dish (no. 3002; Falcon, Becton-Dickinson)
- Pasteur pipette
Solutions - Salt solution containing antibiotics (SA; see Appendix 1)
- SH-7 medium (see Appendix 1)
Miscellaneous - Filter paper
Animals - Land slug (1. frukstorferi) reared on a piece of sweet potato on wet
filter paper
• • Procedure
Cleaning 1. Wipe the mucus off the skin of slug with tissue paper.
2. Anesthetize the slug by placing it on ice.
3. Wipe the body surface of the slug with sterile paper.
Dissection 1. Cut out the ventral foot and dorsal skin (Fig. 7).
2. Keep the excised tissues in saline containing antibiotics for 30 min at
room temperature.
Dissociation 1. Mince the tissue into small fragments.
2. Incubate the minced tissue fragments in the saline containing antibiotics for 10 min.
Culture 1. Transfer 10 fragments into a plastic petri dish, and add 2 ml culture
set-up
medium (SH-7).
2. Culture at 25°C in air containing 5% CO 2 •