16 Annelida
201
Procedure
1. Keep the worms in moistened filter paper for 2-3 days before the ex- Sterilization
periment in order to empty their gut.
2. Wash the worms in running water.
3. Surface sterilize the worms with 0.1 % mercuric chloride and wipe them
with iodine solution.
1. Fix the worms on a sterilized thick cork sheet.
Dissociation
2. Cut and remove necessary tissues with sterilized tools.
3. Keep the cut fragments in sterile Ringer's solution.
1. Cut the fragments into the desired size.
Culture
2. Transfer the fragments into a petri dish, and rinse 15 times with ster- set-up
ilized Ringer's solution.
3. Transfer the fragments, with one drop of culture medium, into a culture vessel.
Results
According to Krontowsky and Rumianzew (1922), cells migrated from
cultured blood vessels with chloragogic tissue, and formed a cell sheet
around the explants. By the 2nd day of culture, the cell sheet had increased in size. On the 3rd day, cell growth continued and some differentiation of the cells was observed. The migrated cells did not exhibit mitosis. The cell culture that showed relatively active cell migration deteriorated after 7-9 days, while the cell culture that showed rather poor
cell migration survived for 3 weeks. Using a similar technique, Battaglia
and Davoli (1997) cultured minced earthworm tissue in Hansen's S-301
medium (see Appendix I). Battaglia and Davoli (1997) were able to maintain the cells for longer than 12 months. The cultured cells consisted of
spherical cysts with vibratile cilia, which showed rapid rotating and peristaltic movements, round nonadherent cells, which often clustered in
small clumps, and cells that adhered to the surface of the culture flask,
forming incomplete monolayers.
References
Battaglia, M. and Davoli, C. (1997) Long-term tissue culture of the earthworm Eisenia foetida
(Annelida, Oligochaeta). In: Maramorosch, K. and Mitsuhashi, J. (eds) Invertebrate Cell Culture: Novel Directions and Biotechnology Applications. Science Publishers, Enfield, pp 261268.
Dietzel, I.D., Drapeau, P. and Nicholls J.G. (1986) Voltage dependence of 5-hydroxytryptamine release at a synapse between identified leech neurons in culture. J. Physiol. 372,191-205.
Fretter, V. and Graham, A. (1976) A Functional Anatomy of Invertebrates. Academic Press, London,
pp.589.
Heacox, A.E., Fischer, A. and Frangenberg, H.R. (1983) Development of a medium for in vitro culture of oocyte from the polychaete, Nereis virens. In Vitro 19,825-832.
Krontowsky, A. and Rumianzew, A. (1922) Zur Technik der Gewebs-kulturen von Regenwiirmern in
vitro. Pflugers Arch. Gesamte Physiol. Menschen Tiere 195,291-299.
201
Procedure
1. Keep the worms in moistened filter paper for 2-3 days before the ex- Sterilization
periment in order to empty their gut.
2. Wash the worms in running water.
3. Surface sterilize the worms with 0.1 % mercuric chloride and wipe them
with iodine solution.
1. Fix the worms on a sterilized thick cork sheet.
Dissociation
2. Cut and remove necessary tissues with sterilized tools.
3. Keep the cut fragments in sterile Ringer's solution.
1. Cut the fragments into the desired size.
Culture
2. Transfer the fragments into a petri dish, and rinse 15 times with ster- set-up
ilized Ringer's solution.
3. Transfer the fragments, with one drop of culture medium, into a culture vessel.
Results
According to Krontowsky and Rumianzew (1922), cells migrated from
cultured blood vessels with chloragogic tissue, and formed a cell sheet
around the explants. By the 2nd day of culture, the cell sheet had increased in size. On the 3rd day, cell growth continued and some differentiation of the cells was observed. The migrated cells did not exhibit mitosis. The cell culture that showed relatively active cell migration deteriorated after 7-9 days, while the cell culture that showed rather poor
cell migration survived for 3 weeks. Using a similar technique, Battaglia
and Davoli (1997) cultured minced earthworm tissue in Hansen's S-301
medium (see Appendix I). Battaglia and Davoli (1997) were able to maintain the cells for longer than 12 months. The cultured cells consisted of
spherical cysts with vibratile cilia, which showed rapid rotating and peristaltic movements, round nonadherent cells, which often clustered in
small clumps, and cells that adhered to the surface of the culture flask,
forming incomplete monolayers.
References
Battaglia, M. and Davoli, C. (1997) Long-term tissue culture of the earthworm Eisenia foetida
(Annelida, Oligochaeta). In: Maramorosch, K. and Mitsuhashi, J. (eds) Invertebrate Cell Culture: Novel Directions and Biotechnology Applications. Science Publishers, Enfield, pp 261268.
Dietzel, I.D., Drapeau, P. and Nicholls J.G. (1986) Voltage dependence of 5-hydroxytryptamine release at a synapse between identified leech neurons in culture. J. Physiol. 372,191-205.
Fretter, V. and Graham, A. (1976) A Functional Anatomy of Invertebrates. Academic Press, London,
pp.589.
Heacox, A.E., Fischer, A. and Frangenberg, H.R. (1983) Development of a medium for in vitro culture of oocyte from the polychaete, Nereis virens. In Vitro 19,825-832.
Krontowsky, A. and Rumianzew, A. (1922) Zur Technik der Gewebs-kulturen von Regenwiirmern in
vitro. Pflugers Arch. Gesamte Physiol. Menschen Tiere 195,291-299.