Chapter 16
Annelida
Subprotocol 1
Oocytes of Nereis virens (Polychaeta)
Oocytes cultures have been made using the medium of Heacox et aI.,
modified Cecil's medium, or modified TC-199 medium (see Appendix
1). The following is the method of Heacox et aI. (1983) to cultivate 00cytes of the polychaete Nereis virens.
Materials
PROTOCOL
- Clinical centrifuge and lO-ml Pyrex centrifuge tube
Equipment
- Nylon mesh (35 and 75 Ilm)
- Syringe with a hypodermic needle
- Hemocytometer
- Sterilized seawater
Solutions
- Nereis balanced salt solution (NBSS; see Appendix 1)
- Antibiotic mixture 1 (100 IV/ml penicillin and 129 Ilg/ml dihydrostreptomycin sulfate in sterile seawater)
- Antibiotic mixture 2 (100 IV/ml penicillin, 129 Ilg/ml dihydrostreptomycin sulfate, and 100 Ilg/ml gentamicin in sterile NBSS)
- Ficoll (Pharmacia Fine Chemicals)
Chemicals
- Matured Nereis worms
Animals
Procedure
1. Wash the worms several times with filter-sterilized seawater.
2. Keep the worms in sterile seawater containing antibiotic mixture 1 for
12-24 h before use.
Sterilization
1. Using a hypodermic needle, collect 0.5-1.0 ml coelomic fluid contain- Bleeding
ing oocytes, avoiding damage to the gut and allowing the internal pressure of the body cavity to fill the syringe.
2. Transfer the contents of the syringe to a sterile lO-ml Pyrex centrifuge
tube and centrifuge the coelomic fluid in a clinical centrifuge for lOIS s at 300 g. This process is sufficient to separate the coelomic fluid
from coelomic cells.
3. Discard the supernatant.