20 Porifera
235
Subprotocol 3
Embryos of Hermaphroditic Sponges (Demospongia)
Materials
- Dissecting tray
Equipment
- Forceps
- Plastic vials (15 ml)
- Fine needles (21 G)
- 24-Well flat-bottom culture plates
- Sterile seawater (SSW; 0.22 11m filtered, supplemented with 1 % v/v gen- Solutions
tamicin from an original stock solution of 50 mg/ml)
- Culture medium
- Hermaphroditic viviparous sponge Negombata magnifica (gravid Animals
colonies)
Procedure
1. Remove 800-1,000 embryos, which are bright orange in color and are Collection
embedded within the sponge mesohyl, from the sponges using for- of embryos
ceps.
2. Transfer the embryos to 15-ml plastic vials containing Ssw.
3. Wash the embryos several times with Ssw.
4. Remove the outer rigid envelope of ciliated cells of the embryos using
fine needles, remove the internal softer mass of cells, and mince this
mass using the needles.
1. Transfer a single minced embryo into the well of 24-well culture plate Culture
with 0.5 ml culture medium.
set-up
Results
According to Rinkevich et al. (1998), cells in long-term primary culture
are always in an arrested form. Generally, four major cell types are observed: blastomeres, choanocytes, spherulous cells, and chromocytes.
Blastomeres were abundant in the first 1.5 months after dissociation,
during which time they became markedly reduced in number and disappeared. Chromocytes were abundant immediately after dissociation
but, within the following 2 weeks, their number and pigmentation were
significantly reduced. Spherulous cells, especially choanocytes, increased
in number during the first 1.5 months. Over a period of 9 months culture, no rapid cell proliferation was observed.
235
Subprotocol 3
Embryos of Hermaphroditic Sponges (Demospongia)
Materials
- Dissecting tray
Equipment
- Forceps
- Plastic vials (15 ml)
- Fine needles (21 G)
- 24-Well flat-bottom culture plates
- Sterile seawater (SSW; 0.22 11m filtered, supplemented with 1 % v/v gen- Solutions
tamicin from an original stock solution of 50 mg/ml)
- Culture medium
- Hermaphroditic viviparous sponge Negombata magnifica (gravid Animals
colonies)
Procedure
1. Remove 800-1,000 embryos, which are bright orange in color and are Collection
embedded within the sponge mesohyl, from the sponges using for- of embryos
ceps.
2. Transfer the embryos to 15-ml plastic vials containing Ssw.
3. Wash the embryos several times with Ssw.
4. Remove the outer rigid envelope of ciliated cells of the embryos using
fine needles, remove the internal softer mass of cells, and mince this
mass using the needles.
1. Transfer a single minced embryo into the well of 24-well culture plate Culture
with 0.5 ml culture medium.
set-up
Results
According to Rinkevich et al. (1998), cells in long-term primary culture
are always in an arrested form. Generally, four major cell types are observed: blastomeres, choanocytes, spherulous cells, and chromocytes.
Blastomeres were abundant in the first 1.5 months after dissociation,
during which time they became markedly reduced in number and disappeared. Chromocytes were abundant immediately after dissociation
but, within the following 2 weeks, their number and pigmentation were
significantly reduced. Spherulous cells, especially choanocytes, increased
in number during the first 1.5 months. Over a period of 9 months culture, no rapid cell proliferation was observed.