20 Porifera
233
- Seawater
- Filtered seawater
- Sponges Microciona, Clathrina, and Polymastia
- Heat-killed Escherichia coli
- Gauze
Procedure
In order to dissociate sponge tissues, the technique used is essentially
the same as that developed by Wilson (1907,1910; see Comments).
Solutions
Animals
Miscellaneous
1. Cut the lobes of sponge into small pieces using scissors.
Mechanical
2. Strain the tissue fragments through gauze. To do this, fold a square dissociation
piece of cloth around the bits of sponge like a bag, and immerse the
fragments in the cloth bag in a saucer of filtered sea water.
3. While the bag is kept closed with the fingers of one hand, squeeze the
sponge fragments in the bag between the arms of a small pair of forceps. The pressure and the elastic recoil of the skeleton of the sponge
will break up the living tissue of the sponge into its constituent cells.
4. Pass the cells through a nylon sieve.
1. Maintain the cell suspension in sterile seawater
Culture
2. Feed the cells daily by the addition of heat-killed E. coli from the 2nd set-up
week onward.
Comments
Wilson's method (1907) results in a mixture of single cells and cell masses.
In order to get more uniform single cells, Humphrey et al. (1960) devised
a chemical method of dissociation. Their method is as follows:
- Immerse 1 g blotted sponge lobe tips in 80 ml Ca 2 + - and Mg2+ -free
seawater (CMF-SW) at O°C (NaCI27.0 g, KCI 8.0 g, Na 2 S0 4 1.0 g, and
NaHC0 3 0.04 g in 11 H 2 0, pH 7.2)
Cut the lobe tips into 2 mm fragments
Soak the fragments in CMF-SW for 30 min
Squeeze the fragments through a no. 25 standard-quality bolting cloth
(Marco Specialty Steel, Inc.) into 80 ml fresh cold CMS-SW
Centrifuge at 150 g for 5 min.
Resuspend the cells at a density of 2x10 7 cells/ml in CMF-SW containing 0.1 % trypsin at O°C
Rotate 15-ml aliquots of this suspension in 100-ml beakers on a gyratory shaker at 80 r.p.m. for 6 h at 5°C
Transfer 3 ml of the resulting suspension into a conical centrifuge tube
233
- Seawater
- Filtered seawater
- Sponges Microciona, Clathrina, and Polymastia
- Heat-killed Escherichia coli
- Gauze
Procedure
In order to dissociate sponge tissues, the technique used is essentially
the same as that developed by Wilson (1907,1910; see Comments).
Solutions
Animals
Miscellaneous
1. Cut the lobes of sponge into small pieces using scissors.
Mechanical
2. Strain the tissue fragments through gauze. To do this, fold a square dissociation
piece of cloth around the bits of sponge like a bag, and immerse the
fragments in the cloth bag in a saucer of filtered sea water.
3. While the bag is kept closed with the fingers of one hand, squeeze the
sponge fragments in the bag between the arms of a small pair of forceps. The pressure and the elastic recoil of the skeleton of the sponge
will break up the living tissue of the sponge into its constituent cells.
4. Pass the cells through a nylon sieve.
1. Maintain the cell suspension in sterile seawater
Culture
2. Feed the cells daily by the addition of heat-killed E. coli from the 2nd set-up
week onward.
Comments
Wilson's method (1907) results in a mixture of single cells and cell masses.
In order to get more uniform single cells, Humphrey et al. (1960) devised
a chemical method of dissociation. Their method is as follows:
- Immerse 1 g blotted sponge lobe tips in 80 ml Ca 2 + - and Mg2+ -free
seawater (CMF-SW) at O°C (NaCI27.0 g, KCI 8.0 g, Na 2 S0 4 1.0 g, and
NaHC0 3 0.04 g in 11 H 2 0, pH 7.2)
Cut the lobe tips into 2 mm fragments
Soak the fragments in CMF-SW for 30 min
Squeeze the fragments through a no. 25 standard-quality bolting cloth
(Marco Specialty Steel, Inc.) into 80 ml fresh cold CMS-SW
Centrifuge at 150 g for 5 min.
Resuspend the cells at a density of 2x10 7 cells/ml in CMF-SW containing 0.1 % trypsin at O°C
Rotate 15-ml aliquots of this suspension in 100-ml beakers on a gyratory shaker at 80 r.p.m. for 6 h at 5°C
Transfer 3 ml of the resulting suspension into a conical centrifuge tube