222
Methods for Setting Up Primary Cultures Specific to Animal Groups
4. Centrifuge the supernatant again at 540 g for 15 min.
5. Suspend the sediment in hydra culture solution.
6. Centrifuge at 700 g.
7. Suspend the cell pellet in culture solution containing a 10-fold concentration of all the antibiotics.
8. Shake the mixture occasionally for 1 h.
9. Wash the mixture three times with cold culture solution containing
Fumidil B at 200 mg/l without any other antibiotics.
10. Ascertain the complete removal of protozoa under a microscope.
Culture 1. Adjust the cell density to 64 x 10 3 cells/ml.
set-up 2. Seed 1 ml cell suspension into either a 125-ml culture flask, and add
5 ml growth medium, or into a Leighton tube containing 1 ml growth
medium.
3. Culture at 22°C.
Comments
- Hydra culture: hydra can be cultured as follows according to the methods of Loomis and Lenhoff (1956)
A stock solution of saturated NaCl (360 g/I,OOO ml) is made by dissolving 2.27 kg commercial table salt in a 7.561 bottle of hot tap water
and allowing it to cool.
This stock solution is diluted 100-fold with tap water to make the brine
solution in which the eggs are hatched (see following Artemia preparation).
500 ml of this diluted salt solution is poured into each of five hatching
dishes; in general, one dish of brine shrimp provides food for two dishes
of hydra.
- Artemia preparation
The aforementioned hatching dishes are seeded with 3-4 ml Artemia
eggs. If many eggs are stuck onto the wall of the container after they
have been seeded, wash them into the salt solution by agitating the
dish, because the eggs will fail to hatch if attached to the wall.
The dishes are incubated for 48 h at room temperature (approximately
21°C), after which time the brine shrimps are ready for feeding to Hydra. If the seeding is performed daily, then living larvae are available
daily. Allowance for hatching over a weekend may be made by varying
the temperature; Artemia hatches in 1,2, and 3 days at temperatures
of 30°, 21 0, and 15°C, respectively.
Living larvae are separated from unhatched eggs by a combination of
flotation, settling and phototropic migration. For this, the contents of
the five hatching dishes are poured into one hatching dish that has
been bound with black tape on all sides except for one corner. A small
microscope lamp is placed at this corner and the whole set up covered
with a large flat cardboard box, the edges of which have 5 cm of black
cloth stapled to them.
Methods for Setting Up Primary Cultures Specific to Animal Groups
4. Centrifuge the supernatant again at 540 g for 15 min.
5. Suspend the sediment in hydra culture solution.
6. Centrifuge at 700 g.
7. Suspend the cell pellet in culture solution containing a 10-fold concentration of all the antibiotics.
8. Shake the mixture occasionally for 1 h.
9. Wash the mixture three times with cold culture solution containing
Fumidil B at 200 mg/l without any other antibiotics.
10. Ascertain the complete removal of protozoa under a microscope.
Culture 1. Adjust the cell density to 64 x 10 3 cells/ml.
set-up 2. Seed 1 ml cell suspension into either a 125-ml culture flask, and add
5 ml growth medium, or into a Leighton tube containing 1 ml growth
medium.
3. Culture at 22°C.
Comments
- Hydra culture: hydra can be cultured as follows according to the methods of Loomis and Lenhoff (1956)
A stock solution of saturated NaCl (360 g/I,OOO ml) is made by dissolving 2.27 kg commercial table salt in a 7.561 bottle of hot tap water
and allowing it to cool.
This stock solution is diluted 100-fold with tap water to make the brine
solution in which the eggs are hatched (see following Artemia preparation).
500 ml of this diluted salt solution is poured into each of five hatching
dishes; in general, one dish of brine shrimp provides food for two dishes
of hydra.
- Artemia preparation
The aforementioned hatching dishes are seeded with 3-4 ml Artemia
eggs. If many eggs are stuck onto the wall of the container after they
have been seeded, wash them into the salt solution by agitating the
dish, because the eggs will fail to hatch if attached to the wall.
The dishes are incubated for 48 h at room temperature (approximately
21°C), after which time the brine shrimps are ready for feeding to Hydra. If the seeding is performed daily, then living larvae are available
daily. Allowance for hatching over a weekend may be made by varying
the temperature; Artemia hatches in 1,2, and 3 days at temperatures
of 30°, 21 0, and 15°C, respectively.
Living larvae are separated from unhatched eggs by a combination of
flotation, settling and phototropic migration. For this, the contents of
the five hatching dishes are poured into one hatching dish that has
been bound with black tape on all sides except for one corner. A small
microscope lamp is placed at this corner and the whole set up covered
with a large flat cardboard box, the edges of which have 5 cm of black
cloth stapled to them.