224
Methods for Setting Up Primary Cultures Specific to Animal Groups
Allow mixing for 10 min, and then decant the suspension and save the
supernatant.
Add fresh trypsin solution to the tissues remaining in the blender.
Operate the blender for 10 min and save the supernatant. This procedure should be repeated between eight and 10 times.
Centrifuge the turbid trypsinized cell suspension at 150 g for 5 min
Resuspend the sediment in medium, and centrifuge at 100 g for 2 min
Repeat washing of the cell suspension three times by centrifugation
100 g for 10 min.
Harvest single cells from the final centrifugation.
Troubleshooting
Hydra seem to carry the intracellular parasite Microsporadia. One source
of infection is the Artemia used for feeding the hydra. Although
Microsporadia-infected hydra continue to bud and regenerate normally
and show no significant physiologic or morphologic changes, the spores
of the Microsporadia rapidly germinate, multiply, and destroy the host
cells when the hydra are cultured in vitro. Therefore, it is imperative that
the spores of the parasite be removed from the hydra cells. Fumidil B
was found to be effective for this purpose (Yu-Ying et aI., 1963).
Results
According to Yu-Ying et al. (1963), the initial cell population consists of
interstitial cells, gastrodermal cells, epitheliomuscular cells, and very
young cnidoblasts, but only interstitial cells remain after a few passages.
It is essential to add the extract of hydra buds into the culture medium
for successful growth in vitro.
Rannou (1968) cultured dissociated cells from Eunicella stricta in a TC199-based medium (organic fraction of fivefold concentrated TC-199
medium 20 ml, penicillin G 20,000 IV, dihydrostreptomycin 5 mg,
colimycine 25,000 IV, and bacitracine 500 IV, in seawater to 100 ml, FBS
10%; pH adjusted to 7.0 with sodium bicarbonate), and could maintain
the cells for several months.
Subprotocol 2
Coenosarc of Tubu/orio (Hydrozoa)
The following procedure is based on the methods of Burnett et aI. (1968).
Materials
Equipment - Petri dishes
- Microforceps
- Maximov slides
- Cataract knives
- Cover glass
Methods for Setting Up Primary Cultures Specific to Animal Groups
Allow mixing for 10 min, and then decant the suspension and save the
supernatant.
Add fresh trypsin solution to the tissues remaining in the blender.
Operate the blender for 10 min and save the supernatant. This procedure should be repeated between eight and 10 times.
Centrifuge the turbid trypsinized cell suspension at 150 g for 5 min
Resuspend the sediment in medium, and centrifuge at 100 g for 2 min
Repeat washing of the cell suspension three times by centrifugation
100 g for 10 min.
Harvest single cells from the final centrifugation.
Troubleshooting
Hydra seem to carry the intracellular parasite Microsporadia. One source
of infection is the Artemia used for feeding the hydra. Although
Microsporadia-infected hydra continue to bud and regenerate normally
and show no significant physiologic or morphologic changes, the spores
of the Microsporadia rapidly germinate, multiply, and destroy the host
cells when the hydra are cultured in vitro. Therefore, it is imperative that
the spores of the parasite be removed from the hydra cells. Fumidil B
was found to be effective for this purpose (Yu-Ying et aI., 1963).
Results
According to Yu-Ying et al. (1963), the initial cell population consists of
interstitial cells, gastrodermal cells, epitheliomuscular cells, and very
young cnidoblasts, but only interstitial cells remain after a few passages.
It is essential to add the extract of hydra buds into the culture medium
for successful growth in vitro.
Rannou (1968) cultured dissociated cells from Eunicella stricta in a TC199-based medium (organic fraction of fivefold concentrated TC-199
medium 20 ml, penicillin G 20,000 IV, dihydrostreptomycin 5 mg,
colimycine 25,000 IV, and bacitracine 500 IV, in seawater to 100 ml, FBS
10%; pH adjusted to 7.0 with sodium bicarbonate), and could maintain
the cells for several months.
Subprotocol 2
Coenosarc of Tubu/orio (Hydrozoa)
The following procedure is based on the methods of Burnett et aI. (1968).
Materials
Equipment - Petri dishes
- Microforceps
- Maximov slides
- Cataract knives
- Cover glass