246
Organ Culture
2. Rinse the ganglia in two changes of culture medium for a total of 1 h
and leave in a third change of medium overnight at 29°C.
3. After digestion, place the ganglia in dishes coated with a layer of insect blood cells (see Comments).
Culture 1. Remove cell bodies on the surface of the ganglion.
set-up 2. Tease apart the outer layer of the ganglion.
3. After removal of most of the ganglionic debris, push the cells toward
the center of the dish using a Pasteur pipette.
4. Keep the dish in a humid (80% relative humidity) incubator at 29°C,
and leave the dish undisturbed for 36 h.
5. Change half the culture medium once a week with freshly prepared
medium.
Comments
In order to coat a dish with insect blood, cut a cockroach leg at the base
of the femur. Press the animal gently, and squeeze the blood out until a
drop of the blood flows into the dish. Spread and settle the blood for 2 h.
Rinse three times with fresh culture medium. (If the washing is not sufficient, melanization of the blood cells may occur.)
Results
According to Howes et al. (1991), neurons excised from the meso- and
metathoracic ganglia are predominantly rounded, although some cells
retained normal stumps. The migrated cells from neurons attached
loosely to the bottom of the dish within 48 h, and outgrowth usually
started at 5 or 6 days. If the dish was not coated with cockroach blood
cells, the neurons did not attach to the bottom, and failed to grow. Growing cells showed a variety of development of processes. On the basis of
the presence of voltage-dependent calcium channels on neuronal processes, neurons were ascertained to maintain a functional state for several weeks.
Subprotocol 6
Stomatogastric Ganglia of Nymphal Cockroaches (Blatta ria)
Materials
Equipment - Dissecting microscope
- Scissors
- Forceps
- Needles
- Pasteur pipettes
- Maximov slides
Organ Culture
2. Rinse the ganglia in two changes of culture medium for a total of 1 h
and leave in a third change of medium overnight at 29°C.
3. After digestion, place the ganglia in dishes coated with a layer of insect blood cells (see Comments).
Culture 1. Remove cell bodies on the surface of the ganglion.
set-up 2. Tease apart the outer layer of the ganglion.
3. After removal of most of the ganglionic debris, push the cells toward
the center of the dish using a Pasteur pipette.
4. Keep the dish in a humid (80% relative humidity) incubator at 29°C,
and leave the dish undisturbed for 36 h.
5. Change half the culture medium once a week with freshly prepared
medium.
Comments
In order to coat a dish with insect blood, cut a cockroach leg at the base
of the femur. Press the animal gently, and squeeze the blood out until a
drop of the blood flows into the dish. Spread and settle the blood for 2 h.
Rinse three times with fresh culture medium. (If the washing is not sufficient, melanization of the blood cells may occur.)
Results
According to Howes et al. (1991), neurons excised from the meso- and
metathoracic ganglia are predominantly rounded, although some cells
retained normal stumps. The migrated cells from neurons attached
loosely to the bottom of the dish within 48 h, and outgrowth usually
started at 5 or 6 days. If the dish was not coated with cockroach blood
cells, the neurons did not attach to the bottom, and failed to grow. Growing cells showed a variety of development of processes. On the basis of
the presence of voltage-dependent calcium channels on neuronal processes, neurons were ascertained to maintain a functional state for several weeks.
Subprotocol 6
Stomatogastric Ganglia of Nymphal Cockroaches (Blatta ria)
Materials
Equipment - Dissecting microscope
- Scissors
- Forceps
- Needles
- Pasteur pipettes
- Maximov slides