12 Arthropods Other than Insects
131
Fig. 7. The cephalothorax area of Penaeus monodon. The lymphoid tissue (Oka
organ) is exposed (white arrows) when the hepatopancreas (HP) is removed (black
arrows). x 1.25. From Chen S.N. and Kou G.H.(1989) Infection of cultured cells
from the lymphoid organ of Penaeus monodon Fabricius by mono don-type
baculovirus (MBV), Journal of Fish Disease Vo1.12., pp 73-76, with the permission
of Blackwell Science Ltd.
Procedure
1. Wash shrimps with clean seawater passed through a filter paper.
2. Surface sterilize the shrimps several times with 2% tincture of iodine
and 75% ethanol.
1. Fix the sterilized shrimp to the bottom of a dissecting tray.
2. Remove the hepatopancreas with scissors and forceps, care being taken
not to injure the digestive system (Fig. 7).
3. Rinse the hepatopancreas extensively in cold divalent cation-free saline.
1. Cut the tissue further into small fragments of approximately 1 mm 3 •
2. Wash the tissue fragments several times in saline.
3. Transfer the fragments into a beaker filled with saline containing
10 mM EDTA and 0.1 mM diisopropyl fluorophosphate.
4. Stir the suspension with a magnetic stirrer for 20-25 min at 4°C,
5. Filter the suspension through a nylon mesh.
6. Centrifuge the suspension at 400 g at 4°C for 10 min.
7. Repeat step 6.
1. Suspend the cell pellet in culture medium.
2. Culture the cells at 27°-28°C.
Results
Cancre et al. (1995) have reported that, in medium 199 containing 20%
low-protein replacement serum (LPSR-l; Sigma), the viability of cells
was 48% after a period of 25 h.According to Ke et al. (1990), cell migraSterilization
Dissection
Dissociation
Culture
set-up
131
Fig. 7. The cephalothorax area of Penaeus monodon. The lymphoid tissue (Oka
organ) is exposed (white arrows) when the hepatopancreas (HP) is removed (black
arrows). x 1.25. From Chen S.N. and Kou G.H.(1989) Infection of cultured cells
from the lymphoid organ of Penaeus monodon Fabricius by mono don-type
baculovirus (MBV), Journal of Fish Disease Vo1.12., pp 73-76, with the permission
of Blackwell Science Ltd.
Procedure
1. Wash shrimps with clean seawater passed through a filter paper.
2. Surface sterilize the shrimps several times with 2% tincture of iodine
and 75% ethanol.
1. Fix the sterilized shrimp to the bottom of a dissecting tray.
2. Remove the hepatopancreas with scissors and forceps, care being taken
not to injure the digestive system (Fig. 7).
3. Rinse the hepatopancreas extensively in cold divalent cation-free saline.
1. Cut the tissue further into small fragments of approximately 1 mm 3 •
2. Wash the tissue fragments several times in saline.
3. Transfer the fragments into a beaker filled with saline containing
10 mM EDTA and 0.1 mM diisopropyl fluorophosphate.
4. Stir the suspension with a magnetic stirrer for 20-25 min at 4°C,
5. Filter the suspension through a nylon mesh.
6. Centrifuge the suspension at 400 g at 4°C for 10 min.
7. Repeat step 6.
1. Suspend the cell pellet in culture medium.
2. Culture the cells at 27°-28°C.
Results
Cancre et al. (1995) have reported that, in medium 199 containing 20%
low-protein replacement serum (LPSR-l; Sigma), the viability of cells
was 48% after a period of 25 h.According to Ke et al. (1990), cell migraSterilization
Dissection
Dissociation
Culture
set-up