12 Arthropods Other than Insects
133
- Clorox (Clorox)
Detergent
Procedure
1. Surface sterilize shrimps by immersion in freshly prepared 5% Clorox Sterilization
(containing 5.25% sodium hypochlorite) for 10 min.
1. Remove lymphoid organs (Oka organs) and ovaries (Fig. 7).
Dissection
2. Wash the excised organs in double-strength Leibovitz's L-15 medium.
1. Cut the organs into fragments smaller than 1 mm.
Culture
2. Wash the tissue fragments several times with double-strength L-15 set-up
medium.
3. Place the fragments obtained from 50 mg lymphoid (Oka) tissues or
1 g ovaries in a 25-cm 2 plastic flask containing STCM.
4. Maintain the cultures at 28°C.
Comments
- Shrimp muscle extract
- Homogenize 15 g fresh muscle of P. penicillatus in sterile 50% seawater 0:1 v/v mixture of distilled water and normal seawater)
- Use the shrimp muscle extract at a concentration of 10%
- Shrimp head extract
- Suspend the shrimp head in Leibovitz's L-15 medium (2x; 10 ml/g
tissue) and homogenize
- Centrifuge the homogenate at 3,000 g for 30 min
- Centrifuge the supernatant again at 10,000 g for 50 min
- Centrifuge the resulting supernatant at 330,000 g for 1 h
- The extract is sterilized by passing it through a membrane filter
(0.2I1m pore)
- Use the extract at a concentration of 8%
The spiny lobster Panulirus homarus (approximately 300 g) is used for
the preparation of lobster hemolymph. The procedure following refrigeration should be completed within 10 min in order to avoid melanization of the hemolymph.
- Wipe the lobster with 70% ethanol-saturated absorbent cotton, and
dry
- Keep the lobster in a refrigerator at 4°C for 1 h
- Prepare a 15-ml syringe with a no. 19 needle containing 5 ml cold
normal saline (0.9 g NaCl/100 ml distilled water, 4°C)
- Suck 5 ml hemolymph from the joint between the basiopodite and
coxopodite of the fifth leg (Fig. 8)
- Mix the hemolymph and normal seawater
- Centrifuge at 3,000 g for 5 min
- Discard the precipitates
- Store the supernatant at -70°C until use
Shrimp
extract
Lobster
hemolymph
133
- Clorox (Clorox)
Detergent
Procedure
1. Surface sterilize shrimps by immersion in freshly prepared 5% Clorox Sterilization
(containing 5.25% sodium hypochlorite) for 10 min.
1. Remove lymphoid organs (Oka organs) and ovaries (Fig. 7).
Dissection
2. Wash the excised organs in double-strength Leibovitz's L-15 medium.
1. Cut the organs into fragments smaller than 1 mm.
Culture
2. Wash the tissue fragments several times with double-strength L-15 set-up
medium.
3. Place the fragments obtained from 50 mg lymphoid (Oka) tissues or
1 g ovaries in a 25-cm 2 plastic flask containing STCM.
4. Maintain the cultures at 28°C.
Comments
- Shrimp muscle extract
- Homogenize 15 g fresh muscle of P. penicillatus in sterile 50% seawater 0:1 v/v mixture of distilled water and normal seawater)
- Use the shrimp muscle extract at a concentration of 10%
- Shrimp head extract
- Suspend the shrimp head in Leibovitz's L-15 medium (2x; 10 ml/g
tissue) and homogenize
- Centrifuge the homogenate at 3,000 g for 30 min
- Centrifuge the supernatant again at 10,000 g for 50 min
- Centrifuge the resulting supernatant at 330,000 g for 1 h
- The extract is sterilized by passing it through a membrane filter
(0.2I1m pore)
- Use the extract at a concentration of 8%
The spiny lobster Panulirus homarus (approximately 300 g) is used for
the preparation of lobster hemolymph. The procedure following refrigeration should be completed within 10 min in order to avoid melanization of the hemolymph.
- Wipe the lobster with 70% ethanol-saturated absorbent cotton, and
dry
- Keep the lobster in a refrigerator at 4°C for 1 h
- Prepare a 15-ml syringe with a no. 19 needle containing 5 ml cold
normal saline (0.9 g NaCl/100 ml distilled water, 4°C)
- Suck 5 ml hemolymph from the joint between the basiopodite and
coxopodite of the fifth leg (Fig. 8)
- Mix the hemolymph and normal seawater
- Centrifuge at 3,000 g for 5 min
- Discard the precipitates
- Store the supernatant at -70°C until use
Shrimp
extract
Lobster
hemolymph