Chapter 38
PROTOCOL
Virus Inoculation-Plaque Assay
Subprotocol 1
Preparation of Inoculum from Hemolymph
Materials
- Centrifuge and centrifuge tubes
Equipment
- Disposable fIlter unit (e.g., Minisart, with a 0.22 11m pore (Sartorius
AG), and a disposable syringe)
- Physiological solution
Solutions
- Culture media
- Phenyl thiourea
Chemicals
- L-Cysteine
- Reduced glutathione
- Virus-infected insects
Animals
Procedure
1. Surface sterilize infected insects by submersion in 70% ethanol for Sterilization
5 min.
2. Rinse the insects with sterile distilled water.
3. Wipe off any water remaining on the surface of the insects using sterilized fIlter paper.
1. Cut or puncture the insect, and gently squeeze out the hemolymph Bleeding
into a centrifuge tube containing culture medium.
2. Centrifuge the fluid at 150 g for 15 min.
1. Pass the supernatant through a 0.22-l1m fIlter.
Filter
2. If the fluid becomes dark, add a phenol oxidase inhibitor, such as phe- sterilization
nyl thiourea, cysteine, or reduced glutathione, to the supernatant.
3. Store the fluid at -80°C until use.