38 Virus Inoculation-Plaque Assay
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1. Pass the supernatant through 0.22-flm filter for decontamination.
2. Store the filtrate at -80°C until use.
Results
The same results as given for subprotocoll are obtained.
Subprotocol 3
Preparation of Inoculum from Isolated Occlusion Bodies
Occlusion bodies, such as polyhedra, can be purified by homogenization
and differential centrifugation from virus-infected insects.
The occluded virus is relatively stable, and can be stored at 4°C for
several months. The occlusion bodies themselves cannot be used as an
inoculum. Cells usually cannot digest the protein of the occlusion bodies, and infection does not occur even if the cells phagocytose the occlusion bodies in their cytoplasm. Therefore, it is necessary to release the
occluded viruses before inoculation.
Materials
- Centr fuge and centrifuge tubes
- Membrane filter
- 0.5% Sodium carbonate
- 1 N HCI
- Occlusion bodies
Procedure
Filter
sterilization
Equipment
Solutions
Viruses
1. Dissolve the occluded bodies in alkaline solution. For example, the Dissolution
polyhedra of nuclear polyhedrosis virus can be dissolved in 0.5% sodium carbonate (pH approximately 10.0).
2. Adjust the pH to 7.0 with 1 N HCl.
3. Centrifuge at 1,500 g for 15 min.
1. Pass the supernatant through a 0.22-flm filter for decontamination.
Filter
2. Store the filtrate at -80°C until use.
sterilization
Results
The results are the same as those given for subprotocol 1.