Materials and Methods for the Detection of Viral Inclusions
- A compound light microscope with an oil immersion objective ( > 1000X ).
- Calcomine orange / 2-methoxyethanol
- Brilliant green / 2- methoxyethanol
- Azure A / 2-methoxyethanol + 0.2M disodium phosphate
- Clearing agent: 5% Triton X-100*
- Rinse: 70% 2-methoxyethyl acetate: 30% ethanol (70/30; 2MEA:ETOH)
- Fix: (optional) 2-methoxyethyl acetate (2MEA)
- Mounting Agent: Euparal or Euparal Vert or LR white
- Materials for the mechanics of the technique: glass slides, cover slips, containers for stains, dropping bottles, scalpel, very fine tipped forceps, etc.
Methods** - Quick and Standard Techniques
Standard Format (without microwave)
*Triton X-100 treatment - When the O-G combination is used, stained plastids often obscure small inclusions. The plastids can be dissolved by treating tissue pieces with a 2% solution of Triton X-100 (Rohm and Haas Co., Philadelphia, PA 19105) for 5 min before staining. This treatment is especially useful for detecting the cylindrical inclusions of potyviruses, particularly during their early stages of development when these small inclusions are located at the cell periphery.
**The techniques, with some modifications, was taken from: Christie, R.G., J.W. Edwardson. 1977." Light and electron microscopy of plant virus inclusions", Fla. Agric. Exp. Stn. Monogr. No.9.150pp.