Materials and Methods for the Detection of Viral Inclusions
	Materials 1. A compound light microscope with an oil immersion objective ( > 1000X ). 2. Stains: a. Calcomine orange / 2-methoxyethanol b. Brilliant green / 2- methoxyethanol c. Azure A / 2-methoxyethanol + 0.2M disodium phosphate 3. Clearing agent: 5% Triton X-100 * 4. Rinse: 70% 2-methoxyethyl acetate: 30% ethanol (70/30; 2MEA:ETOH) 5. Fix: (optional) 2-methoxyethyl acetate (2MEA) 6. Mounting Agent: Euparal or Euparal Vert or LR white 7. 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
Quick Format 1. Set-up: a) Premixed O-G stain in dropper bottle ( 8 parts Brilliant Green: 1 part Calcomine Orange; 1 part H2O) b) Mix Azure A ( 9 parts Azure A + 1 part 0.2M Na2HPO4) as needed. (Do not premix!) c) Premixed 70% 2-methoxyethyl acetate: 30% ethanol as a clearing / fixing solution. d) Dispense stains and Triton X-100 clearing agent into appropriate containers. e) Utilize 10 -15 sec. microwave sessions (enclose beaker of water during use). 2. Pull epidermal strips ( or cut appropriate tissue sections) and place into: a) Triton X-100 for 10 min* and then Orange-Green stain, or b) Azure A stain c.)Microwave 10 -15 seconds. 3. Remove Orange-Green stain (2a) and Azure A stain (2b) with disposable pipettes. Replace with premixed clear / fix solution for 10-60 seconds. 4. Mount tissue pieces from (2a) and (2b) onto a clean slide in Euparal Vert and Euparal respectively. 5. Remove Triton X -100 (2b) with pipette. Replace with Orange-Green stain. Microwave 10 -15 seconds. 6. Remove stain (5) and replace with clear / fixing solution for 10 -60 seconds. Remove solution. 7. Mount tissue from (2b) into Euparal Vert. 8 Examine.	Standard Format (without microwave)   1. Set-up: a. Mix O-G stain ( 8 drops Brilliant Green, 1 drop Calcomine Orange, and 1 drop distilled water) or use premixed O-G stain. b. Mix Azure A ( 9 drops Azure A +1 drop 0.2M Na2HPO4) c. Dispense stains, clearing agent, rinse, and fixing agent into appropriate containers. 2. Pull epidermal strip (or appropriate tissue) and place it into; a) stain or b) clearing agent (10-15 min.). After clearing; stain (10-15 min.). 3. Transfer tissue to 70:30;2MEA:ETOH to decolorize ( < 1 min.). 4. Fix (15-30 min.) (optional). 5. Mount 6. Examine for inclusions Questions Where do I obtain the staining material? How do I mix the stains? Is there someplace to learn how to do this technique?
*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.
A cooperative project of the University of Florida Institute of Food and Agricultural Sciences, Plant Pathology Department, Florida Extension Plant Disease Clinic and the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Bureau of Entomology, Nematology and Plant Pathology. Both are located in Gainesville FL.	..