This technique can be used to distinguish two (or more) morphologically similar viruses within the same sap extract (2,10).
1. Using a variable volume pipet dilute 2 (or more) specific antisera (see Notes 2 and 16) to 1:500 (see Note 3) in phosphate buffer, pH 6.5 (see Note 4), in 0.5-mL microfuge tubes.
2. In a Parafilm lined Petri dish place 20-pL drops of the diluted antiserum (see Note 5) and, using the watchmaker's forceps, float carbon-coated grids (see Note 6) carbon-side down, on them. Incubate at room temperature for 15 min (see Notes 7 and 8).
3. Grind infected material (see Note 9) in a muslin-lined bag in 0.06 M phosphate buffer, pH 6.5, with a hand grinder (see Note 10) to a dilution of 1:10.
4. Place 20-pL drops of sap extract on Parafilm in a Petri dish.
5. Hold the antiserum coated grids in the forceps and wash with 20 drops of phosphate buffer, pH 6.5, in a constant stream using a Pasteur pipet, on the treated side (see Note 11). Drain excess liquid with filter paper and place on sap for 15 min at room temperature (see Note 7).
6. Dilute one of the specific antisera to 1:100 (see Note 3) in a 0.5-mL Microfuge tube and place 20 pL drops on Parafilm in a Petri dish.
7. Wash the grids with 20 drops of buffer as before and drain as before. Place them on the diluted antibody and incubate for 15 min at room temperature (see Note 16).
8. Wash the grids with 20 drops of distilled water and stain with three to five drops of 2% uranyl acetate (see Note 12), draining as before (see Note 14).
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