By using different sizes of colloidal gold particles, conjugated using protein A (see Note 20) to an antibody, it is possible to differentiate different virus particles by labeling with different size gold particles, that is, 5 nm, 10 nm, 20 nm, and so on. Thus it is possible to distinguishing two (or more) different viruses within a sap extract on the same grid clearly and unequivocally.
1. Using a variable volume pipet dilute required specific antiserum to 1:500 in phosphate buffer pH 6.5 in a 0.5-mL microfuge tube.
2. In a Parafilm lined Petri dish, place 20-^L drops of the diluted antiserum and, using the watchmaker's forceps, float carbon coated grids, carbon side down, on them. Incubate at room temperature for 15 min.
3. Grind infected material in a muslin-lined bag in phosphate buffer, pH 6.5, with a hand grinder, to a dilution of 1:10.
4. Place 20 ^L 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. Drain excess liquid with filter paper and place on sap for 15 min at room temperature.
6. Dilute specific antiserum to 1:100 in a 0.5-mL microfuge tube and place 20-^L 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 at room temperature for 15 min.
8. Select a suitable size of PAG (see Note 20) to 1:50 in phosphate buffer pH 6.5 on the Parafilm. Wash grids as before and place them on the PAG and incubate at room temperature for 15 min.
9. Wash the grids with 20 drops of distilled water and stain with three to five drops of 2% uranyl acetate, drain as before.
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