Reagent preparation for the wet-mount virus enumeration protocol

ReagentPrepn method
SYBR gold working stockDilute SYBR gold (Invitrogen) (10,000× stock) into PBS to prepare a 1,000× solution
Ascorbic acid antifade solutionDissolve ascorbic acid into PBS to create a 10% (wt/vol) solutiona
Working bead solutionThoroughly vortex the stock bead solution (2.34-μm silica spheres) (catalog no. SS04N/4186; Bangs Laboratories), dilute it 10-fold into PBS to obtain a concn of ∼108 beads ml−1, and store it at 4°C
Iron chloride solutionDissolve FeCl3·6H2O into ultrapure water to form a solution of 10 g Fe liter−1; the solution has expired if a cloudy precipitate formsb
Ascorbate-EDTA bufferCombine equal parts of 0.4 M Mg2EDTA and 0.8 M ascorbic acid and adjust with 10 N NaOH to reach a pH of 6–7; prepare fresh within 48 h of usec
  • a See reference 11.

  • b See reference 10.

  • c An alternative ascorbate-EDTA buffer can be made with MgCl2 and Na2EDTA if Mg2EDTA is unavailable (10).