Preparation of Uranyl Formate Stain

Scope

This protocol describes the preparation of a 0.75% uranyl formate solution in water for use as a negative stain. The procedure is adapted from Ohi, et al's excellent 2004 paper "Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy" which is a nice introductory overview of some of the techniques and issues in negative stain.

Please note that all weighing of negative stains must be done on the dedicated scale in the Cohen lab area. Any trash generated during this procedure must be disposed of as a combined hazardous waste in the EM Prep area. The container label should read "Uranyl Formate" and the "Toxic" and "Other - Radioactive" hazard boxes should be checked.

Materials

1. Uranyl formate: found in the heavy metals box in the EM Prep area.
   
   
2. 5M NaOH: working solutions of base are kept above the pH meter.

Procedure

1. Weigh out 37.5 mg of uranyl formate into a 20 ml scintillation vial, and wrap with foil to keep dark.
   
   
2. Add 5 ml of deionized water and bring to a consistent boil, stirring until all of the uranyl formate has been dissolved. If chunks remain, it may be helpful to break them up by brief immersion in a sonicating bath.
   
   
3. Add drops of 5 M NaOH until the stain solution becomes slightly darker yellow (too much NaOH will precipitate the stain) and stir for another 5 min in the dark. The objective here is to titrate the pH to a reasonable extent. pH can be checked with paper strips (NOT a meter!). The issue is that the uranyl formate will precipitate out of solution as the pH rises. 4.5 is about as high as one can reasonably achieve.
   
   
4. Filter the solution with a 0.2 μm syringe filter into a Falcon tube wrapped with aluminum foil and add deionized water to a final volume of 5 ml. Several members of the lab are of the opinion that this solution can be frozen in aliquots at -80C until it is needed. The original paper suggests making the solution fresh for each use.