Negative stain EM refers to the practice of applying a heavy metal salt stain to a sample for observation. The stain enhances contrast and allows for initial visualization of specimens. Typically uranium-based stains (acetate or formate) are used, but others such as ammonium molybdate or phosphotungstic acid can be used instead, if preferred.
A small amount (~5uL) is typically applied on to a freshly plasma-cleaned grid, which then sits for a few minutes, followed by washing and staining steps. Samples prepared by negative stain can be visualized immediately on the microscope, thus allowing fast quality assessment of samples.
Negative stained samples are used to evaluate conditions such as: homogeneity (is the protein(s)/complex in one state or is it in pieces and following apart?), dispersity (monodispersity is our friend), size and shape of the protein/complex (are there features on the sample or is it a dot or an extended blob?) and quantity of protein (is the sample on the grid too crowded or can particles be clearly identified and selected?). Depending on the type of protein/complex, a concentration of ~1mg/mL is typically applied to an EM grid. The goal of starting with negative stain is to verify that the project is worth pursuing and/or what modifications may need to be made to ensure a uniform array of particles. CryoEM is time consuming and optimization is key. Too much or too little protein/complex can hinder successful data collection and timely data processing.
Once sample homogeneity, quality, and quantity have been reliably established, the user can begin cryo sample trials.
For in depth knowledge of the inner works of the electron microscope and how to make the most of it for a biological system, Prof Grant Jensen’s tutorial/class is highly recommended.