Uranly acetate solutions with a pH between 4.2 and 4.5 are useful in staining different types of microbial samples with good resolution. A 1 to 3 percent solution is prepared, filtered using a 0.22 micron filter, and stored at 4 degrees Celsius in the dark. When stored in this manner, the stain keeps for at least one year. Uranyl acetate gives good image contrast and produces a fine-grained image that helps visualize small particles with greater clarity. Although this solution can also be used with viruses, it should not be used with samples that cannot withstand acidic conditions.
A neutral solution of phosphotungstic acid at a concentration of 1 to 3 percent is useful when staining viruses that are unstable in acidic conditions. Sodium and potassium salts of phosphotungstic acid are often used because their activity is unaffected by biological media formulations. However, these salts may damage certain viruses and their reaction with lipoproteins of the cell wall may cause an appearance of thin fibers.
Ammonium molybdate is used in staining organisms that are sensitive to changes in osmotic pressure. A neutral solution at a concentration between 1 to 2 percent is used in most cases. However, care should be taken to avoid pH rising above 7.0 because it leads to crystallization of the ammonium molybdate which interferes with visualization of specimens.
Phosphotungstic acid and its sodium and potassium salts tend to damage certain fragile structures of the specimens they stain. This effect can be avoided by using methylamine tungstate as a negative staining agent to provide reasonably good resolution. A 2-percent solution at a pH of 6.5 is generally used. The disadvantage of using this stain is that it should be freshly prepared before every staining because it degrades very fast in storage.
Stains such as uranyl oxalate, formate and sulfate, gold thioglucose, sodium zirconium glycollate and aluminum formate are negative stains that are occasionally used to stain certain samples with specific requirements.