Negative stain

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Negative stain is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid.

For transmitted bright field light microscopy, negative staining is typically performed using a black ink fluid such as nigrosine. The specimen (say a wet bacterial culture spread on a glass slide) is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells (and perhaps their spores) appear light against the dark surrounding background.

In the case of transmission electron microscopy, opaqueness to electrons is related to the atomic number. Some suitable negative stains include ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, auroglucothionate etc. These have been chosen because they absorb electrons well and also adsorb to biomaterials well. The structures which can be negatively stained are much smaller than those studied with the light microscope. Here, the method is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power. The choice of negative stain in electron microscopy can be very important. A study of plant viruses from negatively-stained leaf dips of a diseased plant showed only spherical viruses with one stain and only rod-shaped viruses with another! The (verified) conclusion was that this plant suffered from a mixed infection by two separate viruses.

Warning: negative staining should never be performed with infectious organisms unless stringent safety precautions are followed. Negative staining is a very mild preparation method and does not reduce the possibility of operator infection!