Negative staining is an established method, often used in diagnostic microscopy

Microscopy

Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...

, for contrasting a thin specimen with an optically opaque fluid.

For bright field microscopy

Bright field microscopy

Bright field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample...

, negative staining is typically performed using a black ink fluid such as nigrosin

Nigrosin

Nigrosin is a mixture of synthetic black dyes made by heating a mixture of nitrobenzene, aniline and aniline hydrochloride in the presence of a copper or iron catalyst. Its main industrial uses are as a colorant for lacquers and varnishes and in marker-pen inks...

. The specimen, such as a wet bacteria

Bacteria

Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...

l culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope

Microscope

A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 the bacterial cells, and perhaps their spore

Spore

In biology, a spore is a reproductive structure that is adapted for dispersal and surviving for extended periods of time in unfavorable conditions. Spores form part of the life cycles of many bacteria, plants, algae, fungi and some protozoa. According to scientist Dr...

s, appear light against the dark surrounding background. An alternative method has been developed using an ordinary waterproof marking pen to deliver the negative stain.

In the case of transmission electron microscopy

Transmission electron microscopy

Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...

, opaqueness to electron

Electron

The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...

s is related to the atomic number

Atomic number

In chemistry and physics, the atomic number is the number of protons found in the nucleus of an atom and therefore identical to the charge number of the nucleus. It is conventionally represented by the symbol Z. The atomic number uniquely identifies a chemical element...

, i.e., the number of protons. Some suitable negative stains include ammonium molybdate

Ammonium molybdate

Ammonium heptamolybdate is an odourless crystalline compound ranging in colour from white to yellow-green. It is usually encountered as the tetrahydrate, whose chemical formula is 6Mo7O24·4H2O...

, uranyl acetate

Uranyl acetate

Uranyl acetate is the acetate salt of uranium and is a yellow crystalline solid made up of yellow rhombic crystals and has a slight acetic odor. Uranyl acetate is slightly radioactive, the precise radioactivity depends on the isotopes of uranium present...

, uranyl formate

Uranyl formate

Uranyl formate is a fine yellow free-flowing powder occasionally used in transmission electron microscopy.It is occasionally used as a 0.5% or 1% aqueous negative stain in transmission electron microscopy because it shows a finer grain structure than uranyl acetate...

, phosphotungstic acid

Phosphotungstic acid

Phosphotungstic acid , tungstophosphoric acid , is a heteropoly acid with the chemical formula 31240. It normally present as a hydrate. EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly...

, osmium tetroxide, osmium ferricyanide and auroglucothionate. These have been chosen because they scatter electrons well and also adsorb to biological matter 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 virus

Virus

A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...

es, bacteria, bacterial flagella, biological membrane

Biological membrane

A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content...

 structures and protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s or protein aggregates, which all have a low electron-scattering power. Some stains, such as osmium tetroxide and osmium ferricyanide, are very chemically active. As strong oxidants, they cross-links lipids mainly by reacting with unsaturated carbon-carbon bonds, and thereby both fix biological membranes in place in tissue samples and simultaneously stain them.

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.

Negative staining at both light microscope and electron microscope level should never be performed with infectious

Infection

An infection is the colonization of a host organism by parasite species. Infecting parasites seek to use the host's resources to reproduce, often resulting in disease...

organisms unless stringent safety precautions are followed. Negative staining is a very mild preparation method and does not reduce the possibility of operator infection.