microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of mic ...

, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin

specimen Specimen may refer to: Science and technology * Sample (material), a limited quantity of something which is intended to be similar to and represent a larger amount * Biological specimen or biospecimen, an organic specimen held by a biorepository f ...

with an optically opaque

fluid In physics, a fluid is a liquid, gas, or other material that continuously deforms (''flows'') under an applied shear stress, or external force. They have zero shear modulus, or, in simpler terms, are substances which cannot resist any shear ...

. In this technique, the background is

stained A stain is a discoloration that can be clearly distinguished from the surface, material, or medium it is found upon. They are caused by the chemical or physical interaction of two dissimilar materials. Accidental staining may make materials app ...

, leaving the actual specimen untouched, and thus visible. This contrasts with positive staining, in which the actual specimen is stained.

Bright field microscopy

For

bright-field microscopy Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by at ...

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

nigrosin In staining dyes, nigrosin (CI 50415, Solvent black 5) is a mixture of black synthetic dyes made by heating a mixture of nitrobenzene, aniline, and hydrochloric acid in the presence of copper or iron. Related to induline, it is a mixture of phena ...

and India ink. The specimen, such as 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

spore In biology, a spore is a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plants, algae, ...

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.

Transmission electron microscopy

In the case of

transmission electron microscopy Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a gr ...

, opaqueness to

electron The electron ( or ) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary particles because they have no kno ...

s is related to the

atomic number The atomic number or nuclear charge number (symbol ''Z'') of a chemical element is the charge number of an atomic nucleus. For ordinary nuclei, this is equal to the proton number (''n''p) or the number of protons found in the nucleus of every ...

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

ammonium molybdate Ammonium molybdate can refer to: * Ammonium orthomolybdate, (NH4)2MoO4 *Ammonium heptamolybdate, (NH4)6Mo7O24, usually encountered as the tetrahydrate * Ammonium phosphomolybdate, (NH4)3PMo12O40 * Ammonium tetrathiomolybdate, (NH4)2MoS4 this chemica ...

uranyl acetate Uranyl acetate is the acetate salt of uranium oxide, a toxic yellow-green powder useful in certain laboratory tests. Structurally, it is a coordination polymer with formula UO2(CH3CO2)2(H2O)·H2O. Structure In the polymer, uranyl (UO22+) c ...

, uranyl formate,

phosphotungstic acid Phosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula . It forms hydrates . It is normally isolated as the ''n'' = 24 hydrate but can be desiccated to the hexahydrate (''n'' = 6). EPTA is the na ...

osmium tetroxide Osmium tetroxide (also osmium(VIII) oxide) is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite its toxicity and the rarity of osmium. It also has a number of unusual properties, one being that the ...

, osmium ferricyanide and auroglucothionate. These have been chosen because they scatter electrons strongly 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 A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's ...

es, bacteria, bacterial

flagella A flagellum (; ) is a hairlike appendage that protrudes from certain plant and animal sperm cells, and from a wide range of microorganisms to provide motility. Many protists with flagella are termed as flagellates. A microorganism may have fro ...

biological membrane A biological membrane, biomembrane or cell membrane is a selectively permeable membrane that separates the interior of a cell from the external environment or creates intracellular compartments by serving as a boundary between one part of th ...

structures and proteins 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-link 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 An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...

can be very important. An early study of plant viruses using negatively stained leaf dips from 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 An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable dis ...

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

Other applications

Negative staining transmission electron microscopy has also been successfully employed for study and identification of aqueous lipid aggregates like

lamellar A ''lamella'' (plural ''lamellae'') is a small plate or flake, from the Latin, and may also be used to refer to collections of fine sheets of material held adjacent to one another, in a gill-shaped structure, often with fluid in between though s ...

liposome A liposome is a small artificial vesicle, spherical in shape, having at least one lipid bilayer. Due to their hydrophobicity and/or hydrophilicity, biocompatibility, particle size and many other properties, liposomes can be used as drug deliver ...

s (le), inverted spherical

micelle A micelle () or micella () (plural micelles or micellae, respectively) is an aggregate (or supramolecular assembly) of surfactant amphipathic lipid molecules dispersed in a liquid, forming a colloidal suspension (also known as associated coll ...

s (M) and inverted

hexagonal In geometry, a hexagon (from Greek , , meaning "six", and , , meaning "corner, angle") is a six-sided polygon. The total of the internal angles of any simple (non-self-intersecting) hexagon is 720°. Regular hexagon A '' regular hexagon'' has ...

HII cylindrical (H) phases (see figure).

References

External links

* * {{DEFAULTSORT:Negative Stain Electron microscopy stains Electron microscopy Microscopy Staining