Reticulocytes Human Blood Supravital Stain

Supravital staining is a method of

staining Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissues), in cytology (microscopic study of cells), and in th ...

used in

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 micr ...

to examine living cells that have been removed from an organism. It differs from intravital staining, which is done by injecting or otherwise introducing the stain into the body. Thus a supravital stain may have a greater

toxicity Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subs ...

, as only a few cells need to survive it a short while. The term "

vital stain A vital stain in a casual usage may mean a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties. In supravital staining, living cells ...

" is used by some authors to refer specifically to an intravital stain, and by others interchangeably with a supravital stain, the core concept being that the cell being examined is still alive. As the cells are alive and unfixed, outside the body, supravital stains are temporary in nature. The most common supravital stain is performed on

reticulocyte Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow and then circulate for about a day in the blood stream before developing into ma ...

s using

new methylene blue is an organic compound of the thiazine class of heterocycles. It is used as a stain and as an antimicrobial agent. It is classified as an azine dye, and the chromophore is a cation, the anion is often unspecified. Applications NMB is a stainin ...

brilliant cresyl blue Brilliant cresyl blue is a supravital stain used for counting reticulocyte Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow a ...

, which makes it possible to see the reticulofilamentous pattern of

ribosome Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to fo ...

s characteristically precipitated in these live immature

red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "hol ...

s by the supravital stains. By counting the number of such cells the rate of red blood cell formation can be determined, providing an insight into

bone marrow Bone marrow is a semi-solid biological tissue, tissue found within the Spongy bone, spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It i ...

activity and

anemia Anemia or anaemia (British English) is a blood disorder in which the blood has a reduced ability to carry oxygen due to a lower than normal number of red blood cells, or a reduction in the amount of hemoglobin. When anemia comes on slowly, t ...

. This is in contrast to

ing, when the dye employed is one that is ''excluded'' from the living cells so that only dead cells are stained positively. (Vital stains include dyes like trypan blue and

propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluo ...

, which are either too bulky or too charged to cross the cell membrane, or which are actively rapidly pumped out by live cells.) Supravital staining can be combined with cell surface

antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of t ...

staining (

immunofluorescence Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to spe ...

) for applications such as FACS analysis. Immunofluorescence can also be done within the interior of live cells by reversible

cell permeabilization Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...

using the detergent Triton X-100. Adjusted carefully to the appropriate concentration for the number of cells, the pretreatment can permit access of molecules between 1 and 150

kilodalton The dalton or unified atomic mass unit (symbols: Da or u) is a non-SI unit of mass widely used in physics and chemistry. It is defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at ...

s to the interior of the cell. Although antibodies may be used in a similar way in this context, the term "supravital stain" is typically reserved for smaller chemicals which possess suitable properties intrinsically.

Examples of common supravital dyes

Supravital dyes for RBCs:2011 Hematology, Clinical Microscopy, and Body Fluids Glossary by CAP
/ref> *

New methylene blue is an organic compound of the thiazine class of heterocycles. It is used as a stain and as an antimicrobial agent. It is classified as an azine dye, and the chromophore is a cation, the anion is often unspecified. Applications NMB is a stainin ...

Brilliant cresyl blue Brilliant cresyl blue is a supravital stain used for counting reticulocyte Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow a ...

Crystal violet Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. Crystal violet has antibacterial, antif ...

Methyl violet Methyl violet is a family of organic compounds that are mainly used as dyes. Depending on the number of attached methyl groups, the color of the dye can be altered. Its main use is as a purple dye for textiles and to give deep violet colors in pai ...

* Nile blue * Hoechst stain

References

Staining