stain (biology)

Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of a light microscope. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells. Biological stains are also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.

In vitro staining

In vitro staining involves colouring cells or structures that are no longer living. (In vitro means literally "in glass"; compare with in vivo.)

Preparation

The preparatory steps involved depend on the type of analysis planned; some or all of the following procedures may be required. Permeabilization involves treatment of cells with (usually) a mild detergent. This detergent treatment will dissolve the cell membranes, and allow larger dye molecules access to the cell's interior. Fixation–which may itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Most fixatives (chemicals causing fixation) generate chemical bonds between proteins and other substances within the sample, increasing their rigidity. Common fixative solutions often include formaldehyde, ethanol, methanol, and/or picric acid. Pieces of tissue may be embedded in paraffin wax to increase their mechanical strength and stability and to make them easier to cut into thin slices. Mounting usually involves attaching the samples to a glass microscope slide for observation and analysis. In some cases, cells may be grown directly on a slide. For samples of loose cells (as with a blood smear or a pap smear) the sample can be directly applied to a slide. For larger pieces of tissue, thin sections (slices) are made using a microtome; these slices can then be mounted and inspected.

Staining

At its simplest, the actual staining process may involve immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation. Many dyes, however, require the use of a mordant: a chemical compound which reacts with the stain to form an insoluble, coloured precipitate. When excess dye solution is washed away, the mordanted stain remains. Some staining protocols may require multiple steps of fixating, staining, and mordanting in order to produce distinct images of different structures. A counterstain is stain added which makes visible cells or structures not coloured by the principal stain. (For example, crystal violet stains only Gram-positive bacteria in Gram staining. A safranin counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well.)

In vivo staining

In vivo staining is the process of dyeing living tissues—in vivo means "in life" (compare with in vitro staining). By causing certain cells or structures to take on contrasting color(s), their form (morphology) or position within a cell or tissue can be readily seen and studied. The usual purpose is to reveal cytological details that might otherwise not be apparent; however, staining can also reveal where certain chemicals or specific chemical reactions are taking place within cells or tissues. Often these stains are called vital stains. They are introduced to the organism while the cells are still living. However, these stains are eventually toxic to the organism, some more so than others. To achieve desired effects, the stains are used in very dilute solutions ranging from 1:5,000 to 1:500,000 (Howey, 2000). Note that many stains may be used in both living and fixed cells.

Basic biological stains

Different stains react or concentrate in different parts of a cell or tissue, and these properties are used to advantage to reveal specific parts or areas. Some of the most common biological stains are listed below. Unless otherwise marked, all of these dyes may be used with fixed cells and tissues; vital dyes (suitable for use with living organisms) are noted.

Bismarck brown

Bismarck brown (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid mucins. Bismarck brown may be used with live cells.

Carmine

Carmine is an intensely red dye which may be used to stain glycogen, while Carmine alum is a nuclear stain. Carmine stains require the use of a mordant, usually aluminum.

Coomassie blue

Coomassie blue (also brilliant blue) nonspecifically stains proteins a strong blue colour. It is often used in gel electrophoresis.

Crystal violet

Crystal violet, when combined with a suitable mordant, stains cell walls purple. Crystal violet is an important component in Gram staining.

DAPI

DAPI is a fluorescent nuclear stain, excited by ultraviolet light and showing strong blue fluorescence when bound to DNA. DAPI is not visible with regular transmission microscopy. It may be used in living or fixed cells.

Eosin

Eosin is most often used as a counterstain to haematoxylin, imparting a pink or red colour to cytoplasmic material, cell membranes, and some extracellular structures. It also imparts a strong red colour to red blood cells. Eosin may also be used as a counterstain in some variants of Gram staining, and in many other protocols. There are actually two very closely related compounds commonly referred to as eosin. Most often used is eosin Y (also known as eosin Y ws or eosin yellowish); it has a very slightly yellowish cast. The other eosin compound is eosin B (eosin bluish or imperial red); it has a very faint bluish cast. The two dyes are interchangeable, and the use of one or the other is more a matter of preference and tradition.

Ethidium bromide

Ethidium bromide intercalates and stains DNA. It is a fluorescent red-orange stain. Although it will not stain healthy cells, it can be used to identify cells in the late stages of apoptosis; such cells have much more permeable membranes. Consequently, ethidium bromide is often used as a marker for apoptosis in populations of cells. Ethidium bromide is also used to locate bands of DNA in gel electrophoresis.

Fuchsin

Fuchsin may be used to stain collagen, smooth muscle, or mitochondria. It is frequently used as part of Masson's trichrome.

Haematoxylin

Haematoxylin is a nuclear stain. Used with a mordant, haematoxylin stains nuclei blue-violet or brown. It is most often used with eosin in H&E (haematoxylin and eosin) staining—one of the most common procedures in histology.

Hoechst stains

Hoechst 33258 and Hoechst 33342 are two closely related fluorescent stains. They fluoresce strongly when bound to DNA, but are not visible under transmitted light. The two compounds are functionally very similar, and both may be used in living cells.

Iodine

Iodine is used in chemistry as an indicator for starch. When starch is mixed with iodine in solution, an intensely dark blue color develops, representing a starch/iodine complex. Starch is a substance common to most plant cells and so a weak iodine solution will stain starch present in the cells. Iodine is one component in the staining technique known as Gram staining, used in microbiology. Lugol's solution or Lugol's iodine (IKI) is a brown solution that turns black in the presence of starches and can be used as a cell stain, making the cell nuclei more visible.

Malachite green

Malachite green (also known as diamond green B or victoria green B) can be used as a blue-green counterstain to safranin in the Gimenez staining technique for bacteria. It also can be used to directly stain spores.

Methyl green

Methyl green is chemically related to crystal violet, sporting an extra methyl or ethyl group.

Methylene blue

Methylene blue is used to stain animal cells, such as human cheek cells, to make their nuclei more observable.

Neutral red

Neutral red (or toluylene red) stains nuclei red. It is usually used as a counterstain in combination with other dyes.

Nile blue

Nile blue (or Nile blue A) stains nuclei blue. It may be used with living cells.

Nile red

Nile red (also known as Nile blue oxazone) is formed by boiling Nile blue with sulfuric acid. This produces a mix of Nile red and Nile blue. Nile red is a lipophilic stain; it will accumulate in lipid globules inside cells, staining them red. Nile red can be used with living cells.

Rhodamine

Rhodamine is a fluorescent stain.

Safranin

Safranin (or Safranin O) is a nuclear stain. It produces red nuclei, and is used primarily as a counterstain. Safranin may also be used to give a yellow colour to collagen.