Staining

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
In biochemistry, it involves adding a class-specific dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis. Light microscopes are used for viewing stained samples at high magnification, typically using bright-field or epi-fluorescence illumination.
Staining is not limited to only biological materials, since it can also be used to study the structure of other materials; for example, the lamellar structures of semi-crystalline polymers or the domain structures of block copolymers.

''In vivo'' vs ''In vitro''

In vivo staining is the process of dyeing living tissues. By causing certain cells or structures to take on contrasting colours, their form 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.
In vitro staining involves colouring cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone. Combined with specific protocols for fixation and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools. A counterstain is stain that makes cells or structures more visible, when not completely visible with the principal stain.

Crystal violet stains both Gram positive and Gram negative organisms. Treatment with alcohol removes the crystal violet colour from gram negative organisms only. Safranin as counterstain is used to colour the gram negative organisms that got decolorised by alcohol.

While ex vivo, many cells continue to live and metabolize until they are "fixed". Some staining methods are based on this property. Those stains excluded by the living cells but taken up by the already dead cells are called vital stains. Those that enter and stain living cells are called supravital stains. However, these stains are eventually toxic to the organism, some more so than others. Partly due to their toxic interaction inside a living cell, when supravital stains enter a living cell, they might produce a characteristic pattern of staining different from the staining of an already fixed cell. To achieve desired effects, the stains are used in very dilute solutions ranging from to . Note that many stains may be used in both living and fixed cells.

Preparation

The preparatory steps involved depend on the type of analysis planned. Some or all of the following procedures may be required.
Wet mounts are used to view live organisms and can be made using water and certain stains. The liquid is added to the slide before the addition of the organism and a coverslip is placed over the specimen in the water and stain to help contain it within the field of view.
Fixation, which may itself consist of several steps, aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and alter the specimen so it accepts stains. Most chemical fixatives generate chemical bonds between proteins and other substances within the sample, increasing their rigidity. Common fixatives 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.
Mordants are chemical agents which have power of making dyes to stain materials which otherwise are unstainable
Mordants are classified into two categories:
a) Basic mordant: React with acidic dyes e.g. alum, ferrous sulfate, cetylpyridinium chloride etc.
b) Acidic mordant : React with basic dyes e.g. picric acid, tannic acid etc.
Direct Staining: Carried out without mordant.
Indirect Staining: Staining with the aid of a mordant.

Sr No.	Name of Indirect Staining Technique	Name of mordant applied
1.)	Gram's Staining	Gram's iodine
2.)	Cell Wall Staining a.) Ringer's method b.) Dyar's method	10% Tannic acid 0.34% C.P.C
3.)	Flagella Staining a.) Leifson's method b.) Loeffler's method	Tannic acid in Leifson's stain Loeffler's mordant	Spirochete Staining a.) Fontana's method b.) Becker's method	Fontana's mordant Fontana's mordant

Permeabilization involves treatment of cells with a mild surfactant. This treatment dissolves cell membranes, and allows larger dye molecules into the cell's interior.
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 the sample can be directly applied to a slide. For larger pieces of tissue, thin sections are made using a microtome; these slices can then be mounted and inspected.

Standardization

Most of the dyes commonly used in microscopy are available as BSC-certified stains. This means that samples of the manufacturer's batch have been tested by an independent body, the Biological Stain Commission, and found to meet or exceed certain standards of purity, dye content and performance in staining techniques ensuring more accurately performed experiments and more reliable results. These standards are published in the commission's journal Biotechnic & Histochemistry. Many dyes are inconsistent in composition from one supplier to another. The use of BSC-certified stains eliminates a source of unexpected results.
Some vendors sell stains "certified" by themselves rather than by the Biological Stain Commission. Such products may or may not be suitable for diagnostic and other applications.

Negative staining

A simple staining method for bacteria that is usually successful, even when the positive staining methods fail, is to use a negative stain. This can be achieved by smearing the sample onto the slide and then applying nigrosin or India ink. After drying, the microorganisms may be viewed in bright field microscopy as lighter inclusions well-contrasted against the dark environment surrounding them. Negative staining is able to stain the background instead of the organisms because the cell wall of microorganisms typically has a negative charge which repels the negatively charged stain. The dyes used in negative staining are acidic. Note: negative staining is a mild technique that may not destroy the microorganisms, and is therefore unsuitable for studying pathogens.

Positive staining

Unlike negative staining, positive staining uses basic dyes to color the specimen against a bright background. While chromophore is used for both negative and positive staining alike, the type of chromophore used in this technique is a positively charged ion instead of a negative one. The negatively charged cell wall of many microorganisms attracts the positively charged chromophore which causes the specimen to absorb the stain giving it the color of the stain being used. Positive staining is more commonly used than negative staining in microbiology. The different types of positive staining are listed below.

Simple versus differential

Simple Staining is a technique that only uses one type of stain on a slide at a time. Because only one stain is being used, the specimens or background will be one color. Therefore, simple stains are typically used for viewing only one organism per slide. Differential staining uses multiple stains per slide. Based on the stains being used, organisms with different properties will appear different colors allowing for categorization of multiple specimens. Differential staining can also be used to color different organelles within one organism which can be seen in endospore staining.

Types

Techniques

Gram

is used to determine gram status to classifying bacteria broadly based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine, and a fuchsin or safranin counterstain to. Gram status, helps divide specimens of bacteria into two groups, generally representative of their underlying phylogeny. This characteristic, in combination with other techniques makes it a useful tool in clinical microbiology laboratories, where it can be important in early selection of appropriate antibiotics.
On most Gram-stained preparations, Gram-negative organisms appear red or pink due to their counterstain. Due to the presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex can pass through. Thus, the primary stain is not retained. In addition, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide.

Endospore

is used to identify the presence or absence of endospores, which make bacteria very difficult to kill. Bacterial spores have proven to be difficult to stain as they are not permeable to aqueous dye reagents. Endospore staining is particularly useful for identifying endospore-forming bacterial pathogens such as Clostridioides difficile. Prior to the development of more efficient methods, this stain was performed using the Wirtz method with heat fixation and counterstain. Through the use of malachite green and a diluted ratio of carbol fuchsin, fixing bacteria in osmic acid was a great way to ensure no blending of dyes. However, newly revised staining methods have significantly decreased the time it takes to create these stains. This revision included substitution of carbol fuchsin with aqueous Safranin paired with a newly diluted 5% formula of malachite green. This new and improved composition of stains was performed in the same way as before with the use of heat fixation, rinsing, and blotting dry for later examination. Upon examination, all endospore forming bacteria will be stained green accompanied by all other cells appearing red.