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 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 (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells), or organelles within individual cells.
In biochemistry, it involves adding a class-specific (DNA, proteins, lipids, carbohydrates) 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 (also called vital staining or intravital staining) is the process of dyeing living tissues. By causing certain cells or structures to take on contrasting colours, 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.
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 (e.g. trypan blue or propidium iodide for eukaryotic cells). Those that enter and stain living cells are called supravital stains (e.g. New Methylene Blue and brilliant cresyl blue for reticulocyte staining). 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 (e.g. "reticulocyte" look versus diffuse "polychromasia"). To achieve desired effects, the stains are used in very dilute solutions ranging from to (Howey, 2000). 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 (chemicals causing fixation) 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. +Table represents Indirect Staining Techniques and mordants applied in each:Sr No.Name of Indirect Staining TechniqueName of mordant applied1.)Gram's StainingGram's iodine2.)Cell Wall Staining a.) Ringer's method
b.) Dyar's method10% Tannic acid
0.34% C.P.C3.)Flagella Staining a.) Leifson's method
b.) Loeffler's methodTannic acid in Leifson's stain
Loeffler's mordant (20%Tannic acid )4.)Spirochete Staining a.) Fontana's method
b.) Becker's methodFontana's mordant(5%Tannic acid)
Fontana's mordant(5%Tannic acid) Permeabilization involves treatment of cells with (usually) 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 (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.
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 (BSC), 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 (a black synthetic dye) or India ink (an aqueous suspension of carbon particles). 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 (for positive stains) or background (for negative stains) 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 + Types of staining techniquesSr. No.Staining TechniquePreparationApplicationResult1.Simple (Monochrome)Smear stain with single dye . e.g. Methylene blue, Safranin°≤×←→ etc.Used to highlight microbes and illustrate cellular shapes and arrangementsOrganisms are stained in the color of applied stain2.Negative (Relief)Smear mixed with Nigrosin and spread into thin filmStudy cell morphologyOrganism is stained, the background is black3GramPrimary stain: Crystal violet applied to film then treated with iodine (mordant), alcohol (decolourizer) and counter stained with safraninCharacterizes bacteria in one of two groups, Gram positive or Gram negativeGram positive appears purple in color Gram negative appears pink in color4Acid fast (Ziehl-Neelsen technique)Film stained with hot Z.N.C.F. decolourised (acid-alcohol) and counter stain with methylene blueSeparate non-decolorized acid fast bacteria that are not decolorized from colorized non-acid fast bacteriaAcid fast bacteria:Red Non acid fast: Blue5Endospore (Dornor's method)Primary stain Malachite green heat fixed to penetrate spores; vegetative cells are counterstained with SafraninDetects the presence of endospores in six genera of bacteriaEndospores: Green Vegetative cells: Red6Capsule A: Hiss method (Positive technique)
B: Manevals's technique (Negative)Smear stained with Hiss stain following treatment with copper sulphate
Bacterial suspension smeared along with Congo red and the Maneval's stain is appliedCapsules can be observed as clear zones surrounding cells of capsulated bacteria and are used to demonstrate the presence of capsules.Capsule: Light violet/pale mauve color Bacteria: Purple capsule, bacterial cell, stands out against dark background7Cell wall (Dyar's method)Smear treated with C.P.C. which dissociates to form positively charged cetyl pyridinium and negatively charged chloride ions. Positively charged ions are adsorbed on negatively charged cell wallStains cell wall of bacteriumCell wall: Red Cytoplasm: Blue8Flagella (Leifson's method)Mordant acts to thicken flagella before staining and increases visibility microscopically when stained with Leifson stainDemonstrates presence of flagellaFlagella: Red Vegetative cells: Blue9Nuclear material (Feulgen technique)Smear is treated for hydrolysis to release purines from DNA, purines to cause shift form furanose to aldehyde. Aldehyde groups are available to react with schiff's reagent to form addition compounds.To demonstrate the presence of DNA in cell. But for detection of the DNA, RNA should be selectively destroyed by acid hydrolysis without affecting DNANuclear material- pinkish purple,