Staining for Light Microscopy

Staining The chemicals used for colouring are called dyes or stains. Stains are generally coloured, organic, aromatic substances. The choice of a stain depends upon the cell component under investigation, the fixative used and its reaction with the stain. Stains are defined as, “aromatic, salt-like, crystalline solids that dissolve in water in the form of coloured cations or anions or both. The ions combine with the cell components and give colour to them”. STAINING FOR LIGHT MICROSCOPY Each stain has two kinds of active chemical groups – chromophoric and auxochromic. The chromophoric group imparts colour to the stain. Some examples are: Nitro group Carboxyl group Azo group Indamin group Quinoid group --NO2 --COOH --N=N---N= Auxochromic group dissolves and dissociates in water and helps in the attachment to the cell component. Thus, with the attachment of the auxochrome group, the chromophoric group also gets attached giving the cell component the colour of the stain. Example: Hydroxyl (-OH) group. The staining property of picric acid is due to its nitro group (chromophore) which gives yellow colour to it. However, the attaching property is due to the hydroxyl group (auxochrome) which actually forms a salt with a base. Structural formula of picric acid Classification of Stains Depending upon the colour radical being anion or cation or both, stains are classified into three types: [A] ACIDIC STAINS The chromophoric group of the acidic stains is anion. The net charge on the anion of acidic stain is negative. Such stains have a greater combining capacity at acidic pH. They are used for staining cytoplasmic components like proteins. Acidic stains are used as salts of sodium or potassium and are neutral or slightly alkaline in reaction. These may contain nitro or quinoid groups, as chromophores. The auxochrome groups which actually combine with the proteins of the cytoplasm may be hydroxyl (-OH), carboxyl (-COOH), or sulphonic (HSO4) groups. The reaction here is said to be basic. 1 Examples: Acid Fuchsin, Methyl Blue, Eosin, Picric Acid, Orange G, Congo Red, Bismarck Brown, Fluorescein and Aniline Blue. A substance which shows affinity for acidic stain is termed acidophilic and the phenomenon is termed acidophilia. The acidophilic substances include the cytoplasmic components, especially proteins. [B] BASIC STAINS The chromophoric group of the basic stain is a cation. The net charge on the cation of the basic stain is positive. Such stains have a greater combining capacity at alkaline pH. They are generally used for staining the nucleus, especially nucleic acids. Basic stains stain the acidic substances and are dye salts of colourless acids. Examples: Basic Fuchsin, Thionine, Methyl Green, Acridine Red, Azure B, Crystal Violet, Haematoxylin and Safranine. A substance which shows affinity for basic stain is termed basiphilic and the property is termed as basiphilia. The basiphilic substances include the nucleic acids and chromosomes. [C] NEUTRAL STAINS The neutrals stains are similar to the other types of stains but their both anionic and cationic groups consist of chromophoric groups. Thus, these stains have a combined property of the acidic and basic stains. Examples: Janus Green B and Methylene Blue PROPERTIES OF STAINS The staining capacity of a stain depends on many factors. Two important ones are pH and composition of fixative. Formaldehyde as a fixative increases the tendency of proteins to accept basic stains. Mercuric compounds increase the tendency of proteins to accept acidic stains. MORDANTS Certain stains stain better with the use of mordants. A mordant is a chemical which binds with the cell component on one side and the stain on the other. Thus, it facilitates adsorption of the stain to the cell component for better staining. This phenomenon of combining with cell component and the stain is called lake formation. The mordants are generally double salts of potassium or ammonium with aluminium or ferric sulphate. Ferric ammonium sulphate is called ferric alum and is a common mordant. METACHROMATIC STAINS Some stains of the thiazine group stain various cell components in colours different from the colour of the stain itself. Such stains are called metachromatic stains and are mostly basic in nature. The phenomenon of colour change is called metachromasia. 2 Example: • • • Azure B is blue in colour. It stains the nucleus blue and the cytoplasm pink. Toluidine Blue stains nuclei blue, RNA of cytoplasm pink and glycoproteins red. Thionine The change in colour is due to the action of the dye on various cell components like nucleic acids, mucopolysaccharides and acidic lipids. The reaction is particularly strong in cells containing sulphate groups such as chondroitin sulphate present in cartilage and connective tissues. The action of the stain is due to the substrates which bind to the stain and convert it into dimer or polymer. The polymerized form is responsible for the change in colour. The change is unidirectional. This means, a green stain becomes bluer and a blue stain becomes reddish. The red stain changes into orange or yellow. VITAL STAINS These are used to stain living cells. Eg.: Janus Green B is a vital stain for staining mitochondria in living cells. This stain is green in colour in its oxidized state but becomes colourless in its reduced state. It is this property of Janus Green which is used for staining mitochondria. When cells are exposed to Janus Green B, the stain gets reduced in the cytoplasm but gets oxidized in the mitochondria by the enzyme cytochrome oxidase. Thus, mitochondria appear green in the colourless cells and are maintained in this state as long as cells remain alive. Neutral Red and Methylene Blue are two other vital stains. Neutral Red is used for demonstrating plant vacuoles. Methylene Blue stains nuclei and chromosomes in dividing cells. STAINING OF NUCLEIC ACIDS Methyl Green, Pyronin, Toluidine Blue and Azure B – stain both DNA and RNA. Feulgen stain is used for staining DNA. Methyl green stains DNA green and Pyronin stains RNA pink. The two stains are used either separately or as a mixture while staining the nucleic acids. The phosphoric acid of the nucleic acids carries a negative charge which reacts with the cationic group of basic dyes. The Schiff’s reagent for demonstrating DNA is called Feulgen Stain. The reaction is called Feulgen reaction or nuclear reaction. When DNA is subjected to mild hydrolysis with 1N HCl at 60˚C for 10-15 minutes, aldehyde groups are exposed from deoxyribose sugar by the removal of purine bases. Feulgen gives magenta colour to the DNA. The magenta colour is due to the presence of aldehyde groups which react with Schiff’s reagent. STAINING OF PROTEINS Those proteins which have a high proportion of acidic amino acids with negatively charged side chains, eg., aspartic acid and glutamic acid, are called acidic proteins. 3 Those proteins which have a predominating amount of basic amino acids like histidine, arginine and lysine with positively charged side chains are called basic proteins. Proteins with a balanced amount of acidic and basic amino acids are called neutral proteins. The acidic proteins bind with basic dyes while the basic proteins bind with acidic dyes. However, since the proteins are amphoteric in nature, i.e., the proteins can ionize as acids or bases according to the medium in which they are present, an acid protein can bind with an acid dye and a basic protein can bind with a basic dye. Protein staining is also affected by pH and composition of the fixatives. Stain Naphthol Yellow S Mercuric Bromophenol Blue Fast Green Millon Reagent Sakaguchi Reagent Mercury Orange Diazonium Hydroxide Reacts with Lysine, Arginine, Histidine Lysine, Arginine, Histidine Total proteins or Histones alone (basic) Tyrosine Arginine --SH group containing proteins Histidine, Tryptophan, Tyrosine STAINING OF CARBOHYDRATES Polysaccharides like starch, cellulose, hemicellulose, mucoproteins and chitin are demonstrated by Periodic Acid-Shiff’s (PAS) reaction. The staining is based on the principle of exposing aldehyde groups from polysaccharides which react with Schiff’s reagent giving magenta colour. Periodic acid attacks carbon chains at sites of adjacent hydroxy, hydroxyamino, and hydroxy-imino groups. The adjacent groups (1,2-glycol) are oxidized to aldehydes with concomitant breaking of the carbon chain. STAINING OF LIPIDS • • • • Eg.: Sudan Black B, Sudan Red III, Sudan IV, Oil Red O, Osmium Tetroxide. Sudan Black B is specific for phospholipids. Golgi complex containing phospholipids stain well with this stain. Sudan III or Sudan IV give scarlet red colour to the lipids. Osmium tetroxide stains fats by reacting with unsaturated fatty acids forming black oxides of osmium. Schiff’s reagent reacts with stearic and palmitic acids – Plasmal Reaction. STAINING OF ENZYMES Enzymes get inactivated by chemical fixation. So, they have to fixed by special methods like freezing (non-chemical fixation). Gomori method and alkaline phosphatase reaction are the two methods used to demonstrate enzymes. QUANTITATIVE DETERMINATION BY STAINING METHODS Nucleic acids have characteristic wavelength of absorption in the UV region of the spectrum (260 nm). This property is used for identification of DNA and RNA. The specific absorption is due to the purine and pyrimidine bases in the nucleic acids. 4 DNA in cells is stained with Feulgen reaction and photometric techniques are applied for its quantitative determination. According to Lambert’s law, the proportion of incident light absorbed by a stained material is directly related to its thickness. The Beer’s law states that the proportion of light absorbed is directly related to the concentration of the absorbing molecules. The two laws together are called Beer-Lambert’s law. So the magenta colour with Feulgen stain depends upon the amount of DNA present, i.e., more DNA means more colour, and hence, more absorption of light. Hence, one can determine the amount of DNA present in the cell by using spectrophotometric techniques. STAINING FOR ELECTRON MICROSCOPY Electron microscopy requires no stains as the image is formed by scattering of electrons and not by the absorption of light rays. The biological materials contain C, H, O and N atoms, which are very poor electron scatterers. Therefore, electron micrographs (pictures) will show poor contrast. To increase contrast, heavy metal salts are used. They increase contrast due to increased electron scattering. Examples: Lead Acetate, Lead Citrate, Uranyl Acetate, Phosphotungstic Acid. Such chemicals are called as electron stains. They do not give coloured pictures but do combine with cell components as histological stains and provide good contrast to various cell organelles. 5