Identification of Microbes : Stains and types of staining techniques << Back

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Identification of Microbes : Stains and types of staining techniques


What is staining?

·Colored compound is used to develop a contrast between the specimen and the background. A process of imparting colour to the cell  is known as staining.


What is the difference between stain and dye?

·         A dye is colouring agent used for general purposes and a stain is used for any biological specimen staining. Also dye is crude and stain is purified form. 

·         Dyes are the textile colouring agents that have been prepared with lesser specifications and they may contain  the impurities . Stains are the biological colouring agents that are more pure and prepared with greater care and specification.


Why stain cells?

·         Cells are stain to reveal the size and shape of microorganisms.

·         The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope.

·         Cells may also be stained to highlight metabolic processes.

·         To differentiate between live and dead cells in a sample.

·         Cells may also be enumerated by staining cells to determine biomass in an environment of interest.

·         Cells are stained to demonstrate the presence of  internal and external structures.

·         Cells are stained to distinguish between different types of organisms.




Chemistry of Dye


Definition of Dye

dye is a coloured substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber.

Chemically a dye (stain) may be defined as an organic unsaturated cyclic compound with chromophore and auxochrome group. The colour is usually due to chromophore and dyeing property of salt formation is due to auxochrome.

A chemical possessing only chromophore group may be a good chromogen (Colored compound) but may not be a good stain/dye unless and until it has an auxochrome group. Without an auxochrome group the chromogen is not able to bind to cells or tissues or fibers. The ability of a stain to bind to macromolecular cellular  components depend on the electrical charge found on the chromogen portion as well as on the cellular components to  be stained.

Both dyes and pigments appear to be colored because they absorb some wavelengths of light more than others. In contrast with a dye, a pigment generally is insoluble, and has no affinity for the substrate. Some dyes can be precipitated with an inert salt to produce a lake pigment, and based on the salt used they could be aluminum lake, calcium lake or barium lake pigments.



Simple Stains
They are also referred as monochrome stains, since only one dye is employed for the colouration of bacterial smear (The act of taking bacteria taken from a lesion or area, spreading them on a slide, and staining them for microscopic examination.). The surface of a bacterial cell has an overall acidic characteristic because of large amount of carboxyl groups located on the cell surface de to acidic amino acids. Therefore, when ionization of carboxyl groups takes place it imparts negative charge to the cell surface as per the following equation.

COOH → COO- + H+

H+ is removed and the surface of the bacteria becomes  negatively charged and a positively charged dye like (methylene blue) attaches to the negatively surface and gives it a coloured appearance.

Methylene blue chloride →  Methylene Blue++ Cl‑

Negative Stains
Negative staining is a technique (used mainly in electron microscopy) by which bacterial cells are not stained, but are made visible against dark background. 
Acidic dyes like eosin and nigrosin are employed for this method. Though, this staining technique is not very popular, it has an advantage over the direct or positive staining methods for the study of morphology of cells. This is because of the fact that the cells do not receive vigorous physical or chemical treatments.

The colouring power of acidic dye e.g. eosin in sodium eosinate is having negative charge, therefore, it does not combine with the negatively charged bacterial cell surface. On the other hand, it forms a deposit around the cell, resulting into appearance of bacterial cell colourless against dark background.  Some suitable negative stains include ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osmium ferricyanide and auroglucothionate. These have been chosen because they scatter electrons well and also adsorb to biological matter well. The method is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power.

Differential Stains
Staining procedure which differentiates or distinguishes between types of bacteria is termed as differential staining technique. Methods for simple staining impart same colour to all bacteria and other biological material, may be slight variation in shade. On the other hand, differential staining methods impart distinctive colour only to certain types of bacteria.

The basic principle underlying this differentiation is due to the different chemical and physical properties of cell and as a result, they react differently with the staining reagents. Differential staining procedure utilizes more than one stain. In some techniques the stains are applied separately, while in other as combination. There are two most important differential stains, namely, (A) Gram stain and (B) Acid-fast stain.

(A) Gram Stain

Gram stain is one of the most important and widely used differential stains. It has great taxonomic significance and is often the first step in the identification of an unknown prokaryotic organism. This technique divides bacteria into two groups (i) Gram positive those which retain primary dye like crystal violet and appear deep violet in colour and (ii) Gram negative, which lose the primary dye on application of decolourizer and take the colour of counterstain like safranin or basic fuchsin.

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stains purple while gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer membrane which contains lipids. There are four basic steps of the Gram stain, which include applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, followed by the addition of a trapping agent (Gram’s iodine), rapid decolorization with alcohol or acetone, and counterstaining with safranin. Basic fuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.

Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color the cell.

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell will lose its outer membrane and the lipopolysaccharide layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or red color.

Acid Fast Stains
Acid fast staining is another widely used differential stain­ing procedure in bacteriology. This stain was developed by Paul Ehrlich in 1882, during his work on etiology of tuber culosis (5). Some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms. Acid alcohol is very intensive decolourizer. This staining technique divides bacteria into two groups (i) acid-fast and (ii) non acid-fast. This procedure is extensively used in the diagnosis of tuberculosis and leprosy.

Acid-fastness property in certain Mycobacteria and some species of Nocardia is correlated with their high lipid content. Due to high lipid content of cell wall, in some cases 60% (w/w), acid-fast cells have relatively low permeability to dye and hence it is difficult to stain them. For the staining of these bacteria, penetration of primary dye is facilitated with the use of 5% aqueous phenol which acts as a chemical intensifier. In addition, heat is also applied which acts as a physical intensifier. Once these cells are stained, it is difficult to decolourize
Ziehl-Neelsen method:
Ziehl (6) and Neelsen (7) independently proposed acid fast stain, in 1882-1883 is commonly used today. The staining reagents are much more stable than those described by Ehrlich.
The procedure for staining is as follows. Prepare a smear and fix it by gentle heat. Flood the smear with carbol fuchsin (S19) and heat the slide from below till steam rise for 5 minutes.
Do not boil and ensure that stain does not dry out. Allow the’ slide to cool for 5 minutes to prevent the breakage of slide in the subsequent prevent step. Wash well with water. Decolourize the smear till red colour no longer comes out in 20% sulphuric acid. Wash with water. Counterstain with 1% aqueous solution of malachite green or Loeffler’s methylene blue (S18) for 15-20 seconds. Wash, blot dry and examine under oil-immersion objective
Endospore staining

Bacterial endosporesare metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions.  The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state.  The primary stain applied is malachite green, which stains both vegetative cells and endospores.  Heatis applied to help the primary stain penetrate the endospore.    The cells are then decolorized with water, which removes the malachite green from the vegetative cell but not the endospore.  Safraninis then applied to counterstainany cells which have been decolorized.  At the end of the staining process, vegetative cells will be pink, and endospores will be dark green.