Staining techniques used in diagnostic microbiology

Staining techniques used in diagnostic microbiology

Staining techniques used in diagnostic microbiology

Bacteria can be studied either in unstained (wet mount) or stained preparation. The wet mount preparation is useful for demonstration of motility of bacteria by light microscopy or demonstration of spirochetes by dark-ground microscopy. Stained preparations are used to demonstrate structural details of the bacteria by producing color contrast.

Staining Methods

 Simple stains

Methylene blue and basic fuchsin are the simple stains that provide color contrast but impart the same color to all the bacteria in a smear.

Negative staining

Bacteria are mixed with dyes, such as Indian ink that produces a uniform dark-colored background against which the unstained organisms stand out in contrast. This is used for demonstration of bacterial capsule that are usually not stained by simple stains. India ink method for demonstration of the fungus Cryptococcus neoformans is a common example.

Impregnation stains

Cells and structures that are too thin to be visualized by the light microscope can be rendered visible by impregnation of silver on their surface. Silver impregnation method is a common method used for staining spirochetes, such as T. pallidum, Leptospira, Borrelia, etc.

Differential stains

Differential stains impart different colors to different bacteria or different bacterial structures. The commonly used differential stains include Gram stain, acid-fast stain, and Albert’s stain.

Gram stain: Gram stain was devised by Christian Gram, a Danish microbiologist, in 1884, as a convenient method for classifying bacteria. Gram staining is an essential procedure that is used in the identification of bacteria. The stain differentiates bacteria into two broad groups:

1. Gram-positive bacteria are those that resist decolorization and retain the primary dye-iodine complex, appearing violet. They have a relatively thick amorphous wall and more acidic protoplasm which are believed to retain the basic violet dye and iodine complex within the cell.

2. Gram-negative bacteria are decolorized by organic solvents and take counterstain, appearing red. The decolorizing agent, such as acetone or ethanol, used during staining disrupts this membran-ous envelope, and the dye and iodine complex is washed out of Gram-negative bacteria.

The Gram staining method essentially consists of four steps:

(i) Primary staining with basic dyes, such as methyl violet, crystal violet, etc.

(ii) Application of mordant in the form of dilute solution of iodine.


(iii) Decolorization with ethanol, acetone, or aniline. (iv) Counterstaining with acidic dyes, such as carbol fuchsin, safranine, or neutral red.

There are certain groups of bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae that cannot be considered typical Gram-negative or Gram-positive bacteria. This is because these bacteria either do not take up the Gram stain or they have a different type of envelope. These mycobacteria possess a waxy envelope containing complex glycolipids that make them impervious to the Gram stain. Gram stain also provides useful information on the structure of bacterial cell envelope.

Acid-fast stain: The acid-fast stain was discovered by Ehrlich, who found that after staining with aniline dyes, tubercle bacilli resist decolorization with acid. The method, subsequently, was modified by Ziehl and Neelsen, hence is widely known as Ziehl– Neelsen (ZN) stain. Acid fast staining method consists of following methods:

Fixed smears are first stained by a strong carbol fuchsin with the application of heat. Heating facilitates entry of phenolic carbol fuchsin stain into the bacteria.

It is then decolorized with 5–20% (depending on the bacteria to be stained) sulfuric acid.

It is then counterstained with a contrasting dye, such as methylene blue. The acid-fast bacilli (AFB) retain the red color of carbol fuchsin and appear bright red in stained smears. Pus cells and epithelial cells present in the smear, on other hand, take up the blue color of the counterstain and appear blue.

Acid fastness is due to the (a) high content of lipids, fatty acids, components of mycolic acid, and (b) higher alcohols found in the cell wall of the Mycobacterium. Acid fastness also depends on integrity of the cell wall. 

The ZN smear is best used for staining:

M. tuberculosis, M. leprae, Nocardia, Actinomyces, and Oocysts of intestinal coccidian parasites (such as Cryptosporidium, Cyclospora, Isospora, etc.).

Albert’s stain: Albert’s stain is used for staining the volutin granules of C. diphtheriae. These granules have an affinity for basic dyes and are called metachromatic granules. These granules are stained bluish-black against green protoplasm on staining by Albert’s stain.

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