Staining: Definition and Types

Table of Contents

What is Staining?

Staining, in microbiology, can be defined as a technique which is used to enhance and contrast a biological specimen at the microscopic level. Stains and dyes are used to highlight the specimen at the microscopic level to study it at higher magnification for histopathological studies and diagnostic purposes.

However, staining is not just limited to biological specimens, it can also be used to study the structure of crystalline polymers.

Preparation of the Biological Specimen

The preparation of the biological specimen to be analysed under a microscope depends on the type of staining. Given below are some procedures that are carried out.

  • Wet Mounting: Living biological specimens are mounted on a glass slide with water and specific stains.
  • Fixation: It is a multi-step process which is done to preserve the shape of cells and tissues. Heat fixation is done to kill and adhere the specimens. Chemical fixation is done to generate strong bonds and increase the rigidity of the samples. Common chemical fixatives used are formaldehyde, picric acid, methanol and ethanol.
  • Mordant: Mordants are chemical agents that are used along with dye to make the specimen stainable, which otherwise is unstainable. Mordants are of two types:
  • Basic Mordants: They react with acidic dyes.
  • Acidic Mordants: They react with basic dyes.

When staining is done with the help of mordants, it is known as indirect staining. On the other hand, when staining is done without the help of mordants, it is known as direct staining.

  • Permeabilisation: This procedure involves treating the specimen with a surfactant that dissolves the cell membrane allowing easy staining with the dye.

Types of Staining Techniques

  1. Gram’s staining: This staining procedure is used to identify bacteria based on their cell wall composition. There are two types of Gram’s staining, and the bacteria can be divided into gram positive or gram negative bacteria. It uses crystal violet for the staining of cell walls, iodine as the mordant and safranin or fuchsin as the counterstain.
  2. Endospore staining: This technique is used to find out the presence of endospores in bacteria. Endospores are tough, dormant structures that are less permeable to dyes and stains. Their presence makes the bacteria difficult to kill. The process involves heat fixing the specimen and dying it with a blend of safranin and 5% diluted malachite green.
  3. Ziehl-Neelsen staining: This technique is used to stain acid-fast bacteria such as Mycobacterium tuberculosis that do not stain with Gram’s staining. Carbol fuchsin is used as a stain, and methylene blue is used as a counterstain.
  4. Haematoxylin and Eosin staining: This staining procedure is used in histopathological studies to observe thin tissue sections. The hematoxylin stain colours the nuclei in blue and the cytoplasm and its components in red or pink.
  5. Periodic Acid-Schiff (PAS) staining: PAS staining is used to stain carbohydrate molecules. It is used on liver tissues, kidney, pancreas and ovaries to detect the presence of glycogen and help in the diagnosis of diseases.
  6. Masson staining: Masson’s trichrome is a three-colour staining procedure which is used for differentiating cells from connective tissues. It colours the muscle fibres and keratin to red, collagen to blue-green, cytoplasm to red-pink and nuclei into black colour.

Biological Stains

Some of the most commonly used biological stains are listed below:

  • Acridine orange: It is a fluorescent cationic dye that is selective to nucleic acids. It is used during the cell cycle to analyse DNA molecules.
  • Coomassie blue: It is used in gel electrophoresis to stain the proteins blue.
  • Crystal violet: It is used in Gram’s staining along with iodine to stain the bacterial cell wall in purple colour.
  • Eosin: It is used as a counterstain to hematoxylin that imparts a red colour to the cytoplasm and its components.
  • Ethidium bromide: It provides a red-orange fluorescent stain to the DNA after intercalating with the molecule.
  • Iodine: It is used as a mordant in Gram’s staining.
  • Malachite green: It gives a blue-green colour when used as a counterstain against safranin that is used in endospore staining.
  • Methylene blue: It is used to stain animal cells because it enhances the nuclei.
  • Safranin: It is a red cationic dye that is used as a counterstain in both Gram’s staining and endospore staining.

This sums up staining in general. Read more at BYJU’S Biology.

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