Staining in microbiology, and Its techniques

 What is the staining technique?

              Staining in microbiology involves adding dyes to the smear that are either charged-attractive (cationic dyes like methylene blue or crystal violet) or charged-repelling (anionic dyes like eosin or India ink). Bacterial cells are bound by cationic dyes, making them visible against the light background.

What are the types of staining techniques?

         The following are the types of staining techniques:

What is the preparation of biological specimens?

The type of staining determines how the biological specimen is prepared for microscopy analysis.

Wet mounting:

           It is the process of mounting living biological specimens on a glass slide using water and certain dyes.


          It is a multi-step procedure used to maintain the structure of tissues and cells. To destroy and adhere to the specimens, heat fixation is used. Chemical fixation creates solid connections and boosts the samples’ stiffness. For example, Chemical fixatives include formaldehyde, picric acid, methanol, and ethanol.


         A mordant, also known as a dye fixative, is a material used to set (or bind) dyes on textiles by forming a coordination complex with the dye, which subsequently adheres to the textile (or tissue). It can be applied to color fabrics or make cell or tissue preparations’ stains more intense. Even though few small batch dyers still employ mordants, directors have mostly taken their place in the industry.  There are two types of mordants:

Indirect staining is the process of staining with the aid of mordants. Direct staining, on the other hand, is the process of staining without the aid of mordants.


Using a surfactant to prepare the specimen will disintegrate the cell membrane, allowing for simple dye staining.

 What are the types of staining techniques?

Gram staining:

               By using this staining technique, bacteria can be distinguished depending on the makeup of their cell walls. Bacteria can be classified as gram-positive or gram-negative based on the two different forms of Gram staining. It uses safranin or fuchsin as the counterstain, iodine as the mordant, and crystal violet to stain the cell walls.

Endospore staining:

                The form of staining used to identify the presence of endospores in bacterial vegetative cells is called endospore staining. The bacterial endospores require a dye that can penetrate the spore bacteria’s strong wall. Using Malachite Green, the Schaeffer Fulton method is one way to stain endospores.

Ziehl-Neelsen staining:

                A bacteriological stain called Ziehl-Neelsen (also known as acid fast staining) is used to identify organisms that grow quickly in acid, primarily Mycobacteria. It bears the names of two German medical professionals who improved the stain: pathologist Friedrich Neelsen and bacteriologist Franz Ziehl

Haematoxylin and Eosin staining:

                 Hematoxylin and eosin staining offer crucial details regarding the distribution, morphology, and structure of cells in a tissue sample in addition to aiding in the identification of various cell and tissue types. It aids in the diagnosis of illnesses like cancer. known as H and E stains as well.

Periodic Acid-Schiff (PAS) staining:

                 The staining of carbohydrate molecules is done using PAS. To find glycogen and aid in illness diagnosis, it is applied to the tissues of the kidney, pancreas, liver, and ovaries.

Masson staining:

            The three-color staining technique known as Masson’s trichrome is employed to distinguish cells from connective tissues. Muscle fibers and keratin turn red, collagen turns blue-green, cytoplasm turns red-pink, and nuclei turn black when exposed to it. 

What are biological stains?

Below is a list of some of the most popular biological stains:

Acridine orange:

         The fluorescent cationic dye acridine orange is specific for nucleic acids. DNA molecules are examined while the cell cycle is in progress.

Coomassie blue:

        In gel electrophoresis, Coomassie blue is used to color the proteins blue.

Crystal violet:

        When Gram’s staining bacteria, crystal violet, and iodine are employed to give the bacterial cell wall a purple color.


        This counterstain to hematoxylin gives the cytoplasm and its constituent parts a crimson hue.

Ethidium bromide:

          After intercalating with the molecule, it gives the DNA a red-orange fluorescent stain.


        Iodine is a mordant used in the Gram staining process.

Malachite green:

        When used as a counterstain to safranin, which is employed in endospore staining, it produces a blue-green tint.

Methylene blue:

        Because it highlights the nuclei, it is used to stain animal cells.


          Safranin is a red cationic dye that is employed as a counterstain for endospore and Gram stains.


Rimsha Bashir
Rimsha Bashir

Rimsha Saith is a highly knowledgeable microbiologist with a keen interest in the field. Her expertise and passion are in her writing for Microbiology. As a writer, Rimsha has authored numerous articles that have been well-received by both health and medical students and industries.

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