AAT Bioquest

What are the top types of staining techniques in microbiology?

Posted September 18, 2023


Staining helps to increase the contrast between microorganisms and the background, enabling researchers to study the structural details of the microbe at higher magnification. 

Based on the types and number of dyes used, the top staining can be categorized into the following types: 

  1. Simple Staining: Simple staining uses only a single dye to determine the size, shape and arrangement of cells in the microorganisms. Crystal Violet, Methylene Blue, and Basic Fuchsin are the top three stains used in the simple staining technique. They produce color contrast between the microbes and the background but impart all microbes with the same color. 
  2. Negative Staining: This staining technique is used when a specimen or part of it does not take up simple stains. India Ink is generally used for negative staining. It stains the background black so the unstained capsule stands out in contrast. 
  3. Flagella Stain: This staining technique is used to determine the presence, number, and arrangement of flagella, which is necessary for identifying species of motile bacteria. Because flagella are too thin to be visible under a light microscope, flagella stains are especially formulated with silver salts, which coat the surface of the flagella, thickening the structures so they can be seen and studied more easily.  
  4. Differential Staining: Differential staining involves the use of more than one dye to differentiate microorganisms based on their staining properties. Some of the differential staining techniques include: 
    • Gram stainingGram staining uses two stains to differentiate bacteria into two categories - Gram positive and Gram negative. It serves as a vital tool in the initial characterization and classification of bacteria, aiding in the identification of bacterial pathogens through their Gram reaction (positive or negative) and morphological traits (cocci/rod).

    • Acid-fast staining - Acid-fast staining using the Ziehl-Neelsen technique is used to stain Mycobacterium species. It helps to differentiate between acid-fast bacteria and non-acid fast bacteria, which do not stain well by the Gram staining.  

    • Endospore staining – This differential staining technique helps to distinguish the relatively few species of endospore-producing bacteria from those that don’t produce endospores. Endospore staining highlights the spore structure as well as free spores. 

    • Acridine orange staining – Acridine orange binds to nucleic acid and stains them, enabling the detection of mycoplasmas or bacteria that do not have a cell wall. This staining technique is useful for detecting the presence of bacteria in blood cultures when Gram stain results are difficult to interpret or when light microscopy results are indistinguishable. 

    • Capsule staining – Capsule staining confirms the presence of capsules in certain species of bacteria and yeasts. 

    • Cytoplasmic inclusion staining – This staining technique is used to detect the presence of intracellular deposits of substances such as glycogen, starch, hydroxybutyrate, and polyphosphates among others. 

Additional resources

Development of a standardized Gram stain procedure for bacteria and inflammatory cells using an automated staining instrument

Gram Staining

MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit