Types of Staining techniques used in Microbiology and their applications

Spore of Clostridium botulinum Source: ASM

Acid fast stain (Ziehl-Neelsen technique):

Acid fast bacillus
Acid-fast bacillus

It distinguishes acid-fast bacteria such as Mycobacterium spp from non-acid fast bacteria; which do not stain well by the Gram staining.  It is used to stain Mycobacterium species  (Mycobacterium tuberculosis, M. ulcerans, and M. leprae)

Acridine Orange Stain: This staining method is used to confirm the presence of bacteria in blood cultures when Gram stain results are difficult to interpret or when the presence of bacteria is highly suspected but none are detected using light microscopy. Acridine orange binds to nucleic acid and stains them. It is also used for the detection of mycoplasmas (cell wall deficient bacteria)

Auramine-Rhodamine technique: This fluorochrome staining method is used to enhance the detection of mycobacteria directly in patient specimens and initial characterization of cells grown in culture.

Calcofluor White Staining: It is commonly used to directly detect fungal elements and to observe the subtle characteristics of fungi grown in culture. The cell walls of fungi will bind the stain calcofluor white, which greatly enhances the visibility of fungal elements in tissue or other specimens.

Capsule stain: It helps to demonstrate the presence of capsule in bacteria or yeasts. Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae are common capsulated bacteria.

Staphylococcus in Gram Stain
Staphylococcus in Gram Stain

Cytoplasmic inclusion stains: Identifies intracellular deposits of starch, glycogen, polyphosphates, hydroxybutyrate, and other substances.  E.g. Albert staining is used to stain the volutin or metachromatic granules of C. diphtheriae.

Endospore stain: It demonstrates spore structure in bacteria as well as free spores. Relatively few species of bacteria produce endospores, so a positive result from endospore staining methods is an important clue in bacterial identification. Bacillus spp and Clostridium spp are the main endospores producing bacterial genera.

Gram staining:

Gram stain is a very important differential staining technique used in the initial characterization and classification of bacteria in microbiology. Gram staining helps to identify bacterial pathogens in specimens and cultures by their Gram reaction (Gram-positive and Gram-negative) and morphology (cocci/rod).  

Spore of Clostridium botulinum Source: ASM
Spore of Clostridium botulinum
Source: ASM

Flagella stain: Demonstrate the presence and arrangement of flagella. Flagellar stains are painstakingly prepared to coat the surface of the flagella with dye or a metal such as silver.

The number and arrangements of flagella are critical in identifying species of motile bacteria.

India ink Preparation (Negative staining):

Negative stains are used when a specimen or a part of it, such as the capsule resists taking up the stain.  India Ink preparation is recommended for use in the identification of Cryptococcus neoformans.

Giemsa stain:  Giemsa stain is a Romanowsky stain. It is widely used in the microbiology laboratory for the staining of:

  1. Malaria and other blood parasites
  2. Chlamydia trachomatis inclusion bodies
  3. Borrelia species
  4. Yersinia pestis
  5. Histoplasma species
  6. Pneumocystis jiroveci cysts (formerly Pneumocystis carinii)

lactophenol cotton blue (LPCB) wet mount

LPCB mount is the most widely used method of staining and observing fungi.

About Acharya Tankeshwar 460 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Plz help me to find da answer abt,different types of media for cultivation of microorganism?,I cnnt undrstnd n am confuse

    • You can find this information easily in our site. Please see the respective sections.

    • Dear Anonymous
      You can click on the hyperlinks above to read more about each staining techniques. I hope this helps.

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