The staining techniques in microbiology: An overview

The staining techniques in microbiology.

Microbiology is the scientific study of microorganisms such as bacteria, viruses, fungi, and protozoa. Staining techniques are important in the field because they aid in the detection and identification of microorganisms. 

This article will give an overview of the most commonly used staining techniques in microbiology, as well as their benefits and drawbacks. 

Furthermore, the article will distinguish between Gram staining, acid-fast staining, and endospore staining.

What is staining in microbiology?

Staining is a process that involves the application of dyes or chemicals to a sample of microbial cells in order to make it more visible under a microscope. Different techniques and microscopes are used depending on the type of microbial cell sample.

Staining is so essential in the field of microbiology because it allows scientists to study bacteria in greater detail thereby enabling them to better understand their functions.

Importance of staining in microbiology

  • The fundamental importance of staining in microbiology is that it gives a visualization of the cellular components of bacterial cells under the microscope.
  • Staining techniques are vital for the identification and classification of microorganisms. The process helps microbiologists to distinguish between different types of microbes and to understand how they interact with their environment and subsequently helps us to understand their roles in the various environments.
  • Staining techniques aid scientists in the discovery of new species as well as the investigation of their genetic composition and evolutionary history. A breakthrough that could lead to the development of novel agricultural and industrial products.
  • One of the importance of staining in microbiology is that it is critical for the diagnosis and treatment of bacterial infections because it enables microbiologists to accurately identify the pathogenic bacteria responsible for the infection before a doctor can suggest and appropriate treatment.
  • Staining techniques can provide valuable insights into the structure of the cell wall of an antibiotic-resistant bacteria. This information can be used to determine effective antibiotics against these resistant bacteria.
  • For instance, fluorescent staining of biomolecules is done to study the metabolic pathways of microorganisms. A proper understanding of the pathways of these microorganisms can enable us to synthesize important molecules in a controlled environment.
  • Staining techniques are used for differentiating between the living and the dead cells. This is important when scientists want to accurately monitor and control microbial populations – hitherto this is important for antibiotics production.

Types of Staining Techniques in Microbiology – staining techniques for bacteria

staining techniques in microbiology

There are broadly four different types of bacterial staining. These stains are used in microbiology to visualize the cellular components of microorganisms and they include;

  • Simple staining
  • Negative staining
  • Differential staining
  • Special staining

Simple staining

Simple staining is the use of just a single dye to identify a microorganism. This process is so basic that a smear is fixed to a slide either by air drying or heat-fix and it’s treated (allowed to bind) with a basic dye like safranin, crystal violet, or methylene blue then the solution is washed off with water.

The microbial cell absorbs the dye, resulting in a color change that allows for identification and differentiation. This type of staining technique in microbiology does not provide any complex information about the biochemical or genetic makeup of the cells.

Negative staining

In negative staining, the microbial cell remains unstained against a dark background. The dark background dye is an acidic stain that the microbial cells do not attach to or bind to. This technique is useful for identifying small objects that would be difficult to see and study their biological macromolecules. Due to the higher image contract, much smaller molecules can be visualized by negative staining.

Special staining

As the name indicates, it is a process of using a variety of stains and dye that has an affinity for the particular tissue component of a microbial cell sample. Special stains are used to identify different types of cells such as bacteria and fungi and even tissues such as connective tissue.

Special stains are often used in combination with other laboratory tests to give a comprehensive diagnosis. 

This staining technique has different types namely both for connective tissues and microorganisms; for microorganisms, we have;

  • Brown and Hopps
  • Acid-fast bacilli stain
  • Gomori methenamine silver stain
  • Periodic acid-Schiff

Differential staining

Unlike the simple staining technique that uses just a single dye, differential staining uses more than one chemical stain. This allows different types of microorganisms to be differentiated leading to their easy identification.

Because differential staining involves the use of multiple dyes, the staining process, therefore, requires several steps that involve the use of dyes, decolorizers, and counterstains.

Differential staining combined with genetic analysis and microscopy provides scientists with loads of valuable information regarding the microbial cell sample. 

This staining technique in microbiology has different types namely;

  • Gram staining
  • Acid-fasting staining
  • Structural staining

Gram staining

The gram staining procedure involves the use of dyes, stains, and reagents to distinguish between two types of bacteria: gram-positive and gram-negative. Since Gram-positive bacteria have thicker cell walls and gram-negative bacteria have thinner cell walls, the staining exploits this difference.

First, a smear of the bacteria sample to be identified is mixed with crystal violet dye. Then iodine is added to the slide to form an insoluble complex that traps the crystal violet dye in the cell wall.

Alcohol is used as a decolorizer to remove the primary stain from the gram-negative bacterial cell because the gram-negative bacterial cell has higher lipid content than a gram-positive bacterial cell.

The higher amount of lipids in the cell wall can be readily dissolved by alcohol, creating larger pores in the wall. This allows the crystal–violet–iodine (CV-I) complex to escape, resulting in the decolorization of the bacterial cell.

Finally, a counterstain is added to the slide. The gram-positive cells retain the purple color of the primary stain while the gram-negative cells adopt the red or pink color of the counterstain.

Acid-fast staining

Acid fasting staining is a specialized staining for two genera of bacteria that do not take up the gram stain. This method of staining is used on Mycobacterium spp. and Nocardia. These bacteria have a very high concentration of lipids composed of mycolic acid.

This particular acid prevents the cell wall of these bacteria from binding to the dye used in gram staining.

The Carbolfuschsin method also known as the Ziehl-Neelsen stain is used for staining acid-fast bacteria. This process uses heat to attach stains to the cell wall of these bacteria.

This method of staining cannot substitute for gram staining and vice versa.

Structural staining

Structural staining is a type of differential staining used to check for the antigenic and non-antigenic structures of bacteria. It uses two dyes, an acidic dye, and a basic dye to observe the bacterial structures such as endospores, capsules, and flagella.

The acidic dye will stain the acidic parts of the bacterial cells while the basic dye will stain the basic parts.

This is sometimes called the impregnation method because the process is used to view bacterial cells and structures that are too thin to be seen under the light microscope.

There are a few types of structural staining namely;

  • Endospore staining
  • Flagella staining
  • Capsule staining
Endospore staining

An endospore is a non-vegetative structure of a group of bacteria that helps them to survive in unfavorable conditions.

These spores are difficult to stain with basic dyes. The endospore stain which is characterized by a special dye with heat is used to measure the cell sizes and shapes of these structures.

The Schaffer-Fulton procedure is the most preferred endospore staining method. The first step is the use of a primary stain, malachite green along with the use of steamed heat to penetrate the dye into the spore.

Then the spore is washed with water to decolorize the vegetative cell but the endospore remains stained with the malachite green.

Flagella staining

The most commonly used flagella stain is Hardy Diagnostics. The essence is to visualize the presence and arrangement of bacterial flagella under a light microscope.

The specimen is first smeared on a clean slide, then stained with the flagella stain, and finally observed under the microscope for the arrangement of the flagella to be observed.

Capsule staining

Capsule staining is a technique used to visualize the presence of a bacterial capsule, which is a gelatinous outer layer that surrounds some bacterial cells. The technique is often used during phacoemulsification surgeries to help differentiate between capsular material and bacterial cells.

What Next?

The staining techniques in microbiology are quite many however each technique is important relative to its purpose of use. So, which staining technique do you want to use today?


Basic dyes that are used in negative staining

Basic dyes such as nigrosin, India ink, and nigrosin are commonly used in negative staining. These dyes are acidic, which means that they do not penetrate the cell wall and instead are repelled by the cell wall. 

This results in the formation of a halo of dye that surrounds the cell, allowing it to be visible under the microscope.

What is a structural stain?

Structural staining is a type of differential staining used to check for the antigenic and non-antigenic structures of bacteria. It uses two dyes, an acidic dye, and a basic dye to observe the bacterial structures such as endospores, capsules, and flagella.

Types of stains in microbiology

The types of stain include; Simple staining, Negative staining, Differential staining, Special staining

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