AAT Bioquest

What are the common types of staining?

Posted March 9, 2023


The common types of staining include: simple stains, gram stains, endospore stains, acid-fast stains, capsule stains, flagella stains, and spirochete stains. 

In simple staining, only one stain is utilized, and is used to increase contrast between organisms and the environment so that they are more easily visualized in light microscopes. Examples of some commonly used simple stains are methylene blue, crystal violet, and safranin. These types of stains are only useful for observing a microbial cell's arrangement and morphology, and cannot differentiate between different types of species.

Gram staining is a type of differential stain that uses more than one stain. It displays distinct visualization based on their chemical or structural properties under a microscope. For example, gram stains can visualize differences in cell wall composition. Gram positive cells will show thick layers of peptidoglycan in its cell walls, while gram negative bacteria will show very little peptidoglycan. The primary stain is crystal violet, and the mordant is iodine. Iodine binds to the crystal violet and sits for 30 seconds until the decolorizer is added. After safranin counterstains the decolorized bacteria, gram + bacteria can easily be differentiated from gram - bacteria. 

Endospore staining is a differential stain utilized to visualize bacterial endospores. Endospores are highly resistant to normal staining procedures, as they have durable protein coats composed of keratin. The stains used in the procedure are malachite green as the primary stain and safranin as the counterstain. Malachite green is the ideal stain because it is water soluble and doesn’t adhere to the cell. Thus, it rinses easily from vegetative cells, and  these cells easily take up the counterstain safranin. Endospores resist staining with basic stains.    

Capsule staining is a type of differential stain used to identify capsules of bacterial cells. The cells within the capsule are stained with basic stains (e.g. safranin or crystal violet), while the background (the slide) is stained with acidic stains  (e.g. Congo red or Nigrosin). After staining, capsules appear as a clear ring around the bacterial cells. This procedure is done without fixing the bacteria with heat because their capsules can be destroyed by heat.

Acid-fast stain is a differential stain used to identify acid-fast bacterial cells. Acid-fast organisms typically have almost impermeable cell walls that contain mycolic acid, and abundant quantities of waxes, lipids, and fatty acids. This kind of cell wall is resistant to the majority of compounds, and is the reason why acid-fast bacterial cells require this type of staining technique. Carbolfuchsin is the primary stain used, as it assists in penetrating the cell wall. Heat may also be involved to loosen the waxy layer and allow for better penetration of the primary stain inside the cell. The smear is then rinsed with a decolorizer acid alcohol, which removes the stain from all non-acid-fast cells but doesn’t penetrate the cell wall of acid-fast cells. The decolorized non-acid-fast cells then take up the counterstain methylene blue. Acid-fast stains were made to identify the bacterial species that cause tuberculosis.  

The flagella stain allows for observation of bacterial flagella under a light microscope. Flagella stains, such as silver nitrate stain, use a mordant to coat the flagella with the stain until they are thick enough to be seen under the microscope. Silver nitrate also makes flagella appear larger than they are. It can also be used to visualize arrangement of flagella for identification (e.g. Proteus vulgaris has peritrichous flagella).

Spirochetes are spiral or corkscrew-shaped bacteria. Silver stains like silver nitrate are used to identify thin spirochetes like Treponema pallidum; giemsa stains can also be used to stain spirochetes blue. Warthin-Starry silver stain visualizes spirochetes by staining them black.  

Additional resources

Histology, Staining

Gram Staining

MycoLight™ Rapid Fluorescence Gram-Positive Bacteria Staining Kit