Staining Methods

1. Gram Staining

The Gram staining procedure is a common and important type of differential staining. It classifies bacteria into one or two groups based on the characteristics of their cell walls. This procedure was developed by Danish microbiologist Hans Christian Gram in 1884.

step by step

1. The first stain applied is called the primary stain. Crystal violet is added. This stains all of the cells purple.
2. Next, Gram’s iodine is added as a mordant (a substance used to set or stabilize stains or dyes). Gram’s iodine complexes with the crystal violet to form a crystal violet–iodine clump that is harder to remove from thick layers of peptidoglycan (a carbohydrate) in cell walls.
3. Next, the slide is rinsed with a decolorizing agent that helps wash out the stain. Ethanol or an acetone/ethanol solution is most commonly used. Cells with thick peptidoglycan layers retain the original crystal violet stain much more easily during this step than cells with thin peptidoglycan layers.
4. Finally, a secondary counterstain (a stain added after another stain to contrast with it in some way) is added. Safranin is most commonly used, and it stains the decolorized cells pink. The cells that have retained the crystal violet dye are affected much less noticeably.

Cells that retain crystal violet and remain purple are called gram-positive, and cells that lose crystal violet and become pink when dyed with safranin are called gram-negative. These differences reflect the differences in the peptidoglycan layer, but there are other important differences between gram-positive and gram-negative cells. You will learn more about these in the lessons about prokaryotic cells and the characteristics of taxonomic groups of bacteria.

IN CONTEXT

There are several considerations to remember when using the Gram staining procedure. First, it is important to use young, healthy colonies because older bacterial cells may have damaged cell walls that do not retain the primary stain as well as healthy cells do. Second, errors such as leaving the decolorizer on too long can affect the result. In some cases, most cells will appear gram-positive, while a few will appear to be gram-negative. This suggests damage to the individual cells or that the decolorizer was left on too long. The cells should still be classified as gram-positive if they are all the same species.

Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have implications with clinical relevance. Therefore, Gram staining is a valuable clinical tool, and knowing how to use it is an important skill.

EXAMPLE

Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. This can be helpful in selecting an appropriate antibiotic. You will learn more about the characteristics of gram-positive and gram-negative bacteria and about antibiotics in other lessons.

reflect

Most people have taken antibiotics at some point. Can you remember some of the antibiotics that you have been given? What do you think the doctor considered when deciding which antibiotics to use? Think about this, and pay attention the next time you or someone you know is given antibiotics.

Gram Staining Process
Gram Staining Steps	Cell Effects	Gram-Positive	Gram-Negative
Step 1 Crystal violet The primary stain is added to the specimen smear.	The cells are stained purple or blue.
Step 2 Iodine The mordant makes the dye less soluble, so it adheres to cell walls.	The cells remain purple or blue.
Step 3 Alcohol/Acetone The decolorizer washes away the stain from gram-negative cell walls.	Gram-positive cells remain purple or blue. Gram-negative cells are colorless.
Step 4 Safranin The counterstain allows dye adherence to gram-negative cells.	Gram-positive cells remain purple or blue. Gram-negative cells appear pink or red.

In the specimen shown in the image below, the gram-positive bacterium Staphylococcus aureus retains the crystal violet dye even after the decolorizing agent is added. Gram-negative Escherichia coli, the most common Gram stain quality-control bacterium, is decolorized and is only visible after the addition of the pink counterstain safranin.

EXAMPLE

Gram staining is often used as a first step in identifying bacteria in the lab. Knowing whether a bacterial infection is gram-positive or gram-negative can be helpful in choosing an effective antibiotic.

The micrograph shows Pseudomonas aeruginosa. Is this species gram-positive or gram-negative? Because the rods are light pink, the color of the safranin counterstain, this micrograph shows that P. aeruginosa is gram-negative.

try it

After the Gram staining technique is used on a sample of bacteria, the cells appear light pink. However, the bacterial species is known to be gram-positive.

What is a possible explanation for this finding?

To stain gram positive, the cell walls of the bacteria in the sample must hold the crystal violet primary stain. If the stain is not held, then the cells will show the safranin counterstain. Young, healthy bacterial cells hold the primary crystal violet stain most effectively, because older cells may have damaged cell walls. Additionally, excessive use of the decolorizer could potentially remove the primary stain. Either of these factors could cause the cells to appear gram-negative even if the species is gram-positive.

terms to know

Primary Stain

The first stain added in a series of stains.

Mordant

A substance used to set or stabilize stains or dyes.

Decolorizing Agent

A chemical that helps wash out a stain.

Counterstain

A stain added after another stain to contrast with it in some way.

watch

Gram Stain Procedure

2. Acid-Fast Staining

Acid-fast staining differentiates between two types of gram-positive cells: those that have waxy mycolic acid in their cell walls and those that do not. It is another commonly used, differential staining technique that can be an important diagnostic tool.

Two major techniques for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin and appear red or pink even after a decolorizing agent (an acid-alcohol solution) is added. A secondary counterstain, methylene blue, is used and this stains non-acid-fast cells blue. The techniques differ in that the Ziehl-Neelsen method uses heat to infuse the carbolfuchsin and the Kinyoun technique does not.

Acid-fast staining is used to differentiate red or pink acid-fast bacteria (AFB) from blue non-acid-fast bacteria. This distinction can have clinical importance.

EXAMPLE

Acid-fast staining is important clinically to detect AFB such as Mycobacterium tuberculosis, which causes tuberculosis.

In the micrograph below, Ziehl-Neelsen staining has rendered these M. tuberculosis cells red and the surrounding growth indicator medium blue.

3. Endospore Staining

Some bacterial cells can form endospores that allow them to survive harsh conditions while remaining dormant. You will learn more about endospores in other lessons. They are particularly difficult to kill and important to consider when trying to remove or kill microbes that may cause harm.

Endospore staining uses two stains to differentiate endospores from the rest of the cell. The most commonly used approach is the Schaeffer-Fulton method. The primary stain, malachite green, is pushed by heat into the endospore. Next, water is used to decolorize the cell, while the endospore remains green. A safranin counterstain is used. If endospores are present, they will be stained green whether they are inside or outside of a cell. The vegetative (growing) cells will be stained pink.

Endospores are formed by some pathogens of considerable clinical significance.

IN CONTEXT

Important pathogens that form endospores include the following:
Bacillus anthracis (causes anthrax and raises concern as a possible bioterrorism agent)
Clostridium tetani (causes tetanus)
C. botulinum (causes botulism)
Clostridiodes (Clostridium) difficile (causes a potentially severe intestinal infection)

did you know

Long-term and inappropriate use of certain antibiotics is associated with an increased risk of C. difficile infection. This is one reason it is so important to select antibiotics carefully and use them as prescribed.

The micrograph below is a stained preparation of Bacillus subtilis with the endospores appearing green and the vegetative cells pink.

terms to know

Endospore

A structure formed by some bacteria that allows them to survive harsh conditions while remaining dormant.

Vegetative Cell

An actively growing cell.

4. Flagella Staining

Many organisms use long, thin structures called flagella for movement. These structures are different in Bacteria, Archaea, and Eukarya and are called archaella in Archaea. Flagella are very thin, which makes them difficult to see under a light microscope unless specialized flagella staining techniques are used.

Flagella staining thickens the flagella by applying a mordant that thickens the flagella. The mordant is generally tannic acid, but sometimes potassium alum is also used. Next, the specimen is stained with pararosaniline (most commonly) or basic fuchsin.

This approach is often used by microbiologists to aid in understanding, classifying, and identifying bacteria. However, it is not commonly used in clinical settings.

To stain flagella, it is important to treat specimens carefully. Flagella are delicate and can be easily broken off, as shown in the image below of a flagella stain of Bacillus cereus, a common cause of foodborne illness. The stain reveals that the cells have numerous flagella, which are used for locomotion.

term to know

Flagella

Long, thin structures used by many microbes for movement. The flagella of Bacteria, Archaea, and Eukarya differ.

5. Capsule Staining

Certain bacteria and yeasts have a protective outer structure called a capsule. A capsule is an organized structure found outside of the cell wall. The capsule may be associated with increased virulence (ability to cause disease). You will learn more about capsules in other tutorials, including the lesson on prokaryotic cells.

Because capsules do not absorb most basic dyes, capsule staining generally involves a negative staining technique. The dye stains the background but is not taken up by the capsules. This produces a clear region where the capsule is located.

One common negative staining technique used to visualize capsules involves adding a few drops of India ink or nigrosin to a specimen. The specimen does not need to be fixed. A positive stain may also be used to stain the cell, leaving a clear halo indicating the location of the capsule.

In the micrograph image (a) below, India ink was used to stain the background around the cells of the yeast Cryptococcus neoformans. The halos surrounding the cells are the polysaccharide capsules. In image (b), crystal violet and copper sulfate dyes cannot penetrate the encapsulated Bacillus cells in this negatively stained sample. The encapsulated cells appear to have a light-blue halo.

terms to know

Capsule

An organized structure found outside of the cell wall.

Virulence

The ability to cause disease.

REFERENCES

Beeby, M., Ferreira, J. L., Tripp, P., Albers, S. V., & Mitchell, D. R. (2020). Propulsive nanomachines: the convergent evolution of archaella, flagella and cilia. FEMS Microbiology reviews, 44(3), 253–304. doi.org/10.1093/femsre/fuaa006

Khan, S., & Scholey, J. M. (2018). Assembly, Functions and Evolution of Archaella, Flagella and Cilia. Current Biology : CB, 28(6), R278–R292. doi.org/10.1016/j.cub.2018.01.085

Merriam-Webster. (n.d.). Motility. In Merriam-Webster.com Dictionary. Retrieved August 1, 2022, from www.merriam-webster.com/dictionary/motility

Parker, N., Schneegurt, M., Thi Tu, A.-H., Lister, P., & Forster, B. (2016). Microbiology. OpenStax. Access for free at openstax.org/books/microbiology/pages/1-introduction