Gram Staining: Principle, Procedure, Interpretation, Examples, And Animation

What does gram staining mean? How do I perform Gram Staining? What are the advantages and disadvantages of Gram Staining? 

Gram Staining: Principle, Procedure, Interpretation, Examples, And Animation

If you have been wondering about the answers to these questions, then read on for all you need to know about gram staining, including the basics of the procedure.

What Is Gram Staining?

Gram staining is a method used to identify bacteria or other microorganisms in clinical specimens such as blood, urine, sputum, pus, tissue biopsies, etc.

The technique was developed by Dr. Hans Christian Gram in 1884. Gram staining is also known as Gram’s stain or simply Gram’s test.

Gram staining is performed using a special stain called Gram’s stain. This stain helps us distinguish between live and dead cells.

Live cells appear blue/green, whereas dead cells appear red/pink.

Gram staining is a quick and simple way to identify bacteria in a sample. It can be done at any time during the course of treatment.

The principle behind Gram staining involves using a combination of dyes that selectively bind to certain components of the cell wall of bacteria.

These dyes are then used to highlight specific structures on the surface of the cell walls. 

The stains used are generally divided into two groups: basic (or cationic) dyes and acid dyes. Basic dyes are positively charged, while acid dyes have negative charges.

Most of the basic dyes contain iodine. Acid dyes usually contain bromine.

Why Do We Perform Gram Staining?

The most common reason why we perform Gram staining is to determine whether there are any pathogenic organisms present in a patient’s body.

If so, it will help doctors decide what antibiotic should be administered. 

In addition, if the organism is resistant to antibiotics, it may require additional treatment options.

How Does Gram Staining Work?

The basic principle behind Gram staining is that living organisms (bacteria) contain certain chemicals which react with specific dyes.

When these chemicals interact with the dye, they change color. For example, when the chemical reacts with crystal violet, it turns purple. 

Similarly, the reaction with safranin O gives the cell wall a pinkish-red color. Thus, the presence of bacteria in a specimen can be detected by observing the color changes.

In order to perform Gram staining, first, prepare a slide containing a drop of the specimen under examination. Then add a few drops of the Gram stain solution onto the slide.

Next, cover the slide with a coverslip. Finally, observe the slide under a microscope.

If the specimen contains bacteria, the bacteria will show up as colored dots against a clear background.

The number of bacteria present in the specimen can be determined by counting them.

If no bacteria are found in the specimen, the specimen will appear clear.

Why Does Gram Stain Work?

Gram stain works because of the principle of differential interference contrast microscopy.

This principle states that cells with an intact cell membrane produce a bright field image.

In contrast, cells without a cell membrane do not reflect light and therefore appear dark.

Differential interference contrast microscopy uses two beams of light. One beam passes through the sample and reflects off the cell wall.

The second beam passes through the sample but does not reflect off the cell wall. This second beam produces a bright-field image.

The difference between the bright field image and the dark field image allows us to see the cell wall.

If the cell wall has been damaged, the bright field image will appear brighter than the dark field image.

The Bright Field Image

In addition to identifying the presence of bacteria, gram staining can also reveal information about the type of bacteria. Different bacteria have unique cell wall characteristics.

The differences in cell wall structure allow us to distinguish one species from another. 

If the cell walls appear smooth, the specimen contains Gram-negative bacteria. On the other hand, if the cell walls appear rough, the specimen contains Gram-positive bacteria.

For example, if you were looking at a colony of E. coli (a Gram-negative bacterium) and noticed it was growing very slowly, you could use gram staining to determine whether the slow growth is due to low nutrient levels or to the presence of antibiotics.

If the colony appears to grow normally, you would know that the slow growth is likely caused by antibiotic resistance.

However, if the colony looks like it’s dying, then you’d know that the problem is probably due to low nutrients.

Interpretation Of Gram Stained Micrographs

To interpret a Gram-stained micrograph, first identify the location of the nucleus.

Then count how many cells there are in each area – this helps us understand what kind of bacteria are present.

The shape of the cell wall is important as it helps us distinguish between different types of bacteria such as Gram-negative and Gram-positive.

Once we have counted the total number of cells in a certain area, we can look for any abnormalities such as clumping, lack of separation, and irregular shapes indicating that something is wrong with the culture.

Once we’ve identified the abnormality, we can decide which type of bacteria is causing the problem.

Different types of bacteria have different cell walls, and so knowing the type of bacteria can help us figure out why the bacteria are behaving differently. 

For example, if the cells are clumped together, we might suspect that the cells are dead. Dead cells don’t have a cell wall, so they appear dark on a gram stain.

If the cells are separated, we might suspect that they’re alive. Living cells have a cell wall, and therefore they appear white on a gram stain.

If the cells are irregularly shaped, we might suspect that these are abnormal cells. Cells that aren’t normal may be dividing incorrectly. 

These cells may also be dead.

We then look at the number of flagella present on the surface of the cell.

Flagella are microscopic structures found on the surface of most bacteria that help them move through their environment. They are responsible for swimming, twitching, and rotating.

We also examine the shape of the cell. Cells with long, slender shapes have a higher chance of containing Gram-negative bacteria.

Cells with round shapes usually contain Gram-positive bacteria.

Finally, we look at the arrangement of the nucleoid inside the cell. Nucleoids are DNA molecules located within the cytoplasm of the bacterial cell.

They are responsible for carrying out the instructions encoded in genes.

Nucleoids can either be arranged linearly or circularly. Linear arrangements occur when the nucleoid lies along the long axis of the cell.

Circular arrangements occur when the nucleoids lie around the center of the cell.

Linear arrangements are more common among Gram-negative bacteria, while circular arrangements are more common among bacteria that belong to the genus Streptococcus.

Advantages Of Gram Staining

Advantages Of Gram Staining

There are several benefits associated with performing Gram staining:

Aiding Antibiotics

Gram staining is also very useful in determining the sensitivity of antibiotics, which can be crucial to successful diagnosis and treatment.

Gram staining is also considered to be a rapid method of identification. 

It takes only about 15 minutes to get results from a sample. This makes it ideal for use in emergency situations where antibiotics must be administered quickly.

Finding Types of Bacteria

Gram staining is helpful in identifying unusual pathogens and can be used to differentiate between different types of bacteria.

The technique is also used to detect bacteria in various samples including blood, pus, sputum, urine, stool, throat swabs, etc. 

It is also used to find out whether the microorganisms are alive or not, and can correctly identify more than 99% of all bacteria accurately.


Gram staining is one of the oldest methods available for diagnosing infectious diseases, and the practice has been around since the late 1800s.

Today, this technique is still popular among medical practitioners because of its simplicity and effectiveness, and its high success rate when it comes to finding bacteria and identifying the right strain and type.


Gram staining is also easy to do and simple to learn, requiring minimal training – even those new to microbiology should be able to handle the technique quickly and easily.

There is also no need for expensive equipment; all you need is a light microscope and a glass slide.

Gram staining is also inexpensive. A single gram of stained cells costs less than $1.00.


Gram staining is non-destructive, so there is no need to worry about specimens being destroyed or compromised during the testing process, which can be important.

Final Thoughts

Gram staining is an important tool for every microbiologist. It’s a quick, reliable, and cost-effective way of detecting bacteria, and has a number of benefits and uses for scientists and in the wider medical world. 

Learning more about it is an important element of becoming a better microbiologist, and there are a number of applications that make this a super useful technique to master.

Jennifer Dawkins