Classifying Bacteria with a Gram Stain

The Gram stain procedure is a differential staining procedure that involves adding different stains to bacteria to make them visible. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls. Still today the Gram stain remains one of the most frequently used staining techniques and is an effective way to identify bacteria.


“I have therefore published the method, although I am aware that as yet it is very defective and imperfect; but it is hoped that also in the hands of other investigators it will turn out to be useful. “

– Dr. Hans Christian Gram



  • Bacteria are a type of prokaryotic organism that have cell structures that differ from eukaryotic cells.
  • Differences in the cell membrane and cell wall distinguishes two large classes of bacteria that can be identified by Gram staining.
  • Gram positive bacteria are stained purple; Gram negative stained pinkish red.
  • Difference between Gram positive and Gram negative bacteria influence pathogenicity and responsiveness to antibiotics.


Brief Overview of Bacteria Cell Structure

Bacteria are a class of unicellular organisms know as prokaryotes. A distinguishing feature of these single celled organisms is that lack membrane-bound organelles and structures.

Lacking these membrane bound organelles requires the bacteria to store their chromosome — usually a single of a piece of circular, double-stranded DNA  — to be located in an area of the cell called the nucleoid. Unlike eukaryotic cells, bacteria also have extra-chromosomal,circular DNA called plasmids. Plasmids usually carry advantageous survival genes.

Most prokaryotes also have a cell wall outside the plasma membrane. The cell wall functions as a protective layer and give the organism shape. The capsule is an outermost layer that  enables the organism to attach to surfaces, protects it from dehydration and attack by phagocytic cells, and makes pathogens more resistant to our immune responses.

Some species of bacteria also have flagella — or a single flagellum — used for locomotion, and pili/ fimbriae used for attachment to surfaces including the surfaces of other cells.

Author: OpenStax

Gram Stain: Reagents and Procedure

Reagents Needed for a Gram Stain

        • ✓ Crystal Violet (purple/violet dye)
        • ✓ Iodine (dye stabilizer)
        • ✓ Ethanol (destain)
        • ✓ Safranin (pink/redish dye)

Gram Staining Procedure

        1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.
        2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet–iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls.
        3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless.
        4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye.
Table showing the steps of the Gram staining used to identify gram positive and gram negative bacteria.
Author: OpenStax

The Cell Wall: The Reason for Different Stains

Gram positive (purple stained) and Gram negative (pink stained) bacteria are differential stained  because each class of bacteria has a structurally different composition.

  • Gram positive bacteria have a thick peptidoglycan (cell wall) layer that retains the crystal violet layer. 
  • Gram negative bacteria have a thin peptidoglycan layers sandwhiched between an inner and outer plasma membrane.


Things to Consider for Gram Staining

The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative. However, there are several important considerations in interpreting the results of a Gram stain:

      1. Older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive. It is a good idea to use fresh bacterial cultures to prevent a false negative.
      2. Leaving on decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative. If the decolorizer is left on for too long, cells that should appear gram-positive may not retain the crystal violet stain.

Gram Negative and Gram Positive Bacteria

Gram Stain Identifies Differences in the Bacterial Cell Wall

The Gram staining protocol is used to differentiate two common types of cell wall structures. Gram-positive bacteria have a cell wall consisting of many layers of peptidoglycan totaling 30–100nm in thickness. On the other hand, Gram-negative cells have a much thinner layer of peptidoglycan (no more than about 4 nm thick) than gram-positive cells and the overall structure of their cell envelope is more complex.

While the Gram stain distinguishes between two different classes of bacteria based on their cell wall, the cell wall isn’t the only feature that differs between Gram-positive and Gram-neagtive bacteria.

Author: BiomedGuide

Porins are large membrane proteins found on the outermembrane of gram negative bacteria. They are large enough to allow for passive diffusion through a central pore.

 Differences Between Gram Positive and Gram Negative Bacteria

Gram Negative Bacteria Have an Outer Membrane

Gram-negative also have a second lipid bilayer called the outer membrane which sandwiches the peptidoglycan layer. The outer leaflet of the outer membrane contains the molecules called  lipopolysaccharides (LPS). These LPS molecules are bacetrial endotoxins that are a main cause of infections cause by gram-negative bacteria. They can cause symptoms such as fever, hemorrhaging, and septic shock. The composition of the LPS molecule varies between different species and strains of bacteria. Some variants of LPS are deadly, like the ones found on a pathogenic strain of  Escherichia coli which can cause severe diarrhea and kidney failure.


Gram Positive Teichoic Acid and Lipoteichoic Acids

Techoic acid and Lipotechoic acis are thought to stabilize peptidoglycan by increasing its rigidity. Teichoic acids that are are anchored to the bacterial cell membrane are considered to be lipoteichoic acids. This acids can also be antigenic, meaning they can interpreted as an antigen by the immune system. As a result, teichoic and lipoteochoic acids sometimes have a role in the ability of pathogenic gram-positive bacteria.

One example where teichoic and lipoteichoic acids play a role in pathogenicity is with bacterial species from the Streptococcus genus. Some of these bacteria can bind to proteins on the surface of host cells, increasing their ability to cause infection.

Clinical Relevance of the Gram Stain

Identification of Potential Pathogens

Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance. Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. For example, Gram-negative bacteria tend to be more resistant to certain antibiotics than gram-positive bacteria.

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