Why are the differences in the cell walls between Gram positive and Gram-negative bacteria important for differential staining?

Table of Contents

Bacteria are a large group of minute, unicellular, microscopic organisms, which have been classified as prokaryotic cells, as they lack a true nucleus. These microscopic organisms comprise a simple physical structure, including cell wall, capsule, DNA, pili, flagellum, cytoplasm and ribosomes.

Bacteria can be gram-positive or gram-negative depending upon the staining methods. Let us have a detailed look at the difference between the two types of bacteria.

Gram Staining

This technique was proposed by Christian Gram to distinguish the two types of bacteria based on the difference in their cell wall structures. The gram-positive bacteria retain the crystal violet dye, which is because of their thick layer of peptidoglycan in the cell wall.

This process distinguishes bacteria by identifying peptidoglycan that is found in the cell wall of the gram-positive bacteria. A very small layer of peptidoglycan is dissolved in gram-negative bacteria when alcohol is added.

Gram-Positive and Gram-Negative Bacteria – Overview

The gram-positive bacteria retain the crystal violet colour and stain purple whereas the gram-negative bacteria lose crystal violet and stain red. Thus, the two types of bacteria are distinguished by gram staining.

Gram-negative bacteria are more resistant to antibodies because their cell wall is impenetrable.

Gram-positive and gram-negative bacteria are classified based on their ability to hold the gram stain. The gram-negative bacteria are stained by a counterstain such as safranin, and they are de-stained because of the alcohol wash. Hence under a microscope, they are noticeably pink in colour. Gram-positive bacteria, on the other hand, retains the gram stain and show a visible violet colour upon the application of mordant (iodine) and ethanol (alcohol).

Gram-positive bacteria constitute a cell wall, which is mainly composed of multiple layers of peptidoglycan that forms a rigid and thick structure. Its cell wall additionally has teichoic acids and phosphate.  The teichoic acids present in the gram-positive bacteria are of two types – the lipoteichoic acid and the teichoic wall acid.

In gram-negative bacteria, the cell wall is made up of an outer membrane and several layers of peptidoglycan. The outer membrane is composed of lipoproteins, phospholipids, and LPS. The peptidoglycan stays intact to lipoproteins of the outer membrane that is located in the fluid-like periplasm between the plasma membrane and the outer membrane. The periplasm is contained with proteins and degrading enzymes which assist in transporting molecules.

The cell walls of the gram-negative bacteria, unlike the gram-positive, lacks the teichoic acid. Due to the presence of porins, the outer membrane is permeable to nutrition, water, food, iron, etc.

Difference between Gram-Positive and Gram-Negative Bacteria – Key Points

  • The cell wall of gram-positive bacteria is composed of thick layers peptidoglycan.
  • The cell wall of gram-negative bacteria is composed of thin layers of peptidoglycan.
  • In the gram staining procedure, gram-positive cells retain the purple coloured stain.
  • In the gram staining procedure, gram-negative cells do not retain the purple coloured stain.
  • Gram-positive bacteria produce exotoxins.
  • Gram-negative bacteria produce endotoxins.

Difference between Gram-Positive and Gram-Negative Bacteria

Following are the important differences between gram-positive and gram-negative bacteria:

Why are the differences in the cell walls between Gram positive and Gram-negative bacteria important for differential staining?

Difference between Gram-Positive and Gram-Negative Bacteria

Gram-Positive bacteria Gram-Negative bacteria
Cell Wall
A single-layered, smooth cell wall  A double-layered, wavy cell-wall
Cell Wall thickness
The thickness of the cell wall is 20 to 80 nanometres The thickness of the cell wall is 8 to 10 nanometres
Peptidoglycan Layer
It is a thick layer/ also can be multilayered It is a thin layer/ often single-layered.
Teichoic acids
Presence of teichoic acids Absence of teichoic acids
Outer membrane
The outer membrane is absent The outer membrane is present (mostly)
Porins
Absent Occurs in Outer Membrane
Mesosome
It is more prominent. It is less prominent.
Morphology
Cocci or spore-forming rods Non-spore forming rods.
Flagella Structure
Two rings in basal body Four rings in basal body
Lipid content
Very low 20 to 30%
Lipopolysaccharide
Absent Present
Toxin Produced
Exotoxins Endotoxins or Exotoxins
Resistance to Antibiotic
More susceptible More resistant
Examples
Staphylococcus, Streptococcus, etc. Escherichia, Salmonella, etc.
Gram Staining 
These bacteria retain the crystal violet colour even after they are washed with acetone or alcohol and appear as purple-coloured when examined under the microscope after gram staining. These bacteria do not retain the stain colour even after they are washed with acetone or alcohol and appear as pink-coloured when examined under the microscope after gram staining.

For more information on the differences between gram-positive and gram-negative bacteria, keep visiting BYJU’S website or download the BYJU’S app for further reference.

Further Reading:

  • Microorganisms – Useful Or Harmful
  • Gram-Positive Bacteria

Gram-positive bacteria include the bacteria of genre Staphylococcus, Streptococcus, Enterococcus. These bacteria are the most common cause of clinical infections.

Gram-negative bacteria are more harmful and cause certain diseases. Their outer membranes are hidden by a slime layer that hides the antigens present in the cell.

The cell wall of the gram-positive bacteria absorbs antibiotics and cleaning products. Because of the outer peptidoglycan layer, they are easier to kill. Gram-negative bacteria cannot be killed easily.

Gram-positive bacteria usually cause Urinary Tract Infections. These are caused commonly in people who are more prone to urinary tract infections or are elderly or pregnant.

The gram-negative bacteria cause various infections in humans such as indigestion, food poisoning,  pneumonia, meningitis and other bacterial infections in the blood cells,  bloodstream, wound infections, etc. The infections are caused by Acinetobacter, Pseudomonas aeruginosa and E.coli.

Why are the differences in the cell walls between Gram positive and Gram-negative bacteria important for differential staining?

Put your understanding of this concept to test by answering a few MCQs. Click ‘Start Quiz’ to begin!

Select the correct answer and click on the “Finish” button
Check your score and answers at the end of the quiz

Visit BYJU’S for all Biology related queries and study materials

0 out of 0 arewrong

0 out of 0 are correct

0 out of 0 are Unattempted

View Quiz Answers and Analysis

Most bacteria are classified into two broad categories: Gram positive and Gram negative. These categories are based on their cell wall composition and reaction to the Gram stain test. The Gram staining method, developed by Hans Christian Gram, identifies bacteria based upon the reaction of their cell walls to certain dyes and chemicals.

The differences between Gram positive and Gram negative bacteria are primarily related to their cell wall composition. Gram positive bacteria have cell walls composed mostly of a substance unique to bacteria known as peptidoglycan, or murein. These bacteria stain purple after Gram staining. Gram negative bacteria have cell walls with only a thin layer of peptidoglycan and an outer membrane with a lipopolysaccharide component not found in Gram positive bacteria. Gram negative bacteria stain red or pink after Gram staining.

The cell walls of Gram positive bacteria differ structurally from the cell walls of Gram negative bacteria. The primary component of bacterial cell walls is peptidoglycan. Peptidoglycan is a macromolecule composed of sugars and amino acids that are assembled structurally like woven material. The amino sugar component consists of alternating molecules of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). These molecules are crosslinked together by short peptides which help give peptidoglycan strength and structure. Peptidoglycan provides protection for bacteria and defines their shape.

This image shows the cell wall composition of Gram positive bacteria. CNX OpenStax/Wikimedia Commons/CC BY-SA 4.0

The Gram positive cell wall has several layers of peptidoglycan. The thick layers of peptidoglycan help to support the cell membrane and provide a place of attachment for other molecules. The thick layers also enable Gram positive bacteria to retain most of the crystal violet dye during Gram staining causing them to appear purple. Gram positive cell walls also contain chains of teichoic acid that extend from the plasma membrane through the peptidoglycan cell wall. These sugar-containing polymers assist in maintaining cell shape and play a role in proper cell division. Teichoic acid helps some Gram positive bacteria to infect cells and cause disease.

Some Gram positive bacteria have an additional component, mycolic acid, in their cell walls. Mycolic acids produce a waxy outer layer that provides additional protection for mycobacteria, such as Mycobacterium tuberculosis. Gram positive bacteria with mycolic acid are also called acid-fast bacteria because they require a special staining method, known as acid-fast staining, for microscope observation.

Pathogenic Gram positive bacteria cause disease by the secretion of toxic proteins known as exotoxins. Exotoxins are synthesized within the prokaryotic cell and released into the exterior of the cell. They are specific to certain bacterial stains and can cause serious damage to body organs and tissues. Some Gram negative bacteria also produce exotoxins.

Gram positive cocci refer to Gram positive bacteria that are spherically shaped. Two genera of Gram positive cocci noted for their role as human pathogens are Staphylococcus and Streptococcus. Staphylococcus are spherical in shape and their cells appear in clusters after they divide. Streptococcus cells appear as long chains of cells after division. Examples of Gram positive cocci that colonize the skin include Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes.

Staphylococcus aureus is a Gram-positive coccus (round) bacteria that is found on the skin and mucous membranes of humans and many animals. The bacteria are usually harmless, but infections can occur on broken skin or within a blocked sweat or sebaceous gland, resulting in boils, pustules and abscesses. Paul Gunning/Science Photo Library/Getty Images

While all three are part of the normal human microbiota, they can cause disease under certain conditions. Staphylococcus epidermidis form thick biofilms and can cause infections associated with implanted medical devices. Some Staphylococcus aureus strains, such as methicillin-resistant Staphylococcus aureus (MRSA), have become resistant to antibiotics and can lead to the development of serious illness. Streptococcus pyogenes can cause strep throat, scarlet fever, and flesh-eating disease.

Like Gram positive bacteria, the Gram negative bacterial cell wall is composed of peptidoglycan. However, the peptidoglycan is a single thin layer compared to the thick layers in Gram positive cells. This thin layer does not retain the initial crystal violet dye but picks up the pink color of the counterstain during Gram staining. The cell wall structure of Gram negative bacteria is more complex than that of Gram positive bacteria. Located between the plasma membrane and the thin peptidoglycan layer is a gel-like matrix called periplasmic space. Unlike in Gram positive bacteria, Gram negative bacteria have an outer membrane layer that is external to the peptidoglycan cell wall. Membrane proteins, murein lipoproteins, attach the outer membrane to the cell wall.

This image shows the cell wall composition of Gram negative bacteria. CNX OpenStax/Wikimedia Commons/CC BY-SA 4.0

Another unique characteristic of Gram negative bacteria is the presence of lipopolysaccharide (LPS) molecules on the outer membrane. LPS is a large glycolipid complex that protects bacteria from harmful substances in their environment. It is also a bacterial toxin (endotoxin) that can cause inflammation and septic shock in humans if it enters the blood. There are three components of the LPS: Lipid A, a core polysaccharide, and an O antigen. The lipid A component attaches the LPS to the outer membrane. Attached to the lipid A is the core polyssaccharide. It is located between the lipid A component and the O antigen. The O antigen component is attached to the core polyssaccharide and differs between bacterial species. It can be used to identify specific strains of harmful bacteria.

Gram negative cocci refer to Gram negative bacteria that are spherically shaped. Bacteria of the genus Neisseria are examples of Gram negative cocci that cause disease in humans. Neisseria meningitidis is diplococcus, meaning that its spherical cells remain in pairs after cell division. Neisseria meningitidis causes bacterial meningitis and can also cause septicemia and shock.

Neisseria meningitidis are spherical, Gram negative bacteria which cause meningitis in humans. The bacteria are typically seen in pairs, each one concave on the side facing its partner. Health Protection Agency/Science Photo Library/Getty Images

Another diplococcus bacterium, N. gonorrhoeae, is the pathogen responsible for the sexually transmitted disease gonorrhea. Moraxella catarrhalis is a Gram negative diplococcus that causes ear infections in children, upper respiratory system infections, endocarditis, and meningitis.

Gram negative coccobacillus bacteria have bacterial shapes that are in between spherical and rod shaped. Bacteria of the genus Haemophilus and Acinetobacter are coccobacilli that cause serious infections. Haemophilus influenzae can cause meningitis, sinus infections, and pneumonia. Acinetobacter species cause pneumonia and wound infections.

  • Most bacteria can be broadly classified as Gram positive or Gram negative.
  • Gram positive bacteria have cell walls composed of thick layers of peptidoglycan.
  • Gram positive cells stain purple when subjected to a Gram stain procedure.
  • Gram negative bacteria have cell walls with a thin layer of peptidoglycan. The cell wall also includes an outer membrane with lipopolysaccharide (LPS) molecules attached.
  • Gram negative bacteria stain pink when subjected to a Gram stain procedure.
  • While both Gram positive and Gram negative bacteria produce exotoxins, only Gram negative bacteria produce endotoxins.
  • Silhavy, T. J., et al. "The Bacterial Cell Envelope." Cold Spring Harbor Perspectives in Biology, vol. 2, no. 5, 2010, doi:10.1101/cshperspect.a000414.
  • Swoboda, Jonathan G., et al. "Wall Teichoic Acid Function, Biosynthesis, and Inhibition." ChemBioChem, vol. 11, no. 1, June 2009, pp. 35–45., doi:10.1002/cbic.200900557.