What is the difference between Gram-positive and Gram-negative bacteria?

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The other day in class, our professor asked what really separates Gram-positive from Gram-negative bacteria, and while we all vaguely remembered something about cell walls, the detailed differences weren’t so clear. I later looked it up and found this helpful breakdown. Turns out, it’s mostly about the structure of the cell wall and how each type reacts to the Gram stain, which also affects things like antibiotic resistance and clinical treatment strategies.

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Alicia 2025-06-14T19:27:32+00:00 1 Answer 2 views
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  1. fleming
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    2025-06-14T19:29:32+00:00
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    Gram Staining Procedure

    The classification is based on the outcome of the Gram stain, developed by Hans Christian Gram in 1884:

    1. Primary Stain: Bacterial smear is stained with crystal violet (a purple dye).
    2. Mordant: Iodine solution is added, forming a complex with crystal violet (CV-I complex).
    3. Decolorization: An alcohol or acetone-alcohol mixture is applied. This is the critical differential step.
    4. Counterstain: Safranin (a red dye) is applied.

    Outcome

    • Gram-positive bacteria: Retain the crystal violet-iodine complex after decolorization and appear purple or blue-black under the microscope.
    • Gram-negative bacteria: Lose the crystal violet-iodine complex during decolorization and take up the safranin counterstain, appearing pink or red under the microscope.

    Key Differences in Cell Wall Structure

    Gram-positive Bacteria

    • Peptidoglycan Layer: Thick (20–80 nm), multilayered peptidoglycan layer that traps the CV-I complex.
    • Teichoic Acids: Present; contribute to negative charge and aid in immune recognition.
    • Outer Membrane: Absent.
    • Periplasmic Space: Narrow or virtually absent.

    Gram-negative Bacteria

    • Peptidoglycan Layer: Thin (2–7 nm), single-layered peptidoglycan within periplasmic space.
    • Teichoic Acids: Absent.
    • Outer Membrane: Present; includes:
      • Lipopolysaccharide (LPS): Includes Lipid A (endotoxin), core polysaccharide, and O-antigen.
      • Porins: Protein channels for small molecules.
    • Periplasmic Space: Wide; contains thin peptidoglycan and various enzymes.

    Consequences of Structural Differences

    Feature Gram-positive Bacteria Gram-negative Bacteria
    Gram Stain Purple/Blue Pink/Red
    Peptidoglycan Layer Thick Thin
    Outer Membrane Absent Present
    LPS (Endotoxin) Absent Present (Lipid A component)
    Teichoic Acids Present Absent
    Periplasmic Space Narrow/Absent Prominent
    Porins Absent Present (in outer membrane)
    Antibiotic Susceptibility Generally more susceptible to Penicillins, Lysozyme Generally more resistant to Penicillins, Lysozyme (due to outer membrane barrier)
    Toxin Production Primarily produce exotoxins Produce both endotoxins (LPS) and exotoxins
    Resistance to Drying Generally more resistant Generally less resistant
    Susceptibility to Detergents Generally more resistant Generally more susceptible

    Examples

    Gram-positive Bacteria

    • Cocci: Staphylococcus (e.g., S. aureus), Streptococcus (e.g., S. pneumoniae, S. pyogenes), Enterococcus
    • Bacilli (Rods): Bacillus (e.g., B. anthracis), Clostridium (e.g., C. tetani, C. botulinum), Listeria, Corynebacterium (e.g., C. diphtheriae)

    Gram-negative Bacteria

    • Cocci: Neisseria (e.g., N. gonorrhoeae, N. meningitidis), Moraxella
    • Bacilli (Rods): Escherichia coli, Salmonella, Shigella, Pseudomonas, Klebsiella, Enterobacter, Proteus, Haemophilus influenzae, Bordetella pertussis, Vibrio cholerae
    • Coccobacilli: Haemophilus, Bordetella
    • Spiral: Helicobacter pylori, Campylobacter jejuni

    Clinical Significance

    The Gram stain is often the first step in identifying bacteria in clinical samples (e.g., blood, urine, sputum). Knowing whether an infecting bacterium is Gram-positive or Gram-negative helps guide initial antibiotic therapy choices, as susceptibility patterns often differ between the two groups. The presence of LPS (endotoxin) in Gram-negative bacteria is also clinically significant, as its release can trigger sepsis and septic shock.

    Exceptions

    • Some bacteria do not stain well with the Gram stain (e.g., Mycobacterium species, which require an acid-fast stain due to their waxy cell walls).
    • Some bacteria lack a cell wall entirely (e.g., Mycoplasma) and therefore cannot be Gram-stained.
    • Some bacteria may be Gram-variable, showing inconsistent staining results.

    The fundamental difference between Gram-positive and Gram-negative bacteria lies in the structure of their cell walls, particularly the thickness of the peptidoglycan layer and the presence or absence of an outer membrane containing LPS. This structural variation dictates their Gram stain reaction and influences many other biological properties, including their interaction with antibiotics and the host immune system.

    Source

    Madigan, M.T., et al. Brock Biology of Microorganisms; Beveridge, T. J. (2001). Use of the Gram stain in microbiology. Biotechnic & Histochemistry.

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