Gram-positive vs gram-negative cell walls
The Gram stain looks like a dye test, but it is a wall test. Compare the two cell envelopes and use them to predict the stain and antibiotic susceptibility.
Compare gram-positive and gram-negative cell envelopes and explain how the differences determine Gram staining and antibiotic susceptibility.
The short version
- Gram-positive means a thick peptidoglycan wall and no outer membrane, so the cell stays purple.
- Gram-negative means a thin wall plus an outer membrane carrying lipopolysaccharide, so the cell stains pink.
- The Gram stain reads the wall, not the dye.
- The outer membrane keeps vancomycin and other large drugs out of gram-negative cells.
- Stain colour, not shape, decides gram-positive versus gram-negative.
Gram-positive vs gram-negative cell walls: same stain, two outcomes
A laboratory technician stains one smear with the same dye, the same iodine and the same alcohol. Some bacteria stay deep purple. Others wash out and turn pink. What is different about the cells?
The answer is in the gram-positive vs gram-negative cell walls. This lesson compares the two cell envelopes and uses them to predict the Gram stain and antibiotic susceptibility.[1][2]
Peptidoglycan: the mesh both envelopes share
Peptidoglycan is a mesh of alternating N-acetylglucosamine and N-acetylmuramic acid chains, cross-linked by short peptides.[1]
It exists only in bacteria, which is why so many antibiotics aim at it.[1][3]
The gram-positive and gram-negative envelopes compared
A gram-positive cell has a thick wall: many layers of peptidoglycan, threaded with teichoic acids, outside the plasma membrane. There is no outer membrane.[1]
A gram-negative cell is built differently. Its peptidoglycan is a thin layer, and outside it sits a second lipid bilayer, the outer membrane. The gap between the two membranes is the periplasm.[1]
The outer leaflet of that membrane carries lipopolysaccharide (LPS), made of lipid A, a core polysaccharide and the O side chain.[1][4]
| Feature | Gram-positive | Gram-negative |
|---|---|---|
| Peptidoglycan | Thick, many layers | Thin layer |
| Teichoic acids | Present in the wall | Absent (a gram-positive feature) |
| Outer membrane | Absent | Present, with LPS in the outer leaflet |
| Periplasm | No true periplasm (no outer membrane) | Gap between the two membranes |
| Gram stain | Purple | Pink |
The Gram stain, step by step
- Crystal violetApplied first as the primary stain.
- IodineLocks the dye in as a crystal violet and iodine complex.
- Alcohol or acetoneWashes over the smear as the decolouriser.
- SafraninA pink counterstain, applied last.
In a gram-positive cell, the thick wall holds the complex, and the cell stays purple. In a gram-negative cell, alcohol strips away the outer membrane, the thin wall cannot hold the dye, and the cell takes up pink safranin.[2][5]
So the stain is reading the wall, not the dye.
Try it: reading a smear
- Purple cocci in clusters: thick wall, no outer membrane, so gram-positive
- Pink rods: outer membrane, thin wall, so gram-negative
- Shape alone does not decide it; stain colour does
Why the drugs behave differently: vancomycin and porins
Beta-lactams and vancomycin block peptidoglycan synthesis, so they must reach the wall's synthesis machinery. In a gram-positive cell the wall is at the surface. In a gram-negative cell, the outer membrane stands in the way.[3][6]
Vancomycin is a large molecule that binds the D-alanyl-D-alanine end of wall precursors. It cannot cross the outer membrane, so it is not active against gram-negative bacteria.[3][6][8]
Smaller drugs enter through porins, channels in the outer membrane. Penicillin G is mainly active against gram-positives, and broader penicillins reach more gram-negatives.[3]
The outer membrane is a barrier, and that is why gram-negative bacteria are often more resistant.[3][6]
Two exceptions
Practice question
Original · USMLE styleA 67-year-old man develops fever and low blood pressure two days after abdominal surgery. Temperature is 38.9 °C and blood pressure is 88/52 mmHg. Blood cultures grow gram-negative rods. In the laboratory, the isolate is not inhibited by vancomycin, although vancomycin inhibits peptidoglycan synthesis.
Common questions
What is the difference between gram-positive and gram-negative cell walls?
A gram-positive cell has a thick peptidoglycan wall with teichoic acids and no outer membrane. A gram-negative cell has a thin peptidoglycan layer and an outer membrane carrying lipopolysaccharide.
Why do gram-positive cells stain purple and gram-negative cells pink?
The thick wall of a gram-positive cell holds the crystal violet and iodine complex. In a gram-negative cell, alcohol strips away the outer membrane and the thin wall cannot hold the dye, so the cell takes up pink safranin.
Why is vancomycin not active against gram-negative bacteria?
Vancomycin is a large molecule that cannot cross the outer membrane, so it cannot reach the peptidoglycan it acts on.
What is lipopolysaccharide made of?
Lipopolysaccharide sits in the outer leaflet of the gram-negative outer membrane. It is made of lipid A, a core polysaccharide and the O side chain.
Can a gram-positive organism look gram-negative on a stain?
Yes. If the alcohol is left on too long, even gram-positive cells wash out pink, so a pure culture can look mixed.
Transcript
Read the full narration, by chapter713 words
0:00Same stain, two outcomes
A lab technician stains one smear with the same dye, the same iodine and the same alcohol. Some bacteria stay deep purple. Others wash out and turn pink. Same slide, same stain, two outcomes. What is different about the cells? Today: compare gram-positive and gram-negative cell envelopes, and use them to predict the Gram stain and antibiotic susceptibility.
0:26Peptidoglycan: the shared mesh
Start with what they share. Peptidoglycan is a mesh of alternating N-acetylglucosamine and N-acetylmuramic acid chains, cross-linked by short peptides. It exists only in bacteria, which is why so many antibiotics aim at it.
0:43The gram-positive envelope
A gram-positive cell has a thick wall: many layers of peptidoglycan, threaded with teichoic acids, outside the plasma membrane. There is no outer membrane.
0:54The gram-negative envelope: outer membrane, periplasm, LPS
A gram-negative cell is built differently. Its peptidoglycan is a thin layer, and outside it sits a second lipid bilayer, the outer membrane. The gap between the two membranes is the periplasm. The outer leaflet carries lipopolysaccharide, made of lipid A, a core polysaccharide and the O side chain. Positive: one membrane, thick wall. Negative: two membranes,
1:22The Gram stain, step by step (and the over-decolourising trap)
thin wall. Now the Gram stain. Crystal violet goes on first. Iodine locks it in as a crystal violet and iodine complex. Alcohol or acetone washes over the smear. Last comes safranin, a pink counterstain. In a gram-positive cell, the thick wall holds the complex, and the cell stays purple. In a gram-negative cell, alcohol strips away the outer membrane, the thin wall cannot hold the dye, and the cell takes up pink safranin. So the stain is reading the wall, not the dye. One trap: leave the alcohol on too long, and even gram-positive cells wash out pink, so a pure culture can look mixed.
2:06Try it: purple cocci, pink rods
Try it. A smear shows purple cocci in clusters: thick wall, no outer membrane. A smear shows pink rods: outer membrane, thin wall. Shape alone does not decide it; stain colour does.
2:23Why the drugs behave differently: vancomycin and porins
Now the drugs. Beta-lactams and vancomycin block peptidoglycan synthesis, so they must reach the wall's synthesis machinery. In a gram-positive cell the wall is at the surface. In a gram-negative cell, the outer membrane stands in the way. Vancomycin is a large molecule that binds the D-alanyl-D-alanine end of wall precursors. It cannot cross the outer membrane, so it is not active against gram-negative bacteria. Smaller drugs enter through porins, channels in the outer membrane. Penicillin G is mainly active against gram-positives, and broader penicillins reach more gram-negatives. The outer membrane is a barrier, and that is why gram-negative bacteria are often more resistant.
3:13Two exceptions: mycobacteria and Mycoplasma
Two exceptions. Mycobacteria have a waxy wall that stains poorly with Gram, and that has its own lesson. Mycoplasma has no cell wall at all, so a wall-targeting drug has nothing to hit.
3:26Practice question
Your turn. A 67-year-old man develops fever and low blood pressure after abdominal surgery. Blood cultures grow gram-negative rods. In the laboratory, the isolate is not inhibited by vancomycin, although vancomycin inhibits peptidoglycan synthesis. Which structural feature of the organism best explains this finding? A, teichoic acids in the cell wall that bind the drug and prevent it reaching its target. B, an outer membrane with a lipopolysaccharide layer that blocks entry of large molecules. C, complete absence of peptidoglycan from the cell envelope, leaving no target for the drug. D, a thick peptidoglycan layer that traps the drug before it can reach the plasma membrane. E, a mycolic acid layer outside the cell wall that blocks entry of the drug into the cell. Pause here and decide.
4:29Answer and explanation
A is out: teichoic acids belong to gram-positive walls, not gram-negative ones. C is out: gram-negative bacteria do have peptidoglycan, only a thin layer, and vancomycin acts on it. D is out: a thick wall is the gram-positive pattern, and these are gram-negative rods with a thin wall. E is out: mycolic acids are the waxy wall of mycobacteria, not of gram-negative rods. So the answer is B. The outer membrane keeps large molecules like vancomycin away from the peptidoglycan.
5:06Recap and next lesson
Three things to keep. One: gram-positive means a thick wall and no outer membrane, so purple. Gram-negative means a thin wall plus an outer membrane with lipopolysaccharide, so pink. Two: the Gram stain reads the wall. Three: the outer membrane keeps vancomycin and other large drugs out of gram-negative cells. Next, the cell-wall antibiotics: beta-lactams and vancomycin.
Sources
Numbers in the text, e.g. [1], point here- OpenStax Microbiology, 3.3 Unique Characteristics of Prokaryotic Cells. openstax.org/books/microbiology/pages/3-3-unique-characteristics-of-prokaryotic-cells
- OpenStax Microbiology, 2.4 Staining Microscopic Specimens. openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens
- OpenStax Microbiology, 14.3 Mechanisms of Antibacterial Drugs; 14.5 Drug Resistance. openstax.org/books/microbiology/pages/14-3-mechanisms-of-antibacterial-drugs · openstax.org/books/microbiology/pages/14-5-drug-resistance
- OpenStax Microbiology, 15.3 Virulence Factors of Bacterial and Viral Pathogens (lipid A). openstax.org/books/microbiology/pages/15-3-virulence-factors-of-bacterial-and-viral-pathogens
- Gram stain: decolourisation and pitfalls. Europe PMC PMC10679832. europepmc.org/article/PMC/PMC10679832
- Antibacterial mechanisms of action (cell wall synthesis inhibitors). Europe PMC PMC6604941. europepmc.org/article/PMC/PMC6604941
- Mycoplasma and the absence of a cell wall. Europe PMC PMC12689956. europepmc.org/article/PMC/PMC12689956
- Gram stain; Vancomycin (secondary). Wikipedia. en.wikipedia.org/wiki/Gram_stain · en.wikipedia.org/wiki/Vancomycin
The practice question is original and its patient is synthetic. Every fact is checked against the sources above in independent accuracy reviews. Always follow your local guidelines and your own laboratory’s ranges.
Image credits. Image credits Microscope: Servier Medical Art (smart.servier.com), CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/), adapted (cropped, recoloured). Bacteria illustrations (Staphylococcus aureus, gram-negative bacterium, tuberculosis bacilli): NIAID NIH BioArt Source (bioart.niaid.nih.gov), public domain. Hospital bed icon: Health Icons (healthicons.org), CC0. Blood sample tube: Marcel Tisch via bioicons.com, CC0. The envelope sections, the Gram-stain strip, the peptidoglycan mesh, the smears and all handwriting are drawn in code (original).
Corrections. None so far. Spotted an error? Email info@medservatory.com with the lesson code MS-S1-MICRO-001.
For exam study only, not medical advice. Read the medical disclaimer.
In this series
Playlist on YouTube- EP 01Gram-positive vs gram-negative cell walls5:39
- EP 02Acid-fast stain and the mycobacterial cell wall5:01
- EP 03Endotoxin vs exotoxin: lipid A, A-B toxins and toxoids5:46
- EP 04Bacterial toxins that raise cAMP: cholera, pertussis, anthrax5:54
- EP 05Clostridial toxins compared: tetanus, botulinum, alpha, C. difficile5:22
- EP 06Staph vs strep: catalase, coagulase and haemolysis5:23
- EP 07Encapsulated organisms and the spleen: asplenia and conjugate vaccines5:08
- EP 08Bacterial genetics: how bacteria swap genes5:13
- EP 09Beta-lactamase and antibiotic resistance: three ways bacteria win5:13
- EP 10DNA vs RNA virus replication: which enzyme each genome needs5:48
- EP 11HIV life cycle and drug targets, step by step5:39
- EP 12Hepatitis B serology: reading the panel5:17
- EP 13Antifungal drugs explained: mechanisms of action5:26
- EP 14Bacterial meningitis organisms by age and risk group5:19