Microbiology · Bacterial cell structure
Gram-positive vs gram-negative bacteria
Gram-positive bacteria have a thick peptidoglycan wall and stain purple. Gram-negative bacteria have a thin peptidoglycan layer plus an outer membrane, and stain pink after the counterstain.
Side-by-side comparison
| Feature | Gram-positive | Gram-negative |
|---|---|---|
| Peptidoglycan | Thick, many layers | Thin, one or a few layers |
| Outer membrane | None | Yes, with lipopolysaccharide (LPS) and porins |
| Teichoic acids | Present | Absent |
| Periplasmic space | Small or absent | Present, between the membranes |
| Endotoxin | No LPS | Lipid A of LPS |
| Gram stain color | Purple (keeps crystal violet) | Pink or red (safranin) |
| Examples | Staphylococcus aureus, Streptococcus pyogenes, Bacillus | Escherichia coli, Pseudomonas aeruginosa, Neisseria |
The Gram stain in four steps
- Crystal violet (primary stain): all cells turn purple.
- Iodine (mordant): forms a crystal violet-iodine complex.
- Alcohol or acetone (decolorizer): the key step. The thick wall of gram-positive cells holds the complex; gram-negative cells lose it.
- Safranin (counterstain): colorless gram-negative cells turn pink.
Over-decolorizing can make gram-positive cells look gram-negative, and old cultures can stain unevenly.
Bacteria that do not fit
Mycobacterium has waxy mycolic acids and stains poorly, so an acid-fast stain is used. Mycoplasma has no cell wall at all.
Practice questions
1. After the decolorizer step, which cells are colorless?
Show answer
Gram-negative cells. They pick up the pink safranin in the last step.
2. Why is penicillin G usually more active against gram-positive bacteria?
Show answer
It blocks peptidoglycan cross-linking. Gram-positive peptidoglycan is exposed, while the gram-negative outer membrane limits how much drug reaches its target.
3. Which part of the gram-negative envelope causes endotoxin effects such as fever?
Show answer
Lipid A, part of the lipopolysaccharide in the outer membrane.
Frequently asked questions
What does lysozyme do?
It breaks the bonds between NAM and NAG sugars in peptidoglycan. Gram-positive cells are more exposed to it because they lack an outer membrane.
Is the Gram stain color the same as the species?
No. It is one clue. Shape, arrangement and biochemical tests are needed to identify a species.
Reference: OpenStax Microbiology, sections 2.4 (Staining Microscopic Specimens) and 3.3 (Unique Characteristics of Prokaryotic Cells).