# Gram-positive bacteria
Gram-positive bacteria are bacteria that give a positive result in the [Gram stain](https://www.edgepedia.org/gram-stain) test, retaining the crystal violet dye and appearing purple or dark blue under the light microscope; the category is defined by a thick peptidoglycan cell wall and the absence of an outer membrane.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018) The Danish bacteriologist Hans Christian Gram developed the staining procedure in 1884 while working in Berlin, and it has since become the most important stain in microbiology and a standard first step in identifying bacterial infections.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018) • [2](https://www.nature.com/articles/151671a0)
| Key fact | Detail |
|---|---|
| Defining test | Retain the crystal violet:iodine complex after decolorization and appear purple; Gram-negative cells lose the complex and take up the pink counterstain[3](https://pubmed.ncbi.nlm.nih.gov/8724440/) |
| Cell wall | Thick peptidoglycan layer, 30 to 100 nm or thicker, compared with a layer of only a few nanometers in Gram-negative bacteria[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018) |
| Outer membrane | Absent; instead of lipopolysaccharides, the wall contains teichoic acid and lipoteichoic acid[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018) |
| Wall accessory polymers | Teichoic and teichuronic acids may account for up to 50% of the dry weight of the wall and 10% of the dry weight of the total cell[4](https://repository.stikesbcm.ac.id/id/eprint/581/1/Medical%20Microbiology.pdf) |
| Typical examples | *Staphylococcus aureus*, *Streptococcus pneumoniae*[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC13501153/) |
| Key resistance mechanisms | Target-site alterations such as PBP2a-mediated methicillin resistance in *S. aureus*; vancomycin-resistant enterococci are a monitored healthcare threat[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC13501153/) • [6](https://www.cdc.gov/antimicrobial-resistance/data-research/threats/update-2022.html) |
| Staining exception | Mycobacteria are classified as Gram-positive but stain poorly because their lipid-rich wall requires Ziehl-Neelsen (acid-fast) staining[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC11086966/) |
## The Gram stain and why it works
The staining procedure uses gentian violet, which penetrates the cell wall and cytoplasmic membrane of heat-fixed bacteria. After iodine is added, an insoluble crystal violet:iodine complex forms. In Gram-positive cells the thick cell wall retains this complex when a decolorizer such as ethanol is applied, so the cells appear almost purple. Gram-negative cells retain the dye to a lesser extent or not at all and must be counterstained with safranin or fuchsine, appearing pink or reddish.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018)
The mechanism was understood only about a century after the stain was introduced. Work summarized by Popescu and Doyle, microbiology researchers at the [University of Louisville](https://www.edgepedia.org/university-of-louisville), showed that the cell wall of Gram-positive organisms is responsible for retaining the crystal violet:iodine complex, while in Gram-negative cells the staining procedure damages the cell surface and the dye complexes are lost. A relatively thick cell wall is required to retain the dye, so Gram-stainability is a function of the wall itself rather than the chemistry of cell constituents.[3](https://pubmed.ncbi.nlm.nih.gov/8724440/)
The stain has known limits. Old cultures lose peptidoglycan, so Gram-positive cells can stain Gram-negative or Gram-variable, and organisms without a cell wall, such as *Mycoplasma*, or too small to stain reliably, such as *[Chlamydia](https://www.edgepedia.org/chlamydia-genus)* and *Rickettsia*, cannot be classified this way.[8](https://www.ncbi.nlm.nih.gov/books/NBK562156/)
## Cell envelope structure
A Gram-positive cell has three compartments: the cytosol, a single cytoplasmic membrane, and the surrounding cell wall. The wall is a peptidoglycan macromolecule, a mesh of repeating N-acetylglucosamine and N-acetylmuramic acid disaccharides cross-linked through peptide stems, with attached accessory molecules such as teichoic acids, teichuronic acids, polyphosphates, and carbohydrates.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC98962/) The main structural difference from [Gram-negative bacteria](https://www.edgepedia.org/gram-negative-bacteria) is the thickness of this peptidoglycan layer, which in Gram-positive cells reaches 30 to 100 nm or more.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018)
**Teichoic acids.** Two abundant wall polymers characterize the Gram-positive envelope: wall teichoic acids, covalently bound in the peptidoglycan, and lipoteichoic acids, anchored via lipid domains in the cytoplasmic membrane. Both are phosphodiester-linked polyol repeat units, typically glycerol phosphate or ribitol phosphate, and their multiple phosphate groups give the cell surface a strongly negative charge. They protect against environmental stress and antimicrobial peptides, control autolysin activity and cation concentrations, mediate biofilm formation and binding to medical devices, and in *S. aureus* loss of wall teichoic acid reduces colonization and infection in animal models.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018)
**Surface proteins and capsules.** The wall also serves as an attachment site for proteins that interact with the environment and host tissues. Surface proteins are covalently attached to the peptidoglycan through a C-terminal sorting signal containing the consensus LPXTG sequence; more than 100 such proteins have been identified, including the M proteins of *Streptococcus pyogenes*, protein A of *Staphylococcus aureus*, and several internalins of *Listeria monocytogenes*.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC98962/) Many Gram-positive bacteria also synthesize a polysaccharide capsule, the outermost wall layer, which protects against phagocytosis and complement-mediated lysis; in pneumococci, different capsule polysaccharides serve as vaccine antigens.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018)
**Wall turnover.** Gram-positive bacteria continuously remodel their wall: newly synthesized peptidoglycan is delivered to the inner face, and older material at the outside is hydrolyzed by autolysins. An estimated 50% of the total cell wall mass is turned over within one generation, and these bacteria recycle the hydrolyzed fragments, a process that becomes important for survival in the stationary phase.[1](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018)
## The mycobacterial exception
Mycobacteria are classified as Gram-positive, but their walls contain three major macromolecules, peptidoglycan, arabinogalactan, and mycolic acids, and the high density of lipids prevents accurate Gram staining. They are instead described as acid-fast bacteria and are stained with the Ziehl-Neelsen method. This lipid-rich arrangement forms an efficient permeability barrier, particularly against drugs, and underlies the pathogenicity of mycobacteria.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC11086966/)
## Antibiotic resistance in Gram-positive pathogens
Resistance mechanisms differ between the two Gram categories in ways that matter for diagnosis. Gram-negative bacteria combine an outer membrane barrier with porin changes and multidrug efflux pumps, whereas in Gram-positive organisms phenotypic resistance more directly reflects target-site alterations, such as PBP2a-mediated methicillin resistance, because there is no analogous outer permeability barrier.[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC13501153/) Methicillin-resistant *Staphylococcus aureus* (MRSA) and vancomycin-resistant *Enterococcus* (VRE) are among the seven antimicrobial-resistant healthcare-associated pathogens for which the CDC updated U.S. burden estimates using 2021 and 2022 data.[6](https://www.cdc.gov/antimicrobial-resistance/data-research/threats/update-2022.html) In the 2024 WHO bacterial priority pathogens list, antibiotic-resistant *Staphylococcus aureus* is ranked as high priority for research and public health action, while the critical-priority category is occupied by Gram-negative pathogens and rifampicin-resistant *Mycobacterium tuberculosis*.[10](https://iris.who.int/bitstream/handle/10665/376776/9789240093461-eng.pdf)
Susceptibility remains high to several last-line agents. [In vitro](https://www.edgepedia.org/in-vitro) testing of Gram-positive bacteria causing microbial keratitis found susceptibility rates exceeding 98% to linezolid, vancomycin, tigecycline, quinupristin/dalfopristin, and rifampin.[11](https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2024.1363437/pdf) Infections caused by aerobic Gram-positive rods are comparatively rare, and few large-scale susceptibility studies exist for these organisms; some species have predictable susceptibilities, for example to penicillin, but a correct species or genus diagnosis is required before assuming predictable activity.[12](https://onlinelibrary.wiley.com/doi/10.1046/j.1469-0691.2001.00057.x/full)
## Clinical and diagnostic relevance
The Gram stain gives early, actionable information. In bloodstream infections, Gram stains of positive blood cultures classify organisms into categories such as Gram-positive cocci in clusters, Gram-positive cocci in pairs or chains, and Gram-positive rods, and rapid reliable analysis has been associated with better clinical outcomes.[13](https://arxiv.org/abs/2409.15546) Conventional culture-based antimicrobial susceptibility testing remains the reference standard but typically requires 24 to 72 hours, while molecular assays targeting specific determinants such as *mecA* reduce time to result at the cost of detecting only known resistance genes.[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC13501153/)
Gram-positive bacteria also dominate some infection sites. Across studies of microbial keratitis, Gram-positive bacteria caused 48% to 89% of infections, compared with 11% to 50% for Gram-negative bacteria, a distribution that varies with geography.[11](https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2024.1363437/pdf)
## References
1. [Rohde, M. (2019). The Gram-Positive Bacterial Cell Wall. Microbiology Spectrum.](https://journals.asm.org/doi/full/10.1128/microbiolspec.gpp3-0044-2018)
2. [Histochemistry of the Gram-staining Reaction for Micro-organisms, Nature (1943)](https://www.nature.com/articles/151671a0)
3. [Popescu, A. and Doyle, R. J. (1996). The Gram stain after more than a century. Biotech Histochem 71(3):145-51.](https://pubmed.ncbi.nlm.nih.gov/8724440/)
4. [Medical Microbiology, Section I: Fundamentals of Microbiology](https://repository.stikesbcm.ac.id/id/eprint/581/1/Medical%20Microbiology.pdf)
5. [Molecular mechanisms of antibiotic resistance in Gram-positive and Gram-negative bacteria: a narrative review, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC13501153/)
6. [Antimicrobial Resistance Threats in the United States, 2021-2022, CDC](https://www.cdc.gov/antimicrobial-resistance/data-research/threats/update-2022.html)
7. [The Gram-Positive Bacterial Cell Wall, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11086966/)
8. [Gram Staining, StatPearls, NCBI Bookshelf](https://www.ncbi.nlm.nih.gov/books/NBK562156/)
9. [Navarre, W. W. and Schneewind, O. (1999). Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope. Microbiol Mol Biol Rev.](https://pmc.ncbi.nlm.nih.gov/articles/PMC98962/)
10. [WHO Bacterial Priority Pathogens List, 2024](https://iris.who.int/bitstream/handle/10665/376776/9789240093461-eng.pdf)
11. [Bacterial profiles and antibiotic sensitivity in microbial keratitis, Frontiers in Cellular and Infection Microbiology (2024)](https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2024.1363437/pdf)
12. [von Graevenitz, A. (2001). Antimicrobial therapy of infections with aerobic Gram-positive rods. Clinical Microbiology and Infection.](https://onlinelibrary.wiley.com/doi/10.1046/j.1469-0691.2001.00057.x/full)
13. [A Novel Framework for the Automated Characterization of Gram-Stained Blood Culture Slides Using a Large-Scale Vision Transformer, arXiv](https://arxiv.org/abs/2409.15546)
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*Topic: Encyclopedia › Life and health › Microorganisms and fungi › Bacteria › Gram classification and staining*
*Initially written Sep 17, 2026 · Reviewed: Oct 21, 2026 · Edited: Oct 15, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*
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