Skip to Content

Which bacteria will stain gram-negative?

Bacteria can be divided into two major groups based on the chemical and physical properties of their cell walls. These groups are Gram-positive and Gram-negative bacteria. The Gram stain test, developed in 1884 by Danish bacteriologist Hans Christian Gram, differentiates bacteria based on cell wall structure.

Gram Staining Technique

The Gram stain procedure involves applying a series of stains to a smear of bacteria on a microscope slide:

  1. Primary stain – Crystal violet dye stains all bacteria purple
  2. Mordant – Iodine binds to crystal violet, trapping it in the cell
  3. Decolorizing agent – Alcohol rinses away crystal violet-iodine complex from gram-negative cells
  4. Counterstain – Safranin stains decolorized gram-negative cells pink

After staining, gram-positive bacteria remain purple while gram-negative bacteria appear pink under the microscope. This difference in color is due to differences in cell wall structure.

Cell Wall Structure

Gram-positive cell walls have a thick peptidoglycan layer which retains the crystal violet dye after decolorization. Gram-negative cell walls have a thinner peptidoglycan layer surrounded by an outer membrane which does not retain the violet stain.

Cell Wall Feature Gram Positive Gram Negative
Peptidoglycan layer Thick (makes up 90% of cell wall) Thin (makes up 10% of cell wall)
Outer membrane Absent Present

The Gram staining result thus reflects differences in cell wall structure between Gram positive and Gram negative bacteria.

Gram Negative Bacteria

Gram negative bacteria have an outer membrane composed of lipopolysaccharides and phospholipids surrounding a thin peptidoglycan layer. This structure does not retain the crystal violet-iodine complex and readily decolorizes during the Gram staining procedure. Here are some examples of Gram negative bacteria:


  • Escherichia coli
  • Salmonella
  • Shigella
  • Klebsiella
  • Proteus
  • Enterobacter
  • Serratia

The Enterobacteriaceae are facultative anaerobic, rod-shaped bacteria that live in soil, water, and the intestinal tracts of animals. They ferment lactose and produce gas. Many are part of the normal gut flora but some can cause food poisoning and other infections.


  • Pseudomonas
  • Moraxella
  • Acinetobacter

Pseudomonadaceae are aerobic, rod-shaped bacteria found widely in soil, water, and plants. Pseudomonas aeruginosa is an opportunistic pathogen that can infect immunocompromised individuals. Other species can cause spoilage of food.


  • Vibrio
  • Aeromonas
  • Plesiomonas

Vibrionaceae are curved gram negative rods found in marine and freshwater environments. Vibrio cholerae causes the diarrheal illness cholera. Other Vibrio species can cause food poisoning.


  • Neisseria

Neisseria includes cocci that appear in pairs. N. gonorrhoeae causes the STD gonorrhea and N. meningitidis causes bacterial meningitis.

Other Gram Negative Bacteria

  • Haemophilus influenzae
  • Bordetella pertussis
  • Brucella abortus
  • Helicobacter pylori
  • Campylobacter jejuni
  • Bacteroides fragilis
  • Fusobacterium nucleatum
  • Prevotella melaninogenica

This list of gram negative pathogenic bacteria is not comprehensive but illustrates the variety of shapes (rods, cocci, curved rods) and arrangements (single, pairs, chains) that can occur among gram negative bacteria.

Clinical Relevance of Gram Staining

Gram staining remains one of the most useful diagnostic tests in medicine and microbiology. It provides valuable information about bacteria to help guide treatment:

  • Aids in tentative identification of pathogen
  • Indicates suitable antibiotics for treatment
  • Gram positives are generally more susceptible to antibiotics that inhibit cell wall synthesis like penicillin and vancomycin
  • Gram negatives are generally more susceptible to antibiotics that disrupt membranes like polymyxins

The Gram stain provides a quick, inexpensive way to gain information about an unknown bacterial isolate from a patient specimen. It can help distinguish normal flora from potential pathogens and allow proper antibiotic selection while awaiting definitive bacterial identification and susceptibility testing.


The Gram staining procedure differentiates bacteria based on cell wall properties. Gram positive bacteria retain crystal violet dye and stain purple while Gram negative bacteria are decolorized and take up the pink counterstain. This is due to structural differences in the cell wall.

Gram negative bacteria have an outer membrane that prevents retention of the crystal violet complex during decolorization. Examples of gram negative bacteria come from a diverse set of families and include both rods and cocci.

The Gram stain remains highly useful clinically for its ability to provide information about unknown bacterial isolates from patient specimens. It can guide treatment approaches while awaiting definitive identification and antibiotic susceptibility results.