Caister Academic Press

Gram-negative Bacteria

Molecular Diagnostics: Current Research and Applications
Edited by: Jim Huggett and Justin O'Grady
"I would highly recommend this book" (Doodys); "useful new insights" (Aus. J. Med. Sci.)
Gram-negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain protocol. Gram-negative bacteria will thus appear red or pink following a Gram stain procedure due to the effects of the counterstain (for example safranin).

The Gram Stain

In microbiology, the visualization of bacteria at the microscopic level is facilitated by the use of stains, which react with components present in some cells but not others. This technique is used to classify bacteria as either Gram-positive or Gram-negative depending on their colour following a specific staining procedure originally developed by Hans Christian Gram. Gram-positive bacteria appear dark blue or violet due to the crystal violet stain following the Gram stain procedure; Gram-negative bacteria, which cannot retain the crystal violet stain, appear red or pink due to the counterstain (usually safranin).

The reason bacteria are either Gram-positive or Gram-negative is due to the structure of their cell envelope. (The cell envelope is defined as the cell membrane and cell wall plus an outer membrane, if one is present.) Gram-positive bacteria, for example, retain the crystal violet due to the amount of peptidoglycan in the cell wall. It can be said therefore that the Gram-stain procedure separates bacteria into two broad categories based on structural differences in the cell envelope.

Cell envelope of Gram-negative Bacteria

The Gram negative cell envelope contains an additional outer membrane composed by phospholipids and lipopolysaccharides which face the external environment. The highly charged nature of lipopolysaccharides confer an overall negative charge to the Gram negative cell wall. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains. Many species of Gram-negative bacteria are pathogenic. This pathogenicity is often associated with the lipopolysaccharide (LPS) layer of the Gram-negative cell envelope.


Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. Mycobacteria have a cell envelope which is not typical of Gram positives or Gram negatives. The mycobacterial cell envelope does not consist of the outer membrane characteristic of Gram negative bacteria, but has a significant peptidoglycan-arabinogalactan-mycolic acid wall structure which provides an external permeability barrier.

Characteristics of Gram-negative Bacteria

Gram-negative bacteria have a characteristic cell envelope structure very different from Gram-positive bacteria. Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the loose network of peptidoglycan chains referred to as the peptidoglycan layer.

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Gram-negative bacteria

Further reading