Gram Negative Bacteria - Overview

Introduction

Gram negative bacteria stain pink following a Gram stain, whereas Gram positive bacteria stain purple.

The Gram stain is an empirical procedure, but this reaction is a manifestation of fundamental differences in the properties of the bacterial species. Gram positives and negatives have a different cell wall structure, different nutritional requirements, different types of toxins and different resistance to chemotherapeutic agents, disinfectants and enzymes.

Gram Stain

This is the most widely used differential stain. The primary stain (crystal violet) is applied. This is then washed away and a mordant (iodine solution) is applied to complex the crystal violet to the bacterial cell. This is removed and a decolourising agent, usually acetone, is applied. This removes the purple stain only from the Gram negative bacteria, while Gram positive organisms retain it. The unstained Gram negatives are then visualised with a red counterstain, usually Carbol Fuchsin, which will not alter the purple appearance of Gram positives.

Dead bacteria, cells and organic debris will stain pink. In older cultures of Gram positive bacteria there are likely to be dead cells which stain pink. Since Gram negative bacteria do not give false positive reaction it is usually safe to assume that bacteria giving a doubtful reaction are Gram positive.

Cell Wall Structure

The cell wall of Gram negative bacteria has some special features:

thin peptidoglycan layer just outside the cytoplasmic membrane

Outer membrane, similar to the cytoplasmic membrane but containing different proteins, specifically lipopolysaccharide, located in the outer leaflet of the outer membrane

Lipopolysaccharide (LPS)

There are three regions to this molecule:

The lipid region, lipid A, contains 3-hydroxy fatty acids linked to a diglucosamine backbone. These fatty acids are hydrophobic and intercalate into the phospholipid bilayer of the outer membrane.

The LPS core region is linked to lipid A and is a short oligosaccharide which is variable between bacterial type and contains unusual sugar residues. Bacteria with a LPS which stop at this point are called R-form, or rough bacteria, and they will autoagglutinate in saline and are of low virulence.

The third region is the O-side chain, which is a repeating oligosaccharide with 50 or 80 repeat units extending into the external environment of the bacterium. This makes the surface of the bacterium hydrophilic and the O-side chain is highly antigenic.

Bacteria with an O-side chain are termed smooth and are usually virulent.

The LPS also has biological properties. The lipid A part disrupts mammalian cell membranes, and causes pyrexia. Gram negative septicaemia leads to circulatory collapse. The LPS is also known as endotoxin because of these properties.

Gram Negative Bacteria

Individual bacteria have characteristic shapes. The cells may be spherial (coccus), rod shaped (bacillus), comma shaped (curved rod), spiral (spirochaete) or filamentous. The shape can differ to some degree with the growth conditions, but usually the shape of most bacteria is an important clue to their identity.

See a list of the Gram negative bacteria.

References

Rycroft, A. (2008) Fundamentals of veterinary bacteriology RVC student notes

Ryan, K. (2010) Sherris medical microbiology McGraw Hill Professional

Black, J. (2008) Microbiology: principles and explorations John Wiley and Sons