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Effective utilisation and understanding of the clinical bacteriology laboratory can greatly aid in the diagnosis of infectious diseases. Although described more than a century ago, the Gram stain remains the most frequently used rapid diagnostic test, and in conjunction with various biochemical tests is the cornerstone of the clinical laboratory. First described by Danish pathologist Christian Gram in 1884 1 2 and later slightly modified, the Gram stain easily divides bacteria into two groups, Gram positive and Gram negative, on the basis of their cell wall and cell membrane permeability to organic solvents (box 1). 3-5 Information derived from a Gram stain and several simple biochemical tests can be enormous, often providing a presumptive diagnosis and significantly influencing patient care. Unfortunately the training for correctly interpreting the Gram stain is disappearing. 6 The goal of this article is to review the utility of the clinical bacteriology laboratory and discuss its role in the diagnosis of common clinical pathogens.
Gram stain basics
Box 1: Gram stain technique
Air dry specimen and fix with methanol or heat.
Add crystal violet stain.
Rinse with water to wash unbound dye, add mordant (for example, iodine: potassium iodide).
After waiting 30-60 seconds, rinse with water.
Add decolorising solvent (ethanol or acetone) to remove unbound dye.
Counterstain with safranin.
Gram positive bacteria stain blue (retained crystal violet).
Gram negative bacteria stain red (decolorised and then counterstained).
The present hypothesis for the mechanism of the Gram stain states the cell wall acts as a physical permeability barrier restricting diffusion of the stain complex, 7 and any micro-organism with a cell wall able to retard the efflux of the crystal violet-iodine complex should be Gram positive. The mechanism further implies that solvent decolorisation causes significant damage to the cell surfaces of Gram negative bacteria, and only limited damage to Gram positive bacteria. This suggests Gram negative bacteria are more "leaky", causing these thin walled lipid-rich cells to lose their crystal violet stain and appear red from the counterstain. Gram positive cells, thick walled and lipid-poor, appear blue from retaining the original crystal violet.
Gram stain utility
Gram stain interpretation gives immediate information regarding the presence or absence of bacterial disease and can guide initial antibiotic treatment. Additionally, epithelial and inflammatory cells are stained...