Use of the diagnostic bacteriology laboratory: a practical review for the clinician
- Lucile Salter Packard Children's Hospital at Stanford, Stanford University School of Medicine, 725 Welch Road, Palo Alto, California, USA 94304, USA
- Dr Steinbach
- Received 27 March 2000
- Accepted 5 June 2000
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 18841 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
The Gram stain classifies bacteria (fig 1) phenotypically based on differences in cell wall thickness with differing glycosaminopeptide and lipoprotein compositions: Gram positive bacteria have a peptidoglycan layer 10–15 times thicker than Gram negative bacteria. The cell wall, synonymous with the peptidoglycan layer, is a rigid framework of cross linked peptidoglycan forming the outermost component of the cell. The more complex Gram negative bacteria also have an outer membrane beyond the peptidoglycan layer that consists of lipopolysaccharide (endotoxin), lipoprotein, and phospholipids. In some Gram negative species there also exists a periplasmic space between the outer membrane and the inner cytoplasmic membrane with β-lactamases that degrade β-lactam antibiotics.
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). …