The most fundamental technique for classifying bacteria is the gram stain, developed in 1884 by Danish scientist Christian Gram. It is called a differential stain because it differentiates among bacteria and can be used to distinguish among them, based on differences in their cell wall.
In this procedure, bacteria are first stained with crystal violet, then treated with a mordant—a solution that fixes the stain inside the cell (e.g., iodine-KI mixture). The bacteria are then washed with a decolorizing agent, such as alcohol, and counterstained with safranin, a light red dye.
The walls of gram positive bacteria (for example, Staphylococcus aureus) have more peptidoglycans (the large molecular network of repeating disaccharides attached to chains of four or five amino acids) than do gram-negative bacteria. Thus, gram-positive bacteria retain the original violet dye and cannot be counterstained.
Gram negative bacteria (e.g., Escherichia coli) have thinner walls, containing an outer layer of lipopolysaccharide, which is disrupted by the alcohol wash. This permits the original dye to escape, allowing the cell to take up the second dye, or counterstain. Thus, gram-positive bacteria stain violet, and gram-negative bacteria stain pink.
The gram stain works best on young, growing populations of bacteria, and can be inconsistent in older populations maintained in the .
Microbiologists have accumulated and organized the known characteristics of different bacteria in a reference book called Bergey's Manual of Systematic Bacteriology (the first edition of which was written primarily by David Hendricks Bergey of the University of Pennsylvania in 1923).
The identification schemes of Bergey's Manual are based on morphology (e.g., coccus, bacillus), staining (gram-positive or negative), cell wall composition (e.g., presence or absence of peptidoglycan), requirements (e.g., aerobic, facultatively anaerobic) and biochemical tests (e.g., which sugars are aerobically metabolized or fermented).
In addition to the gram stain, other stains include the acid-fast stain, used to distinguish Mycobacterium species (for example, Mycobacterium tuberculosis, the cause of tuberculosis); endospore stain, used to detect the presence of endospores; negative stain, used to demonstrate the presence of capsules; and flagella stain, used to demonstrate the presence of flagella.