Culture specimens that are over 24 hours old may stain incorrectly, losing their ability to hold on to the crystal violet stain. They may appear as gram-negative organisms when they are actually gram-positive, or the specimen may show a gram-variable result.
Gram stains reveal the structural differences in the cell walls of organisms in the laboratory. Gram-positive organisms have a thick cell wall containing up to 90 percent peptidoglycan and appear blue or purple under the microscope after staining with crystal violet. Gram-negative organisms have a much thinner cell wall containing up to 10 percent peptidoglycan and a high lipid content. These organisms appear red or pink under the microscope after staining with crystal violet.
The gram stain is a critical laboratory test that is integral to the study and treatment of infectious disease. Many antibiotics work on either gram-negative or gram-positive organisms because of the way they break down the cell walls. Broad-spectrum antibiotics work on both gram-negative and gram-positive organisms.
The gram stain was created by Hans Christian Gram in 1884. He was searching for a way to visualize cocci in lung tissue samples of those who had died from pneumonia. He used crystal violet as his primary stain, followed by iodine as a fixer and ethanol as a decolorizer. The decolorizing step creates the loss of the blue-purple coloration that differentiates gram-positive and negative organisms from one another.