Gramstain

Gram staining, also known as Gram stain, is a differential staining technique used in microbiology to classify bacteria into two broad groups: Gram-positive and Gram-negative. It was developed by Danish bacteriologist Hans Christian Gram in 1884. The method relies on differences in the structure of bacterial cell walls, particularly the thickness of the peptidoglycan layer and the presence of an outer membrane in Gram-negative bacteria.

The procedure consists of a sequence of steps: fixation, application of crystal violet, treatment with iodine as a mordant, decolorization with alcohol or acetone, and counterstaining with safranin. Gram-positive organisms retain the crystal violet-iodine complex and appear purple under a light microscope, whereas most Gram-negative organisms lose the dye during decolorization and appear pink to red after counterstaining.

Interpretation and limitations: Some organisms are Gram-variable or do not stain well. Mycobacteria and other organisms with lipid-rich cell walls may resist Gram staining and require acid-fast or other specialized stains. Age of culture, suboptimal technique, and over- or under-decolorization can lead to misclassification. The Gram result is an initial diagnostic aid and is used in conjunction with morphology, culture characteristics, and biochemical tests to identify organisms.

Applications and significance: The Gram status informs initial antibiotic decisions, as Gram-positive and Gram-negative groups differ in cell wall structure and permeability to many antibiotics. It remains a standard first step in routine clinical microbiology laboratories for isolates from sterile sites, blood cultures, and other specimens, as well as in educational settings.

The method was introduced by Hans Christian Gram in 1884 and has since become the cornerstone of bacterial taxonomy and diagnostic microbiology.