Gramstaining

Gram staining, or Gram stain, is a differential staining technique that classifies bacteria into Gram-positive and Gram-negative groups based on cell wall properties. Developed by Hans Christian Gram in 1884, it remains a standard first step in bacterial identification used in clinical, environmental, and research laboratories.

The procedure typically comprises four steps: applying crystal violet as the primary stain, adding iodine as a mordant to form a crystal violet–iodine complex, decolorizing with alcohol or acetone, and counterstaining with safranin. Gram-positive cells retain the violet dye and appear purple; Gram-negative cells take up the counterstain and appear pink or red.

The distinction reflects structural differences: Gram-positive bacteria have a thick peptidoglycan layer with teichoic acids, which traps the dye; Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane that facilitates decolorization.

Limitations include variability with culture age, smear thickness, and reagent quality; some organisms stain inconsistently (Gram-variable) or require different methods (e.g., acid-fast staining for Mycobacteria). Atypical wall can yield misleading results, and results should be interpreted with other tests.

Clinical relevance: Gram staining helps guide initial treatment decisions by indicating likely Gram-positive versus Gram-negative infection, but it is not definitive and should be confirmed with culture and additional tests.

Quality control and safety: the technique uses reference controls and is performed under standard biosafety guidelines to ensure accuracy and safety.