gramkleuring

Gramkleuring, also known as Gram staining, is a differential staining technique used in microbiology to classify bacteria into Gram-positive and Gram-negative based on differences in cell wall structure. The method involves fixing a bacterial smear to a slide, staining with crystal violet, adding iodine as a mordant, decolorizing with alcohol or acetone, and counterstaining with safranin. After staining, Gram-positive bacteria appear purple, while Gram-negative bacteria appear red or pink under a light microscope.

Rationale: Gram-positive bacteria have a thick peptidoglycan layer that retains the crystal violet-iodine complex after decolorization. Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane; the decolorizer removes the violet-iodine complex, allowing the counterstain to be seen.

Procedure notes: Typical steps are brief, around 30–60 seconds per stain, with careful timing and proper fixation to ensure accuracy. The age of the culture, cell clumping, and technique can influence results. Some bacteria are Gram-variable and may not fit cleanly into either category, especially if the culture is old or poorly prepared.

Applications and limitations: Gramkleuring is a foundational step in bacterial identification and helps guide initial antibiotic choices because Gram status relates to cell wall characteristics and drug susceptibility. Limitations include variability for certain species, poor reliability for atypical organisms (e.g., Mycobacteria or intracellular bacteria), and artifacts from over- or under-decolorization that can misclassify cells.

History: The technique was developed by Hans Christian Gram in 1884 and remains a central tool in clinical and research microbiology.