Gram stain
The Gram stain (also known as Gram's stain or Gram's method) is a laboratory technique in microbiology, which causes bacteria with certain biologically and medically important characteristics to be colored violet or red when viewed under a microscope. Gram-positive bacteria are those that are stained dark blue or violet by the process. Organisms that are Gram-positive have cell walls containing multiple layers of peptidoglycan bound together by amino acid bridges.
Gram-negative organisms do not take up the peptidoglycan-selective coloring, and are stained red by the default second dye.
Not all bacteria reliably take either stain. Mycobacteria, for example, tend to need the Ziehl-Neelsen stain, which uses heat and acid to drive a stain into dye-resistant cell walls.
Technique
- Prepare a heat-fixed smear of the material containing the bacteria of interest
- Stain with crystal violet
- Treat with iodine solution, usually Lugol's solution. Its mordant solution fixes the crystal violet in Gram-positive cell walls.
- Decolorize with an alcohol or acetone-alcohol
- Counterstain with safranin, a red dye
Clinical correlations
Broad-spectrum antibiotics affect both Gram-positive and Gram-negative bacteria. No antibiotic attacks all Gram-positive, all Gram-negative, or any other total population of bacteria. Gram staining is a guide to antibiotic selection, but bacterial culture and antibiotic sensitivity testing as well as clinical experience — especially local to a given area — remain the gold standard of selecting antibiotics for treatment.
Gram-positive
The first clinically useful antibiotic, penicillin, of the beta-lactam class, tended to attack only Gram-positive bacteria.
Gram-negative
Other classes, such as streptomycin, a member of the aminoglycoside class, tended to attack Gram-negative bacteria.
Broad-spectrum
Broad-spectrum antibiotics, the earliest representatives of which were tetracyclines, attack both Gram-positive and Gram-negative bacteria.