Gram stain

The Gram stain 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

1. Prepare a heat-fixed smear of the material containing the bacteria of interest
2. Stain with crystal violet
3. Treat with iodine solution, usually Lugol's solution. Its mordant solution fixes the crystal violet in Gram-positive cell walls.
4. Decolorize with an alcohol or acetone-alcohol
5. 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 selectng 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 negative bacteria.