Methylene Blue (0.065 g/L) — Selective inhibitor: Inhibits the growth of most Gram-positive organisms by interfering with their cell membrane. Gram-negative bacteria are largely unaffected at this concentration, providing selectivity in mixed-flora samples.

Eosin Y (0.4 g/L) — pH indicator + inhibitor: At neutral pH, Eosin Y is soluble and produces pink/amber coloration. As organisms ferment carbohydrates and produce acid, Eosin Y combines with Methylene Blue to form an insoluble complex that precipitates onto colonies — producing the purple, blue-black, or characteristic metallic green sheen depending on the degree of acid production.

The metallic green sheen mechanism: In strong acid producers such as E. coli, the pH around the colony drops sharply. The dye complex precipitates in such large amounts that it forms a reflective layer on the colony surface, producing the iridescent metallic green appearance when viewed under oblique reflected light.

Dipotassium Phosphate (2.0 g/L): Buffers the medium to maintain pH 7.1 ± 0.2 — essential for consistent dye behaviour and reproducible colony differentiation.

Lactose (5.0 g/L): Primary fermentable carbohydrate. Differentiates lactose fermenters (coliforms) from non-fermenters (Salmonella, Shigella). The original Levine formula used lactose alone at 10 g/L.

Sucrose (5.0 g/L): Added per the Holt-Harris and Teague formulation. Some organisms ferment sucrose more readily than lactose — notably Proteus species, which are sucrose-positive but lactose-negative. The presence of sucrose enables differentiation of sucrose-positive non-lactose-fermenters from pathogens such as Salmonella and Shigella, which ferment neither carbohydrate and remain colourless.