Peptone Water works by providing the minimum nutritional support necessary to prevent osmotic lysis or death of microorganisms during transfer, dilution, and transport, without introducing nutrients that could bias downstream quantitative results. The peptone hydrolysate supplies free amino acids and short oligopeptides that act as osmotic buffers, stabilising cell membranes of stressed and injured cells. Critically, the absence of fermentable carbohydrates prevents acid accumulation during holding periods, and the absence of selective agents or dyes ensures no organism group is suppressed. When used as a basal medium for the indole test, the tryptophan content of peptone is metabolised by tryptophanase-positive organisms (e.g. E. coli) to produce indole, detected by Kovacs' reagent. For carbohydrate fermentation tests, a specific sugar plus a pH indicator (e.g. phenol red) is added to 1 % Peptone Water to provide both the nitrogen base and the indicator system.

Reference: ISO 6579-1:2017 Annex D. E. coli ATCC 25922 = positive control; Klebsiella pneumoniae ATCC 13883 = negative control.