Phosphatase activity is a constant feature of all isolates of all major species of the family Enterobacteriaceae.

In this study we evaluated phosphatase activity in members of the family Enterobacteriaceae by conventional methods and by a novel method. The novel method is based on the formation of bright-green-strained colonies by phosphatase-positive, but not phosphatase-negative, strains in the presence of a phosphate substrate, such as phenolphthalein monophosphate or 6-benzoylnaphthyl phosphate (6-BNP), and methyl green. A total of 1,055 strains belonging to 65 different species of Enterobacteriaceae were tested for green staining of the colonies in the presence of methyl green and either phenolphthalein monophosphate or 6-BNP and for phosphatase activity by three different conventional methods. With the sole exception of one Leminorella richardii type strain, all isolates of all of the species formed green-stained colonies in the presence of the substrate 6-BNP. All strains were phosphatase positive by all of the conventional methods.