Phosphatase activities were assayed at 37°C as
described [17] using x-naphthylphosphate as a
substrate and tetrazotized-o-dianisidine as a post-
coupling reagent. Protein was determined accord-
ing to Bradford [18] using bovine serum albumin
as a standard. Activity units are expressed as
pmol naphthol produced per h. Staining of
colonies for phosphatase activity at pH 9.5 was
performed as follows. A drop (2 ~1) of 1 mg ml ~ ’
a-naphthylphosphate in 0.2 M glycine-NaOH
buffer, pH 9.5, was applied to each colony. Five
min later, the plate was flooded with 10 ml of 2
mg ml - ’ tetrazotized-o-dianisidine in 0.2 M Na
acetate buffer, pH 4.8, which resulted in the im-
mediate appearance of purple staining at the level
of the colony.