ALKALINE PHOSPHATASE C ASSAY FOR P. aeruginosa
 

PURPOSE: Can be used to test for the induction of the pho-regulon by monitoring the time of appearance of alkaline phosphatase in the culture medium. Although this assay could theoretically be used as a measure of outer membrane permeability, linearity of the initial rate of hydrolysis can not be obtained.

MATERIALS: The substate is 1 para-nitrophenyl phosphate (PNPP) tablet in 5ml of 0.1M Tris-HCl, pH 8.0 as a stock solution.

METHODS: Put 500µl of the substrate solution plus 500µl of the cells to be tested in a cuvette, mix, and record the hydrolysis at OD₄₀₅ on a strip chart recorder. The rate of hydrolysis is proportional to the amount of enzyme which has been synthesized and released from the cells.