A rapid chromogenic microtitre assay of arginine aminopeptidase activity in Mycoplasma strains

Arginine-utilizing strains of Mycoplasma can be screened by assay of their arginine aminopeptidase activity. A standardized chromogenic method is described that enables enzyme detection in small volumes of cell suspension in less than 3 h. Cell suspensions (10 μl) in 96-well microtitre plates are incubated at 37 °C, pH 8.0, with 0.1 mM arginyl-β-naphthylamide (100 μl). This is hydrolysed to release β-naphthylamine, which gives a coloured product on diazotization with fast garnet. M. alkalescens can be detected in this way with as few as 1.1×105 viable cells and M. fermentans with 2.3×106 cells. The method has been shown to enable division of 28 strains into three groups of fermentative and arginine-hydrolysing mycoplasmas. This procedure has potential for routine laboratory use.