A QUANTITATIVE MICROTITER PLATE HEMOLYSIS ASSAY FOR LISTERIA MONOCYTOGENES

ABSTRACT All virulent strains of L. monocytogenes produce the extracellular SH‐activated hemolysin, listeriolysin O, while nonhemolytic strains of L. monocytogenes are avirulent suggesting that the expression of this hemolysin is necessary for virulence of L. monocytogenes. Hence, testing for hemolysis becomes clinically relevant for an isolate identified as L. monocytogenes. However, the quantification and interpretation of this characteristic on blood agar poses several problems. Hence we have proposed a simple quantitative microtiter plate hemolysis assay. The assay is made of SRBC (3% SRBC in PBS) in microtiter plates to which CASO‐cultured cell‐free supernatants of L. monocytogenes or other test cultures are added. After mixing, the plates are incubated at 37C for 15–30 min and the hemolysis is visually read as CHU and MHU, as well as by a 620 nm scan for absorbancy. Percent hemolysis is calculated. We feel that this assay should prove to be of significance in a microbiology laboratory in which routine hemolysis assays are conducted.