A simple and exact two-point interpolation method for determination of haemolytic activity in microtiter plates

A new haemolytic test based on direct reading of the optical density of unlysed erythrocytes was developed and adapted for use with microtiter plates and an appropriate reader. The relationship between the concentration of erythrocytes and the optical density was not strictly linear, but a high reproducibility of the curve was noted. There was a linear relation between the optical density of unlysed erythrocytes and -log2 of the haemolysin dilution in a wide range. The correlation coefficient was higher than 0.99. Because of this strong linearity, only two points of the two-fold dilution series, representing the dilutions before and after 50% lysis, were used to interpolate the exact haemolysin titer. These calculations were preferably performed automatically by a connected personal computer. The titers obtained by this method were identical with those obtained by the traditional tube test based on measurement of the absorbance of released haemoglobin. The standard deviations obtained for different haemolysin preparations were found to be 3.7-8%.