Plate assay methods for amino acids. 2. Factors affecting the cup plate assay for lysine with Streptococcus faecalis ATCC 6057

A number of factors was found to affect the cup plate assay for lysine with Streptococcus faecalis ATCC 6057. Hydroxylysine, aspartic acid, and Tween 80 were found to be among the essential components of the assay medium. Removal of any of these three components produced diffuse growth zones or otherwise unsatisfactory plates. The inclusion of asparagine and additional dipotassium phosphate to the medium was found to be advantageous but not an absolute requirement. It was important not to autoclave the hydroxylysine and the additional dipotassium phosphate together with the rest of the assay medium, but to add these components to the sterilized medium immediately before pouring the plates. With certain concentrations of hydroxylysine in the medium, the addition of L ‐glutamine was found to potentiate the effect of hydroxylysine and thereby improve the lysine plates. The effect of graded amounts of the above mentioned medium components and of the pH of the assay medium was also studied. Increasing the sterilization time of the medium from 5 to 10 min. at 120°C. improved the readability of the lysine plates and led to more easily reproducible results. Factors influencing the preparation of the inoculum for the lysine plates were also studied: the age of the stab culture, the amount of inoculum used per plate, and the influence of the nature of the suspension medium used for washing the inoculum.