Dye-diffusion test for semiquantitative staphylocoagulase assay.

Dye-diffusion test for semiquantitative staphylocoagulase assay. Ever since 1903, when Leob reported the ability of staphylococci to produce coagulase, an enzyme capable of clotting plasma, numerous workers have demonstrated the practical applicability of this phenomenon as an indicator of potential pathogenicity of these organisms. Two general procedures for coagulase determination are the tube method (2) and the slide method (1). Recently, a capillary tube method (3) and a nephelometric method (5) were introduced. The common weakness of these methods is the difficulty in detecting various degrees of coagulation. Since different degrees of coagulase activity exist among staphylococcal strains, it would be helpful to establish a more quantitative method of coagulase assay. A rapid, simple dye-diffusion test for semiquantitative estimation of coagulase activity is described. Two drops (ca. 0.1 ml) of Staphylococcus aureus culture were added by means of a sterile Pasteur pipette to a glass tube (5 X 15 mm), sealed at one end and containing 4 drops (ca. 0.2 ml) of reconstituted plasma solution (Difco). The mixture was stirred with a toothpick (previously sterilized by autoclaving) to ensure homogeneity. The tube with plasma and culture was placed in an incubator at 37 C for 2 hr to permit coagulation to occur. A drop of crystal violet solution (4) was added to the tube after the reaction time and allowed to diffuse for an arbitrary standard diffusion time of 1.5 hr. A basic assumption of this test is that the rate of diffusion of the dye is inversely proportional to the amount of coagulation. A scale was established to calibrate the degree of coagulation of plasma by S. aureus. From the bottom of a number of tubes, measurements were made of the length of clear zone, representing clots formed by different amounts of activity of coagulase produced (Fig. 1). This eight-point scale is more meaningful and reproducible than the existing 0, +, + +, + ++, + + + + designation proposed by F. J. Turner