Serum Dilution Test for Bactericidal Activity. II. Standardization and Correlation with Antimicrobial Assays and Susceptibility Tests

Abstract

The serum dilution tes,t for bactericidal activity is widely used and recommended despite variations in method. A standardized serum dilution test is a necessary pre. requisite to meaningful comparisons of results among laboratories and reproduci. bility within laboratories. Therefore, we evaluated both micro- and macrotechniques for a standardized serum dilution test in which Mueller-Hinton broth supplemented with physiologic concentrations of Mg⁺⁺ and Ca⁺⁺ and combined with 50% pooled human serum (MHB-S/HS) was used as the diluent. Serum dilution tests were done with 13 antimicrobial agents against strains of Staphylococcus aureus, Streptococcus sanguis, Streptococcus mutans, Streptococcus faecalis, Pseudomonas aeruginosa, and Serratia marcescens isolated from the blood of patients with bacterial endocarditis or septicemia. The physiologic diluent MHB-S/HS performed like human serum in the serum dilution test and gave results comparable to those obtained by determination of minimal inhibitory and minimal lethal concentrations and assay of antimicrobial agents in test serum. Since the bactericidal end point was defined as ⩾99.9% killing of the initial inoculum of 5 × 105 colony-forming units/ml, similar results were obtained with different subculture techniques. When dilution tests are required to guide therapy of systemic infections, we recommend a standardized method in which MHB-S/HS is used as the diluent.