Catalase Assay for Bacterial Quantification

We used the catalase assay developed by Iwase et al.^{16 (link)} with slight modifications. Bacterial strains were grown overnight in 1% soytone at 37°C with shaking. Optical density at 600 nm (OD₆₀₀) was recorded to estimate bacterial concentration and 2 mL of culture (6–8 × 10⁹ bacteria) was centrifuged; the supernatant was removed; and the cell pellet was resuspended in 100 μL of normal saline (9 g/L sodium chloride; Quality Biological, Gaithersburg, MD) and transferred into 13 × 100 mm borosilicate glass tubes. One-hundred microliters of 1% Triton X-100 and 100 μL of 35% stabilized hydrogen peroxide were added. The tubes were left untouched at room temperature until the foaming stopped (about 2 minutes). The height of the foam in the tube was then recorded. A standard curve was also created using dilutions of commercially available bovine catalase (Sigma-Aldrich; 3,000 units/mg), and the final results were expressed as units of catalase (U) per 10¹⁰ bacterial cells, assuming an OD₆₀₀ of 1.25 corresponds to 10⁹ bacteria/mL of culture for all strains tested.