To determine bacterial growth and dissemination levels, the aerobic bacteria in blood, peritoneal lavage fluids, and spleens were assessed.
Six hours after CLP or sham surgery, blood was collected in sterile tubes with previously added EDTA for anticoagulation. Whole blood was diluted 10- to 104-fold with sterile PBS, and 20 μL was immediately plated on 5% solid agar medium for bacterial culture. The remaining portion was centrifuged at 3,000 rpm for 8 min, and the plasma was then isolated for further experiments.
Mice peritoneum lavage fluids were obtained in 5 mL of ice-cold PBS. Lavage fluids were collected and filtered through a 70-μm nylon filter (BD Biosciences) to remove debris. Peritoneal lavage fluids from mice that underwent sham surgery were diluted by 10-fold, whereas fluids from mice that underwent CLP surgery were diluted by 102–104-fold with sterile PBS and 20 μL of each were immediately plated on solid medium for bacterial culture. The remaining portions were stored at −80°C for future experiments (21 (link)).
To evaluate the bacterial seeding in organs, spleens were excised and homogenized in 1 mL sterile PBS. After vortexing, 20 μL of the fluids were immediately used for bacterial culture (21 (link)).
All solid media were incubated overnight at 37°C, and colony-forming units (CFUs) were calculated according to the dilution fold.
Six hours after CLP or sham surgery, blood was collected in sterile tubes with previously added EDTA for anticoagulation. Whole blood was diluted 10- to 104-fold with sterile PBS, and 20 μL was immediately plated on 5% solid agar medium for bacterial culture. The remaining portion was centrifuged at 3,000 rpm for 8 min, and the plasma was then isolated for further experiments.
Mice peritoneum lavage fluids were obtained in 5 mL of ice-cold PBS. Lavage fluids were collected and filtered through a 70-μm nylon filter (BD Biosciences) to remove debris. Peritoneal lavage fluids from mice that underwent sham surgery were diluted by 10-fold, whereas fluids from mice that underwent CLP surgery were diluted by 102–104-fold with sterile PBS and 20 μL of each were immediately plated on solid medium for bacterial culture. The remaining portions were stored at −80°C for future experiments (21 (link)).
To evaluate the bacterial seeding in organs, spleens were excised and homogenized in 1 mL sterile PBS. After vortexing, 20 μL of the fluids were immediately used for bacterial culture (21 (link)).
All solid media were incubated overnight at 37°C, and colony-forming units (CFUs) were calculated according to the dilution fold.
