Effect of dilution on sedimentational separation of bacteria from blood.

Bacteria must be separated from septic whole blood in preparation for rapid antibiotic susceptibility tests. This work improves upon past work isolating bacteria from whole blood by exploring an important experimental factor: Whole blood dilution. Herein, we use the continuity equation to model red blood cell sedimentation and show that overall spinning time decreases as the blood is diluted. We found that the bacteria can also be captured more efficiently from diluted blood, up to approximately 68 ± 8% recovery (95% confidence interval). However, diluting blood both requires and creates extra fluid that end users must handle; an optimal dilution, which maximizes bacteria recovery and minimizes waste, was found to scale with the square root of the whole blood hematocrit. This work also explores a hypothesis that plasma backflow, which occurs as red cells move radially outward, causes bacterial enrichment in the supernatant plasma with an impact proportional to the plasma backflow velocity. Bacteria experiments carried out with diluted blood demonstrate such bacterial enrichment, but not in the hypothesized manner as enrichment occurred only in undiluted blood samples at physiological hematocrit.

Factors affecting sedimentational separation of bacteria from blood.

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.