Differential Centrifugation – Isolation of Rickettsia from Red Abalone Post-Esophagus

Post-esophagus tissue was dissected and weighed (0.423g) by Lisa from a large Red Abalone (###) that had been previously infected with Withering Syndrome. Tissue was homogenized in ice cold 3mL Tris-Sucrose buffer (TSB; ) with a glass, 7mL homogenizer on ice. Homogenate was transferred to a 15mL conical tube. Homogenizer components were rinsed with 1mL TSB and transferred to the same 15mL conical tube. The homogenate was spun 10mins, 250g, 4C. Supe (dark brown, milky) was evenly split onto two Percol gradients (35mL; made with TSB); 30% and 50%. Gradients were spun 1hr, 25,000g, 4C in a Sorval T-21 centrifuge with a SL-50T rotor using the “SoftSpin” feature and the brake off.

Results:

Gradients didn’t show any distinct layers/bands beside a layer that seemed to reside on top of each of the Percoll gradients, as though the sample never entered the gradient. This layer seemed to contain larger cell debris which was brown in color and a hazy “sub” layer. However, there was also a small amount of cellular debris suspended (not pelleted) near the very bottom of each tube.

The top and bottom fractions were collected separately from each of the gradients. The top fractions were combined and the bottom fractions were combined. These fractions were transferred to new high-speed centrifuge tubes. The samples were washed with 1x PBS (Lisa’s recipe?) by filling each tube with 1x PBS and inverting numerous times. The tubes were then centrifuged @ 20,000g, 4C, 30mins in the same centrifuge and rotor described above. Supe was removed and the pellets (which were mostly loose) were resuspended in ~1.2mL of 1x PBS. These samples were transferred to snap cap tubes and stored temporarily @ 4C.

The pellet from the top fractions contained a significant amount of insoluble, cellular debris which settles relatively quickly to the bottom of the tube, leaving a brown, turbid supernatant. The pellet from the bottom fractions is mostly brown and turbid, with virtually no visible cellular debris.