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

Post-esophagus tissue was dissected and weighed (1.9g) by Lisa from a large Red Abalone (11-8-2) 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 (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.

Results:

Tubes were destroyed during spinning! Tubes were Beckman-Coulter Quick-Seal RoundTop Centifuge Tubes (Cat#: 244236). Called Beckman-Coulter. Turns out that these tubes require a special heat-sealing device AND specialized aluminum adapters for use in the rotors. Will order “normal” high speed centrifuge tubes and repeat experiment.

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