Disaggregate mechanically

Ultrasound bath

• Use 37-80 KHz cavitation frequency as lower frequency ultrasound (typically 20khz) results in aggressive cavitation that may induce lysis.

• Use glass vessels for optimal propagation of ultrasound as the conventional plastic test tubes dampens ultrasound.

• Make sure ultrasound bath is clean and that vessel is placing at the recommended water level and position in the bath

• Typical protocol: 5 mL sample, 5 min @40 KHz in a glass vessel. If the ultrasound bath is equipped with a sweep functionality, use this.

Ultrasound probe

• Typically, the rod-type sonicator probes are more aggressive than the above bath types, but amplitude, wattage and frequency parameters can be tailored.

• Use probes for small volumes and higher frequencies than 20khz.

• Cool sample in ice bath when performing homogenization

• Typical protocol: 1mL sample on ice. Example: 10 cycles of 30 sec pulses at 20 watts, with 30 sec idle time.

Rotor/stator blender

• Handheld rotor/stator equipment, e.g. tissueruptor or ultra-turrax types. Preferentially choose disposable probes to minimize contamination

• Walk-away platforms like the tube drive and the DT-20M tubes are sealed to minimize aerosol formation.

• Add antifoam agents, e.g. 1 µL Sigma antifoam 204 to 5 mL of culture

• Typical protocol: Add 0.005-0.01% antifoam. 5-10mL sample. Homogenize for e.g. 2-5 min at max speed.

Syringe homogenization

Protocol: Passage 200 µL sample through 27G cannula 20 times.

Bead beating

Protocol: 5 mL sample + 1 mL 3 mm glass beads. 1 min at max speed.

Vortex with glass beads

Protocol: 5 mL sample + 1 mL 3 mm glass beads. 3 min at max speed.