Continuous Spore Disruption Using Radially Focused, High-Frequency Ultrasound

Abstract

We report on the development of a novel, continuous-flow, radially focused ultrasonic disruptor capable of lysing Bacillus spores in the absence of added chemical denaturants, enzymes, or microparticles. Greater than 99% disruption was achieved for Bacillus globigii spores and Escherichia coli and Bacillus subtilis vegetative cells with sample residence times of 62, 12, and 12 s, respectively. Microscopic and SEM images indicated that at equivalent power levels, the incidence of cell death or loss of viability typically exceeded the efficiency of (visible) cell lysis. However, semiquantitative PCR showed up to a 1,000-fold increase in intracellular DNA availability from ultrasonically disrupted spores, and liberated DNA was intact and available for subsequent detection.

Microalgal cell disruption in a high-power ultrasonic flow system

“…Because of its unique advantages, continuous-flow, radially focused ultrasonic systems have been used to disrupt microorganisms, such as in lysing Bacillus spores (Chandler et al, 2001). However, to the best knowledge of the authors, continuous ultrasonic flow systems have never been applied in microalgal cell disruption for lipid extraction, although the flow cells for such operations are commercially available.…”

A Flow-Through Ultrasonic Lysis Module for the Disruption of Bacterial Spores

“…These transducers are inexpensive and the supporting electronics are simpler than the laboratory test and measurement systems we have created in the past. 4 Dual transducers clearly performed spore lysis more rapidly than a similarly configured single transducer system.…”

“…This gene was chosen based on our previous work where dilution-to-extinction PCR was used to test our previous bench top cylindrically focused ultrasound system. 4 Real-time PCR was performed in an optically clear 96-well plate designed for the fast cycling block. Each well had a final volume of 20 mLd5 mL of sample template, 4 mL of nuclease free water (Ambion, Austin, TX), 10 mL TaqMan Fast Universal Master Mix (2Â) (Applied Biosystems, Foster City, CA), 900 nM of the forward and reverse primer, and 250 nM of the fluorogenic probe (0.25 mL of each primer and 0.5 mL of probe, respectively).…”

Comparisons of protein extraction procedures and quantification methods for the proteomic analysis of Gram-positive Paenibacillus sp. strain D9

“…Among the methods available for cell disruption at the laboratory scale, sonication is the most widely employed for cell lysis (Belgrader et al 1999;Chandler et al 2001;Feliu et al 1998;Kuboi et al 1995;Lanigan et al 2004;Tkac et al 2004). Chemical cell disruption using detergents is a milder and simpler alternative compared to mechanical methods.…”

“…Nowadays, it is possible to find ready to use commercial kits for every cell type (Caňas et al 2007). During (or after) cell lysis, interfering compounds (e.g., proteolytic enzymes, salts, lipids, polysaccharides and nucleic acids) need to be inactivated and/or removed (Chandler et al 2001;Cilia et al 2009;Middelberg 1995). The two most important interfering parameters are salt and proteolysis.…”