Sonication of bacterial suspensions at a frequency of 20 kHz induces an initial decline in cell numbers for E. coli and Kl. pneumonia during the first 5 min, which can be ascribed to the sensitivity of bacterial strains to ultrasonic treatment. There was a continuous reduction in the number of live cells over 15 min sonication (Joyce et al. 2003). However, at the higher frequency of 580 kHz, an initial increase was observed which was more pronounced in Kl. pneumonia, which is because of the deagglomeration effects of power ultrasound (Duckhouse et al. 2004).
Results presented in Figs 3 and 4 illustrate a significant effect for low-frequency ultrasound on bacterial cell viability (Joyce et al. 2003). Tests with a 20 kHz probe illustrate a greater effect on cell viability compared with a 40 kHz bath.
It has been widely reported that the acoustic frequency is an important factor which influences size and formation of cavitation bubbles (Mason 1999; Mason and Lorimer 2001). For low ultrasonic frequencies (20 kHz) entering a reaction solution, large cavitation bubbles will form which on collapse generate high energy. However, at higher frequencies (580 kHz), the acoustic cycle is shorter thus giving less time for cavitation bubble formation. Hence, the bubbles are smaller and collapse with less energy (Joyce et al. 2003). Therefore, bacterial suspensions treated with 20 kHz ultrasound experience significantly larger mechanical and thermal effects than those treated at 580 kHz, whereas the reverse is true for the chemical effects arising from free radical formation.
The sensitivity of bacteria to sonication is affected by medium composition, viscosity, sound transfer and power distribution within the reaction solution. However, other solution components such as organic solvents will slowly decompose during sonication, providing minor contributions to sonochemical reactions (Tiehm et al. 2001; Lee et al. 2009). It has also been reported that the shape of bacteria has a significant effect on their sensitivity to ultrasonic treatments. Generally, large bacteria are more sensitive to sonication than small bacteria because of the large surface area subjected to ultrasound. Cocci/spherical bacteria are more resistance to sonication treatments than bacilli/rod shaped bacteria.
The results herein indicate a significant reduction in live/viable bacterial cell numbers after 15 min treatment at low frequencies. The inactivation with a 20 kHz probe was greater than with a 40 kHz bath. This indicates that the direct insertion of an ultrasonic probe tip into the reaction suspension provides more direct and powerful energy to the system. Although flow cytometry data were comparable to viable plate count techniques, the percentage of live cells appeared higher. This observation is considered to be almost certainly because of the ability of flow cytometry to identify and count bacteria as single cells, whereas viable plate counts only enumerate colony forming units (CFUs) which can be either single bacterial cells or agglomerates of cells.
Flow cytometry has an additional advantage over more traditional viable plate counts; in that, this method quantifies bacterial cells into different population (live/viable and dead). This increases our understanding on bacterial viability in terms of metabolic activity and cell wall integrity i.e. viable but nonculturable populations.