On 2014 Nov 21, Gerhard Nebe-von-Caron commented:

This work is unfortunatelly missing appropriate revision. There is no evidence that the authors observed single bacteria due to the fact that no pictures of scatter versus nucleic acid fluorescence are shown. It would be recommended that the authors submit the raw data listmode files to http://flowrepository.org/ for validation. The fact that the picture in their 2010 poster "Flow cytometry as a means of observing the effects of ultrasound on bacteria" shows scatter data and antibody fluorescence of white blood cells instead of bacteria and regions of interest that indicate a lack understanding of the data analysis made me search for a follow up paper from the authors and my suspicion was confirmed in this paper. For example there is no distinction made between bacteria and noise signals. Figure 5 and 7 show the settings of quadrants cutting across clusters which is scientifically incorrect, and some of the clusters are in part likely to be caused by trigger artefacts. Figure 5e include permeabilised cells and DNA negative events in the lower left quadrant which could be pumping cells, cell debris as well as bubbles and micelles, distinguishable by appropriate data analysis.

The conclusion "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" indicates a lack of understanding of the measurement principles. Apart from the problems of colony formation, discussed in several of my publications, when measuring bacteria, cell aggregation can only be detected if genome equivalents are measures or one realises that double positive cells in case of energy transfer staining combinations represent two cells with one red and permeabilised and another green and intact, either coincident or still attached to each other. Most instruments unless aligned and maintained to the standards required for microbial analysis will not detect bacteria adequately when triggering on forward scatter. Thus the separation of bacteria from instrument noise needs to be demonstrated and a comprison of the data should be based on absolute counts. Also the use of aggregated bacteria would have been preferential to compare the effect of disaggregation compared to cellular injury as an indicator for the delivered energy on the membrane.

On 2014 Nov 21, Gerhard Nebe-von-Caron commented:

This work is unfortunatelly missing appropriate revision. There is no evidence that the authors observed single bacteria due to the fact that no pictures of scatter versus nucleic acid fluorescence are shown. It would be recommended that the authors submit the raw data listmode files to http://flowrepository.org/ for validation. The fact that the picture in their 2010 poster "Flow cytometry as a means of observing the effects of ultrasound on bacteria" shows scatter data and antibody fluorescence of white blood cells instead of bacteria and regions of interest that indicate a lack understanding of the data analysis made me search for a follow up paper from the authors and my suspicion was confirmed in this paper. For example there is no distinction made between bacteria and noise signals. Figure 5 and 7 show the settings of quadrants cutting across clusters which is scientifically incorrect, and some of the clusters are in part likely to be caused by trigger artefacts. Figure 5e include permeabilised cells and DNA negative events in the lower left quadrant which could be pumping cells, cell debris as well as bubbles and micelles, distinguishable by appropriate data analysis.

The conclusion "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" indicates a lack of understanding of the measurement principles. Apart from the problems of colony formation, discussed in several of my publications, when measuring bacteria, cell aggregation can only be detected if genome equivalents are measures or one realises that double positive cells in case of energy transfer staining combinations represent two cells with one red and permeabilised and another green and intact, either coincident or still attached to each other. Most instruments unless aligned and maintained to the standards required for microbial analysis will not detect bacteria adequately when triggering on forward scatter. Thus the separation of bacteria from instrument noise needs to be demonstrated and a comprison of the data should be based on absolute counts. Also the use of aggregated bacteria would have been preferential to compare the effect of disaggregation compared to cellular injury as an indicator for the delivered energy on the membrane.