Parker, Albert E.Miller, LindseyAdams, JacobPettigrew, CharlesBuckingham-Meyer, KelliSummers, SummersChristen, AndresGoeres, Darla2023-01-312023-01-312022-12Albert E. Parker, Lindsey Miller, Jacob Adams, Charles Pettigrew, Kelli Buckingham‐Meyer, Jennifer Summers, Andres Christen, Darla Goeres, Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo‐activated chlorine dioxide treatment, Journal of Applied Microbiology, Volume 133, Issue 6, 1 December 2022, Pages 3413–3423, https://doi.org/10.1111/jam.157651365-2672https://scholarworks.montana.edu/handle/1/17665This is a pre-copyedited, author-produced PDF of an article accepted for publication in Journal of Applied Microbiology following peer review. The version of record [Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo-activated chlorine dioxide treatment. Journal of Applied Microbiology 133, 6 p3413-3423 (2022)] is available online at: https://doi.org/10.1111/jam.15765.Aim. To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. Methods and results. A photo‐activated chlorine dioxide treatment (Photo ClO2) was tested in two independent experiments against thick (>100 μm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. Conclusions. Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z‐slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. Significance and impact of study. There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.en-UScopyright Oxford University Press 2022http://web.archive.org/web/20191107025238/https://academic.oup.com/journals/pages/access_purchase/rights_and_permissionsbeer's lawbiovolumeconfocal scanning laser microscopeexperimental designs for imagingphoto-activated chlorine dioxideArticle