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. 2017 Sep 6;7:10697. doi: 10.1038/s41598-017-10543-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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diNn induces outer and inner membrane permeabilization in addition to a decrease of bacterial cell length. Time lapse studies were conducted for 5 hours at 37 °C in CaMHB agarose pad supplemented with NPN, PI, and diNn when needed. Fluorescence and wide field images were analyzed in order to evaluate outer and inner membrane permeabilization and length of the cells. An increase in fluorescence indicates membrane permeabilization. Kinetics of the (a) outer and (b) inner bacterial membrane permeabilization in the presence of increasing concentrations of diNn. (c) Slopes of the sigmoidal curves fitting of outer and inner bacterial membrane permeabilization. The rate of permeabilization process is inversely proportional to the slope. According to one way ANOVA’s test and Tukeys comparison test, outer membrane permeabilization at 5 µM was significantly different (p < 0.001) than permeabilization at 1 µM and 3 µM whereas no statistically significant difference was observed in inner membrane permeabilization between the three concentrations (N = 3, values are mean ± SEM). (d) Outer (green) and inner (red) membrane permeabilization and bacterial length (blue) evaluation as a function of time and in the presence of diNn at 5 µM (N = 3).