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. 2018 Sep 25;5:180184. doi: 10.1038/sdata.2018.184

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Copyright © 2018, The Author(s)

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/ The Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files made available in this article.

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(a) Syto9 versus PI plot of the unstained sample, indicating a low signal therefore minimal auto fluorescence or background fluorescence. (b) Same sample stained only with PI. It can only penetrate cells with a damaged membrane either dead or not, which may explain the low signal since samples derived from actively growing cultures. (c) Sample stained with only Syto9. It penetrates every cell irrespective of their viability, which may explain the high signal. (d) Manual gating of population sub-sets using FlowJo of actively growing mycobacterial cultures stained with PI and Syto9. The population with a high Syto9 and PI signals was defined as the population of live cells with an intact membrane (LCIM). The population with a high PI signal and a relatively low Syto9 signal was defined as the population of dead cells (DC), while the population with a relatively low Syto9 and PI was considered as the population of live cells with a damaged membrane (LCDM). Every sample was analysed following that pattern. (e) Samples treated with 0.05% SDS for ~1 h displayed a slight increase of LCDM, indicating that a higher concentration of SDS or a longer exposure may damage a higher proportion of cell membrane. (f) When heat-treated, the DC population increased significantly.