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. 2019 Jun 17;9:8667. doi: 10.1038/s41598-019-45164-5

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© The Author(s) 2019

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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TAG accumulation under the form of ILI is driven by carbon excess and nitrogen starvation in stationary phase. (A) Mycobacterial cultures were grown in different media containing increasing glycerol concentrations (1%, 2% and 5%) and were collected at 24 h or 48 h, corresponding to exponential or stationary phase, lyophilized and finally the same amount of dry cell weight was used for apolar lipid extraction. Left panel corresponds to TLC plate analysis of TAG extracted from exponential and stationary-phase cultures with increasing concentrations of Gly, with triolein as standard. Right panel corresponds to TLC densitometric analysis of the relative TAG level in each sample with cultures in classic 7H9 (7H9_Exp) used as reference. (B) Cultures grown in minimal salt medium containing either 1 g/L or 0.05 g/L of NH₄Cl and 1% Gly as carbon source were collected after 8 h, 24 h or 48 h incubation periods, lyophilized and equal weights of dry cells used for apolar lipid extraction. TAG levels from each culture were analysed by TLC with triolein as standard. The TLC plate (left panel) is representative of two individual experiments. TLC densitometric analysis of relative TAG levels in each sample with cultures in exponential phase in classic 7H9 (7H9_Exp) used as reference (right panel). All results are expressed as mean values ± SD of two independent experiments. TAG band intensities were compared using a one-way ANOVA test where * corresponds to a p-value < 0.05. (C) Average Nile-Red fluorescence intensity determined for 6 different 126 µm² fields containing between 50 and 150 cells each. Fluorescence intensities were compared using a two-tailed Mann-Whitney test where * corresponds to a p-value < 0.05. (D) Phase contrast, Fluorescence and Merge channels of M. smegmatis cells grown for 24 h in MSM NL Gly 1% medium. Cells harbour distinct morphologies and contain ILI occupying most of the cytoplasm space. Scale bars represent 2 µm. Cells were fixed with glutaraldehyde and processed for EM. (E) Thin section of an in vitro culture of M. smegmatis in classical 7H9 medium. The scale bar represents 1 µm. (F) Thin section of an in vitro culture of M. smegmatis in MSM NL Gly medium. Right panel is a zoomed-in picture providing a better view and resolution of ILI. Scale bars represent 2 µm.