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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Extraction of the total non-covalent-linked lipids from mycobacteria (Figure 1)
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	<li>Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a polytetrafluoroethlene (PTFE) liner screw caps. Add a solution consisting of 5 mL of chloroform and 10 mL of methanol (chloroform:methanol, 1:2). 
	NOTE: Wh...

thin layer chromatography-based separation of mycolic acid variants: a method to separate mycobacterial cell wall lipids based on polarity

Source: Guallar-Garrido, S., et al.&#160; <a href="https://www.jove.com/v/62368">Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography</a>. J. Vis. Exp. (2021).
This video demonstrates the thin-layer chromatography-based separation of mycolic acids. This technique separates the different mycolic acids&#8212;long-chain lipids present in the cell wall of mycobacteria&#8212;based on their polarity.

<img alt="Figure 1" src="/files/ftp_upload/21094/21094_Figure1.jpg" width="700px" /> 
Figure 1: Scheme of the procedure of extracting lipid content of mycobacteria grown on solid media. Main steps to decipher lipids present on mycobacteria cells.
<img alt="Figure 2" src="/files/ftp_upload/21094/21094_Figure2.jpg" width="700px" /> 
Figure 2: Scheme of the procedure for extracting mycolic acid content of mycobacteria grown on solid media. Main steps to...

Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

Source: Guallar-Garrido, S., et al.&#160; Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography. J. Vis. Exp. (2021).
This video ...

The mycobacterial cell wall contains different variants of mycolic acids - long-chain fatty acids with conserved alpha chains. Structural differences exist in their beta chains, resulting in the variations in the polarity of different mycolic acids.
To separate the variants using thin layer chromatography, or TLC - a liquid chromatography technique - take mycobacterial cell wall lipids in the desired organic solvent. Spot the lipids near the base of a TLC plate precoated with a thin layer of adsorbent material, which functions as the stationary phase.
Place the plate inside a jar containing an organic solvent - the mobile phase - ensuring the liquid level is below the sample spots to prevent premature diffusion of the mycolic acids.
During the run, the mobile phase rises through the tiny pores of the adsorbent layer on the plate via capillary action. Eventually, the mycolic acids dissolve in the solvent and travel upward.
More polar mycolic acid variants are transiently adsorbed by the polar stationary phase via intermolecular forces, hindering their upward movement. Less polar mycolic acids remain in the non-polar mobile phase and migrate further.
The differences in migration result in the variants of mycolic acid separating into discrete bands.
Upon completion, spray the dried plate with a phosphomolybdic acid stain. Heat the plate to reduce the stain in the presence of the lipids, imparting a dark green color to the bands.