Mycobacteria Lipids are an important characteristic of the genus and are critical in host Mycobacteria interactions. Speed and versatility are the main advantages of this procedure. This method can be used to understand the role of lipids and the pathogenicity of mycobacteria and their immunostimulatory effect.
For the non-covalent linked lipids extraction, add 15 milliliters of one to two chloroform to methanol solution to a glass tube with a PTFE liner screw cap under a laminar flow hood. Then, scratch 200 milligrams of mycobacteria from a solid medium, and add the mycobacteria to the glass tube. Incubate the tube overnight with constant stirring.
On the next day, filter the organic solvents through a glass funnel lined with filter paper into a glass tube. After using nitrogen gas flux, to evaporate the liquid phase in the tube, fill the tube with nitrogen gas, to store the tube at four degrees Celsius. Then, add 15 milliliters of a two to one chloroform to methanol solution to the cellular debris and incubate the tube overnight with constant stirring.
On the next morning, allow the mixture to rest for one hour, before using a Pasteur pipette to transfer the organic solvents into a filter paper lined glass funnel into the same glass collection tube. Then, evaporate the liquid phase as demonstrated, before refilling the tube with nitrogen gas for four degrees Celsius storage. For mycolic acid extraction, add two to five milliliters of a stirifying solution and 200 milligrams of mycobacteria biomass to a hermetic glass tube with a PTFE liner screw cap, and to mix the contents by vortexing.
Incubate the mixture inside a dry bath at 80 degrees Celsius overnight. The next day, when the tube has cooled room temperature, add two milliliters of N hexane, and to mix the contents for 30 seconds by vortexing. Allow the tube to settle until two clear phases appear and transfer the upper N hexane phase to a new tube.
Mix two additional milliliters of N hexane with the tube contents and collect the upper layer again. Then, evaporate the tube contents by nitrogen flow and store the sample covered in nitrogen at four degrees Celsius as demonstrated. To analyze the samples by TLC cover one wall of the TLC chamber with a piece of filter paper, add Vaseline on the chamber corners to seal the chamber, to can the solvent mixture over the filter paper and add the remaining volume of the solvent to the bottom of the TLC chamber.
Close the TLC chamber for at least 20 minutes to saturated. Meanwhile, dissolve the lipids in the glass tube in 200 to 1000 microliters of chloroform and use a capillary glass tube to apply 10 microliters of the lipid chloroform suspension directly onto the TLC plate. After allowing the sample to dry for five minutes, insert the plate into the saturated TLC chamber and allow the mobile phase to run through the TLC.
When the solvent reaches one centimeter from the upper end of the plate, place the plate under laminar flux until the silica is completely dry. Then, spray the dried plate with 15 to 20 milliliters of 10%molar data phosphoric acid hydrate in ethanol until the plate is bright yellow, and heat the plate for two to five minutes at 120 degrees Celsius. In this representative analysis, the mycolic acid extracted from various mycobacteria species, were analyzed through one dimensional TLC using two different elution systems and two dimensional TLC.
Two types of methylation procedures were also performed to confirm the presence of type five mycolic acid. In both elution systems, the mycolic acids were located approximately in the middle of the plate, although, the spot corresponding to type five mycolic acid, was observed only after saponification. Two dimensional TLC also allows the identification of individual mycolic acid types.
For example, mycobacterium brumae expresses only type one mycolic acids, mycobacterium bovis BCG expresses type one and four, mycobacterium fortuitum expresses type one and five, and mycobacterium abcessus expresses type one and two mycolic acid profiles. The total lipid extracts from various mycobacteria species were analyzed in function of their polarity and size. It reveals that most A polar lipids, such as PDIMs, are present in mycobacterium bovis BCG, but not in mycobacterium fortuitum, mycobacterium brumae and the smooth morphotype of M abcessus.
Phenolic GlycoLipids are present only in mycobacterium bovis BCG, while GlycoPeptidoLipids are observed only in samples from mycobacterium abcessus. Similar to mycolic acids, Acylglycerols and PDIMs, PIMs, can also be easily visualized through one dimensional TLC or 2D TLC analysis. Two types of stains were used to reveal PIMs in one dimensional TLC.
The most important thing to remember when attempting this protocol is to not use any plastic instruments throughout the procedure.
