For fatty acid analysis, adult nematodes were washed from plates and allowed to settle. The excess water was removed from the worm pellet and 1 ml of 2.5% methanolic H2SO4 was added and incubated at 80 °C for 1 h to generate fatty acid methyl esters, which were extracted by adding 1.5 ml water and 0.2 ml hexane. The hexane was sampled for determination of fatty acid composition by gas chromatography on an SP-2380 fused silica capillary column (Supelco, Bellefonte, Pennsylvania, United States) using an Agilent (Palo Alto, California, United States) 6890 series gas chromatograph [18 (link)].
For lipid analysis, about 0.5 ml of adult nematodes were collected in a glass tube and frozen. Lipids were extracted by incubation in (1:1) chloroform/methanol overnight at −20 °C. The samples were washed with 2.2 ml Hajra's solution (0.2M H3PO4, 1M KCl) and the chloroform phase containing the lipids was isolated. The silica gel HL plates (Analtech, Newark, Delaware, United States) were activated by incubation at 110 °C for 1 h and 15 min. The samples were loaded onto the thin layer chromatography plates along with lipid standards (Sigma, St. Louis, Missouri, United States). The plates were run with a 65:43:3:2.5 chloroform/methanol/water/acetic acid solvent mixture until the solvent front was three-fourths of the way up the plate. The plate was dried, a new solvent mixture of 80:20:2 hexane/diethyl ether/acetic acid was added, and the plate was run until the solvent front reached the top of the plate. The marker lanes were visualized using iodine vapor and the corresponding bands for triglycerides and individual phospholipids in the silica gel were scraped into individual tubes. To quantitate, 50 μg of 15:0 free fatty acid was added to each tube as an internal standard and fatty acid analysis was performed by gas chromatography as described above [22 (link)].
For lipid analysis, about 0.5 ml of adult nematodes were collected in a glass tube and frozen. Lipids were extracted by incubation in (1:1) chloroform/methanol overnight at −20 °C. The samples were washed with 2.2 ml Hajra's solution (0.2M H3PO4, 1M KCl) and the chloroform phase containing the lipids was isolated. The silica gel HL plates (Analtech, Newark, Delaware, United States) were activated by incubation at 110 °C for 1 h and 15 min. The samples were loaded onto the thin layer chromatography plates along with lipid standards (Sigma, St. Louis, Missouri, United States). The plates were run with a 65:43:3:2.5 chloroform/methanol/water/acetic acid solvent mixture until the solvent front was three-fourths of the way up the plate. The plate was dried, a new solvent mixture of 80:20:2 hexane/diethyl ether/acetic acid was added, and the plate was run until the solvent front reached the top of the plate. The marker lanes were visualized using iodine vapor and the corresponding bands for triglycerides and individual phospholipids in the silica gel were scraped into individual tubes. To quantitate, 50 μg of 15:0 free fatty acid was added to each tube as an internal standard and fatty acid analysis was performed by gas chromatography as described above [22 (link)].
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