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Analysis of Cellular Fatty Acids
Cellular fatty acids are analysed after conversion into fatty acid methyl esters (FAMEs) by saponification, methylation and extraction following the protocol of Sasser (1990). The fatty acid methyl esters mixtures are separated by gas chromatography and detected by a flame ionization detector. In subsequent analysis, fatty acids are identified by a GC-MS run, on an Agilent GC-MS 7000D system (Vieira et. al., 2021). Peaks were identified based on retention time and mass spectra. For comparison with the literature we can still provide the naming of fatty acids according to the MIDI databases.
Further derivatization procedures for structural elucidation of yet unidentified compounds can be performed upon request. The position of single double bounds will be confirmed by a derivatization to the corresponding dimethyl disulfide adduct (Moss and Lambert-Fair, 1989). Branched-chain fatty acid positions, cyclo-positions and multiple double bounds could be determined by derivatization to their 3-pyridylcarbinol (“picolinyl”) and/or 4,4-dimethyloxazoline (DMOX) derivatives (Harvey, 1982, Spitzer, 1997, Yu et al., 1988).
Required material: For analysis of cellular fatty acids 30 mg freeze dried cells are required. Alternatively, an actively growing culture (still in exponential growth phase upon arrival, without signs of sporulation or cell lysis) could be send. Subculturing (if required) will be charged separately. If subculturing is requested by the customer, detailed cultivation conditions (media, temperature, oxygen supply, cultivation duration) must be provided.
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Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. MIDI, Inc., Newark
Vieira S, Huber KJ, Neumann-Schaal M, Geppert A, Luckner M, Wanner G, Overmann J (2021) Usitatibacter rugosus gen. nov., sp. nov. and Usitatibacter palustris sp. nov., novel members of Usitatibacteraceae fam. Nov. within the order Nitrosomonadales isolated from soil. Int J Syst Evol Microbiol 71:004631.
Moss, C.W., Lambert-Fair, M.A. 1989. Location of Double Bonds in Monounsaturated Fatty Acids of Campylobacter Cryaerophila With Dimethyl Disulfide Derivatives and Combined Gas Chromatography-Mass Spectrometry. J Clin Microbiol 27:1467-1470.
Harvey, D.J. 1982. Picolinyl esters as derivatives for the structural determination of long chain branched and unsaturated fatty acids. Biomed Mass Spectrom 9:33-38
Spitzer, V. 1997. Structure analysis of fatty acids by gas chromatography - low resolution electron impact mass spectrometry of their 4,4-dimethyloxazoline derivatives - a review. Prog Lipid Res 35:387-408.
Yu, Q.T., Liu, B.N., Zhang, J.Y. and Huang, Z.H. (1988) Location of methyl branches in fatty acids: Fatty acids in uropygial secretion of Shanghai ducks by gas chromatography-mass spectrometry of 4,4-dimethyloxazoline derivatives. Lipids 23:804-810.