We exposed bacteria to acid stress, and would like to determine if this stress had any effect on the fatty acid content on the bacterial cell.
In case you have enough amount of bacteria, you could try a common fatty acid extraction followed by a methylation step, to separate and quantify fatty acids using gas-chromatography. You can find a high number of papers about those processes in the literature.
You can extract the lipids by protocol suggested by Folch method using Chloroform:methanol = 2:1, once you get the extract, evaporate the solvent to get more concentrated samples. You can followed either by methylation and gas chromatography or TLC if you just want to get general information of the lipid classes. Here are some papers that use similar method.
1. Russel & Harwood (1979) Biochem. J. 181: 339-345. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1161166/pdf/biochemj00458-0095.pdf)
2. Counsell & Murray (1986) International Journal of Systematic Bacteriology. 36: 202-206 (http://ijs.sgmjournals.org/content/36/2/202.full.pdf)
If you want to use GC for your lipid analysis, check the esterification method suggested by Christie (1993) here: http://lipidlibrary.aocs.org/topics/ester_93/file.pdf
Folch reagent extracts virtually all known lipids, although care must be taken in the partitioning step, since salt concentration affects the partitioning of some glycolipids. LC-MS methods are now available for direct analysis of complex lipid classes, including fatty acid compositions, from lipid extracts. These should be particularly useful for bacteria, which have a relatively simple lipidome. I would advise checking all solvents for peroxides with KI/starch solution, since a small amount of peroxide can destroy samples.
Dear Atheesha,
the methodology above commented is right but actually you do not need to isolate your lipid to perform the fatty acid analysis. You can use a direct transesterification/esterification method and bypass the isolation steps. Then you obtain your fatty acids as FAME and you can analyze them by GC. You can determine both profile and concentrations.
Article A high-performance direct transmethylation method for total ...
Since your sample may be small in volume or weight I guess, I will strongly recommend the total lipid extraction method using chloroform-methanol in a ratio of 2:1 then analyze the sample using electrospray-ionization mass spectrometery(ESI-MS). Here, no derivatization or transesterification of the sample is needed. This is a simple, quick, and uses small volume (50 microliters) and accurate in terms of identification and quantification of the fatty acids present. You could look at this paper for the extraction details. Abugri et al. 2012. Comparison of Transesterification Methods for Fatty Acid Analysis in Higher Fungi: Application to Mushrooms. Food Analytical Methods: Volume 5, Issue 5 , pp 1159-1166. If you need further help send me an email and I will walk you through it on Skype. Best regards.
Dear Atheesha, You can please follow the links below to obtain certain valuable informations:-
1. https://books.google.co.in/books?hl=en&lr=&id=yvwDqC0xBuUC&oi=fnd&pg=PA161&dq=simplest+technique+for+lipid+analysis+from+bacterial+cells&ots=-Hx1XXWdax&sig=I8ARcTH2NYWt0CT9ZX5sKULkWsg#v=onepage&q&f=false
2. https://books.google.co.in/books?hl=en&lr=&id=65ip-K-YUzwC&oi=fnd&pg=PA347&dq=simplest+technique+for+lipid+analysis+from+bacterial+cells&ots=7c1MRj_KUR&sig=dEUtDRoifa-LhI02dJ3aQ3HPsIc#v=onepage&q&f=false
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