Analysis of polar lipids
For this method 200 mg freeze dried cells are needed. If the analysis of respiratory quinones is requested in parallel we need 200 mg freeze dried cells in total for both methods.
Extraction of polar lipids
Polar lipids are extracted from 100 mg of freeze dried cell material using a choroform:methanol:0.3% aqueous NaCl mixture 1:2:0.8 (v/v/v) (modified after Bligh and Dyer, 1959). The extraction solvent is stirred overnight and the cell debris pelleted by centrifugation. Polar lipids are recovered into the chloroform phase by adjusting the chloroform:methanol:0.3% aqueous NaCl mixture to a ratio of 1:1:0.9 (v/v/v).
When polar lipids from the same biomass have previously been subjected to respiratory lipoquinone analysis the two stage method described by Tindall (1990a; 1990b) is used to first extract the respiratory lipoquinones followed by the polar lipids.
Analysis of polar lipids
Polar lipids are separated by two dimensional silica gel thin layer chromatography (Macherey-Nagel Art. No. 818 135). The first direction is developed in chloroform:methanol:water (65:25:4, v/v/v), and the second in chloroform:methanol:acetic acid:water (80:12:15:4, v/v/v/v). Total lipid material was detected using molybdatophosphoric acid and specific functional groups detected using spray reagents specific for defined functional groups, Full details are given in Tindall et al. (2007).
The labelled picture contributes the report.
- Bligh, E.G. and Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911-917
- Tindall, B.J. (1990a). A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst. Appl. Microbiol. 13, 128-130
- Tindall, B.J. (1990b). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol. Letts. 66, 199-202
- Tindall, B.J., Sikorski, J., Smibert ,R.M., and Kreig, N.R. (2007) Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Microbiology 3rd edn. Pp. 330-393. Editors: C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. Marzluf, T. M. Schmidt, L. R. Snyder ASM Press, Washington DC, USA
Coordination of the identification services. Identification of aerobic and facultative anaerobic Gram positive and Gram negative bacteria, physiological, biochemical and morphological characterization. Curator for filamentous bacteria belonging to different lines of descent (e.g. Beggiatoa, Thiothrix, Flexibacter, Sphaerotilus)