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Analysis of cellular fatty acidsAnalysis of cellular fatty acids

Analysis of cellular fatty acids

For the analysis of the cellular fatty acids an active growing culture or 30 mg freeze dried cells is required.

This analysis is performed by using the Sherlock MIS (MIDI Inc, Newark, USA) sytem.

Method

Extraction and analysis of fatty acids. Fatty acid methyl esters are obtained from 40 mg cells scraped from Petri dishes by saponification, methylation and extraction using minor modifications of the method of Miller (1982) and Kuykendall et al., (1988). The fatty acid methyl esters mixtures are separated using Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID, Newark, DE 19711 U.S.A.) which consisted of an Agilent model 6890N gas chromatograph fitted with a 5% phenyl-methyl silicone capillary column (0.2 mm x 25 m), a flame ionization detector, Agilent model 7683A automatic sampler, and a HP-computer with MIDI data base (Hewlett-Packard Co., Palo Alto, California, U.S.A.). Peaks are automatically integrated and fatty acid names and percentages calculated by the MIS Standard Software (Microbial ID). The gas chromatographic parameters are as follows: carrier gas, ultra-high-purity hydrogen; column head pressure 60kPa; injection volume 2 µl; column split ratio, 100:1; septum purge 5 ml/min; column temperature, 170 to 270°C at 5°C/min; injection port temperature, 240°C; and detector temperature, 300°C.

References

  • Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Canadian J Microbiloology 42,989-1005.
  • Kuykendall, L.D., Roy, M.A., O'Neill, J.J., Devine, T.E. 1988. Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. International Journal of Systematic Bacteriology 38:358-361.
  • Miller, L.T. 1982. A single derivatization method for bacterial fatty acid methyl esters including hydroxy acids. Journal of Clinical Microbiology 16:584-586.

 

 

Additional informations for interpreting the results and publication

If the data supplied is used in a publication the following acknowledgement should be included in the "Materials and Methods", “Fatty acid analyses were carried out by the Identification Service of the DSMZ, Braunschweig, Germany“. If included in other forms of presentation (e.g. in supplementary data or lectures) the same wording should appear together with the data presented.
Where work has been carried out on cell material or strains supplied by the customer, the DSMZ makes no guarantee concerning the authenticity of the material supplied.
The identification of the fatty acids included in the accompanying report are based on the various peak naming tables supplied by MIDI Inc (Newark, USA) as part of their Sherlock Microbial Identification Ssystem. Different peak naming tables have been developed over the years and may be specific for certain groups of organisms. With an increasing appreciation of lipid diversity (including fatty acids) it is important to realize:

  1. the MIDI Sherlock system is primarily set up to identify organisms against a known range of organisms and makes no claim to be comprehensive
  2. the MIDI Sherlock peak naming tables make no claim to be complete and not all peaks appearing in the chromatogram may be identified
  3. using different peak naming tables the MIDI Sherlock system may identify some peaks with the same retention time differently
  4. not all fatty acids mentioned in the published literature are included in the peak naming tables of the MIDI Sherlock system. These may either not be reported or simply listed according to their equivalent chain lengths (ECL).
  5. in some cases the MIDI Sherlock system lists fatty acids as “summed features” please refer to the notes provided by MIDI Inc
  6. the technical notes provided by MIDI Inc give an introduction to fatty acid analysis and a guide to interpreting the reports generated. See the list of links at the end of this document.
  7. in cases where it is fairly evident that the fatty acids of a novel strain are adequately covered by making reference to a single peak naming table (e.g enterobacteria, bacilli) a single report will be supplied. In cases where a single report may not be sufficient or not necessarily accurate more than one report will be supplied. The peak naming table used to generate the report may be identified as follows:


Volume: DATA File: E113315.41A Samp Ctr: 16 ID Number: 24268
Type: Stat Bottle: 37 Method: TSBA40
Created: 3/31/2011 7:16:38 PM
Sample ID: Your strain designation

in the upper, middle to right hand portion of the header the wording “Method” applies to the method used to do the initial analysis and the peak naming table is the same: eg. Method: TSBA40

If the initial data has been re-analysed using a different peak naming table the header reads:

Volume: DATA File: E113315.41A Samp Ctr: 16 ID Number: 24268
Type: Stat Bottle: 37 Method: TSBA40 Calc. Method: ACTIN6
Created: 3/31/2011 7:16:38 PM
Sample ID: Your strain designation

Calc method indicates which peak naming table has been used in the re-analysis

The DSMZ supplies the fatty acid reports without further comment or interpretation and it is the responsibility of the customer to interpret the results in a fashion relevant to the organism they have supplied. Most peak naming tables use the “omega” (ω) nomenclature for identifying the position of unsaturation, numbering from the methyl end of the fatty acid. However, this is not always the case and the alternative nomenclature for numbering the position of unsaturation from the carboxyl end has also been used both in the Sherlock system as well as in the literature.

At the time of writing the DSMZ uses an Agilent 6890N gas chromatograph and version 6.1 of the MIDI Inc Sherlock MIS software. The software version appears at the bottom left hand corner of the reports. The method of preparation of the samples is the standard method given in MIDI ´Technical Note 101 (and in the handbook). The database used to identify the fatty acids is given on the report sent to you, as explained above. The reference:

Kuykendall, L. D., Roy, M. A. O'Neill, J. J. and Devine, T. E. Fatty Acids, Antibiotic Resistance, and Deoxyribonucleic Acid Homology Groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 1988 38: 358-361.

Also gives details of the method of preparing the fatty acids, but does not give up-to-date information on the gas chromatograph or software currently used in the DSMZ.

Useful links provided by MIDI Inc.

T.N. 101- Microbial ID by FAME Analysis
www.midi-inc.com/pdf/MIS_Technote_101.pdf

Understanding Fatty Acid Naming
www.midi-inc.com/pdf/MIS_Fatty_Acid_Naming.pdf

Example Sherlock MIS Sample Report
www.midi-inc.com/pdf/MIS_Report_Interpretation_Guide.pdf

Understanding Similarity Index (SI)
www.midi-inc.com/pdf/MIS_Similarity_Index.pdf

T.N. 102- Tracking by FAME Analysis
www.midi-inc.com/pdf/MIS_Technote_102.pdf

Photo of Susanne  Verbarg
Dr. Verbarg, Susanne
Phone: +49-531/2616-231

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)