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Analysis of Mycolic Acids

Mycolic acids are extracted from freeze dried or fresh material using minor modifications of the method described by Vilchèze and Jacobs (2007) for analysis of mycolic acids by HPLC. Dried extracts are reconstituted in chloroform:methanol and analyzed on an Agilent QTof mass spectrometer by direct infusion into the ESI source. Mycolic acids were identified based on comparison of exact masses from known mycolic acid structures.

Required material: For this method either 60 mg freeze dried cells or 300 mg fresh cell material is required.

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References

  1. Vilchèze, C. and Jacobs, W.R. (2007), Isolation and Analysis of Mycobacterium tuberculosis Mycolic Acids. Current Protocols in Microbiology, 5: 10A.3.1-10A.2.11.
  2. Bouam, A., Armstrong, N., Levasseur, A., Drancourt, M. (2018) Mycobacterium terramassiliense, Mycobacterium rhizamassiliense and Mycobacterium numidiamassiliense sp. nov., three new Mycobacterium simiae complex species cultured from plant roots. Sci Rep 8, 9309.
  3. Fong-Fu Hsu, Kristina Soehl, John Turk, Albert Haas (2011) Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization. Analytical Biochemistry, 409, 112-122.

 

  

Analysis of Whole-Cell Sugars 

Diagnostic sugars in whole-cell hydrolysates (1N H2SO4, 100 °C, 2 hours) are analyzed by TLC on cellulose plates. The result supports the classification and identification of Actinobacteria.

Required material: Either a culture of the strain or 15 mg lyophilized cells.

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References

  1. Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, 101-129.
  2. Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226-231.

 

  

Detection of isomers of 2,6-diaminopimelic acid (Dpm) and of 2,6-diamino-3-hydroxypimelic acid (OH-Dpm)

Whole-cell hydrolysates (4N HCl, 100°C, 16 hours) are examined by TLC on cellulose plates for the presence of Dpm isomers or OH-Dpm. The result supports the classification and identification of Actinobacteria.

Required material: Either a culture of the strain or 5 mg lyophilized cells.

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References 

  1. Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, 101-129.
  2. Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407-77.
  3. Rhuland, L. E., Work, E., Denman, R. F. & Hoare, D. S. (1955). The behaviour of the isomers of 2,6-diaminopimelic acid on paper chromatograms. J Am Chem Soc 77, 4844-4846.