A Correlative Light-Electron Microscopy (CLEM) Protocol for the Identification of Bacteria in Animal Tissue, Exemplified by Methanotrophic Symbionts of Deep-Sea Mussels

  • Sven R. Laming
  • Sébastien Duperron
Part of the Springer Protocols Handbooks book series (SPH)


Bacterial symbionts associated with animal tissues play major roles in the functioning of various ecosystems. Identification of bacteria often relies on marker gene comparative sequence analysis and fluorescence in situ hybridization (FISH). However, analysis of bacteria and host ultrastructure using transmission electron microscopy (TEM) can be equally important to understand the localization of bacteria and the degree of host-symbiont integration. We here provide a protocol which allows both FISH and TEM to be performed sequentially on a single section of tissue. Observations can then be superimposed, allowing ultrastructural investigation to be coupled with proper FISH-based identification of bacteria.


Correlative microscopy Fluorescence in situ hybridization Symbiosis Transmission electron microscopy 


  1. 1.
    Dubilier N, Bergin C, Lott C (2008) Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat Rev Microbiol 6:725–740CrossRefPubMedGoogle Scholar
  2. 2.
    Pernthaler A, Pernthaler J, Amann R (2002) Fluorescence in situ hybridization and catalysed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68:3094–3101CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Stoecker K, Dorninger C, Daims H, Wagner M (2010) Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) improves signal intensity and increases rRNA accessibility. Appl Environ Microbiol 76:922–926CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Cavanaugh CM, Levering PR, Maki JS et al (1987) Symbiosis of methylotrophic bacteria and deep-sea mussels. Nature 325:346–347CrossRefGoogle Scholar
  5. 5.
    Distel DL, Lee HKW, Cavanaugh CM (1995) Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel. Proc Natl Acad Sci U S A 92:9598–9602CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Halary S, Duperron S, Boudier T (2011) Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans. Appl Environ Microbiol 77(12):4172–4179CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11:36–42Google Scholar
  8. 8.
    Duperron S (2015) Characterization of bacterial symbionts in deep-sea metazoans: protocols for conditioning, fluorescence in situ hybridization, and image analysis. In: Mc Gentity TJ, Timmis KN, Nogales B (eds) Hydrocarbons and lipid microbiology protocols. Springer. doi: 10.1007/8623_2015_73
  9. 9.
    Duperron S, Halary S, Lorion J et al (2008) Unexpected co-occurrence of 6 bacterial symbionts in the gill of the cold seep mussel Idas sp. (Bivalvia: Mytilidae). Environ Microbiol 10:433–445Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Sven R. Laming
    • 1
  • Sébastien Duperron
    • 1
    • 2
  1. 1.Sorbonne UniversitésParisFrance
  2. 2.Institut Universitaire de FranceParisFrance

Personalised recommendations