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Protocol for Laser Scanning Microscopy of Microorganisms on Hydrocarbons

  • Thomas R. NeuEmail author
  • John R. Lawrence
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Microbial communities in their fully hydrated state can be imaged in space and time (4-dimensionally) by laser scanning microscopy using 1-photon or 2-photon excitation. In this protocol, we provide guidance on how to examine microorganisms associated with liquid, viscous and solid hydrocarbons. Practical aspects are discussed including the material and sources, microscopy consumables, software programs and time constraints. The details of mounting samples for the upright and inverted microscope as well as options for fluorescence staining of bacteria and hydrocarbons are presented. Suggestions are made for recording images and subsequent digital image analysis. Finally, notes are added and a guideline for troubleshooting is supplied.

Keywords:

Bacteria Biofilms Colonisation of hydrocarbons Confocal laser scanning microscopy Deconvolution Degradation of hydrocarbons Digital image analysis Fluorescence techniques Fluorochromes Hydrophobicity Image analysis Imaging Imaging techniques Laser scanning microscopy Lectins Microorganisms Quantification Two photon laser scanning microscopy Visualisation 

Notes

Acknowledgements

Image courtesy: A. Macedo and W.-R. Abraham (Fig. 2b), S. Furuno and L. Wick (Fig. 2c). Support of the Canada–Germany collaboration by Environment Canada and Helmholtz Centre for Environmental Research – UFZ. Excellent technical support was provided over many years by Ute Kuhlicke and George Swerhone.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Helmholtz Centre for Environmental Research – UFZMagdeburgGermany
  2. 2.Environment CanadaSaskatoonCanada

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