Abstract
In microbial oceanography, cell size, volume and carbon (C) content of pelagic bacteria and archaea (‘bacteria’) are critical parameters in addressing the in situ physiology and functions of bacteria, and their role in the food web and C cycle. However, because of the diminutive size of most pelagic bacteria and errors caused by sample fixation and processing, an accurate measurement of the size and volume has been challenging. We used atomic force microscopy (AFM) to obtain high-resolution images of pelagic bacteria and Synechococcus. We measured the length, width and height of live and formalin-fixed pelagic bacteria, and computed individual cell volumes. AFM-based measurements were compared with those by epifluorescence microscopy (EFM) using 4′,6-diamidino-2-phenylindole (DAPI). The ability to measure cell height by AFM provides methodological advantage and ecophysiological insight. For the samples examined, EFM (DAPI)-based average cell volume was in good agreement (1.1-fold) with live sample AFM. However, the agreement may be a fortuitous balance between cell shrinkage due to fixation/drying (threefold) and Z-overestimation (as EFM does not account for cell flattening caused by sample processing and assumes that height=width). The two methods showed major differences in cell volume and cell C frequency distributions. This study refines the methodology for quantifying bacteria-mediated C fluxes and the role of bacteria in marine ecosystems, and suggests the potential of AFM for individual cell physiological interrogations in natural marine assemblages.
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Acknowledgements
We thank Maura Manganelli, Jessica Ward and Anne-Clarie Baudoux for kindly providing samples from BWZ II cruise 2006 in Antarctica, CCE LTER Process Cruise 2007 and from the SWAT3 phage experiment. We thank Krystal Rypien for her help on the statistical test. We thank Katherine Barbeau and Melissa Garren for their comments. We thank Asylum Research folks for AFM support. Chlorophyll and temperature data were retrieved from the SCCOOS data archive (www.sccoos.org) supported by NOAA, from the CCE LTER data archive (http://oceaninformatics.ucsd.edu/datazoo/data) supported by the Division of Ocean Sciences, NSF Grant OCE-0417616 and from the BWZ II website supported by NSF Grant ANT- 0444134 to FA. This work was supported by grants from the Gordon and Betty Moore Foundation Marine Microbiology Initiative and from NSF to FA.
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Malfatti, F., Samo, T. & Azam, F. High-resolution imaging of pelagic bacteria by Atomic Force Microscopy and implications for carbon cycling. ISME J 4, 427–439 (2010). https://doi.org/10.1038/ismej.2009.116
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DOI: https://doi.org/10.1038/ismej.2009.116
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