Tuesday, 15 October 2013

Improved techniques for sampling the microbiology of subsurface sediments

Our understanding of microbiota in subsurface marine sediments is somewhat limited. Challenges imposed by the attributes of low metabolic activity, small cell size and low abundances of such organisms make detection and collection very tough. This, alongside problems associated with cell enumeration and the separation of cells from sediment makes current methods for sampling subsurface microbiology lengthy and inefficient. This paper demonstrates new methods that have been developed  to overcome such problems

Cell enumeration
Flow cytometry (FCM) is a powerful and efficient tool in the enumeration of cells yet its use for in sediment cell counts is limited by analytical interference of non-biological particles (NBP). Work by the authors previously demonstrated that when processing the green and red filtered fluorescent images on a FCM cytogram, cells stained with SYBR Green I have a pattern distinguishable from NBP patterns.

Separation from sediment
High biomass samples

Large particles are removed by sieving the sample through a 100┬Ám mesh, and the resultant sample analysed by FCM. This method allows for accurate staining and enumeration of sediment samples providing the ratio between sample amount and dye concentration is optimised.  An incorrect ratio reduces the cell count accuracy as adsorbtion of the dye by NBPs decreases the concentration of free dye available for intracellular DNA staining meaning cells go unnoticed; overadsorbtion alters the NBP fluorescence pattern making it difficult distinguish between cells and NBP on the FCM cytogram.

Low biomass samples
Previous work by Kallmeyer et al, 2008 used bilayer density-based cell separation: a method of separating microbial cells and sediment particles based on density differences. This technique resulted in huge variations in the percentage cell recovery, though consistencies of low cell recovery, particularly with high sediment volumes, and high cell numbers remaining in the layer of precipitated sediment were found throughout all samples. These consistencies were attributed to the co-precipitation of cells with sediment particles via hydrodynamic dragging at the density interface.
Morono et al, modified this technique of density based separation by creating four different density layers using high density solutions. This new multilayer technique demonstrated to be more effective than simple bilayer separation for both natural and control sediments, with an up to 45.6 times higher recovery than found in bilayer separation. Whilst an increase was seen for all samples, deep (100-359m below seafloor (mbsf)) sediment samples had a low recovery (average 39.5%) compared to the relatively high (60.5%) recovery from shallow (0-100 mbsf)sediment.
It was found that a higher density solution was optimum for creating the density layers as the high viscocity and low Reynolds number reduces the effect of turbulent flow particles thus increasing the percentage recovery.  The high density solution also reduces the amount of cells in precipitate by loosening the compactness of the sediment allowing cells to navigate to the correct density layers.
The recovered cells can then be enumerated using FCM.

Overall the authors have been successful in developing and optimising methods for the separation of cells from sediments, improved efficiency of cell recovery and high-throughput cell enumeration. Whilst improvements on these techniques are needed in the future, the new methods that have been shown ensures accurate and repeatable data can be obtained on subsurface microbes within a reasonable time frame enabling us expand our knowledge of the nature of microbial communities in the largely uncharacterised deep sedimentary biosphere.

Morono Y., Terada T., Kallmeyer J. and Inagaki F. (2013) An improved cell separation technique for marine subsurface sediments: applications for high-throughput analyses using flow cytometry and cell sorting. Environmental Microbiology, 15(10), 2841-2849


  1. Hi Marie, do the authors state why they think they are recovering less cells for the deep sea sediments? My guess would be that their method is not as effective with the particularly fine sediments that can be found in the deep sea, making it more difficult to separate the cells, but I would be interested to hear whether the authors make any note of this.
    Thanks, Rachel.

    1. Hi Rachel, thank you for your comment. Your guess is very much correct- separating cells from very light, fine sediment particles is particularly challenging. From the deepest sediment sample (359mbsf) only 7.5% of cells could be recovered which is much less than the average recovery for the deep sediments. The authors also however have made the presumption that the low recovery levels could be attributed to a higher cell density of cells found deeper within sediments. If their presumption is correct, using a higher density solution to separate cells and sediment could help to increase percentage recovery.
      Thanks, Marie