A portable cryo‐plunger for on‐site intact cryogenic microscopy sample preparation in natural environments

Comolli, Luis R.; Duarte, Robert; Baum, D.W.; Luef, Birgit; Downing, Kenneth H.; Larson, David M.; Csencsits, R.; Banfield, Jillian F.

doi:10.1002/jemt.22001

Cited by 34 publications

(28 citation statements)

References 19 publications

(26 reference statements)

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“…Rectangular prism-shaped cells with large vesicles and a three-layer cell wall were evident in cryo-TEM images of planktonic cells recovered from lake water. Based on prior TEM studies (Comolli et al, 2012), we infer that these cells belong to the genus Haloquadratum (Figure 6a). Other prominent morphologies included rod-shaped and triangular cells (Figures 6b and d).…”

Section: Resultsmentioning

confidence: 75%

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Metagenomic and lipid analyses reveal a diel cycle in a hypersaline microbial ecosystem

et al. 2015

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Marine microbial communities experience daily fluctuations in light and temperature that can have important ramifications for carbon and nutrient cycling. Elucidation of such short time scale community-wide dynamics is hindered by system complexity. Hypersaline aquatic environments have lower species richness than marine environments and can be well-defined spatially, hence they provide a model system for diel cycle analysis. We conducted a 3-day time series experiment in a well-defined pool in hypersaline Lake Tyrrell, Australia. Microbial communities were tracked by combining cultivation-independent lipidomic, metagenomic and microscopy methods. The ratio of total bacterial to archaeal core lipids in the planktonic community increased by up to 58% during daylight hours and decreased by up to 32% overnight. However, total organism abundances remained relatively consistent over 3 days. Metagenomic analysis of the planktonic community composition, resolved at the genome level, showed dominance by Haloquadratum species and six uncultured members of the Halobacteriaceae. The post 0.8 μm filtrate contained six different nanohaloarchaeal types, three of which have not been identified previously, and cryo-transmission electron microscopy imaging confirmed the presence of small cells. Notably, these nano-sized archaea showed a strong diel cycle, with a pronounced increase in relative abundance over the night periods. We detected no eukaryotic algae or other photosynthetic primary producers, suggesting that carbon resources may derive from patchily distributed microbial mats at the sediment-water interface or from surrounding land. Results show the operation of a strong community-level diel cycle, probably driven by interconnected temperature, light abundance, dissolved oxygen concentration and nutrient flux effects.

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Section: Resultsmentioning

confidence: 75%

“…Cryogenic TEM Samples for cryo-TEM were prepared on site using a portable cryo-plunge device as described in Comolli et al (2012). The program ImageJ (NIH, http://rsb.info.nih.gov/ij/) was used for analysis of the two-dimensional image projections.…”

Section: Estimation Of Time Series Organism Abundance Patternsmentioning

confidence: 99%

Metagenomic and lipid analyses reveal a diel cycle in a hypersaline microbial ecosystem

et al. 2015

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“…DNA was extracted from both filters for phylogenetic analysis. Cells that passed through the 0.2-mm filter from samples taken 7 and 9 days after acetate injection began were preserved in vitreous ice on site for later ultrastructural characterization using cryo-TEM 17 . Cryogenic preservation eliminates fixation and dehydration artefacts 18 and can provide morphological information with B2-4 nm resolution 19 .…”

Section: Resultsmentioning

confidence: 99%

“…Ten and 250 nm colloidal gold particles (BBInternational, Cardiff, UK) were put on TEM grids for cryo-TEM and SIR spectroscopy, and for correlative FISH and TEM, respectively, and allowed to dry prior to sample addition. Aliquots of 5 ml 0.2-mm-filtered groundwater sample were deposited onto the grids, manually blotted with filter paper and plunged into liquid propane at liquid nitrogen temperature using a portable cryo-plunge device on site 17 . Grids were stored in liquid nitrogen until further analysis.…”

Section: Methodsmentioning

confidence: 99%

“…14 and recovered cells that passed through a B0.2-mm filter for DNA extraction and phylogenetic characterization. We preserved cells in vitreous ice on site for later ultrastructural characterization using cryogenic transmission electron microscopy (cryo-TEM) 17 . Filtration enriched for cells of the WWE3, OP11 and OD1 phyla, a vast phylogenetic radiation that lacks cultivated representatives.…”

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confidence: 99%

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Diverse uncultivated ultra-small bacterial cells in groundwater

et al. 2015

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Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a B0.2-mm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009 ± 0.002 mm 3 ). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms.

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