231 research outputs found
Quantification of microbial cells and virus-like particles in sediments from Atacama Trench
No full textSediment cores were collected using a multicorer (MUC). Microbes and virus-like particles were extracted from sediments in a 3°C room using a modified version of the washing protocol of Danovaro and Middelboe, 2010 (see Schauberger et al., 2021). After the washing procedure, the extracted microbial cells and virus-like particles were fixed with 25% glutaraldehyde (1% final concentration) and stored at −80°C prior to flow cytometry. These samples were measured in triplicates using a BD FACSCanto™ II flow cytometer, after staining with SYBR Green I. Sediment extracts were diluted 1 : 10 in 0.02 μm-filtered TE Buffer prior to all measurements. The flow rate was 5–7 μl/min, as determined by BD Trucount™ Beads. The laser settings and gating examples can be found in the Supporting Information of Schauberger et al. (2021)
Quantification of microbial cells and virus-like particles in sediments from Kermadec Trench
No full textSediment cores from sites K3, K4, K5, and K7 were recovered by subsampling a box corer (50 × 50 cm), while sediment cores from sites K4 and K6 were collected using an autonomous lander system. Microbes and virus-like particles were extracted from sediments in a 3°C room using a modified version of the washing protocol of Danovaro and Middelboe, 2010 (see Schauberger et al., 2021). After the washing procedure, the extracted microbial cells and virus-like particles were fixed with 25% glutaraldehyde (1% final concentration) and stored at −80°C prior to flow cytometry. These samples were measured in triplicates using a BD FACSCanto™ II flow cytometer, after staining with SYBR Green I. Sediment extracts were diluted 1 : 10 in 0.02 μm-filtered TE Buffer prior to all measurements. The flow rate was 5–7 μl/min, as determined by BD Trucount™ Beads. The laser settings and gating examples can be found in the Supporting Information of Schauberger et al. (2021)
Separation of free virus particles from sediments in aquatic systems v1
No full textThisprotocol is for the separation of free virus particles from sediments. It is from: Danovaro, R., and M. Middelboe. 2010. Separation of free virus particles from sediments in aquatic systems, p. 74–81. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. Please see the full chapter for additional details. </p
Quantification of microbial cells and virus-like particles in the water column above Atacama Trench
No full textWater samples were collected using 7.5-Liter Niskin bottles and fixed with 25% electron microscopy graded glutaraldehyde (1% final concentration) and stored at -80°C until quantification by flow cytometry. Samples were measured in triplicates using a BD FACSCanto™ II flow cytometer, after staining with SYBR Green I (Brussaard 2004). The flow rate was 5–7 μl/min, as determined by BD Trucount™ Beads. The laser settings and gating examples can be found in the Supporting Information of Schauberger et al. (2021)
Quantification of microbial cells and virus-like particles in the water column above Kermadec Trench
No full textWater samples were collected using 7.5-Liter Niskin bottles and fixed with 25% electron microscopy graded glutaraldehyde (1% final concentration) and stored at -80°C until quantification by flow cytometry. Samples were measured in triplicates using a BD FACSCanto™ II flow cytometer, after staining with SYBR Green I (Brussaard 2004). The flow rate was 5–7 μl/min, as determined by BD Trucount™ Beads. The laser settings and gating examples can be found in the Supporting Information of Schauberger et al. (2021)
Quantification of Viral and Prokaryotic Production Rates in Benthic Ecosystems: A Methods Comparison
No full textViruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the 3H-thymidine incubations for the determination of viral production rates, and the use of 3H-thymidine versus 3H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 × 107 viruses g-1 h-1, and were similar but overall less variable compared to those obtained by the 3H-thymidine method (0.3 to 9.0 × 107 viruses g-1h-1). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3–35.1 and 9.3–34.6 ngC g-1h-1 using the 3H-thymidine or 3H-leucine method, respectively). These results indicated that viral lysis was responsible for the abatement of 55–81% of the prokaryotic heterotrophic production, corroborating previous findings of the major role of viruses in benthic deep-sea ecosystems. Moreover, our methodological comparison for the analysis of viral production in marine sediments suggests that microscopy-based approaches are simpler and more cost-effective than those based on radiotracers. These approaches also reduce time to results and overcome issues related to generation of radioactive waste
Chapter Eleven. Microbial Disease in the Sea: Effects of Viruses on Carbon and Nutrient Cycling
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