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Published: January 18, 2017
FEMS Microbiology Ecology
Authors: Joy Buongiorno, Stephanie Turner, Gordon Webster, Masanori Asai, Alexander K. Shumaker, Taylor Roy, Andrew Weightman, Axel Schippers, Karen G. Lloyd
C-DEBI Contribution Number: 341
mBio
Authors: Jordan T. Bird, Eric D. Tague, Laura A. Zinke, Jenna M. Schmidt, Andrew D. Steen, Brandi Kiel Reese, Ian P.G. Marshall, Gordon Webster, Andrew Weightman, Hector F. Castro, Shawn R. Campagna, Karen G. Lloyd
Editors: Nicole Dubilier
Published: April 16, 2019
C-DEBI Contribution Number: 471
FEMS Microbiology Ecology
Authors: Joy Buongiorno, Stephanie Turner, Gordon Webster, Masanori Asai, Alexander K. Shumaker, Taylor Roy, Andrew Weightman, Axel Schippers, Karen G. Lloyd
Published: January 18, 2017
C-DEBI Contribution Number: 341
Abstract
Two common quantification methods for subseafloor microorganisms are catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and quantitative PCR (qPCR). Using these methods, we quantified Bacteria and Archaea in Baltic Sea basin sediments (IODP Exp. 347) down to 90 mbsf, testing the following hypotheses in an inter-laboratory comparison: 1) proteinase K permeabilization of Archaeal cell walls increases CARD-FISH accuracy, and 2) qPCR varies by more than an order of magnitude between laboratories using similar protocols. CARD-FISH counts did not differ between permeabilization treatments, demonstrating that proteinase K did not increase accuracy of CARD-FISH counts. However, 91% of these counts were below the quantification limit of 1.3 × 107 cells cm−3. For qPCR, data varied between laboratories, but were largely within the same order of magnitude if the same primers were used, with 88% of samples being above the quantification limit. Copy number values were elevated by preparing a sediment slurry before DNA extraction: 3.88 ×106 to 2.34 ×109 16S rRNA gene copies cm−3 vs. 1.39 × 107 to 1.87 × 109 total cells cm−3. By qPCR, Bacteria were more abundant than Archaea, although they usually were within the same order of magnitude. Overall, qPCR is more sensitive than CARD-FISH, but both require optimization to consistently achieve both precision and accuracy.Related Items
Awards
Award Dates: February 1, 2012 — January 31, 2015
PI: Karen G. Lloyd (University of Tennessee, Knoxville)