Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays

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StandardStandard

Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays. / Dick, Richard; Dick, Linda; Deng, Shiping et al.
Yn: Soil Biology and Biochemistry, Cyfrol 121, 01.06.2018, t. 240-248.

Allbwn ymchwil: Cyfraniad at gyfnodolynErthygladolygiad gan gymheiriaid

HarvardHarvard

Dick, R, Dick, L, Deng, S, Li, X, Kandeler, E, Poll, C, Freeman, C, Jones, T, Weintraube, MN & Saxena, J 2018, 'Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays', Soil Biology and Biochemistry, cyfrol. 121, tt. 240-248.

APA

Dick, R., Dick, L., Deng, S., Li, X., Kandeler, E., Poll, C., Freeman, C., Jones, T., Weintraube, M. N., & Saxena, J. (2018). Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays. Soil Biology and Biochemistry, 121, 240-248.

CBE

Dick R, Dick L, Deng S, Li X, Kandeler E, Poll C, Freeman C, Jones T, Weintraube MN, Saxena J. 2018. Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays. Soil Biology and Biochemistry. 121:240-248.

MLA

Dick, Richard et al. "Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays". Soil Biology and Biochemistry. 2018, 121. 240-248.

VancouverVancouver

Dick R, Dick L, Deng S, Li X, Kandeler E, Poll C et al. Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays. Soil Biology and Biochemistry. 2018 Meh 1;121:240-248. Epub 2018 Maw 31.

Author

Dick, Richard ; Dick, Linda ; Deng, Shiping et al. / Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays. Yn: Soil Biology and Biochemistry. 2018 ; Cyfrol 121. tt. 240-248.

RIS

TY - JOUR

T1 - Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays

AU - Dick, Richard

AU - Dick, Linda

AU - Deng, Shiping

AU - Li, Xiufen

AU - Kandeler, Ellen

AU - Poll, Christian

AU - Freeman, Christopher

AU - Jones, Timothy

AU - Weintraube, Michael N.

AU - Saxena, Jyotisna

PY - 2018/6/1

Y1 - 2018/6/1

N2 - There has been growing interest in fluorescence-based microplate methods to measure enzyme activities due tothe sensitivity of fluorimetric detection and the potential for simultaneous and rapid assaying of multiple enzymeactivities in the same soil suspension. However, micro-scale methods could introduce considerable operator errorsuch as: 1) the requirement to put soil samples into a suspension; 2) the very small amounts of soil placed in eachmicroplate well; 3) pipetting error because μL volumes are required; and 4) the need for standard curve calibrationwith every sample to account for quenching. For valid data comparison and interpretation, there isclearly a need to have a strict and agreed-upon enzyme assay protocol to standardize the microplate-basedmethod. Therefore, the objectives were to: 1) determine the reproducibility and comparability of the standard pnitrophenolbench-scale and 4-methylumbelliferone microplate enzyme assays measured by five laboratories forβ-glucosidase (EC 3.2.1.21) and acid phosphomonoesterase (EC 3.1.3.2) on the same soil samples; and 2) determinethe degree and the sources of variability associated with the assays within and among the laboratories.The results showed that overall variability was highest for replication on the microplate (n=4), whereas suspensionreplication had low CVs. This suggests an important source of variation is from pipetting not variabilityfrom soil suspensions. A major effort was made to control for methodological differences by using air-dried soils(therefore more stable over varying storage periods) and operator consistency for each task across the labs (e.g.preheated reagents, microplate reader sensitivity set to the highest standard, readings taken within an hour ofreaction termination, and controls for substrate autohydrolysis). As a result, the differences among labs weremuch smaller than differences due to soil type for the microplate method, indicating operator error can beminimized by following the same strict protocol. At the molar level, enzyme activity rates measured across thefive labs were not the same between bench and MUF microplate methods (although they were within an order ofmagnitude), but were quite similar in terms of ranking of soil management treatments and soil types (Table 2).Correlations between bench and microplate assays were strong for both enzymes, although slightly stronger foracid phosphomonoesterase (r=0.93) than β-glucosidase (r=0.81). Additionally, for both acid phosphomonoesteraseand β-glucosidase, correlation r values were mostly similar for MUF microplate and PNP benchmethod correlation with EL-FAME biomarkers, suggesting both methods were measuring activity originatingfrom the same microbial groups. We conclude that different labs using the same MUF microplate protocol tested,gives reasonably similar absolute activity values, variability, and ranking of treatments (highest to lowest). Wepropose that the MUF microplate method described in this study be considered as a standard protocol for assayingsoil enzyme activities, providing that the buffer pH for the incubation be adjusted to the optimal pHaccording to the enzyme of interest.

AB - There has been growing interest in fluorescence-based microplate methods to measure enzyme activities due tothe sensitivity of fluorimetric detection and the potential for simultaneous and rapid assaying of multiple enzymeactivities in the same soil suspension. However, micro-scale methods could introduce considerable operator errorsuch as: 1) the requirement to put soil samples into a suspension; 2) the very small amounts of soil placed in eachmicroplate well; 3) pipetting error because μL volumes are required; and 4) the need for standard curve calibrationwith every sample to account for quenching. For valid data comparison and interpretation, there isclearly a need to have a strict and agreed-upon enzyme assay protocol to standardize the microplate-basedmethod. Therefore, the objectives were to: 1) determine the reproducibility and comparability of the standard pnitrophenolbench-scale and 4-methylumbelliferone microplate enzyme assays measured by five laboratories forβ-glucosidase (EC 3.2.1.21) and acid phosphomonoesterase (EC 3.1.3.2) on the same soil samples; and 2) determinethe degree and the sources of variability associated with the assays within and among the laboratories.The results showed that overall variability was highest for replication on the microplate (n=4), whereas suspensionreplication had low CVs. This suggests an important source of variation is from pipetting not variabilityfrom soil suspensions. A major effort was made to control for methodological differences by using air-dried soils(therefore more stable over varying storage periods) and operator consistency for each task across the labs (e.g.preheated reagents, microplate reader sensitivity set to the highest standard, readings taken within an hour ofreaction termination, and controls for substrate autohydrolysis). As a result, the differences among labs weremuch smaller than differences due to soil type for the microplate method, indicating operator error can beminimized by following the same strict protocol. At the molar level, enzyme activity rates measured across thefive labs were not the same between bench and MUF microplate methods (although they were within an order ofmagnitude), but were quite similar in terms of ranking of soil management treatments and soil types (Table 2).Correlations between bench and microplate assays were strong for both enzymes, although slightly stronger foracid phosphomonoesterase (r=0.93) than β-glucosidase (r=0.81). Additionally, for both acid phosphomonoesteraseand β-glucosidase, correlation r values were mostly similar for MUF microplate and PNP benchmethod correlation with EL-FAME biomarkers, suggesting both methods were measuring activity originatingfrom the same microbial groups. We conclude that different labs using the same MUF microplate protocol tested,gives reasonably similar absolute activity values, variability, and ranking of treatments (highest to lowest). Wepropose that the MUF microplate method described in this study be considered as a standard protocol for assayingsoil enzyme activities, providing that the buffer pH for the incubation be adjusted to the optimal pHaccording to the enzyme of interest.

M3 - Article

VL - 121

SP - 240

EP - 248

JO - Soil Biology and Biochemistry

JF - Soil Biology and Biochemistry

SN - 0038-0717

ER -