Introduction to microplate MPN-enumeration of hydrocarbon degraders

A.R. Johnsen

doi:10.1007/978-3-540-77587-4_324

Introduction to microplate MPN-enumeration of hydrocarbon degraders

A.R. Johnsen

Department of Geochemistry

Research output: Chapter in Book/Report/Conference proceeding › Chapter in book › Research › peer-review

Abstract

Most-probable-number (MPN) methods are used for determining the number of specific types of microorganisms in environmental samples or liquid cultures. Here we present three specific methods for applying the MPN method to quantify hydrocarbon-degrading populations in samples from contaminated environments. Two of those use specific coloration of active hydrocarbon-degrading communities enriched in the MPN procedure, namely the INT-method and a protocol that uses the tetrazolium compound WST-1 for detection of growth-positive wells. Finally, we describe an MPN enumeration based on mineralization of ¹⁴C- labeled substrates, which is probably the most reliable quantification method for hydrocarbon degraders. In this method, a ¹⁴C-hydrocarbon is mineralized to ¹⁴CO₂ in growth-positive wells, and the produced ¹⁴CO₂ is captured in alkaline traps and quantified. In the procedure described below, we have modified the method for a 96-well microplate setup, where the ¹⁴CO₂ is quantified simultaneously from all wells by autoradiography followed by digital image analysis.

Original language	English
Title of host publication	Handbook of hydrocarbon and lipid microbiology
Editors	Kenneth N. Timmis
Place of Publication	New York
Publisher	Springer
Pages	4160-4171
Number of pages	12
ISBN (Print)	978-3-540-77584-3
DOIs	https://doi.org/10.1007/978-3-540-77587-4_324
Publication status	Published - 2009

Programme Area

Programme Area 2: Water Resources

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10.1007/978-3-540-77587-4_324

Cite this

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title = "Introduction to microplate MPN-enumeration of hydrocarbon degraders",

abstract = "Most-probable-number (MPN) methods are used for determining the number of specific types of microorganisms in environmental samples or liquid cultures. Here we present three specific methods for applying the MPN method to quantify hydrocarbon-degrading populations in samples from contaminated environments. Two of those use specific coloration of active hydrocarbon-degrading communities enriched in the MPN procedure, namely the INT-method and a protocol that uses the tetrazolium compound WST-1 for detection of growth-positive wells. Finally, we describe an MPN enumeration based on mineralization of 14C- labeled substrates, which is probably the most reliable quantification method for hydrocarbon degraders. In this method, a 14C-hydrocarbon is mineralized to 14CO2 in growth-positive wells, and the produced 14CO2 is captured in alkaline traps and quantified. In the procedure described below, we have modified the method for a 96-well microplate setup, where the 14CO2 is quantified simultaneously from all wells by autoradiography followed by digital image analysis.",

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AB - Most-probable-number (MPN) methods are used for determining the number of specific types of microorganisms in environmental samples or liquid cultures. Here we present three specific methods for applying the MPN method to quantify hydrocarbon-degrading populations in samples from contaminated environments. Two of those use specific coloration of active hydrocarbon-degrading communities enriched in the MPN procedure, namely the INT-method and a protocol that uses the tetrazolium compound WST-1 for detection of growth-positive wells. Finally, we describe an MPN enumeration based on mineralization of 14C- labeled substrates, which is probably the most reliable quantification method for hydrocarbon degraders. In this method, a 14C-hydrocarbon is mineralized to 14CO2 in growth-positive wells, and the produced 14CO2 is captured in alkaline traps and quantified. In the procedure described below, we have modified the method for a 96-well microplate setup, where the 14CO2 is quantified simultaneously from all wells by autoradiography followed by digital image analysis.

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DO - 10.1007/978-3-540-77587-4_324

M3 - Chapter in book

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EP - 4171

BT - Handbook of hydrocarbon and lipid microbiology

A2 - Timmis, Kenneth N.

PB - Springer

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ER -

Introduction to microplate MPN-enumeration of hydrocarbon degraders

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