Introduction to Microplate MPN-Enumeration of Hydrocarbon Degraders
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 po
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K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_324, # Springer-Verlag Berlin Heidelberg, 2010
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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 14 C- labeled substrates, which is probably the most reliable quantification method for hydrocarbon degraders. In this method, a 14C-hydrocarbon is mineralized to 14CO2 in growthpositive 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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Introduction
Most-probable-number (MPN) methods are used for determining the number of specific types of microorganisms in environmental samples or liquid cultures. Most MPN methods are based on growth in liquid medium under selective conditions that only allows growth of the specific target organisms, this is conveniently done in 96-well microplates. Hydrocarbon degraders are generally MPN-enumerated in minimal medium containing one or more hydrocarbons. Cells that can use the hydrocarbons as the sole source of carbon and energy will proliferate, whereas nondegraders will starve and become metabolically inactive. The MPN-methods are based on the dilution-to-extinction principle, where the sample is serially diluted until the target organism is no longer present in the highest dilutions. The presence or absence of the target organism is then determined for several subsamples of each dilution followed by calculation of the MPN from the distribution of positive and negative subsamples. The right way of calculating MPNs, standard errors, confidence limits and test-values for detecting significant differences has been a popular subject for almost a century. Many applied studies using MPN-procedures refer to an article by Cochran (1950). There seems to be a general consensus that many MPN algorithms slightly overestimate actual cell numbers, the bias is, however, negligible when MPNs are expressed as log-values. A single cultivable cell in the highest positive dilution should, in principle, be able to give a positive well for the methods to work. It is therefore important to incubate the plates long enough to allow a single starter cell to multiply to a detectable level. Some people advocate that MPN plates should be incubated
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