A Cell Extraction Method for Oily Sediments

Abstract

To get a first impression of the size of the microbial community present in a sediment sample, the determination of cell abundances in sediments is of great importance for biogeochemical studies. One of the most simple and reliable methods is direct counting, where the cells are stained with a DNA-binding stain and counted under an epifluorescence microscope. However, in oily sediments, DNA-specific stains and molecular probes bind to the hydrocarbons, causing massive background fluorescence, thereby hampering cell enumeration. To overcome this problem, we developed an extraction method in which the hydrocarbons are removed with organic solvents prior to cell extraction. Due to the reduced background fluorescence, the microscopic image becomes clearer, making cell identification and enumeration much easier.

A volumetric ratio of 1:2 to 1:5 between a formalin-fixed sediment slurry and solvent delivers highest cell counts. n-Hexane delivers best results for samples containing less biodegraded oil, whereas methanol turned out to be the most appropriate solvent for samples containing strongly biodegraded oil. The optimal solvent to sample ratio has to be determined prior to analysis for each type of sample.

However, it has to be kept in mind that solvents also tend to lyse cells and that the given protocol has to be adapted to the individual conditions in order to minimize cell lysis and maximize hydrocarbon removal.