Quantitative flow cytometric detection of specific microorganisms in soil samples.
Jean-Christophe Thomas (1), Marcel Desrosiers (2), Richard Villemur (3) and Yves St-Pierre (2) Introduction
Specific detection and accurate enumeration of microbial populations in the environment is crucial during
bioremediation treatments, in which microorganisms are inoculated in soil to breakdown pollutants. Up to now,
enumeration of specific microorganisms in environmental samples relied on counting visible colonies on selective
agar media (CFU) or on epifluorescence microscopic counting.
Flow cytometry (FCM) offers the ability to perform rapid, multiparametric analysis of specific individual cells in
a heterogeneous population. However, the use of FCM to monitor bacteria in soil has been hampered by the
high levels of autofluorescence from environmental samples and by background noise due to non-specific attachment
of the fluorescent probes to debris.
In this work, we report a new multiparametric strategy that allows specific and quantitative detection of
Sphingomonas sp. strain 107 inoculated into soil samples. This method was developed
in five successive steps:
Step 1: Use light scattering profiles for qualitative and quantitative analysis of isolated bacteria.
Step 2: Develop a protocol to determine optimal fixation and permeabilization of cell wall and membrane for microorganism of interest. The protocol must allow efficient entry of a specific fluorescent oligonucleotide probe while maintaining the morphological integrity of the cell.
Step 3: Elaborate a gating strategy to discriminate indigenous microflora and soil particles from microbes specifically stained with FITC-labeled probes.
Step 4: Compare counts obtained by CFU assay and by the FCM method.
Step 5: Apply this method to monitor a pyrene-degrading bacterial species, Sphingomonas sp. strain 107, introduced into a contaminated soil microcosm during an aerobic treatment.
(1) Ecologie Microbienne du Sol
UMR CNRS 5557, Universite Lyon 1
La Doua, Bat. 741
43 bd du 11 Novembre 1918
69622 Villeurbanne Cedex, France
(2) Centre de Recherche en Immunologie
531 Boul. des Prairies
Laval (Quebec), Canada H7V 1B7
(3) Centre de Recherche en Microbiologie Appliquee
531 Boul. des Prairies
Laval (Quebec), Canada H7V 1B7.CA