Routine fluorescence in situ hybridization in soil

Area of Science:

• Environmental microbiology
• Molecular biology techniques

Background:

• Autofluorescence of soil particles and time-consuming manual counting hinder fluorescence in situ hybridization (FISH) for soil bacteria identification.
• Nycodenz has shown potential in concentrating bacteria and reducing soil particle interference for FISH analysis.

Purpose of the Study:

• To present a routine, efficient protocol for FISH in soil using Nycodenz.
• To enable fast and easy enumeration of soil bacteria.
• To develop a semi-automated approach for bacterial counting.

Main Methods:

• Developed a routine protocol for FISH in soil incorporating Nycodenz and silicon grease coated slides for parallel sample processing.
• Implemented Image J macros for a semi-automated bacterial enumeration approach.
• Compared the Nycodenz protocol with a soil slurry method, evaluating extractants like Na(2)EDTA and Tween 20.

Main Results:

• The Nycodenz protocol allowed for fast and easy enumeration of hundreds of bacteria, with software-assisted counts matching eye-counts.
• Fifty-five percent of DAPI-stained bacteria were co-labeled with a Domain Bacteria FISH probe.
• The Nycodenz protocol significantly improved bacterial counts compared to the soil slurry method, which was impaired by soil particles.

Conclusions:

• The proposed Nycodenz-based FISH protocol offers a time-efficient method for characterizing soil bacterial communities.
• The use of Nycodenz effectively concentrates bacteria and mitigates soil autofluorescence issues.
• Na(2)EDTA as an extractant enhanced bacterial observation, while Tween 20 showed no significant benefit.