Chapter Outline: Gold-tagged in situ
hybridization for whole cell detection in environmental samples
Proposed title: Gold-tagged in situ hybridization for whole cell
detection in environmental samples
Chapter
no.: _________
Authors: Hannes Schmidt (MS) and Thilo Eickhorst (PhD)
University
of Bremen
Leobener Str., UFT
28359
Bremen, Germany
Phone: +49(421) 218-63446
Fax: +49(421) 218-9863446
E-mail: eickh@uni-bremen.de
Notes:
· TBD
Proposed chapters
In situ detection of microbes in environmental
microbiology
Aim
Methods
available and their limitations
- Detection of single
microbial cells
- Identification and
visualization in situ
- Quantification of
absolute and relative abundances
- Top-to-bottom approach
Need
for combinative approaches and higher resolution
- Correlative in situ
detection of microbes and living conditions
- Surface structure and
substrate conditions
- Combination with high
resolution microscopy techniques
- Quantification and
localization on different microscopic scales
NanoGold as a marker for in situ detection of
microbes
State
of the art
- Nanogold-labeled
substrates
- Immunological approaches
- ISH-approaches (Table)
- Autometallography
(Silver/Gold)
Problems/drawbacks
- Review of previously
documented problems for ISH techniques
- General problems in
environmental samples
- Need for increased
specificity and signal intensity
Gold-FISH – microscopy/detection of fluorescent and
gold signals
Development
of the new Gold-FISH protocol
- Goal: simultaneous
application/deposition of fluorescent and gold markers
- Signal amplification for
both markers
- Enhancement of nanogold
particles for EM applications
Results/Applications
so far
- pure and mixed bacterial
cultures
- soil and sediment
- plant roots
Technical
aspects/hints /troubleshooting for the application of Gold-FISH to
environmental samples
- Cell wall
permeabilization
- Autometallography
- Surface charge
- Computer-aided analysis
Potential for gold-based detection techniques in
Environmental microbiology
- Correlative microcopy of
Gold-FISH (e.g. FM and SEM-EDS)
- Combination with
nanoSIMS
- Potential for
non-invasive microscopy (e.g. X-ray CT)
Chapter Highlights
The
following concepts will be conveyed in this chapter:
- Novel approach for a simultaneous labeling of microorganisms with a fluorescent dye and nanogold
- Optimization of specific nanogold deposition by tyramide signal amplification
- Possible application of different microscopy techniques for the analysis of gold-FISH labeled single cells
- Microorganism interactions on different surface morphologies and microenvironments in terms of structure and biogeochemical conditions
- Element-microorganism interactions
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