FISH staining of anammox bacteria
| Anammox cell abundance (CARD-FISH) |
|---|
| Approach: catalyzed reporter deposition – fluorescence in situ hybridization (CARD-FISH) |
| Context: lab |
| Spatial scale: point sample |
| Temporal scale: days (DNA; snapshot) |
| Units: cells L-1 |
| Community captured: anammox bacteria |
| Co-measurements: O2, NO2-, NH4+ |
Method Overview
Anammox bacteria are identified and enumerated in fixed seawater samples by catalyzed reporter deposition – fluorescence in situ hybridization (CARD-FISH). Seawater samples are fixed with paraformaldehyde, filtered onto polycarbonate membranes, and hybridized with anammox-specific 16S rRNA probes (e.g., Brod541 for Candidatus Brocadia-like anammox) labelled with horseradish peroxidase. Tyramide signal amplification (CARD) amplifies the fluorescent signal, enabling detection of anammox cells at low abundances. Cells are visualized by epifluorescence microscopy and counted relative to total DAPI-stained cells[1].
CARD-FISH cell counts, when combined with cell-specific anammox rates derived from isotope experiments, provide community-level anammox rate estimates.
Scale of measurement
A snapshot of anammox cell abundance at time of collection.
Data generated
Anammox cell concentrations (cells L-1). Combined with cell-specific rates, volumetric anammox rates can be estimated.
Units & currency
Units are cells L-1. The currency is cell number.
Sample size
Typical samples are < 1 L in volume.
Repositories & databases
Limitations
False positives can arise if the probe cross-hybridizes with non-target organisms. Not all anammox lineages may be detected by a given probe set, leading to underestimates in diverse communities. Fixation and hybridization can reduce signal efficiency. Cell-specific rates used for conversion to volumetric estimates are typically derived from pure cultures and may not represent the activity of environmental anammox bacteria.
Example Applications & Protocols
Classic examples
- Schmid et al. (2003) 16S–23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria [1]
Recent applications
Common calculations/conversions
- Anammox rate = [anammox cells L-1] × cell-specific rate (fmol N2 cell-1 d-1).