Fluorescent in situ hybridization microscope counts
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| Life cycle stage abundance (FISH) |
|---|
| Approach: fluorescence in situ hybridisation (FISH) with life-stage-specific or species-specific probes |
| Context: in situ, lab |
| Spatial scale: point sample |
| Temporal scale: days |
| Units: cells L-1; % of population |
| Community captured: specific species (those with validated FISH probes) |
| Co-measurements: species-specific life stage duration |
Method Overview
Fluorescence in situ hybridisation (FISH) uses rRNA-targeting probes labelled with fluorescent dyes to identify and enumerate specific life cycle stages or species in fixed water samples. For life cycle work, probes are designed to target rRNA transcripts that are expressed at different levels in different life stages, or probes are used in combination with morphological recognition to count distinct stages. Fixed samples are filtered onto polycarbonate membranes, hybridized with fluorescent probes, and cells are counted by epifluorescence microscopy. CARD-FISH (catalyzed reporter deposition) amplifies the signal for low-biomass samples[1].
Scale of measurement
Point samples; days between collection points.
Data generated
Species-specific and life-stage-specific cell abundances (cells L-1) or proportional abundances (%). When combined with total DAPI or SYBR Green counts, the fraction of the community in each life stage is calculated.
Units & currency
Units are cells L-1 or % of cells in each life stage. The currency is measured abundance of each recognizable life stage.
Sample size
Typical samples are < 1 L in volume.
Repositories & databases
Limitations
FISH is only possible for taxonomic groups for which validated probes exist. Cross-hybridization with non-target organisms can produce false positives; probe specificity must be validated. The method does not distinguish between life cycle stages unless probes specifically target stage-expressed transcripts. Low rRNA content in dormant cells (e.g., resting cysts) can lead to false negatives.
Example Applications & Protocols
Classic examples
- Frada et al. (2006) CaCO3 optical detection with fluorescent in situ hybridization: a new method to identify and quantify calcifying microorganisms from the oceans [1]
Recent applications
Common calculations/conversions
- % of community in stage X = (FISH-positive cells for stage X) / (total DAPI-stained cells) × 100.
References
- ↑ 1.0 1.1 Frada, M., Not, F., de Vargas, C., & Probert, I. (2006). CaCO3 optical detection with fluorescent in situ hybridization: a new method to identify and quantify calcifying microorganisms from the oceans. Journal of Phycology, 42(6), 1162–1169. https://doi.org/10.1111/j.1529-8817.2006.00276.x