Pulse-chase labeling of bacterial prey
Template:BreadcrumbsInteractions
| Bacterivory by mixotrophs (pulse-chase 14C) |
|---|
| Approach: 14C pulse labelling of bacterial prey; radioactivity in FCM-sorted pigmented cells |
| Context: incubation, field |
| Spatial scale: point sample |
| Temporal scale: hours |
| Units: prey cell-1 h-1 |
| Community captured: specific FCM-defined populations (unpigmented heterotrophs; pigmented mixotrophs) |
| Co-measurements: cell abundances |
Method Overview
14C-labelled bacteria (pulse phase) are added to seawater samples and allowed to be ingested by grazers. After a short pulse incubation, samples are fixed and specific cell populations are sorted by flow cytometry based on their optical properties — distinguishing unpigmented heterotrophic protists from pigmented (autofluorescent) cells, which may include constitutive mixotrophs. The radioactivity in each sorted population is measured by liquid scintillation counting. The radioactivity per sorted pigmented cell, relative to the specific radioactivity of the labelled bacterial prey, gives the ingestion rate of bacteria by that population. This approach uniquely identifies bacterivory in autofluorescent (phytoplankton-like) cells, directly demonstrating mixotrophy in situ[1].
Scale of measurement
Hours of incubation required to allow detectable ingestion.
Data generated
Per-cell bacterial ingestion rates (prey cell-1 h-1) for specific FCM-sorted populations, including pigmented mixotrophic cells.
Units & currency
Units are prey cell-1 h-1. The currency is cells.
Sample size
Typical samples are < 1 L in volume.
Repositories & databases
Limitations
The assumption is that all radioactivity in the sorted pigmented cell fraction arose from bacterial ingestion (bacterivory), rather than other reasons for co-associations such as stickiness or bacterial attachment to cell surfaces. Contamination of the sorted fraction by unlabelled bacteria can inflate apparent ingestion rates. FCM sorting throughput limits the number of cells collected per sample.
Example Applications & Protocols
Classic examples
- Zubkov & Tarran (2008) High bacterivory by the smallest phytoplankton in the North Atlantic Ocean [1]
Recent applications
Common calculations/conversions
- Ingestion rate = (cpmsorted pigmented cells / cpmlabelled bacterium-1) / (number of sorted cells × incubation time).
References
- ↑ 1.0 1.1 Zubkov, M. V., & Tarran, G. A. (2008). High bacterivory by the smallest phytoplankton in the North Atlantic Ocean. Nature, 455, 224–226. https://doi.org/10.1038/nature07236