MICRO-CARD-FISH
CAtalyzed Reporter Deposition Fluorescence In Situ Hybridization combined
with MICROautoradiography is currently the method of choice to address
the activity of selected prokaryotic groups. Fluorescence in situ hybridzation
with cyanine 3 labeled probes already has a long history, however, this
approach frequently suffered from low detection rates especially in open
ocean settings with low ribosomal content of the prokayrotes.
Probes for CARD-FISH are labeled with a horseradish peroxidase (HRP) tag.
After hybridization of the probe to the target site a signal amplification
step is introduced where tyramides labeled with FITC or CY3 conjugates
are deposited near the hybridized probe through the enzyme activity of
the horseradish. This makes it possible to detect prokaryotes with very
low ribosomal content and detection efficiencies of up to 80%. Due to rather
large HRP-probes the permeabilization of the prokaryotic cell wall is crucial.
While for bacteria lysozyme treatment is usually sufficient the archaeal
cell wall has to be treated with proteinase K to allow penetration of the
probes.
With microautoradiography it is possible to visualize the uptake of radiolabeled
substrates such as leucine or glucose by the prokaryotes. After the hybridization
step the samples
are developed similar to photographic film resulting in silver grain halos
around those cells which incorporated the radioactive molecules. Thus in
combination with CARD-FISH it is possible to get information on who are
the main microbial groups in a water sample and which ones are predominantly
active.
We developed a thorough protocol specifically for open ocean prokaryotes
which is available here:
Further information on the FISH technology can be found at on the SILVA database project webpage.
Suggested reading:
Teira, E., T. Reinthaler,
A. Pernthaler, J. Pernthaler, and G. J. Herndl. 2004. Combining
catalyzed reporter deposition-fluorescence in situ hybridization and
microautoradiography to detect substrate utilization by bacteria and
archaea in the deep ocean. Applied and Environmental Microbiology 70:
4411-4414.
Reprint (pdf)
Sintes, E., G.J. Herndl. 2006. Quantifying substrate uptake of individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography. Applied and Environmental Microbiology 72: 7022-7028.
Reprint (pdf)
Pernthaler, A., J. Pernthaler, and R. Amann. 2002. Fluorescence in
situ hybridization and catalyzed reporter deposition for the identification
of marine bacteria. Applied and Environmental Microbiology 68: 3094-3101.
Cottrell, M. T., and D. L. Kirchman. 2003. Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware estuary. Limnology and Oceanography 48: 168-178.
Ouverney, C. C., and J. A. Fuhrman. 1999. Combined microautoradiography
16S rRNA probe technique for determination of radioisotope uptake by
specific microbial cell types in situ. Applied and Environmental Microbiology
65: 1746-1752.
Comming soon:
- Protocol for Dissolved Inorganic Carbon Uptake by Prokaryotes in Seawater
Suggested reading:
Herndl, G.J., T. Reinthaler, E. Teira, H. van Aken, C. Veth, A. Pernthaler,
J. Pernthaler, 2005. Contribution of Archaea to total prokaryotic production
in the deep Atlantic Ocean. Applied and Environmental Microbiology 71: 2303-2309
Reprint (pdf)
High Pressure Sampler
Currently we are finishing the development of pressure retaining samplers
which allows to take water samples in the dark ocean without appreciable
pressure loss.
The two other known high pressure systems are designed for
subsampling from one bigger pressure vessel, however, we follow the strategy
of incubations in different samplers opened at the same depth.
The 125
samplers used for bacterial production, bacterial respiration and bacterial
ectoenzyme activity measurements are made from titanium have and have a
volume of 50 mL each. Tests in the pressure tank at the NIOZ and on the test cruise showed a
maximum pressure loss of 4%. Labeled substrates for bacterial activity
measurements are placed on the free floating piston in the sampler prior
to deployment. The samplers are closed during the downcast, opened at a
certain depth of interest and closed again for the upcast. A minimum of
6 samplers is opened per depth with an electrical motor.
The samplers are developed and fabricated at the Marine Technology Department at the NIOZ.
Autoanalyzer for oxygen concentration measurements
Determining oxygen concentrations in seawater via the classical Winkler method is considered labor intensive and tedious. Moreover, producing high quality data usually demands procedural experience. Together with the Marine Technology Department and the Analytical Laboratory at the NIOZ we designed a fully automated spectrophotometric approach to determine oxygen concentrations in seawater that is easy to use and is able to produce WOCE quality data.
The instrument and a dedicated laboratory container are available for seagoing research via the NIOZ research facilities equipment pool.
Suggested reading:
Reinthaler, T., K. Bakker, R. Manuels, J. Van Ooijen, and G. J. Herndl. 2006. Fully automated spectrophotometric approach to determine oxygen concentrations in seawater via continuous-flow analysis. Limnolology and Oceanography: Methods 4: 358-366.
Abstract