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Flow Cytometry in Biological Oceanography

Dr. Frank J. Jochem

last revised 14-May-2013

Objectives of Flow Cytometry in biological oceanography
Lab Facilities
Cruises the Flow Cytometers have been carried to
Special Objective: DNA/Cell Cycle Analysis of Phytoplankton
Special Objective: Cytometric Immuno-detection of Nitrate Reductase
Links to other aquatic flow cytometry sites

Objectives of Flow Cytometry Work in Marine Planktology

Application of flow cytometry was introduced into marine planktology to rapidly and precisely estimate cell numbers of pico- and nanophytoplankton, i.e. algal cells of less than 20 µm in size, within the frame of microbial food web studies in various marine systems. Flow cytometry complemented epifluorescence microscopy in studies on the distribution and composition of pico- and nanophytoplankton populations and allowed higher sampling frequencies along ship transects and during incubation experiments. On board application of flow cytometry allowed distributional studies on prochlorophytes, coccoid cyanobacteria (Synechococcus) and small eukaryotic algae in different oceanic provinces such as the Baltic Sea, the North Atlantic, the Caribbean Sea, the Indian Ocean and in Antarctic waters.

In addition to abundance estimates, flow cytometry allows to quantify changes in cellular pigmentation (chlorophyll and phycoerythrin concentrations) of different distinguished phytoplankton sub-populations in response to the light or nutrient regime, either along a depth profile or in incubation experiments. Thus, information on the physiological status or response upon different treatments (changes in light conditions, nutrient additions) can be easily assessed by flow cytometry.

In addition to abundance estimates, flow cytometry was used to study ecophysiological aspects of pico- and nanophytoplankton by application of specific fluorescent dyes and/or fluorogenic substrates:

Mixotrophic activity of phytoflagellates was assessed by the uptake of fluorescein-labeled polysaccharides (dextranes) and FITC-labeled bacteria.
Dark survival of different phytoplankton species was assessed by the use of FDA (fluorescein diacetate): This non fluorescent substrate penetrates easily into living cells and is cleaved into fluorescent fluorescein by various esterases; the accumulation of fluorescence within the cells to be quantified by flow cytometry is a measure of (catabolic) metabolic activity of the cells. Different dark survival capacities based on different patterns of metabolic adjustments could be distinguished among various studied species.
One major topic was the DNA analysis of phytoplankton to assess in-situ growth and grazing rates. With a new staining technique developed at the Institute of Marine Sciences at Kiel University, Germany, it is now possible to simultaneously measure DNA and chlorophyll fluorescence on argon-laser based bench top cytometers, a prerequisite to apply DNA staining in field samples where chlorophyll fluorescence, together with cell size, is necessary to distinguish sub-populations of phytoplankton.
Nitrate reductase, the key enzyme in the assimilation of nitrate in algae, could be quantified by flow cytometry using polyclonal antibodies raised against this enzyme in the marine bloom-forming diatom Skeletonema costatum. Despite the low concentration of this enzyme in algal cells, we could detect its changes and relative concentrations even at the lowest level upon a shift from ammonia to nitrate nutrition, besides other physiological responses, simulating the initiation of a typical upwelling bloom in a chemostat experiment. This work was performed at and in collaboration with colleagues at the Hopkins Marine Station, Pacific Grove, CA.

Lab Facilities

Becton Dickinson FACSort laser-based (488 nm) cytometer with three channel fluorescence analysis, forward and side scatter, and cell sorting facility. The size resolution of this machine (based on latex particles and in-situ measurements of prochlorophytes of about 0.4 µm in size) is ca. 0.3 µm. Picophytoplankton can be easily detected. Typical measurement volumes range between 20 and 500 µl and cell suspensions of 12,000 to 4,500,000 cells/ml can be processed. The cell sorter allows recovery of sub-populations of interest for further experiments or taxonomic examination. Cell sorting results in a 50 to 100 fold dilution of the sorted cell suspension as compared to the original population; therefore, the cell sorter is equipped with a Becton-Dickinson cell concentrator and a ultra-tangential flow filtration for volumes of 50-150 ml to be concentrated down to 5 ml is available as well. The FACSort has proved convincingly robust and has performed well on several cruises on various research vessels. Since all optical components are standardly mounted on one bench, no problems of alignement or drift in sensitivity was encountered during cruises. At Florida International University, the instrument was expanded with a FACSLoader autosampler and a dual excitation red diode laser option.

A Zeiss Axioskop epifluorescence microscope complements the flow cytometer for visual inspection of plankton samples and staining success. The microscope was equipped with a C-mount triple tube, on which a Nikon Cooplix 990 camera was attached for simple microphotography [Follow this link for examples].

Both instruments were funded by German Research Council grant DFG Jo 192/5-1. After relocation to the Marine Science Institute at The University of Texas at Austin and, subsequently, to the Marine Biology Program at Florida International University in Miami, Florida, the instruments remained at FIU after I left for the private industry in 2007.

Cruises the Flow Cytometers have been carried to:

Research Vessel	Area	Time	Instrument
Meteor	North Atlantic	June 1989	Fluvo
Alkor	Baltic Sea	July 1990	Fluvo
Alkor	Baltic Sea	August 1991	Fluvo
Meteor	North Atlantic	May 1992	Fluvo
Alkor	Baltic Sea	August 1992	Fluvo
Polarstern	Antarctica	September-November 1992	Fluvo
Alkor	Baltic Sea	April/May 1993	FACSort
Meteor	Mediterranean	June 1993	Fluvo
Columbus Iselin	Caribbean Sea	September/October 1993	FACSort
Gyre	Caribbean Sea	May 1994	FACSort
Alkor	Baltic Sea	August 1994	FACSort
Alkor	Baltic Sea	October/November 1994	FACSort
Meteor	Indian Ocean	May 1995	FACSort
Alkor	Baltic Sea	July/August 1995	FACSort
Alkor	Baltic Sea	August 1996	FACSort
Longhorn	Gulf of Mexico	July 1999	FACSort
Longhorn	Gulf of Mexico	October 1999	FACSort
Longhorn	Gulf of Mexico	May 2000	FACSort
Suncoaster	Gulf of Mexico	May 2007	FACSort

Links to other working groups applying flow cytometry in aquatic sciences and related sites:

As the links provided in this section were hopelessly outdated, they were removed for now in order to not lead users into dead links. Links to major current labs and working groups applying flow cytometry in marine ecology (including the new FlowCam technology, which I could use in the Gulf of Mexico and Florida Bay) will be added in the future, as time allows.

Objectives of Flow Cytometry Work in Marine Planktology

Lab Facilities

Cruises the Flow Cytometers have been carried to:

Research Vessel

Area

Time

Links to other working groups applying flow cytometry in aquatic sciences and related sites: