Analytical Techniques

Flow cytometry

Flow cytometry is a laser-based method allowing to analyze multiple cell parameters simultaneously. We use a BD Accuri C6® flow cytometer to determine the cell density of phytoplankton and bacteria cultures as well as to measure cellular properties such as lipid and protein content using staining techniques. In addition, cell size and transparency are also measured. For this purpose, there are two further detectors, the forward scatter and the side-scatter.

 

CASY

The CASY® technology is based on the electrical detection of cells passing through a measuring pore. Cells with intact cell membranes generate a signal with an intensity proportional to the volume of the cell. This device is therefore used to measure culture densities and cell size.

 

Gas chromatography (fatty acid analysis)

To measure phytoplankton and zooplankton fatty acid content, sample solutions are injected into a gas chromatographer (GC Varian 3800) which transports each sample into a column where the various fatty acids are separated based on their retention time. A detector records the exit flow of the column to determine the time at which each fatty acid exits the column and the amount of that fatty acid. The data are presented on a chromatogram of detector response (y-axis) against retention time (x-axis) from which the fatty acid concentrations in the original sample are determined.

 

Elemental Analyses

Elemental analysis of carbon and nitrogen from solid sample material. (CN)

Carbon and nitrogen content of phytoplankton and zooplankton are obtained with a CHNS-analyzer. Samples go through a high-temperature combustion process producing CO2 and N2. These gases are cleaned in various steps to remove any byproducts and separated from each other by a TPD column (temperature-programmed desorption). The individual gases are then detected and quantified by means of a thermal conductivity detector.

 

Photometric analysis of the phosphorus content in solid sample material. (P)

Quantitative and qualitative determination of plankton phosphorus content is performed by the colorimetric analysis of orthophosphate (PO4). The bound phosphorus is converted into phosphate by oxidation and, after staining with molybdate and ascorbic acid, phosphate can be determined quantitatively by photometry at 880 nm.

Elemental analysis of dissolved organic carbon (DOC) and dissolved organic nitrogen from liquid sample material. (DOC)

Dissolved organic carbon is measured by incinerating at 750 °C pre-filtered liquid samples. In this high-temperature combustion process, with subsequent catalytic oxidation, the bound carbon is entirely converted into CO2.  This CO2 is subsequently subjected to a three-stage intensive drying on a carrier gas (synthetic air) and purified. The difference between synthetic air and CO2 is detected with a wide range NDIR. As a result, the elemental carbon concentration which is present in organically bound form (NPOC) DOC is obtained.

 

Fluorometric analysis

Protein Quantification in solid sample material (FP)

Quantitative determination of the total protein content in a sample is carried out by fluorescence measurement at 355 and 590 nm. For this purpose, the sample material is extracted with ultrasound, stained with Sigma's FluoroProfile Protein Quantification Kit, and measured with a fluorometer.

Quantitative RNA:DNA analysis of solid sample material  (RNA/DNA)

The RNA:DNA ratio is a useful indicator of nutritional condition and growth in marine organisms. Quantitative analysis of RNA and DNA is performed by staining the RNA and DNA with ethidium bromide and measuring the total nucleic fluorescence. The RNA is then eliminated by RNase and the remaining DNA is measured. The difference between the two measurements gives the RNA content of the sample.

Luminometric analysis

Alkaline phosphatase measurement in solid sample material (AP)

Alkaline phosphatase is an enzyme which hydrolyzes phosphoric acid ester into phosphate and alcohol. Alkaline phosphatase activity is higher in P-limited organisms to increase P-acquisition and retention from ingested food items. The alkaline phosphatase activity is therefore used as a proxy for P-stress in aquatic consumers. The alkaline phosphatase is extracted from the sample material with ultrasound, and stained with a dyeing reagent before being measured.

 

High-speed camera

Because of their scale and speed, small zooplankton (<500µm) behavior cannot be studied in detail by naked eye or with traditional video methods. Hence, we record and analyze patterns and mechanisms of feeding and moving with a high-speed high-quality PHANTOM Lab110 camera.

 

Technical Assistants:

Saskia Ohse

Julia Haafke

Ursula Ecker