Staining particulate organic matter with DTAF--a fluorescence dye for carbohydrates and protein:a new approach and application of a 2D image analysis system

Abstract

Aggregates consist in many cases of microorganisms attached to an organic matrix. Several attempts have been made to visualise the mucoid components of the matrix by staining (e.g. alcian blue for carbohydrates and coomassie brilliant blue for proteins). Modern equipment, such as epfluorescence microscopes, flow cytometers and confocal laser scanning n?icroscopes, requires or works best with fluorescently marked objects. Therefore, there is a great need for a fluorescent dye to visualise polysaccharides associated with aggregates. Like proteins, polysaccharides are very heterogeneous in their 3-dunensional structure. Depending on the types of interglycosidic bonds and the orders of monosaccharide building blocks, various conformations are possible (i.e. helices or molecule layers). Moreover, saccharides are very hard to modify covalently in water at natural pH levels. To date, the only available fluorescence marker is 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF), which binds covalently to polysaccharides at room temperature when the pH is above 9 This paper compares the results of staining particulate organic matter (POM) with DTAF with the results of staining with the DNA-specific stain propidium iodide, which has been recently introduced to visualise detritus. Results are related to other POM properties, e.g. particle volume measured by coultere Counter and particulate organic carbon (POC). When plankton and floccular sediment layer samples were stained with DTAF, the total particle volume was, in 13 out of 17 samples, 11 to 393% greater than that revealed by propidium iodide. The mucoid matrix of large particles (>50 pm charneter) stained well with DTAF, whereas this material was only weakly detected w t h propidium iodide. In contrast, very small particles such as bacteria were seen better with propidium iohde. The total parbcle volume of a DTAF-stained sample calculated from 2-dimensional image analysis data correlated significantly with POC, total particulate polysaccharides, Coultera-counter-derived total particle volume, and light-microscope-derived summed volumes of bacteria, phytoplankton and protozooplankton. The total biovolumes contributed on average 62% of the Coultera Counter volume and 45% of propidium-iodide-stainable and 50% of DTAF-stainable volume in plankton samples from the Kirr Bucht and 58,59 and 61 % from the Rassower Strom, respectively. The remaining volume must be interpreted as the volume of amorphous detritus and, particularly in the case of the fluorescently stained particles, also as amorphous organic material.