STAINING PARTICULATE ORGANIC-MATTER WITH DTAF - A FLUORESCENCE DYE FOR CARBOHYDRATES AND PROTEIN - A NEW APPROACH AND APPLICATION OF A 2D IMAGE-ANALYSIS SYSTEM

Citation
R. Schumann et D. Rentsch, STAINING PARTICULATE ORGANIC-MATTER WITH DTAF - A FLUORESCENCE DYE FOR CARBOHYDRATES AND PROTEIN - A NEW APPROACH AND APPLICATION OF A 2D IMAGE-ANALYSIS SYSTEM, Marine ecology. Progress series, 163, 1998, pp. 77-88
Citations number
64
Categorie Soggetti
Marine & Freshwater Biology",Ecology
ISSN journal
01718630
Volume
163
Year of publication
1998
Pages
77 - 88
Database
ISI
SICI code
0171-8630(1998)163:<77:SPOWD->2.0.ZU;2-G
Abstract
Aggregates consist in many cases of microorganisms attached to an orga nic matrix, Several attempts have been made to visualise the mucoid co mponents of the matrix by staining (e.g. alcian blue for carbohydrates and coomassie brilliant blue for proteins). Modern equipment, such as epifluorescence microscopes, flow cytometers and confocal laser scann ing microscopes, requires or works best with fluorescently marked obje cts. Therefore, there is a great need for a fluorescent dye to visuali se poly saccharides associated with aggregates. Like proteins, polysac charides are very heterogeneous in their 3-dimensional structure. Depe nding on the types of interglycosidic bonds and the orders of monosacc haride building blocks, various conformations are possible (i.e. helic es 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 (DTA F), which binds covalently to polysaccharides at room temperature when the pH is above 9. This paper compares the results of staining partic ulate organic matter (POM) with DTAF with the results of staining with the DNA-specific stain propidium iodide, which has been recently intr oduced to visualise detritus. Results are related to other POM propert ies, e.g. particle volume measured by Coulter(R) Counter and particula te organic carbon (POC). When plankton and floccular sediment layer sa mples were stained with DTAF, the total particle volume was, in 13 out of 17 samples, 11 to 393 % greater than that revealed by propidium io dide. The mucoid matrix of large particles (>50 mu m diameter) stained well with DTAF, whereas this material was only weakly detected with p ropidium iodide. In contrast, very small particles such as bacteria we re seen better with propidium iodide. The total particle volume of a D TAF-stained sample calculated from 2-dimensional image analysis data c orrelated significantly with POC, total particulate polysaccharides, C oulter(R)-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 Coulter(R) Co unter volume and 45 % of propidium-iodide-stainable and 50 % of DTAF-s tainable 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 i n the case of the fluorescently stained particles, also as amorphous o rganic material.