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