Kopp at MPIL-PLOEN.MPG.DE
Fri Sep 24 11:38:39 EST 1999
Donald O'Malley wrote:
> Does anyone know of a vital stain for paramecium?
> We're trying to image the movements of paramecium and
> want a stain that will provide either intense color or
> high contrast, so as to be easily visible in video frames.
> Longer term (several hours or longer) toxicity to the paramecium
> is not of any concern, but we would like them to be able to
> continue swimming for at least an hour or two,
> before going flagella-up.
IF have the facilities, you can use several fluorescent dyes. The
advantage of fluorescent dyes is that you can work with very low
concentrations. I haven't tried staining Paramecium, but some other
ciliates (Euplotes, Colpidium). I would first try DAPI (will mainly
stain the nucleus, but you can use a combination of epifluorescence and
normal light (from below) to make visible the whole cells) or Acridin
Orange. Molecular Probes Inc. (www.probes.com) sells all kinds of
specialized fluorescent dyes (although rather expensive). I've tried
some of the "Cell Tracker" series (CMFDA, CMTMR), but not all of them
work with all species.
For DAPI, you can try to stain with 1 to 5 µg / ml for 1 to 4 hours. For
Acridin Orange, I think the concentration was 10 to the -7 µMol. Cells
will stay alive and mobile, but I can't guarantee that e.g. swimming
speed remains unchanged.
Alternatively, instead of labeling Paramecium directly, you can also
feed them stained food. This is probably less harmful for them. In the
microbial ecology literature, there's quite a lot of papers on how to
prepare fluorescently labeled bacteria (FLB's), using stains such as
FITC or DTAF. Instead of feeding them bacteria, you could also try to
use of fluorescent microspheres (= little latex balls of the size of a
few µm or so). A classical textbook experiment is feeding them yeast
stained with congo red or neutral red. (Since these are pH-indicators,
they change color as the pH changes in the food vacuoles, but I don't
know whether you want to see that). Maybe some other non-fluorescent
stain can be used as well.
Some literature (neither complete, nor probably the most important):
Verni, F. and P. Gualtieri (1997). "Digestive process of the raptorial
feeder ciliate Litonotus lamella (Radophora, Litostomatea) visualized by
fluorescence microscopy." Micron 28(6): 447-451. - This used Acridin
Lessard, E. J., M. P. Martin, et al. (1996). "A new method for
live-staining protists with DAPI and its application as a tracer of
ingestion by walleye pollock (Theragra chalcogramma (Pallas)) larvae."
Journal of Experimental Marine Biology and Ecology 204: 43-57.
Pfister, G. and H. Arndt (1998). "Food selectivity and feeding behaviour
in omnivorous filter-feeding ciliates: A case study for Stylonychia."
European Journal of Protistology 34(4): 446-457. - Also uses DAPI
Epstein, S. S. and J. Rossel (1995). "Methodology of in situ grazing
experiments: evaluation of a new vital dye for preparation of
fluorescently labeled bacteria." Marine Ecology Progress Series 128:
Dolan, J. R. and D. W. Coats (1991). "A study of feeding in predacious
ciliates using prey ciliates laeled with fluorescent micropheres."
Journal of Plankton Research 13(3): 609-628.
Balczon, J. M. and J. R. Pratt (1995). "A comparison of methods for
estimating short-term feeding rates of algivorous ciliated protozoa."
Archiv für Protistenkunde 146: 49-58.
Li, A., D. K. Stoecker, et al. (1996). "Ingestion of fluorescently
labeled and phycoerythrin-containing prey by mixotrophic
dinoflagellates." Aquatic Microbial Ecology 10: 139-147. - This uses
CMFDA (Cell Tracker Green)
Mischke, U. (1994). "Influence of food quality and quantity on ingestion
and growth rates of three omnivorous heterotrophic flagellates." Marine
Microbial Food Webs 8(1-2): 125-143. - Different methods used
I hope I could help you a bit.
Max-Planck-Institut fuer Limnologie
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