Warning! Toxic dinoflagellate.

[Rene]

Hi guys, here is another example of a dividing dinoflagellate. In a controlled way each of the two daughter cells gets part of the theca. The process is called desmoschisis, and division always follows these suture lines.

Stained with Calcofluor White, an optical brightener which stains the cellulose plates of armoured dinoflagellates:


Here's the corresponding brightfield image:


Admittedliy, it doesn't look very nice, apologies for that. Lugol preservation generally does not give great views, and this is heavily bleached with thiosulphate before staining.

The dinoflagellate comes from a brackish creek in the south of the Netherlands. It is called Alexandrium ostenfeldii, it is one of the toxic species involved in shellfish intoxication. Cells are about 35 um across.

Images: 20x/0.7 Olympus splanapo, cmos cam TIS 5Mp. Stacks of around 15 images.

Best wishes, René

(images posted in http://www.photomacrography.net  Replies in German appreciated)

[TPL]

Hoi René,

danke für die interessanten Bilder von der Teilung.
Wurde dieser Prozess schon eimal so schön mit Bildern belegt? OK, das Hellfeld-Bild ist eher... ein Belegbild, aber ich finde Deine Fluoreszenz-Aufnahmen spektakulär!

Nur schade, dass diese faszinierenden Organismen so hässliche Wirkungen haben. Ich hoffe, die breiten sich nicht aus, denn ansonsten haben wir bald – trotz aller Initiativen zur Gesundung mariner Ökosysteme – ein "hausgemachtes" Problem...

m.v.g. Thomas

[Rene]

Hi Thomas, thanks for the kind words. No, stacking is not much used (yet) in the scientific literature, and that's a shame. Especially for this kind of fluorescence  work, stacking works without hiccups.

Alexandrium is a natural component of the phytoplankton, in brackish creeks it can bloom. There is little toxin, but it accumulates in shellfish. I don't mind swimming in such waters: you would need to drink a considerable amount of water for direct intoxication. Liters of brackish water, that's enough to get diarrhea, even without harmful species ;-)

Best wishes, René

[Bernd]

Hallo Rene,

durch die Calcofluor White-Floureszenz werden die Details des Dinoflagellaten-Panzers hervorragend sichtbar! Im amrikanischen Forum hattest du mal geschrieben, daß du die Dinoflagellaten mit Lugolscher Lösung fixiert hast. Das Hellfeld-Bild sieht so aus, als ob du das hier auch gemacht hast. Wäscht du das Fixiermittel anschließend aus? Welche Konzentration von Calcofluor White gibst du zu? Es wäre schön, wenn du deine Methode ausführlich beschreiben könntest.

Viele Grüße
Bernd

[Rene]

Hi Bernd,

Yes, you guessed right, this is lugol-preserved material. Staining takes place within the sedimentation chamber, simply by replacing half of the supernatant by the stain solution. As there is such a large volume within the chamber, the concentration of Calcofluor is very low (1mg/l). Basically, I've diluted the stain until I couldn't detect a background anymore: within a sedimentation chamber this is at a way lower conc. then I would use for the usual slide+coverslip preparation. I've optimized the mixture for our samples, it contains some thiosulphate (neutralizing free iodine), buffer (pH needs to be >6), formaline (stabilizing the sample) and NaCl (only for marine samples). But it's not very critical.

For imaging I leave it staining for a couple of hours or overnight. Some specimen, or sometimes whole populations don't stain very well, this is to do with maturation of the theca, like the Alexandrium cells above.

Fading is a problem, not more so than with other UV stains. I use a dimmable UV led, and switch over to camera immediately after spotting the cell I want to check. General exposure time is around 1/10 sec. (TIS 5Mp cmos), the UV led at about 1/4th of maximum power suffices. Some (tiny) dino's need be imaged within 30 sec for best results, but if left incubating again, they regain their original quality.

Good luck, René