I was using epifluorencense and i distinguish, like you, heterotrophs from autotrophs and  also cyanobacteria from the rest of autotrophs, but not the others groups.

Maria

Hello,

I did my PhD looking at the different phytoplanktonic group and differentiate autotrophs to heterotrophs. For each samples, I was doing two observations: 1 with the traditional light to identify the species and then switching on the epifluorescence to observe if the organism is auto- or heterotrophic.   I have checked the article you mentioned, and they don't provide a lot information "Each cell was manually identified ".  I am almost sure that they should have a picture  of the organisms without fluoresecence for identification.

Today I work in a company where we do viability count for Ballast Water using epifluorescence, and identification is done by switching off the fluorescence

Bye

Aurore

Thank Maria and Aurore for your replies. 

Yes, that's true, there are a lot of information about how they identified the organisms, thanks again for your suggestion.

Luca

Hi Luca, here's a good starting point on what's possible with epifluorescence on different phytoplankton groups: 

Schubert H, Schumann R (2002) Epifluorescence microscopy: Identification of higher taxa of phytoplankton. In: Pelagic Ecology Methodology. Rao S (ed.) Tokyo, Balkema Publ, 95-108

HTH, René

Thank Rene, this is helpfull

p.s.

Are you able to send the article, please?

Hi Luca,

I can send you a pdf by email, if you write me at [email protected]. I will not post fulltexts, because the copyright issue is not solved for web platforms (in my opinion).

What the cited other authors did, was to check simply for chlorophyll a (chla, all autotrophs), protein (by FITC staining) giving the shape of the whole cell and DAPI for nucleus / DNA. The photographs given are very good, but needed much and skilled image processing. This is an effort in itself.

The differentiation between the flagellate groups is not (much) helped by this procedure, however. You need almost as much taxonomical experience as with traditional (non-fluorescence) microscopy. I give you only some examples:

Cyanobacteria do not have a nucleus. Should help to sort them out. However, larger cells concentrate DNA in the centre, what looks like a nucleus. Moreover, the blue excitation of cyanobacterial chlorophyll a gives such a low signal that it is most likely only in the scattering light range. The photobiology of this so-called blue excitation gap is explained in the dissertation thesis of my co-author.

As far as I can see in the manuscipt of Taylor et al., they do not use special filters for fluorescing pigments (fucoxanthin - diatoms, chl b for green algae, chlc again diatoms, phycocyanin for cyanobacteria and phycoerythrin for cyanobacteria and cryptophytes. That is possible in theory, but only partly in praxi. The content of our book chapter gives some information and some examples. However, they were obtained from cultured species. There is another paper by Geiß et al. 2003 applying that filter combination to Prochlorothrix (chlb containing cyanobacteria). The effort was huge, since the chlb signal is very week and very near to chla. It worked better in nutrient enriched samples (raw cultures).

There is a microscopy PAM on the market adressing several algal group also by fluorescense. I did not check if that works well. However, I have great doubts since the fluorescing pigments are not much in variety. The non fluorescing pigments (carotenoids) are useless here.

I follow the issue not very closely (anymore), because I do not see an advantage in the use of fluorescence except for differentiation of auto- and heterotrophic flagellates. For this I do not stain the samples, just check any flagellate for chla. And with another excitation (UV or green), I check for auto- and heterotrophic procaryotes (I still use DAPI as counterstain, if I want to quantify also bacteria).

Good luck with your projects, Rhena