Automated Identification of Microbial Populations (EU Project AIMS)

Pico- and nanoplankton (0.2 to 20 µm in diameter) often lack morphological features that are taxonomically useful for identification at the light microscopic level. These restraints usually require the use of electron microscopy, pigment analysis with high-performance liquid chromatography (HPLC) or sequencing of conserved genes (e.g., rRNA genes) before a definitive identification can be made of particularly difficult taxa. The identification of phytoplankton using flow cytometry offers a faster alternative for identification of problem taxa than these conventional but often time consuming and laborious methods. However, in some cases, parameters measured by the flow cytometer (i.e., light scatter, absorption) are insufficient for a clear identification. Such problems can be overcome if fluorescently-labelled rRNA probes, specific for different algal groups, are used to confirm the identification provided by the flow cytometer.
 

For probe development, a database of algal 18S rRNA sequences was analysed using the ARB computer program (Ludwig, TU München) to find unique regions that match perfect only to the target species genes and have at least one to two mismatches with those from all other organisms. Oligonucleotides complementary to these sites were labelled with fluorochromes or with enzymes and hybridised to different algae or the PCR products of their 18S or 28S rRNA genes for subsequent analysis using chemiluminescent or fluorescence detection systems (microscopy or flow cytometry).
 

For our investigations, algae were selected for probe development if they met the following criteria:

• relevance for European waters
• availability in culture collections
• distribution of species over taxonomic groups
• different sizes and shapes of cells

Using these criteria as a baseline, we developed a broad range of probes representing different groups, classes, orders, genera and species of algae, i.e., for chlorophytes in general or for Alexandrium tamarense, a toxic dinoflagellate.

 
The development of 18S rRNA probes in our institute is part of an EU project (MAS3-CT97-0080) called AIMS (Automated Identification of Microbial Populations), which has the goal to provide a fast and reliable system for the identification of phytoplankton species using flow cytometry and artificial neural networks (ANNs). The probes developed in our part of the project could be used in two ways:

• Field samples could be hybridised with the probes prior to flow cytometry and identification is made by comparing the fluorescence of labelled target and unlabelled non-target cells.

• Samples were sorted after "normal" flow cytometry, hybridised afterwards and viewed under an epifluorescence microscope to confirm the identification made by the ANN.