Report TSA-FISH and cytometry
I.C. Biegala and D. Marie, SBR

Dec 2000

The aim of November and December was to test if in situ hybridisation with TSA amplification technique was possible when hybridisation is performed in liquid and if the fluorescent signal is sufficiently high to be detected with a flow cytometer.

Tests were performed on Chlamydomonas concordia (Chlorophyta) because of its large size 10 µm and the fact that HRP labelled probes can penetrate easily inside the cell without particular perforation treatment. HRP labelled probe specific for the division chlorophyta was used and negative controls were done without probes.

Successful liquid hybridisation was performed on this species, the ratio signal to noise was even higher than hybridisation performed on filter (Fig. 1). In addition, it was possible to detect with the flow cytometer a signal from hybridised cells 20 times higher than negative control (Fig. 2). This later results was obtained on cells in stationary phase and is expected to be higher in exponential phase.

During these experiments several problems were encountered, the most important are mentioned below:

1. rRNA from Chlamydomonas concordia are degraded much more rapidly than other tested cells such as prokaryotes and dinoflagellates. Thus rapid manipulation of fixed cells is important and –80°C sample storage is probably a minimum.
2. Cells are often packed together which is an important problem for enumeration with flow cytometer and signal identification. However the use of seringe has been tested with success as well as a dehydration with graduation steps both with success.
3. During liquid hybridisation it is possible to loose the cells as they are not easily seen. Different colorant (Comassie blue, Toluidine blue and Evans blue) were used without success as they are washed out during dehydration or hybridisation steps. A solution is to increase the amount of cell in order to see the cell pellet, but the probe availability can become a limiting factor, to solve this problem new probe stock must be done with a higher probe concentration. Other solution will be investigated in the future.
4. Cell loss is an important problem even if it has not been completely evaluated. Several areas are under investigations such as the increase of centrifugation speed and time, the decrease of hybridisation steps, the use of triton.

For the next future fluorescence quantification with flow cytometry should be done at several step of Chlamydomonas concordia growth phase as well as on other cell types such as picoeukaryotes and cyanobacteria. Later cell loss will be carefully evaluated.

 Fig. 1. TSA FISH on Chlamydomonas concordia A hybridisation on filter, B hybridisation in liquid apart from the observation step.

 Control / Chlo-HRP

Fig. 2. Comparison of fluorescent intensity with flow cytometry (top graphic) and fluorescent microscopy (bottom images) between the control and TSA-FISH hybridised Chlamydomonas concordia.