Camila

the cells in fixative solution (methanol-acetil acid 3:1) can be conserved during 2 months alt less

luis

Well, if I were you I would opt for performing FISH on fresh cells or i.e. cells not left too long on ethanol or so. This is because it  could affect the cells morphology as permeablization and preserving steps in way deteriorate the cells.

Try getting ready other things required for the FISH and then maybe you can  worry about cells as Probe is needed!

Ratish

I have do FISH in lymphocytes stored at -20 in fixing solution for a long time without problems

luis

Hi Camila

You did not specify, which cells you want to proceed.

In the case of plankton bacteria, we are fixing in 2% formaldehyde (for G+ 20% ethanol) and within 24h the sample must be harvested onto a polycarbonate membrane (0.2 /um), air-dried and deep-frozen (but even in -18°C it lasts for more than 2 months).

For ciliates/flagellates and their ingested bacteria, Lugol-fixing was used, the sample was filtered through polycarbonate 2/um membrane but before finishing, in small amount of liquid, Lugol was titred with thiosulphate, postfixed with formalin (to 2%) and the harvesting was finished. The membranes were air dried and frozen.

Jezbera J, Horňák K & Šimek K (2005) Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization. FEMS Microbiol Ecol 52: 351–363.

Bautista–Reyes F, Macek M (2012) Ciliate food vacuole content and bacterial community composition in the warm-monomictic crater lake Alchichica (México). FEMS Microbiol. Ecol. 79: 85‑97

Bouin's liquid (to be added in the solution composed to reach [in the sample] minimum 5% of picric acid saturated in concentrated formalin and [without any change] 2% of acetic acid) is being used, too. However, you must be aware to avoid the rest of the fixative: The membrane must be washed (before drying and freezing) to loose yellow colour (it would fade the fluorescence).

Fried J, Ludwig W, Psenner R, Schleifer KH (2002) Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques. Syst. Appl. Microbiol. 25: 555‑571

Generally, agarose-mounting as described in CARD FISH (TSA) method is highly recommended.

Pernthaler J, Glöckner FO, Schönhuber W & Amann R (2001) Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. Methods Microbiol 30: 207–226.

Any direct comment to [email protected]

Cheers

Mirek Macek