I used 16S rRNA probe labelled with Cy3 and flurescence in situ hybridisation for fecal batch culture fermentation.
When I am counting the bacteria from this experiment, using a fluorescence microscope, I find that they have different levels of florescence. Do I count every visible bacteria or just the brightest bacteria? If you have a source of information on this please let me know.
Many thanks.
Hi Florencia,
It is difficult to give you any suggestions without having seen one of your micrographs. However, assuming that (a) you are applying traditional FISH with Cy3-(mono)labeled oligonucleotide probes and (b) you can exclude any form of background fluorescence, I would expect different levels of fluorescent intensity as the number of rRNA molecules will most likely vary in your target cells. So even weak signals should be counted if you are interested in obtaining cell numbers.
Maybe this was helpful & good luck with your FISHing,
Hannes
I agree with Hannes. Additionally, you can search information about fluorescence emission for aspecific binding to organic or inorganic components in your sample, as seen for complex samples which can contain several components besides microbial communities.
- Badges
- Science topic