For fluorescent in situ hybridisation (FISH), how can we fix bacterial samples (both gram +ve and -ve) using ethanol? As I can't use paraformaldehyde (PFA) fixed samples for my experiment.
Sandeep,
would you mind just to clarify why PFA cannot be your fixative? This would perhaps induce a bit more chance of getting a serious reply to your request.
Thanks, Wolfgang
Thanks Wolfgang,
Actually I will be doing liquid-based FISH on those fixed samples. Then bacterial cells will be sorted by using fluorescent signals. Sorted cells will then be used for single cell genomics. In sorted bacterial cells, PFA is likely to interfere with their DNA, that's why I am looking for some other alternative.
Thanks, Sandeep
I have no clear idea about bacterial cell as i have worked with fibroblasts and stem cells in attached cell culture and did it for lymphocytes in suspended culture. if you feel can try the same method if it is for sorting
Dear Sandeep,
don't know whether this would/could be an (real) alternative for your problem, but as NON-FA-containing fixatives there come to my mind the CARNOY's (like) fixatives...
CARNOY ( ) (and therefore perhaps also) METHACARN (where methanol of CARNOY's original is substituted for ethanol) at least in
"Intragastric distribution of Helicobacter pylori during short-term omeprazole therapy: study using Carnoy’s fixation and immunohistochemistry for detection of bacteria" by
Ishihara, S. et al, DOI: 10.1046/j.1365-2036.2001.01067.x in: Alimentary Pharmacol.& Therapeutics 15, Issue 9, pp 1485–1491, Sep 2001
(cf. http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2036.2001.01067.x/full) has been reported to fix H. pylori successfully also for IHC.......Perhaps there can be found other recipes too...(naturally one could try always first ONLY fixation with a solution of 96% EtOH....) best wishes and regards, Wolfgang
Sandeep, that I might be on the right way perhaps can be seen within:
Swidsinski A, Verstraelen H, Loening-Baucke V, Swidsinski S, Mendling W, Halwani Z (2013):
Presence of a Polymicrobial Endometrial Biofilm in Patients with Bacterial Vaginosis. PLoS ONE 8(1): e53997. doi:10.1371/journal.pone.0053997,
(.....@ http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053997)
BR, Wolfgang
Dear Wolfgang,
Thanks you very much for your suggestions. I tried the CARNOY ( 6:3:1 mixture of ethanol, chloroform, and acetic acid...> ) solution fixation method and it gives promising results. Although, I have to play around with the protocol to get the convincing result.
Thanks, Sandeep
Dear Sandeep, you're welcome!
And if my hint to Carnoy's fix yields positive results I can be happy with you
(naturally such recipes always have to be optimized for your own task...) Would be great to read once a time that you were able to get fascinating results...
Best wishes and regards, Wolfgang
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