One of Mycobacteria smegmatis mutant strain I am studying is showing growth defect and elongated cells. We are expecting these elongated cells might be due to failure of cell division. we are curious to know the replication status of chromosome in these cell lines. Is it possible to identify its ploidy status of mycobacteria using flow cytometry ?
I tried doing the same by staining DNA with Hoechst but I got a single broad peak in wild type cell and a much broader peak with extension towards right side in mutant strain. I could not interpret anything.
Like in mammalian cells where different ploidies gives different distinct peaks, Is it possible to get the same in bacteria?
Characterization of DNA-content by FACS has been shown to be possible with bacteria by quite a number of publications (e.g. http://digital.csic.es/bitstream/10261/5293/1/planktonic.pdf).
You could also try fluorescence microscopy to quantify the DNA content of single cells and correlate that with their morphology.
Good luck with your experiments,
Christian
Hello
Yes you can use FACS to describe DNA-content
find attachment here , Good Luck
http://onlinelibrary.wiley.com/book/10.1002/0471142956/homepage/Archive.html
https://archive.org/stream/Protocols_molecular_biology/Vol.263-FlowCytometryProtocols_djvu.txt
https://www.qiagen.com/resources/molecular-biology-methods/dna/
Thank you Dr.Christian Wechselberger for your suggestion.
I will try fluorescence microscopy to quantify the DNA content.
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