Hey Takeshwar, the method provided by Stefan is great for enumeration of total bacterial counts. However, if you want to gather more information about your sample (live/dead bacteria), you can use a combination of DAPI and acridine orange (AO) to differentiate. Since in a native, non-fixed sample, DAPI cannot penetrate the cells it will only stain dead cells. On the other hand, acridine orange can penetrate the cells and will only stain live cells. There is already another discussion here on ResearchGate (https://www.researchgate.net/post/Can_anyone_recommend_a_good_fluorescent_stain_with_which_one_could_stain_bacterial_DNA) and a good article in the Journal of Applied and Environmental Microbiology (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837990/). I hope that helps. Best, Markus

Hi Tarkeshwar,

I found the following protocol (attached) that contains a detailed instruction for staining aquatic bacteria.

Hey Takeshwar, the method provided by Stefan is great for enumeration of total bacterial counts. However, if you want to gather more information about your sample (live/dead bacteria), you can use a combination of DAPI and acridine orange (AO) to differentiate. Since in a native, non-fixed sample, DAPI cannot penetrate the cells it will only stain dead cells. On the other hand, acridine orange can penetrate the cells and will only stain live cells. There is already another discussion here on ResearchGate (https://www.researchgate.net/post/Can_anyone_recommend_a_good_fluorescent_stain_with_which_one_could_stain_bacterial_DNA) and a good article in the Journal of Applied and Environmental Microbiology (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837990/). I hope that helps. Best, Markus

Thank you very much Dr.Stefan and Dr. Markus..

Hello Tarkeshwar

I am attaching the following protocol, this is a protocol described for Philip Kerrison and Michael Steinkei at 2010. This guide refers specifically to the qualitative or quantitative determination of bacterial cells. I hope you serve.

Regards,

Yaima

Thank you very much Yaima

Hello Tarkeshwar

I would recommend the following protocol for specific DNA staining of bacteria using DAPI: http://www.ncbi.nlm.nih.gov/pubmed/23288319. The procedure is described for a flow cytometric approach and includes quantitative staining according to DNA content. But the cells can also be analyzed microscopically.

Regards, Christin

Thanks to all for providing me valuable answer.