Abstract
DiOC2 (Novo et al., 2000) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts towards red emission as the dye molecules self associate at the higher cytosolic concentrations caused by larger membrane potentials. Proton ionophores such as CCCP destroy membrane potential by eliminating the proton gradient. The magnitude of membrane potentials varies with different bacterial species. For many gram-positive species, including Staphylococcus aureus and Micrococcus luteus, the red:green ratio tends to vary with the intensity of the proton gradient while in many gram-negative bacteria such as Escherichia coli and Salmonella choleraesuis, the response of the dye does not appear to be proportional to proton gradient intensity. Mycobacterium tuberculosis itself is a difficult organism to work with because of its rigid cell wall.
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
Dr. Amit Singh is a Wellcome- DBT India Alliance Intermediate Fellow. The work was supported by the Wellcome- DBT India Alliance grant, WTA01/10/355.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Since this kit is specific for bacterial cells, the host cells will not get stained.
Yes this experiment can be carried out with fluorescent microscopy. The green fluorescence of DIOC2(3) requires 488 nm excitation and 530 nm emission while the red fluorescence requires 488 nm excitation and 610 nm emission. Hope that will help!
Many Thanks Manbeena!!