I am using ethidium bromide (EtBr) to stain dead E. coli cells in a broth culture. When visualizing under Olympus IX73 inverted fluorescence microscope, the EtBr-stained cells fluoresced under green excitation light but did not show fluorescence under UV light, which is unusual since EtBr typically fluoresces under UV.
Additionally, I understand that EtBr primarily stains dead cells by intercalating with DNA in cells with compromised membranes. However, I want to confirm:
Thank you
EtBr can enter live cells and is used to reduce mtDNA and mitochondria in live cells. It is also used in veterinary practice to treat trypanasome infectons so is probably not very carcinogenic since that meat enters the food chain
Hello Ayshwarya Ravikumar
When visualizing under Olympus IX73 inverted fluorescence microscope, the EtBr-stained cells fluoresced under green excitation light but did not show fluorescence under UV light, which is unusual since EtBr typically fluoresces under UV.
There is a possibility that the presence of blue green autofluorescence of bacteria may eclipse the red fluorescence light of the intercalated ethidium bromide.
Q1. Does EtBr exclusively stain dead cells, or can it also enter live cells and potentially cause mutations or lead to cell death?
EtBr exclusively stains dead cells. The envelope of intact cells is impermeable to ethidium bromide molecules. EtBr, a DNA-intercalating fluorescent dye, cannot effectively enter bacterial cells as long as the outer membrane barrier is intact. However, upon passing the outer membrane, EtBr rapidly traverses the cytoplasmic membrane, enters the bacterial cytosol and binds to intracellular nucleic acids. The latter can be detected by fluorescence spectrometry, as EtBr displays strongly enhanced fluorescence when intercalating with nucleic acid.
The below attached papers will help!
https://pubmed.ncbi.nlm.nih.gov/6817038/#:~:text=The%20interaction%20of%20ethidium%20bromide,impermeable%20for%20ethidium%20bromide%20molecules.
Article Outer Membrane Permeabilization Is an Essential Step in the ...
LPS molecules on the outer face of the outer membrane are ionically cross-linked to each other by divalent cations (Mg2+or Ca2+) binding to phosphate groups in lipid A, generating a permeability barrier. EDTA is considered a permeabilizer which can chelate and thus displace divalent cations, destabilizing and releasing LPS, thereby increasing the permeability of cell to itself and other compounds.
Following 1 or 3h of growth, if you use 200μM to 500μM EDTA, EtBr accumulates suggesting that EDTA is able to make the outer membrane more permeable to EtBr at these concentrations. But at higher time points, you might not observe the same effect suggesting that the outer membrane may remodel itself during entry into stationary phase and becomes less reliant on cation-mediated cross-linking to maintain its permeability barrier to EtBr.
Q2. If EtBr can mutate or kill live cells, how long does this process typically take, and at what concentrations is it likely to occur?
In the paper attached below, the investigators analyzed the toxic impact of EtBr on pure culture of E.coli KL226 by exposing the culture to a few concentrations of EtBr between 0.2 and 0.8 µM. Secondly, the mutagenic impact of EtBr at 10^-13 M concentration was studied by applying it on the Colony Forming Units (CFUs) of E.coli KL226 using L-rod spread plate method.
They found that EtBr exposure at concentrations 0.2µM, 0.4µM, 0.6µM and 0.8µM induced growth curve pattern of E. coli KL226 by the entry and accumulation of its molecules within it. Not alone the accumulation of EtBr could cause the induction in growth pattern, but necessarily the accumulated EtBr must be in an effective concentration to induce the cellular activity, to consequentially induce overall growth curve pattern.
Secondly, sub picomolar (10^-13 M) concentration of EtBr is known to have the ability to cause mutation in E.coli KL226.
Please refer to the article for more information.
https://rjpn.org/ijcspub/papers/IJCSP10A1249.pdf
Best.
Malcolm Nobre Thank you for your response and for sharing the paper. I will definitely look into it
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