I want to compare the inhibition of bacterial cell death in presence of greenly synthesized nanoparticles and plant extracts. Both nanoparticles and plant extracts are showing the same ROS activity eperimentally but nanoparticles are actually showing inhibition in during growth kinetics experiments. So I am confused and want to prove that it is not only ROS which favor the death but also rupture of membrane.
Reactive oxygen species (ROS) is also responsible for inhibitory activities and bacterial cell killing.Strong oxidants like the various ROS can damage other molecules and the cell structures of which they are a part. Among most important of these are the actions of free radicals on the fatty acid side chains of lipids in the various membranes of the cell, especially mitochondrial membranes (which are directly exposed to the superoxide anions produced during cellular respiration). A hydroxyl radical removes a hydrogen atom from one of the carbon atoms in the fatty acid chain (only a portion of which is shown) forming a molecule of water and leaving the carbon atom with an unpaired electron (in red); thus now a radical. Several possible fates await it. One of the most likely (and shown here) is to react with a molecule of oxygen (O2) forming a peroxyl radical.One of the insidious things about free radicals is that in interacting with other molecules to gain a stable configuration of electrons, they convert that target molecule into a radical. This might then steal a hydrogen atom from a nearby side chain making it now a radical. So a chain reaction begins that will propagate until two radicals meet each other and each contributes its unpaired electron to form a covalent bond linking the two. another peroxyl radical on a nearby side chain crosslinking them with a covalent bond. another nearby carbon-centered radical crosslinking them covalently. In both these latter cases, radical formation comes to an end but with the result that the fatty acid side chains of membrane lipids may have become so deformed as to damage the membrane. The lipofuscin so characteristic of aging cells may be formed by these mechanisms.
Hi Parth,
I don't have a lot of experience with bacterial cell death, but I've worked with mammalian cells and apoptosis assays quite extensively. I think that either propidium iodide (PI) or trypan blue staining may be able to address the experiment you've proposed. Both dyes are excluded from cells with intact membranes, whereas dead cells (with compromised membranes) are permeable to the dyes. Thus, increased staining correlates with membrane damage and cell death. I've always done PI experiments on a flow cytometer, which gives great statistics. For mammalian cells, we always count trypan blue positive cells on a hemacytometer; I'm not sure how that would translate in a bacterial system. Here is some literature that I found regarding PI in bacteria and general trypan blue information that you may find helpful:
http://www.bdbiosciences.com/documents/Bacterial_Detection_Live_Dead.pdf
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Usage/t8154use.pdf
Good luck!
Natalie
Morning, for bacteria PI permeability doesn´t mean cell death. Bacteria can recovery from transient plasma membrane permeability, therefore a good way to detect bacteria death by flow cytometry is double staining with PI and some functional probe as FDA. ROS can induce slow growing speed, without cell death. My best regards, Alberto
I suggest you a double staining with the Sytox green which enter in the bacteria with damage membrane only and bind to the DNA and a staining with the Alexa fluor 633 hydrazide which also enter in the bacteria when the membrane is damage but bind to the carbonylated proteins. (see Saint-Ruf et al, 2010, 2014)
Hi all
Is there any method based on agar incorporated ethidium bromide in a plate for the detection of membrane permeability and damage ?
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