Hello Rasoul,

Are you trying to assess "oxidative" stress or "redox" stress? there is a big difference between the two: oxidative stress, as pointed out by several colleagues involves an unbalance between oxidant production and antioxidant defenses in a biological systems. Redox stress, on the other hand, refers to a disruption of the redox potential of the cell that prevents the physiological function of ROS and RNS as second messengers and the ability of redox sensors to detect and/or react with ROS and RNS (for clearing them out or as redox switches).

Why do I think this is important? because when focusing just on oxidative stress, you only measure oxidative outcomes (i.e. oxidized biomolecules), which have been very well described by several colleagues here. However, it misses all the additional features of redox stress, e.g. reductive stress (when there is such a high redox potential that ROS and RNS cannot act as second messengers), electrophilic stress (when electrophiles consume antioxidants such as glutathione, irreversibly forming glutathione adducts), thiol stress (when critical cysteine residues in proteins are modified and thus, cannot act as redox switches), and more.

Therefore, you may want to look for markers of redox stress rather than just oxidative stress :)

Just my 2 cents

Good luck with your research!

Dear Sir, we can identify the oxidative stress of a bacteria through growth and morphology.

How? could you please explain?

measurement of ROS

Yes it can be said that the cells are in oxidative stress but for that confirmatory test should be done by measuring the ROS produced at the stationary phase

There are many assays for ROS production including scopoletin (Ae Ra Kim et. al

Phytotherapy research : PTR, 18(1), 1-7 (2004)), 2,7-dichlorofluorescein diacetate (Lílian Lund Amado et. al, Science of The Total Environment, 407(6), 9 (2009)), etc. These reagents are pretty easy to find and the assays easy to perform.

Hello

@ Matthieu Depuydt: I have determined Carbonylated proteins by ELISA; but despite the differences in catalase and SOD activities, there is no difference in the content of carbonylated protein. Actually, I think that carbonylation needs strong oxidative conditions. Maybe before carbonylation, some other oxidative reactions (such as s-s formation) occurs for biomolecules,

@ Nicolle Breusing

Suppose that :

in one hand, we have determined ROS

and on the other hand, we have determined catalase, and SOD activities

then the results showed that both ROS and the enzyme activity increased dramatically.

Now, according to the definition of oxidative stress (imbalance between ROS and scavenger enzymes), how can we conclude that this amount of ROS is not in balance with the activity of catalase and SOD?

Can I confirm oxidative stress just by an increase in ROS ?

Dear Rasoul,

there is a new tendency established by Jones that oxidative stress is defined by the thiol redox status (like as GSH/GSSG). You can read "Redefining Oxidative Stress. ANTIOXIDANTS & REDOX SIGNALING. Volume 8, Numbers 9 & 10, 2006." for more information. There are no reference values to the most biomarkers (MDA-TBARS, carbonyl content, nitric oxide, ...) then you must to establish comparisons (before/after or groups).

Hope it's help you.

Guilherme

Sure, ROS are not, in strict terms, the OS marker. You can measure also the reduced glutathione that is exhausted under the OS, but again it is just an indicator of increased ROS levels that can (but not must) result in the OS.

To prove the OS, you have to measure more or less "conventional" OS markers such as products of oxidative damage of cellular constituents, e.g., DNA (like 8-oxoguanine), proteins (like carbonyl groups), or lipids (like malondialdehyde). Only in case you can see these markers you can state that ROS caused the OS.

Hello,

@ David Reyk

About the word "imbalance": How do you judge that the amount of anti-oxidative enzymes is not in balance with the amount of ROS?

Just by determination of modified proteins, or some other biomarker?

Hello Rasoul,

Are you trying to assess "oxidative" stress or "redox" stress? there is a big difference between the two: oxidative stress, as pointed out by several colleagues involves an unbalance between oxidant production and antioxidant defenses in a biological systems. Redox stress, on the other hand, refers to a disruption of the redox potential of the cell that prevents the physiological function of ROS and RNS as second messengers and the ability of redox sensors to detect and/or react with ROS and RNS (for clearing them out or as redox switches).

Why do I think this is important? because when focusing just on oxidative stress, you only measure oxidative outcomes (i.e. oxidized biomolecules), which have been very well described by several colleagues here. However, it misses all the additional features of redox stress, e.g. reductive stress (when there is such a high redox potential that ROS and RNS cannot act as second messengers), electrophilic stress (when electrophiles consume antioxidants such as glutathione, irreversibly forming glutathione adducts), thiol stress (when critical cysteine residues in proteins are modified and thus, cannot act as redox switches), and more.

Therefore, you may want to look for markers of redox stress rather than just oxidative stress :)

Just my 2 cents

Good luck with your research!

You may follow the cell glutathione content, its drop is an early event in many oxidative stress situations

It should be important to assess possible ROS-induced lipid peroxidative damage by measuring Thiobarbituric acid-reactive substances (TBARS)

The measurement of TBARS is a well-established method used to detect lipid peroxidative damage through the photometric detection of the thiobarbituric acid-conjugated adduct. For details see Falone S. et al. PLoS One. 2012; 7(10): e48334

Maybe you can use a Gluthatione Detection Kit. As high GSH (Reduced glutahione) levels are indicative of oxidative stress. I used this one from Sigma (Cat Number: CS0260) and it worked like a charm http://www.sigmaaldrich.com/catalog/product/sigma/cs0260 . It is based on the mCBL method.

However, there are plenty of other options out there to measure GSH such as the propietary ThiolTracker from Invitrogen: http://www.lifetechnologies.com/order/catalog/product/T10095 or DTNB based methods.

http://www.sigmaaldrich.com/catalog/product/sigma/cs0260

I have a document that i made when i was searching for methods to measure GSH just like you. It contains a table with info about 10 GSH-Kit alternatives with a brief description and links to all of them. If you're interested i could search for it on my computer and send to you.

Dear Rasoul

in my opinion before the knowleage about which cells are under Oxidative Stress,primary task is to find the sources and pathway, which cause the Cells Oxidative Stress . The past and recent researches are based on the scientific laboratory methods , neglecting the importance of clinical and epidemiological approach to findi the human biomarkers from environmental toxics and their adverse health effects .

My added paper is moderate contribution to your question, I hope so .

Article Impacts of Exogenous Derived Nitrogen Oxide and Sulfur Compo...

Hello Rasoul, for complementary tests you can try the Lipoperoxidation (or lipid peroxidation), the Determination of oxidized proteins, or even tests that fing oxidative damage in DNA.

we are analyzing oxidative stress on some bacterial species (rhizobia and agrobacteria) antioxidant enzymes' modulation give an idea about oxidative status, but it is not enough to conclude for stress "really"effect . i propose to analyze some oxidative stress indices as lipid peroxidation (MDA), ROS measurement (mainly O2;, H2O2)

Rasoul,

Check out this website http://www.lifetechnologies.com/order/catalog/product/C10422

I think the question is how to measure oxidative stress (OS) in the bacterial population. Although, measuring the status of redox molecules or redox stress in cells is relevant in determining OS, measuring lipid peroxidation (TBARS, MDA), protein carbonylation, DNA adducts or damage (if ROS level is very high), GSH/GSSG ratio and finally ROS level would be more relevant in the present case.

To the main question, only measuring the redox regulatory enzymes, no body can predict the OS condition in cells. At both the lower or higher level of redox state, OS can arrive depending upon the neutralization state of ROS in the cells. For more info answer of Paula Aracena can be followed.

The term "imbalance" discussed above meant for the activity in pool between the redox regulatory molecules (both enzymatic and non-enzymatic) and ROS. If the imbalance falls in favor of the later then damage to biomolecules leads to OS cells.

We would use T-bars or isoprostanes

TBARS is more likely to be easier, cheap, and gives you consistent results. TBARS are widely accepted measures for wide variety of lipid peroxides. The later one also you can measure which will provide you about the free radical-catalyzed peroxidation of essential fatty acids.

I think that overexpression of catalases is an indirect way to measure environmental/endogenous oxidative stress. You can try measuring ROS levels in the supernatant, but i think it's well known that cells entering the stationary phase are stressed by the low nutrient availability or the accumulation of toxic compounds such as H2O2 or other ROS. Both, the nutrient scarcity and the presence of H2O2 induce the expression of the alternative sigma factor RpoS, which controls oxyR and a lot of genes related to protection against a variety of stresses, including oxidative stress. I'm not sure of the purpose of your study, but i think that, maybe, is not necessary to demonstrate oxidative stress in stationary-phase cell cultures.

Hello,

In fact, during the growth of the bacterial strain, cells enter into VBNC state even when there is still enough carbon source in the culture medium. My purpose is to determine if Oxidative stress causes the VBNC formation?

Yes , this is what you asked in the original question and the answers or discussions were made accordingly. Simple measure lipid peroxides first to have a preliminary idea to your assumption.

A simple treatment with H2O2 induces a rapid entry into the VBNC state, and the same happens with treatments with a low dose of chlorine, or copper, or acidic pH, or stressing temperatures, or oligotrophy, or osmotic stress, etc. The problem is that a variety of stresses have common regulation pathways, so it is difficult to say that the VBNC state is only caused by oxidative stress. I think that the key question is if VBNC cells have the mechanisms to face the H2O2 generated on solid media. It has been demonstrated that VBNC cells lose catalase activity during the VBNC response (Oliver, 2010; Kong et al., 2004).

Oxidative or redox should be measured as relative parameters. In fact, markers (peroxidation, reduction, lipid changes, etc.) are relevant if compared after physical or chemical treatment.

It is necessary for the calibration to assess (and fix) the minimal amount of nutriments, fetal calf serum, non-essential amino acids, vitamins, lipids etc. to add to the culture media (they all may interfere with the parameters you want to measure).

The best approach is first to assess the minimal concentrations of the different components to be added to culture media warranting cell viability (base line). Once you establish the minimal requirements, different culture conditions and treatments may allow to determine oxidation or reduction capacities to relate cultures (exposed and not exposed to a treatment,to a physical condition, etc) tested simultaneously.