Team:Fatih Turkey/Experiments5

Fenton Reagent Assay

To make our lab safe and secure; we needed to solve the safety problem caused by B.subtilis endospores. Endospores may gain pathogenicity according to the study done by Imperial College in 2008 iGEM. (http://partsregistry.org/Help:Bacillus_subtilis/Advantages_and_disadvantages)
In order to avoid such dangerous circumstances, we used a chemical solution that kills both original bacterium forms and endospores effectively: Fenton Reagent. The solution decontaminates the media with help of production of oxygen radicals. These radicals damage the DNA content and enzymes in the bacterium that are needed to reform original bacterium body.
We prepared modified Fenton Reagnet solution for our purpose according to J. B. Cross , 2003.1 Normally, Fenton Reagent produces oxygen radicals by the help of hydrogen peroxide.  On the other hand, modified version includes copper ions and dissolved oxygen which boosts killing mechanism. We prepared this solution in two ways: With distilled water (DFR) or oxidized water. (OFR)
With this experiment, we aimed to measure the effectiveness of Fenton Reagent solution on B. Subtilis and E. Coli. We wanted to see the effect of chemical on the biofilm. Therefore, we prepared corn strach including LB Agar for a more qualified biofilm. In order to understand the death in the plates, we added a small amount to the liquid culture and measured its OD value.
Assay contrivance is as follows;
Plate 1: On this plate, OFR is added on the B. Subtilis biofilm.
Plate 2: We formed another B. Subtilis biofilm and added same amount of DFR on it.
Plate 3: One drop of E. Coli with RFP and same amount of DFR added on B. Subtilis biofilm.
Plate 4: It was same with plate 3. Only exception was that we used OFR solution on this plate.
Plate 5: This plate was prepared as a control group of 3rd and 4th plates. One drop of E. Coli with RFP was added. Note that no Fenton Reagent solution was added.
After adding the FR solutions on all of plates, we measured their OD values at the beginning of this experiment. After waiting enough time for the exposure to chemical, we measured them again. We determined that OD values of 1st, 2nd, 3rd and 4th plates did not change. But the the OD value of 5th plate increased. This result shows us both DFR and OFR killed the current bacteria content on the plates.
1 Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions; J. B. Cross, R. P. Currier, D. J. Torraco, L. A. Vanderberg, G. L. Wagner, and P. D. Gladen Chemistry Division1 and Biosciences Division,2 Los Alamos National Laboratory, Los Alamos, New Mexico 87545