A Different Method for AMC

     Throughout this semester, we have been focusing on oxidative stress against the bacteria Deinococcus radiodurans. Due to some very consistent results showing that there was no effect on the bacteria when being exposed to hydrogen peroxide, we decided that we would be focusing on bleach. Bleach has a similar effect on cells so we will be moving forward with that. 

    First, we started by doing a dilution series of bleach. We started with a stock of household bleach that was at 5.25%. We wanted a large range to start with just to see if there was any growth, the range went from 0.10%, 0.25%, and 0.50% of 5.25%. The bleach was diluted using nuclease free water. The goal amount of cells was an OD600 value of 1. We added 50uL of cells in TGY at an OD of 1 to 450uL of the respective amount of NaOCl (bleach). After combining, the solutions were then incubated at 30 degree Celsius for 30 minutes. After the exposure, we plated in triplicates for our concentrations and had one control. The control contained 50uL of our cells and 450uL PBS. 100uL of the exposed cells and control was pipetted into each plate. We used a disposable cell spreader for each plate and spread in  a circular motion, going from the middle to the edges The plates were then left out to dry for an hour and placed in 30 degree celsius incubation for 48 hours.

    48 hours after incubating the plates, we took them out and inspected them. All plates except our control had no growth. But, our control plate growth was discolored. D. radiodurans typically has a orange/pink pigmentation when its grown, the control (which had no exposure to any bleach), was more white. After gram staining our control, we realized that there was staph and E. coli in our control. We had gram stained our grown cells prior to doing the experiment and didnt notice any contamination. This led us to believe that the PBS was contaminated. Once gram staining the PBS, we say that it had evidence of staph and E. coli within it. Due to this, we decided to do one more round of testing. We decided that although we didn't have a reliable control, we would move to concentrations of bleach that was 0.10% and lower for our next experiment. 

First experiment. Control at bottom.

    To try and avoid was happened last time, we decided to stay away from PBS completely this round and have two controls, a negative and a positive. For our new concentrations, we started at 0.10% and went down to 0.075%, 0.050%, and 0.025%. For our controls we did a plate with 50uL cells and 450uL TGY then another plate with 50uL cells and 450uL 0.025% bleach. To dilute our bleach solution back from the 5.25%, we did C1V1 and came up with the following:

    The same method was done for the normalization of the OD and the amounts that were inputted, also everything was exposed/incubated at 30 degree Celsius. The plates were also inoculated the same way and were stored in the same area. The only difference is that they will be incubating for 72 hours due to leaving them over the weekend.