Changing Oxidative Stressor From Hydrogen Peroxide to Sodium Hypochlorite

 

    This week we went back to our own oxidative stress experiment. After a long discussion about our lack of results when using Hydrogen peroxide as a stressor, we decided to switch our stressor from that to Sodium Hypochlorite (NaOCl). Even though we never got to test our results from the exposure where we introduced catalase, Chad was certain they would yield results no different than any of the tests we had done before. Instead it was decided to go with Sodium Hypochlorite, from bleach. 

    Before we could start our actual experiment, we would need to see what percentage of sodium hypochlorite would be ideal. We would be using agar plates, and mixing the cells with different dilutions of NaOCl and inoculating. The plates would than incubate for 48 hours before establishing how each dilution percentage effected the cells. We started by doing a dilution from household bleach at roughly 8% sodium hypochlorite, diluting down to 5.25% originally before doing further dilutions. (From now on any percentages will be referring to the percent of sodium hypochlorite compared to the whole, unless specified otherwise. The bleach is also diluted in nuclease free water.) After diluting to 5.25% we diluted down to 0.5%, 0.25% and 0.1% in order to start with a large range. 


0.5% = 67 mM

0.25% = 34 mM

0.1% = 13 mM


    A few papers I had skimmed over the weekend suggested that the highest millimolar concentration you could go without killing the cells would be 10 mM. However it was decided to start with a larger range just to ensure that was correct. 

    After diluting the cells in TGY to an OD600 of 1, we mixed 50μl of cells with 450μl of the respective diluted sodium hypochlorite. We than incubated the mixture at 30 degrees Celsius for 30 minutes. After incubation, the cells were inoculated onto ager plates in triplicate per dilution, with a single control. However, the control was 50μl of cells added to 450μl of PBS instead of TGY or Nuclease free water. The PBS was also contaminated, which was unnoticed at the time. 

    We inoculated 100μl of sample onto each plate, and spread with a new sterilized cell spreader each time. After drying they were incubated at 30 degrees Celsius for 48 hours. After looking them over none of the plates had growth except for the contaminated control, which was graham stained and revealed to be a mix of staph and e.coli. 



    With no growth we decided to move to another round of dilutions, this time at even smaller percentages. This time we did 0.1%, 0.075%, 0.05% and 0.025%. We did the same methods with the dilution of cells, mixture and plating. However this time we did two controls, one with 50μl of cells and 450μl of TGY for a cell control and one with 500μl of 0.025% of NaOCl for a bleach control. 



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