Finalized UV-Exposure Methodology on D.xinjiangensis and Urease Retesting

 Intro

Previously, cells grown only in R2B were exposed to UV for 2 minutes at two varying energy levels. These energy levels were 50,000 and 100,000μJ/cm² . Dilution factors were not 1:10, and no growth was characterized on any irradiated cells post incubation. As a final attempt to see growth in R2A cells, we will shorten the duration of exposure to 1 minute, keep dilution factors 1:10, and have 8 intervals between the values of 200-25,000/cm² . These specific values will be 200, 400, 800, 1600, 3100, 6300,12500, and 25000μJ/cm² . 

Additionally, previous urease tests yielded negative results which contradict the original paper in 2009 characterizing D.xinjiangensis. To confirm our findings , we will make more urease plates and reinoculate them. 


Methods

3 broths of R2A were inoculated with D.xinjiangensis and grown overnight. Afterwards, they were normalized to an OD value of 1. 24 1.5μL centrifuge tubes were filled with 180μL of R2B. After sterilization , 3 squares were loaded into the UV Crosslinker , then 50μL of the normalized samples were pipetted onto the parafilm squares. Then, they were exposed to a constant energy level for 1 minute, where they were then diluted by transferring 20μL into a corresponding centrifuge tube containing 180μL of R2B. This brought the total volume to 200μL and kept the dilution factor 1:10. We repeated this step for all 8 intervals. For our control, we diluted our normalized cells to a 1:10 dilution factor with the same method. These were then plated on corresponding R2B plates, which made n=3 for each plate. Plates were then incubated at 30 degrees Celcius for 48 hours. 

To make urea media, 450mL of distilled water was mixed with 7.5g of agar. After autoclaving, 50mL of urea base was added once media had reached 55 degrees Celsius or below. When this step occurred, our media was a deep red color. Therefore, we needed to titrate the media with an acid such as HCl. Once a peachy color was reached, 6 plates were poured and 2 were inoculated with S.epidermis as a positive control. Two plates were inoculated with B.subtilis as a negative control, and two plates were inoculated with D.xinjiangensis. Plates were incubated at 30-37 degrees Celsius for 48 hours. 



Results

Positive control S.epidermis , positive result due to pH indicator phenol pink 
Negative result from D.xinjiangensis due to pH indicator phenol pink not changing color
UV test results post incubation

Discussion

No growth of irradiated cells were seen on any R2B plates. Non-irradiated cells showed growth post incubation. This test indicates that cells grown in R2B are extremely sensitive to UV exposure than those grown in TGY. The next steps for UV would be to test cells grown in TGY but exposed in R2B to have equal penetration to R2B cells. Additionally, the urease test confirms D.xinjiangensis as urease negative. However, this still contradicts the original paper written on D.xinjiangensis. 











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