Deinococcus radiodurans UV Treatment

 A Summary of D. radiodurans Applications

November 22nd, 2021

Overview

Deinococcus radiodurans (D. radiodurans) is a very interesting bacterium in many ways, but what makes it stand out, literally, is its ability to withstand huge amounts of ultraviolet (UV) radiation. This week's project was to compare manganese-added (Mn+) and non-added (Mn-) D. radiodurans viability under 500 J/m^2 of UV radiation to a control (from both Mn+ and Mn-) that was not exposed to UV radiation. The procedure is done using two diluted cultures of D. radiodurans obtained from a concentrated broth culture, one washed with a phosphate buffer while the other is unwashed. The hypothesis is that the cells which are exposed to UV radiation from both the Mn+ and Mn- cultures should show less growth (less colonies) than the Mn+ and Mn- control samples, and this is because UV radiation imposes oxidative stress which in great amounts causes "damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species [ROS]" (Slade and Radman). Another hypothesis states that the colonies on the Mn- side of the plates exposed to UV radiation are more numerous than the Mn+ side because high levels of intracellular manganese is believed to increase oxidative stress in D. radiodurans. A third hypothesis narrates that washed D. radiodurans cells should have better UV resistance than unwashed cells, this is because the cells are introduced to a fresh TGY media (containing tryptone, glucose and yeast extract) with less waste materials which can otherwise impact cellular functions. 

Procedure

To begin with, previously grown concentrated D. radiodurans broth cultures (one Mn+ and the other Mn-) are used to obtain diluted broth cultures for manganese-added and non-added cells. First, the Optical Density (OD) of a 1 to 1 diluted cultures is obtained. This is done by placing 200 microliters of concentrated cells (Mn+ and Mn-) into 200 microliters of TGY medium to make the final volume 400 microliters. The OD reading for the Mn+ cells was 3.54 while that of the MN- cells was 4.34. It was not thought that the difference between OD readings would generate an issue at first, so the procedure continued with these two samples of cells. Another 400 microliters of broth culture of both Mn+ and Mn- cell types obtained with the same dilution are kept for washing with a phosphate buffer. 

    The procedure of washing the bacterial cells in a phosphate buffer starts by placing 200 microliters of broth cultures of Mn+ and Mn- D. radiodurans in a clean Microcentrifuge Tube and centrifuging the mixture for 1 minute to obtain a pellet of cells on the bottom of the tube. The remaining medium is discarded and 200 microliters of phosphate buffer are added. The phosphate buffer is a balanced salt solution which gets rid of unwanted extracellular materials present in the medium of the broth cultures. After the cells were mixed with the buffer by vortexing, they are spun down by centrifigation, and the supernatant was discarded. This is done another time by adding 200 microliters of buffer, spinning, and discarding the supernatant. Finally, the cells from both samples are resuspended in 400 microliters of fresh TGY medium and incubated at 28 degrees Celsius to ensure optimal viability. 

    After about 20 minutes, a 1:40,000 x serial dilution is performed on both samples of D. radiodurans (Mn+ and Mn-) from both washed and unwashed broth cultures. To illustrate, a dilution of 1:200 x is done twice to reach the dilution of 1:40,000 x. That is, 5 microliters of both samples of bacteria (Mn + and Mn -) from washed and unwashed cultures are placed into 995 microliters of TGY medium, then 5 microliters of the resulting mixture are placed into another 995 microliters of TGY for a final volume of 1 milliliter. This then creates the desired dilution in all the samples of the washed and unwashed cells. 

    The next step involves plating the broth cultures on Petri dishes containing TGY with agarose added in a solid media consistency. There are 8 Petri dishes total, 4 for washed cells and 4 for unwashed cells. Two of the 4 plates designated to washed and unwashed cells are for exposure to UV while the other two are controls. Each Petri dish is divided into two halves, one for the Mn+ and the other for the Mn- samples. The plating is done with The Spread Plate Technique using a metal spreading tool often referred to as a "hockey stick." On each half of the plates, 200 microliters of the corresponding Mn+ or Mn- samples are pipetted and dispensed to be spread evenly by the "hockey stick" avoiding the depletion of one sample to the wrong half of the Petri dish. After all samples are plated the same way on all of the 8 plates, they are left on top of the lab bench to dry. Consequently, 4 of the 8 plates are placed into a UV-Box one at a time to be exposed to 500 J/m^2 of ultraviolet radiation for about 10 seconds. Ultimately, all 8 dishes are incubated at 28 degrees Celsius for two days. 

Results 

The results of the procedure were not as expected in the hypothesis. After about two days of incubation time, the 8 plates are brought out for performing the Colony-Forming Unit (CFU) Assay. All of the Mn- samples from the 4 plates that were treated with UV showed no growth, while the Mn+ samples had numerous colonies. Also, all of the samples (Mn+ and Mn-) that were plated on the 4 control plates showed numerous colonies as well. The results are shown below.

* These plates are only for the washed cells because the unwashed cells showed identical growth results. 

    As shown above, one plate of UV treated cells shows a 50 % loss of colonies when comparing both Mn+ and Mn- samples. This is very unusual since this procedure yielded conflicting results in the past; therefore, it will be repeated to either confirm or nullify the hypothesis. Moreover, the Colony-Forming Unit of each plate is as follows:

Washed cells:

Control Plate 1 - 682 colonies for Mn+ and 446 colonies for Mn-

Control Plate 2 - 714 colonies for Mn+ and 314 colonies for Mn-

UV Plate 1 - 272 colonies for Mn+ and 0 colonies for Mn-

UV Plate 2 - 255 colonies for Mn+ and 0 colonies for Mn-

Unwashed cells:

Control Plate 1 - 734 colonies for Mn+ and 501 colonies for Mn-

Control Plate 2 - 1190 colonies for Mn+ and 480 colonies for Mn-

UV Plate 1 - 413 colonies for Mn+ and 0 colonies for Mn-

UV Plate 2 - 444 colonies for Mn+ and 1 colony for Mn-

    It is suspected after achieving such results that D. radiodurans relies on manganese to aid in its survival under UV radiation. Although the results do not confirm the original hypothesis which states that high levels of manganese are harmful for D. radiodurans, the other hypothesis that less colonies of both Mn+ and Mn- samples will be shown on UV-exposed plates than control plates still holds true. The third hypothesis that washed cells will show better growth than unwashed cells, however, is refuted, as more CFU's are present on plates from unwashed cells. In the upcoming week, the procedure will be repeated using Mn+ and Mn- starting broth cultures with matched OD reading to get more consistent results. As for now, one could keep thinking that we made a prodigious discovery!

References

Slade, D. and Radman, M. (March 2011) Oxidative stress resistance in Deinococcus radiodurans. Microbial Mol Biol Rev.