S-STEM Scholar Alex Hugenberg-Cox Blog

 Overall the path to getting the poster for the conference was very rocky but the conference ended up going well and I felt confident when presenting. This poster discussed the various characteristics of D. sonorensis, mainly biochemical but also morphological. However a large part of the poster was looking at the differences in plaque structure when grown in different nutrients and looking into what the meant for the cell. There was also a small amount of genomic data and an example of our methods for the UV exposure.  Final Introduction-             Deinococcus sonorensis was isolated from ground soil of the Sonora Desert of Arizona in 2005. Like so many members of this genus, D. sonorensis is radioresistant, but notably presents with a sticky biofilm structure. This  biofilm has made culturing and accessing the cells more challenging and thus relatively little is known about the cell biology of the species or the nature of the biofilm. Our core objective in the current study is und

      As we lead up to the Conference, we are redoing the last few tests that need to be done, running the UV analysis for sonorensis and working on the poster. We redid the MRVP tests, the Citrate tests, and the sucrose test as well as ran the catalase test and two UV tests comparing D. sonorensis on both TGY and R2A, comparing the plaque structure of both and its ability to survive UV radiation.      For the catalase test we simply dropped hydrogen peroxide on the colonies and observed to see it it formed bubbles or not. When it did form bubbles we knew that it has the enzyme catalase. The citrate and sucrose tests were color changes. For the citrate test we could see an obvious difference this time between the positive and negatives, and could tell that it was not able to utilize citrate. The sucrose test did have a slight color change this time but not enough to define it as a positive or negative, so it remains inconclusive. The procedure for the MRVP test was the same as last ti

      This week we started work on the poster as well as were able to finish the sporulation tests. For the sporulation tests we used the two sets of plates that had sat out through spring break in order to truly starve the cells in order to force them to produce spores if they are able. The procedure for a sporulation test is very fundamentally similar to gram staining, using malachite green to distinct vegetative cells from spores.  Sporulation Procedure 1. Bring 250mL of water in a beaker to a slight boil using a hot plate. 2. Use a wax crayon to draw a circle onto six slides.  3. Label two slides each of the positive control , negative control, and tested bacteria (+B. subtilis, - S. epi, ? D. sonorensis.) as well as the date in order to separate the two sets 4. Spread each sample within notated circle and use Bunsen burner to heat fix onto slide.  5. Attach clothing pins on either end of the slide and rest upon the top of the beaker, allowing the steam to hit the sample.

      This week we started a second spore test as well as performed both the oxidase and oxygen requirement tests and made/inoculated urease plates. We also performed another RNA isolation.      Starting another spore test was simple as we only needed to inoculate it, let it incubate for a few days, then leave it to rest with the other spore test. The oxidase test was simple as we only needed to add a few drops of a specialized reagent and look for a color change. For the oxygen requirement test we needed to inoculate our sonorensis  and controls in to  thioglycollate agar tubes . After they incubated we would be able to tell the oxygen requirement based on the location and distribution of the bacteria in these tubes. Then we can categorize it as an obligate aerobe, microaerophile, facultative anaerobe, aerotolerant anaerobe, or obligate anaerobe. For the urease plates we first needed to make a concentrated urea solution and a solution of only agar and water. After autoclaving the agar

    This week we looked at the biochemical tests we inoculated last week. The lactose, sucrose, and citrate plates were all color changes while we had to add iodine to the starch plates and perform a procedure to see the MRVP results.      Deinococcus sonorensis was positive for lactose fermentation, inconclusive for sucrose fermentation, negative for the MR test and inconclusive for the VP test, positive for the starch test and inconclusive for the citrate test. The sucrose, VP and citrate tests all showed no difference between the negative control, positive control and test and will need to be redone. All of f the TGY plates grew, with the 1/2 TGY plate having a smaller amount of growth and the double TGY plate thriving. However the only R2A plate that had growth was the regular recipe.  Methyl red test  1. Transfer 2.5 ml of culture into a new sterile culture tube. 2. Add 5 drops of the methyl red reagent.  3. Compare the test organism to the control cultures to immediately interpre

    This week we learned about microscopy as well as did RNA isolation, qPCR and inoculated a number of biochemical tests.      Chad spent one day teaching us about the florescent microscope and showing us the basic parts and procedures. We learned about the four channels that showed different colors and different parts of cells, like blue for the nucleus and green for the cytoskeleton/cell wall and red for cytosol. He showed us how to prepare the slide then went through the procedure, and showed us around the program.      This week we did the RNA isolation again.  The UV exposure was performed at  70  mJ/cm^2 for two minutes, with the  E.coli  being grown that morning then normalized, exposed and diluted. After the 30 minute recovery at 30 ° C we moved on to RNA isolation. With one round of bead beating we did the procedure like normal with one change. Instead of working out of a normal tube holder i switched to working out of an ice box after the bead beating step and extraction was