Deinococcus Sonorensis Characterization Continuation

 Introduction:

This past week consisted of a series of biochemical tests on Deinococcus sonorensis, which continues the characterization process of the species. After leaving the culture plates within a starved state for a week duration, we completed a sporulation stain to tests our species for a sporulation gene. We found a solution to avoid casein media curdling when placed through an autoclave cycle, then performed casein hydrolysis tests the new recipes. We inoculated the SIM medias, along with the sucrose and MRVP medias. 

Method:

We tested if the species Deinococcus sonorensis enters into a state of sporulation when a stressor is applied to the bacteria's environment. To test this, we pushed D. sono, B. subtilis, and S. epidermidis. into a starved state, by leaving agar plates of a growth culture agar plate out of the incubator for a week, to give the bacteria ample time to consume all nutrients present on plate, and starve. A malachite green stain is performed to distinct vegetative cells from spores. 

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. We selected the following: +B. subtilis, - S. epi, ? D. sonorensis.

4. Inoculate 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.

6. Flood slide with malachite green. 

7. Place a damp paper towel over slide to prevent malachite green from evaporating.

8. Allow steam to permeate sample for 5 minutes.

9. After waiting allotted duration, relocate slide to gram stain tray. Thoroughly rinse slide with DI water.

10. Flood with the counterstain safranin and allow it to sit for 50 seconds.

11. Thoroughly rinse with DI water and blot slide dry. 

12. Repeat procedure with each bacteria being tested. 

13. View stain under microscope to analyze and distinguish vegetative cells from sporulated cells.

Results: 

The results for the sporulation test performed on D. sonorensis were inconclusive. The bacteria used as controls performed as expected. 

Discussion: 

Sporulation is a survival mechanism that some bacteria can enter into under unfavorable conditions. This can be triggered through extreme temperatures, desiccation, lack of nutrients, and exposure to chemicals. When triggered, bacteria with the ability will produce highly resilient spores, which places the cell in a dormant state to ensure long-term viability. When performing microscopy post sporulation, there are a few things to look for. The stain malachite green will bind to the sporulated parts of the cell. The counterstain will bind with the vegetative portions, which provides a contrast that is easy to discern spores from healthy cells. When analyzing the D. sonorensis stain, we found that there were no green portions, which does not align with a positive result for sporulation. Despite this, other factors made the test inconclusive. The stain did not adhere to a commonly seen positive or negative result, so more tests will have to be done to obtain a conclusive answer. There were a few issues along the way that may be responsible for this. Perhaps much of the  malachite green stain evaporated away when placed above the steam. The steam was used to open up the cell and allow the malachite green to permeate, but it is possible that the steam may have evaporated the dye, preventing it from fully coating the spores. A damp paper towel was used in our experiment to prevent this, but I observed that it would dry out through the five minute duration. If redone, I will watch the paper towel and re-dampen in through the process to ensure the dye does not evaporate. My prediction for D. sonorensis was that it would be a spore producing bacteria, since it aligns with the story of sonorensis and it's plaque. If time permits, a redo of this test will be valuable for a good characterization. This next week in lab, I plan on working out the kinks in the casein test, and get it up and running. We have found a casein media that can survive the autoclave, and have control bacteria selected. We will also perform UV tests on D. sonorensis at different strengths, and see if differences arise between the sonorensis grown in TGY versus R2A.

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