Deinococcus Sonorensis Characterization Continuation

Introduction: 

    As we continue our characterization of Deinococcus Sonorensis in preparation to our upcoming conference, our group detailed a schedule to work upon. This past week, we prepped and performed a starch digestion test on the bacteria. We prepared agar plates of our bacteria along with positive and negative controls, starving the colonies, to test sporulation count. Media was prepared to test for urease and protease. From there, we outlined further plans for the SIM test we plan on performing. 

Procedure: 

After initial growth of colonies on starch plates, we as a group decided upon a different strategy, as the growth from previous attempts failed to yield optimal growth required for the iodine portion of the test. 

1. We took one starch plate prepared for the group, and using a sharpie, marked a division of the plate into four even quadrants.

2. We labeled each section with the indicated positive, negative, and unknown bacteria as follows:

 + B. Subtilis, - S. Epidermidis, ? D. Sonorensis 

3. After inoculating the indicated bacteria into a small portion of each quadrant, we place the plate to incubate for two days. 

4. After the colonies have shown growth, we submerge the colonies in iodine solution, an wait up to a minute to look for color change. 

To prepare for the sporulation test, we pick out a bacteria that is positive for sporulation, along with a bacteria known to not produce spores, to create a basis for our unknown variable, D. Sonorensis. To mimic a starved environment that will jumpstart the dormancy process in spore producing bacteria, we performed the following: 

1. We first poured plates of R2A to give our group media to work with, to grow the selected bacteria. 

2. We selected Bacillus Subtilis to use as our positive control, Staphylococcus Epidermidis for the negative control, and Deinococcus Sonorensis for our unknown variable. 

3. After determining media preference, we prepare two plates of R2A medium for B. Subtilis and D. Sonorensis, along with one plate of TGY medium for S. Epidermidis, labeling each respectively. 

4. After inoculating each plate with it's indicated bacteria, we leave the plates out for the next week in order to get the colonies into a state of starvation. 

Media preparations were conducted to prepare for further biochemical tests on D. Sonorensis.

1. Prepared 250 mL of urea broth to test for urease enzyme in our bacteria.

2. We then put it through a filtration system before sending to the autoclave. 

3. To test for the protease enzyme, we create a 100mL milk media, by adding 28.0gm/L to a traditional TGY recipe. 

4. Send to autoclave.

5. After autoclave cycle is complete, we plan to pour the plates and complete the tests at a future date.

We outlined how we plan on performing our SIM tests. Since each test will show results for hydrogen sulfide products, Indole products, and motility, we had to select controls that would cover all three. 

1. We prepare four test tubes of SIM solution. 

2. In the first tube, we will inoculate our bacteria we plan on measuring, D. Sonorensis.

3. For the second tube, we select E.coli, which shows positive results for both indole products and motility but negative for sulfur.

4. For the third tube, we select S. Epidermidis, which gives a negative result for all three variables. 

5. For the fourth tube, we select C. freundii, which is positive for both motility and sulfer production, while being negative for Indole products. 

Results: 

    Observing a formation of a halo around the D. Sonorensis colony post the introduction of iodine flooding indicated a positive test result for starch digestion. We noticed the halo was less vibrant in color in comparison to the positive bacteria. 

(Figure 1. showcasing results of each bacteria post iodine flooding. D. Sonorensis in the right quadrant displays a halo, indicating a positive result for amylase enzyme production.)

Discussion:

    When comparing D. Sonorensis to the positive control bacteria, it seems less vibrant in it's color and appears to have a weaker halo. It is difficult to differentiate between what is the colony and what is the halo, compared to B. Subtilis. This may mean D. Sonorensis just has lower gene expression of the amylase enzyme than our positive. With this information, we now know D. Sonorensis is able to take starch and break it down into simple sugars to be used for energy, perhaps being another reason the bacteria has a reputation of being very resistant, as it gives the bacteria a competitive edge against the rest. 

    Our sporulation test will be the most time consuming in preparation, as it needs to be in a nutrient deprived state for long enough to show signs of spores. In certain bacteria, sporulation acts as a defense from the organism. When the bacteria detects they are in a state they may not survive in, they have a mechanism that creates durable spores throughout its tissues. This will put the organism into a dormant state, allowing them to survive until environmental conditions have adjusted to a sustainable level. Bacillus subtilis is the leading bacteria for sporulation, and was chosen as our positive for this very reason. We were unconfident with what medium S. Epidermidis would be best to use, but I am confident that our TGY should suffice, especially considering how we are trying to starve the colonies. By leaving each plate in a safe but un-incubated setting, we hope as they run out of nutrients, it will kickstart any sporulation mechanism from within the bacteria. After the plates have sat to our set duration, we plan on performing a methylene green stain, to differentiate and quantify the ratio of vegetative portion to spored portion of bacteria. 

    The urea media we created posed several challenges. We had to go back and fix some rations of ingredients after an accidental addition of too much DI water as our solvent. We had to learn how to use the filtration system as well at the end of the recipe. In the end, we successfully created the medium and will use it next week to test for urease enzyme from our bacteria. This test will be conclusive of whether D. sonorensis is able to brake down urea into ammonia and carbon dioxide. As D. Sonorensis is a bacteria residing within soil, it would be beneficial to the bacteria to have this enzyme, due to the high traces of urea found in animal urine, which the bacteria must have to face in it's natural environment. It will be interesting to see what results are shown from this test, due to how much urea it must face day to day. The casein plate was rather simple to make, since it just needed the addition of skim milk powder to the original recipe of TGY. This test will be conducted in similar fashion to the starch digestion test outlined earlier, as it too will form a halo when showing a positive result for protease. 

    For the SIM test, we had to plan carefully using the materials we had. Ideally, it would have been nice to have a bacteria that had a positive for all three, but we had to add an additional tube to account for what was on hand. Since the tests results are clearly displayed in three different portions of the tube, we do not have to worry about the results of one test permitting our ability to read the next. A positive result of sulfide production means that our bacteria can convert the amino acid, cysteine, into hydrogen sulfide, indicated by the formation of a black precipitate. A positive result for indole production means our bacteria can break down the amino acid, tryptophane, into indole, with the help of tryptophanase enzyme. A positive result will show a red color at the top of the medium after the introduction of Kovac's agent. This medium also allows for observation of motility, which when positive, will have the bacteria diffuse throughout, giving a cloudy or opaque appearance. Results of this test along with more information is expected to be followed up on in the future. 

    This week was very much preparing for the future. A lot of different media needed to be prepared in order to fire off tests in a timely fashion. In the next week, my group plans to get started on the tests prepared this past week, along with conducting the catalase and oxidase tests.