Continuation of the biochemical tests of D. sonorensis to gauge metabolic processes

 Introduction:

This week in lab my team furthered our research towards our full characterization of D. sonorensis. After working with different recipes to find a viable casein media, we ran the casein test to see whether sonorensis has an amylase enzyme. My team performed the inoculation procedure for the SIM protocol to test the bacteria for indole products, motility, and hydrogen sulfide products. 

Method:

The casein test procedure is one similar to the previous starch hydrolysis procedure, meaning a clear halo will be formed around the sites of casein hydrolysis.

1. Prepare one casein media agar plate and divide the plate into thirds using a permanent marker. 

2. Label control bacteria and the bacteria to be tested in their respected  portion of the plate. 

3. Inoculate each indicated bacteria to their respected labeled section on the agar plate by streaking a very small portion of the section with bacteria. 

4. Allow agar plate to dry before placing into incubator at 37 C for 24 hours. 

5. Observe for presence of clear halo surrounding colonies. 

The SIM test observes for three factors within one media. The test allows insight on if bacteria can reduce sulfur to produce hydrogen sulfide products along with if the bacteria has motility, and if the bacteria produces indole products. 

1. Prepare SIM agar deep tubes according to the number of tubes needed to give proper controls for experiment. 

2. Use an inoculation needle to stab into the agar deep with culture.

3. Place tubes in incubator for 24 hour duration.

4. Put three drops Kovacs reagent on all vials and wait thirty seconds to observe for red color change at the top of vial. 

5. Observe for black agar and motility.

Results: 

In the casein test, a distinct halo formed around the D. sonorensis colony, indicating a positive for the amylase enzyme in the bacteria. In the SIM test, the lack of color change in the agar and at the top of the vial indicated a negative for indole products and production of hydrogen sulfide. D. sonorensis was nonmotile in our results. 

Discussion:

The casein test proved a slight challenge in preparation. In online forums, recipes included a TGY mixture with skim milk powder for the casein recipe, which did not hold up in the autoclave. With a few test runs of different recipes, a simple skim milk powder and agar recipe sufficed for our trials. In the first run of the casein test, there was a large overgrowth of the colonies due to my streaking technique. A little goes a long way when streaking the culture onto the plate and anything more than a dot will cause an overgrowth of the colonies into the other sectioned off bacteria cultures on the plate. Being unaware of this, I did multiple small streaks in one section since I was unconfident that I had effectively streaked the culture onto the plate. Despite the visible halo clearing around the cultures, another trial had to be performed due to this cross contamination of cultures. This second trial yielded the same results, and correct technique prevented the overgrowth seen before. 

The SIM test provides an efficient biochemical characterization, as it assesses for three distinct biochemical properties within one media. D. sonorensis had no blackening of agar, indicating a negative for the sulfide portion of the test. This indicator tests for the production of hydrogen sulfide gas that some bacteria produces as a byproduct of metabolizing sulfur-containing amino acids. Within the media, the ferrous ammonium sulfate will bind to any hydrogen sulfide produced to form a black precipitate. For the indole portion of the test, no red layer on the surface of the medium was formed after adding the Kovac's agent, indicating a negative result. This test determines whether bacteria contains the enzyme tryptophanase, which hydrolyzes the amino acid tryptophan into indole products. D. sonorensis would not have tryptophanase in this case, since the indole would have reacted with the kovac's agent if so. Motility is a trait within a species that gives a competitive edge, since it means the bacteria has indicators that can sense environmental changes, and allow it to move towards or away from what's detected. D. sonorensis only grew along the stab line, indicating that the species is nonmotile.

This past week in lab we gauged a better understanding of the metabolic processes of D. sonorensis. We had to adjust which incubation temperature we used for the vials in the SIM trials since the growth was very slow. In the future I will place the vials in the warmer temperature to start off. This next week in lab we are finishing off the tests in preparation for a complete poster. We will perform the catalase test and redo the VP portions of the MRVP tests to see if we can get a result that isn't variable, like before. This is a challenge, since in the lab we do not have a control bacteria that shows a clear positive for this result. Updates on the biochemical characterization are to come shortly.

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