Redoing tests and Poster work

     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 time with MR being negative and VP inconclusive

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 interpret the result

Voges-Proskauer test

 1. Use the remaining 2.5 ml of culture grown in MR-VP broth.

 2. Add 0.6 ml (or 12 drops) of Barritt’s reagent A.

 3. Add 0.2 ml (or 4 drops) of Barritt’s reagent B.

 4.Carefully shake the tube for 30 seconds to 1 minute to expose the medium to atmospheric oxygen (necessary for oxidation of acetoin to obtain a color reaction).

 5. Allow the tube to stand for at least 30 minutes.

 6. Within 1 hour, compare the test result to control cultures to determine if the culture is VP positive 

Second sucrose test

citrate test, positive on top and sonorensis below

    We also did UV tests on sonorensis, exposing it just like we would UV then plating it to see its death point. For each one we did three biological reps from each of the two medias, before measuring the OD value and normalizing to 1 +/- .05. Then exposing 50μl to different levels of radiation for 5 minutes before adding to 450μl of media, vortexing and plating on a divided plate of the respective media.

    In the first test we exposed them to 2500, 3000, 3500, 4000, and 5000 (*100μJ/cm^2). The samples grown on R2A didn't grow on any of them while the samples grown on TGY grew on all of them. 

 TGY 5000 plate, three separate biological samples

 TGY 3000 plate, three separate biological samples

    In the second test we started at 3000 for both medias, testing lower for the R2A samples and higher for the TGY samples. The R2A samples were tested at 3000, 2000, 1500, 1250 and 1000 (*100μJ/cm^2). The TGY samples were tested at 3000, 5000, 6000, 7000, 8000 and 9000 (*100μJ/cm^2). 

R2A Blue tube holder, TGY Red tube holder

Energy	TGY	R2A
1000	N/A	No Growth
1250	N/A	No Growth
1500	N/A	No Growth
2000	N/A	No Growth
3000	Growth	No Growth
5000	Growth	N/A
6000	No Growth	N/A
7000	No Growth	N/A
8000	No Growth	N/A
9000	No Growth	N/A

 

        

    Beyond the tests, a majority of time was spent working on the poster, on both the introduction and the rest of the poster. At this point we now have a comprehensive list of all of the results from the tests. 

Enzyme	Results
Urease	Negative (-)
Amylase	Positive (+)
Protease	Positive (+)
Oxidase	Positive (+)
Catalase	Positive (+)
Tryptophanase	Negative (-)
Substrate	Results
Lactose	Positive (+)
Citrate	Negative (-)
Sucrose	Inconclusive
Miscellaneous	Results
Motility	Nonmotile
Production of Hydrogen Sulfide Gas	Negative (-)
Mixed Acid Pathways	Negative (-)
Gram Staining	Positive (+)
Shape	Rod
Sporulation	Negative (-)
Butanediol Pathways	Inconclusive
Media	R2A, TGY, NB
Oxygen Requirement	Aerobe