Lab Blog Week 4
Introduction:
This past week in lab, we took out our plates of E. coli and D. sonorensis we had left to incubate. Alongside Brian and Diana, we prepared three different strengths of R2A and TGY to inoculate our sonorensis in. Our group overviewed the different tests that are involved with a full characterization of a bacteria, and learned the procedures that come with each. As the week ended, we prepared some tests to get us started in the characterization process for the upcoming week.
Method:
Our group prepared 3 flasks containing 250 mL of R2A and 3 flasks same volume of TGY.
1. We first weigh out the initial concentration one would use to prepare 250 mL R2A medium.
2. For the next flask, we prepare a media with half the concentration usually used.
3. For the third flask, we weigh out double concentration one would use.
4. We repeat steps 1-3 creating flasks of TGY at these different concentrations.
5. We used 3.75g agar across each flask and sent to autoclave.
6. We had to remake our media concentrations, as it burst in the autoclave from the settings being off from standard.
7. We were able to pour 6 plates each of the regular concentrations of R2A and TGY medium into agar plates. The remaining concentrations will be left to next week, where then we will inoculate all plates with D. sonorensis to see how growth is impacted.
This past week, we overviewed each structural and biochemical characterization test we plan to perform on D. sonorensis and learned the procedures involved, referring to procedures from microbiology characterization textbook found in lab.
Determining temperature preference:
1. Inoculate three agar slants each with loopful of broth from sonorensis, testing one from 20 degrees, second from 30 degrees, and third from 40 degrees.
2. Place inoculated slants into correct test tube rack.
3. Label appropriately and analyze results in a week.
Cellular morphology tests:
1. Follow gram staining procedures to determine gram status.
2. Measure cell size of rods or spheres by using several cells to establish a typical cell size.
3. Determine shape of cell.
4. Determine arrangements for bacteria cells, the organization of morphology.
5. Analyze results
Colony morphology tests:
1. Inoculate sonorensis on R2A agar plate using a four way streak.
2. Label appropriately and put plate upside down to incubate in 30 degrees.
3. Analyze colony shape and size by looking at the largest colony.
4. Analyze chromogenesis, looking at pigment and how it changes in different conditions.
5. Analyze opacity, surface chroma, and surface consistency.
Oxygen requirement test:
1. Inoculate loopful of sonorensis into a thioglycollate broth tube.
2. Label appropriately and incubate at 30 degrees C.
3. Analyze impact on colonies growth.
Growth in broth:
1. Prepare a liquid R2A nutrient broth.
2. Inoculate culture into broth.
3. Analyze appearance in broth.
Biochemical characterization tests
Acid and IMViC tests:
1. Prepare sugar fermentation tests by preparing three vials each of the following: Glucose, lactose, sucrose, mannitol (Already saw results of mannitol from growth on mannitol media).
2. For IMViC test, prepare three vials each of MRVP and SIM, along with three plates citrate.
3. Do a loop inoculation of sonorensis culture into one of the three vials for each type of test.
4. Streak one plate from each test with sonorensis.
5. For each test, identify a bacteria that shows a positive and one that shows a negative result to compare sonorensis results against. Prepare accordingly.
6. Let sit and analyze results for color change and bubble formation, referring to textbook to know what it means.
Catalase test:
1. Prepare a four-way streak plate of culture
2. Add two drops of 3% H2O2 to a large colony or a streak on the plate.
3. Observe results for bubbles and analyze.
Oxidase test:
1. Take four-way streak plate of culture and add 2 drops of oxidase solution to colony or streak.
2. Observe for any color changes in the next minute and refer to textbook for result analysis.
Hydrogen Sulfide production test:
1. Refer to SIM test for any color changes or black agar present.
2. Refer to textbook for result analysis.
Urease test:
1. To see if Urease is present, perform a needle and loop inoculation on urea slant with sample of sonorensis.
2. Observe for color change in the agar and analyze result meaning.
Starch digestion test:
1. To see if amylase is present, take a starch agar plate and inoculate sample of sonorensis, a positive bacterium, and a negative bacterium.
2. Label appropriately and place to incubate.
3. To analyze, first flood plate with iodine and observe for any iodine-agar color reaction. Analyze result meaning.
DNA digestion test:
1. Inoculate sonorensis on a plate of DNase test agar.
2. Add hydrochloric acid to medium to clear away and see if DNA has been digested.
Sporulation test:
1. Heat fix sonorensis culture onto glass slide.
2. Flood the stain with malachite green, and place a cover slip over.
3. Pass slide through flame to drive stain into spores.
4. Allow slide to cool at room temperature.
5. Remove access stain with DI water.
6. Counterstain with safranin for up to 1 minute.
7. Blot slide dry and analyze ratio of stained green endospores against vegetative pink cells.
Results:
We got our plates back from the 13 different mediums we had tested for growth of sonorensis on. The E. coli grew as expected for each plate, validating the medias we prepared. D. sonorensis was shown to grow on the R2A plates and the TGY plates. No growth was seen from other medias.
We are awaiting results on the growth of D. sonorensis in different concentrations of R2A and TGY broth, as a mistake was made with the autoclave which imploded our broths, preventing most from being poured. Next week will leave the 1/2 concentration as well as the x2 concentration of R2A and TGY left to be poured into agar plates. From there, we will inoculate D. sonorensis, with more information to come in the future.
Results from characterization test will come next week when we test SIM, Citrate, Starch digestion, Glucose fermentation, Sucrose fermentation, MRVP, and lactose fermentation, along with any other tests we have time and materials to perform.
Discussion:
This past week, lots of media was made within our group, with a key takeaway being that more people creates more complication with simple tasks. We expected the process to be performed much faster since we had the manpower of three people behind it, but it instead created more confusion and took up more time due to the back and forth of information sharing between the three of us. I slowed down the process when I mistakenly added too much water when bringing one of the medias up to volume, forcing us to stop and take time to calculate out how much of the ingredients we needed to add back to ensure accurate concentration.
In class, we overviewed the tests we will need to have completed to form a full characterization of Deinococcus sonorensis. We were able to look at its growth on agar and find its preferred media, and are moving forward with using R2A after observations of a more homogenous streak consistency, which will make the stubborn plaque structure easier to work with. I regret not spending more time with the inoculated plates and getting an understanding on not only how growth changes, but how the the selectives and differentials were impacted. I failed to pay close attention to the results of those sonorensis inoculated plates that did not show growth, but had other indications. I think our group will reperform the mannitol test, since we did not at the time understand to what extent it was involved with characterization of the bacteria.
So far, we have prepared citrate and starch plates, as well as 3 test tubes each of SIM, Sucrose, Fructose, Lactose, and MRVP broth. We have these tests ready to fire off when we return next week, since they can be completed and analyzed very quickly. For the other tests, we plan on overviewing procedure together in class next week to ensure everyone understands what is involved, and so we can get a better understanding of what our schedule should look like to get our characterization complete in due time. In preparing procedure, I noticed the textbook we are referring to is often quite vague, and more specificity in procedure will have to be laid out with the help of mentors and technicians.
This past week has been surrounded around getting prepared for the characterization tests we plan to perform on D. sonorensis. Our group worked through a basic understanding of each test, and prepared those which we could do right away. This next week in lab, I plan on obtaining a full understanding of what is going on within the cell during each test, as well as what our results have to say about our bacteria. I plan on outlining in detail the procedure for each test, filling in gaps in my knowledge. We will pick out our positive and negative control bacteria and complete what tests we can during lab.
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