Week 16 (01/30/23 - 02/03/23): Discovering New Concentrations for MIC Testing with D. aquaticus and Tetracycline
Introduction:
This week we are continuing our minimum inhibitory concentration (MIC) testing with D. aquaticus. Based on testing performed last semester, we chose our concentrations of tetracycline to be 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0 ng/ml. We expect to see growth on 0 ng/ml because there is no antibiotic added. As for the plates with antibiotic included, we hope to see growth on the 1 ng/ml plate and no growth on the 10 ng/ml and 100 ng/ml plate. If the results are consistent with our goal for the project, we can correctly identify the MIC for D. aquaticus and move forward with testing on other species of Deinococcus.
Methods:
Our sample of D. aquaticus that was inoculated on 01/26 was contaminated with fungus, so a new sample was inoculated and placed in the incubator on 01/31.
The 4 125 ml flasks were stored to the side after being autoclaved. We collected these flasks and set them up to be heated up. We used a hot plate and placed each of the 125 ml flasks on the plate individually to liquify the media. Once the media was completely liquid, the flask was removed from the hot plate and set to the side to cool. Once the media passed the baby bottle test, tetracycline was added to the media and swirled around to mix. Exact amounts of tetracycline added to each flask is shown in figure 1. Once each flask is mixed, the media is poured onto its designated plate and left out overnight to allow the media to solidify completely.
The next day, we moved on to working with the 4 test tubes containing 2.5 ml of TGY with 0.5% agar. The test tubes were placed in the autoclave again so the media could be liquified. While waiting for the autoclave to finish, gram staining was performed on our D. aquaticus sample to ensure that there was no contamination. Gram stain results are shown in figure 2. The OD600 value was also taken, and because our original sample of D. aquaticus had an OD value that was too high, we diluted the bacteria by adding 100 ul of the culture to 100 ul of TGY. After diluting it down, we had a new OD600 reading that was appropriate to use in our procedure. OD600 results are shown in figure 3. The 4 test tubes were removed from the autoclave and set to the side to allow them to cool. Once the test tubes passed the baby bottle test, it was time to move on to adding tetracycline. The procedure was as follows:
A) 5 ul was removed from the 100 ng/ml tube. 250 ul was removed from the 10 ng/ml and 1 ng/ml tube. This was to ensure that after the serial dilution, we had the exact desired concentration in each tube.
B) The 0.05 mg/ml sample of tetracycline was removed from the refrigerator an placed on the vortexer for 6 seconds.
C) Add 5 ul of the tetracycline to test tube 1, containing 100 ng/ml TGY. Mix tube on vortexer for 6 seconds.
D) Take 250 ml out of the 100 ng/ml test tube and add it to the 10 ng/ml test tube, Mix tube on vortexer for 6 seconds.
E) Take 250 ml out of the 10 ng/ml test tube and add it to the 1 ng/ml test tube, Mix tube on vortexer for 6 seconds.
F) Nothing is added to test tube 4.
After the serial dilution is finished, 25.25 ul of D. aquaticus is added to each of the 4 test tubes and each tube was placed on the vortexer for 6 seconds. Then all 4 test tubes were poured on their designated plate, and a spreader was used to create a lawn. The plates were left out for 15 minutes to allow the samples to dry, and they were eventually placed in the 30o C incubator for 2 days.
Test tube number | Tetracycline concentration | Amount of tetracycline added |
1 | 100 ng/ml | 50 ul |
2 | 10 ng/ml | 5 ul |
3 | 1 ng/ml | 0.5 ul |
4 | 0 ng/ml | 0 ul |
Figure 1: Amount of tetracycline added to each flask before pouring over a plate.
Results:
Sample | A600 | Correction | λ | A(λ) |
1 | 2.78 | 0.10 | 600 | 2.78 |
2 | 0.90 | 0.10 | 600 | 0.90 |
Figure 3: OD600 values on our D. aquaticus sample. Sample 1 was our original sample, sample 2 was our original sample after being diluted with TGY.
| Figure 4: Results of our MIC testing with D. aquaticus. Concentrations of tetracycline are as follows: A has a concentration of 100 ng/ml, B has a concentration of 10 ng/ml, C has a concentration of 1 ng/ml, and D has a concentration of 0 ng/ml. |
Discussion:
Based on our plating results, it looks like the concentrations of antibiotic were too low. This was confusing to me, because in our testing last semester, there was no bacterial growth on the 100 ng/ml plate. It does not look like there was contamination on the plates, so I will need to analyze our concentrations and see if we can look at higher concentrations of tetracycline in our next try. The positive control plate of 0 ng/ml showed the expected results, which leads me to believe the procedure was performed correctly. I will be looking into different ways that we can expedite the procedure so we can finish identifying the MIC for tetracycline while also moving on to antibiotic testing for the other species of D. aquaticus.
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