Week 16: February 13, 2023 - February 17, 2023

 February 13, 2023

Continuing with our MIC testing on D. aquaticus, we picked up where we left off on 2/10. We left the agar plates out to settle before we could continue with our testing. To finish off with the plates, we made soft agar tubes to be able to grow bacteria on the agar plates. 

We started off by heating up our flask of TGY + soft agar until they were liquified. We then pipetted 2.5 ml of the mixture into 6 different test tubes. Since the sterile mixture was transferred into a non sterile container, the tubes were put into the autoclave before we could continue using them. 


February 14, 2023

Since the test tubes of TGY + soft agar had been autoclaved, we were able to begin working with them. Before we began using the tubes, we took an OD600 value of our D. aquaticus sample to make sure it was within our desired range of 0.8 -1.00. The sample that we used was from 12/9/2022 so it had been growing for a while. It had an OD600 value of 2.10 which was more than double what we needed. To fix this problem, we diluted our sample down by mixing 200 μL of D. aquaticus with 200 μL of pure TGY broth (no agar). We added both parts into an eppendorf tube and vortexed it for a few seconds to thoroughly mix it. We checked the OD value again and it came out to 0.76 which was close enough to 0.8 for us to be able to use it. 

Soft Agar Plates 

  1. Create a water bath by heating up a beaker of water on a hot plate

  2. Carefully place agar tubes in the hot water bath until they become liquid

  3. Remove from hot water bath and allow to cool

  4. Once they pass the baby bottle test, begin adding in the different concentrations of antibiotic 

  5. Add in 25. 25 μL of D. aquaticus to each tube 

  6. Vortex tubes using vortexer on level 6 for 6 seconds 

  7. Quickly pour the contents of each tube onto the corresponding plate

  8. Spread the soft agar 

    1. Put the lid of the plate on and move it around until the soft agar is evenly spread

  9. Put the plates in the incubator at 30°C 


                                            Soft agar tubes being heated in a water bath in order to use

February 16, 2023 

The plates all had growth on them except for one of the 1000 ng/ml plates. One of the 10 ng/ml plates had minimal growth. We had made duplicate plates of each concentration in case we had error on one of them. This would also allow us to confirm or deny any results. Since the results were completely different from the results we had gotten from last semester’s microplate reading and agar plating, we were unable to confirm our MIC results for our antibiotic testing on D. aquaticus.

After having a talk with Dr. Tuohy and Jonathan to see what could have gone wrong, we decided to do a gram stain on the culture that grew on the 1,000 ng/ml plate to be sure that it was D. aquaticus growing on there and be able to rule out the possibility of contamination. 

We decided that moving forward, we would look at a higher range of antibiotic concentrations. 1,000 ng/ml will now be our lowest concentration and we will continue our search for D. aquaticus’s MIC. 

 

Results from 2/14/2023 soft agar plates






References 

Panec, M., & Sue Katz. (2006). Plaque Assay Protocols. https://asm.org/ASM/media/Protocol-Images/Plaque-Assay-Protocols.pdf?ext=.pdf

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