Week 17: February 20, 2023 - February 24, 2023

 February 21, 2023 

After our plate results last week, we decided on a higher range of concentrations to use moving forward in our search for D. aquaticus’s MIC. We chose 1,000 ng/ml, 2,000 ng/ml, and 3,000 ng/ml. In order to begin testing the new range of antibiotic concentrations, I first made 400 ml of TGY + 1.5% agar as preparation for another round of plating with a layer of soft agar on top. The autoclaved TGY with agar was then pipetted into individual 125 ml flasks and autoclaved in order to make pouring quicker, more accurate, and more consistent.  

Making plates 

  1. Label plates with concentrations 

    1. 1,000 ng/ml, 2,000 ng/ml, 3,000 ng/ml

    2. Each plate will have a duplicate plate for a total of 6 plates

  2. Remove TGY 

    1. Amounts in table below 

  3. Add in antibiotic 

    1. Amounts in table below 

  4. Vortex on level 6 for 6 seconds 


Concentration 

TGY Removed 

Antibiotic Added 

1,000 ng/ml 

500 μL

500 μL

2,000 ng/ml 

1,000 μL

1,000 μL

3,000 ng/ml 

1,500 μL

1,500 μL


Making Soft Agar 

  1. Remove TGY from tubes containing 2.5 ml of TGY + agar 

    1. Amount in table below 

  2. Add antibiotic into tube 

    1. Amount in table below 

  3. Vortex on level 6 for 6 seconds 

  4. Add in 50 μL of D. aquaticus to each tube 

  5. Vortex the tubes once more

    1. Level 6 for 6 seconds 

  6. Pour the contents of the tube onto the hardened plate

  7. Cover and let the soft agar solidify 

  8. Place plates in the incubator at 30°C  


Concentration 

TGY Removed

Antibiotic Added

1,000 ng/ml

1.25 μL

1.25 μL

2,000 ng/ml

2.5 μL

2.5 μL

3,000 ng/ml

2.75 μL

3.75 μL


February 23, 2023 

We let the plates incubate in the 30°C incubator for 48 hours. The two plates of 1,000 ng/ml concentration were covered in growth. The two plates of 2,000 ng/ml concentration seemed to be clear of any growth. The plates of 3,000 ng/ml had growth but it did not look consistent to the  growth that we had been having with D. aquaticus. There did not appear to be any growth of D. aquaticus so we will need to narrow our range down to be able to get close to the MIC. 

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