Week 13 (11/28/22 - 12/1/22) MIC Testing With a New Antibiotic

 

Introduction:

The Minimum inhibitory concentration of kanamycin was achieved with our previous work, with a concentration of 1.75 ug/ml. The only issue is that there is no kanamycin resistance gene on the plasmid that we will be working with in transformation. Instead of moving forward with kanamycin, we are starting again with tetracycline, as the plasmid we are working with has a tetracycline resistance gene.

 

Tetracycline is a protein synthesis inhibitor that belongs to its own class of antibiotics. It binds to the 30S subunit on the ribosome, preventing protein synthesis from occurring. This antibiotic has been used in our MIC testing before, but the concentrations of tetracycline added were too high to see any growth. The smallest concentration used was 0.0975 ug/ml, and this only gave us an absorbance reading on the spectrophotometer of 0.02. Because of this, we will be testing even smaller concentrations of tetracycline, working with ng/ml. This will be performed with a diluted stock concentration of tetracycline, and using P2, P10, P200, and P1000 pipettes.  

 

 

Methods: 

 

200 mls of TGY was made using the 3:3: 1 method. Then 150 ml of TGY was added to a 500 ml flask, and 2.25 g of agar was added to the TGY to make 1.5% agar. 50 ml of TGY was added into a 125 ml flask, and 0.25 g of agar was added to the flask to make 0.5% agar. Both flasks containing TGY were autoclaved.

 

After autoclaving, the 150 ml flask was heated on a hot plate. Once the TGY was at an adequate temperature, 25 ml of TGY was added into 4 separate 125 ml flasks and autoclaved. The 50 ml flask of TGY was also heated on a hot plate, and 2.5 ml of TGY was added into 4 separate glass test tubes and autoclaved. DI water was also autoclaved for future use in diluting the stock concentration of tetracycline.

 

The flask containing 25 ml of TGY with 1.5% agar were heated on a hot plate after being autoclaved. While waiting for the TGY to cool, the tetracycline stock was diluted. We started with 5 mg/ml of tetracycline and added 30 ul of this sample to 2.97 ml of water, to make the new stock concentration 0.05 mg/ml. The TGY liquified and once it was at cool enough temperature, tetracycline was added to each flask. The specific concentrations and amounts of tetracycline added are shown in figure 1. Each flask was mixed thoroughly and the TGY was poured onto their designated plates and left out to dry overnight. The 4 plates were parafilmed and placed in the refrigerator to store them until we finish the procedure on Monday. 

 

Gram staining was performed on our 2 samples of D. aquaticus, results shown in figure 2.  

 

 

Figure 1: Outline of our MIC experiment using soft agar and plates. The section on the left is to be finished next week, where we are starting off right after the second autoclave label is placed on the image. The section on the right has been accomplished this week, and the plates are poured. The concentrations of tetracycline for arch plate are as follows: 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0 ng/ml as the negative control.

Results: 

 Results from the MIC evaluation of tetracycline are not finished this week due to errors with TGY.

Figure 2: Gram staining results on our D. aquaticus samples. The one on the left is from 10/27, the one on the right is from 9/17. 

 

 

Discussion:

 

We experienced some errors this week working with TGY. The first error began with the 125 ml flask filled with TGY with 0.5% agar. While heating this on the hot plate, the sample bubbled up too quickly and caused us to lose most of our TGY. Since we need exact amounts of TGY for this project since we are working with nanograms, this TGY had to be remade and autoclaved again. The second error was with the TGY with 1.5% agar. The 25ml of TGY added to 50 ml flasks caused the media to overflow in the autoclave. We assumed since we were working with a flask double the amount of TGY it would be fine, but the leakage caused us to lose our TGY. Because we need 25 mls of TGY poured onto each plate, this TGY was unable to be used since we did not know the exact amount in each flask, and that would mess up the concentration of tetracycline we were aiming to have on each plate. This TGY needed to be remade, and we needed to start from the beginning. This used up our entire day on Tuesday and Wednesday, due to the multiple steps of autoclaving needed to make both TGY samples. In the future we will be sure to use a larger flask when working with the autoclave to ensure nothing spills out. 

 

Based on our gram stains, our D. aquaticus does not look contaminated, so these samples are available for us to use next week. On Monday, the test tubes filled with 2.5 ml of TGY with 0.5% agar will be autoclaved again so we can have the TGY in liquid form, then stored in the 42 degrees Celsius water bath. When each tube is ready to be used, tetracycline will be added to each tube at a specific concentration, and then each tube will be poured on top of the designated plate.