Week 20: March 20, 2023 - March 24, 2023

 Continuing Search for D. aquaticus’s MIC

This week we were determined to finish up our work with finding the MIC of D. aquaticus. We are repeating a test from the previous few weeks to help us determine the MIC. We hoped to get conclusive results by retesting D. aquaticus with Tetracycline at three different concentrations. In this trial, we added a negative control and a positive control. The negative control will only have TGY + antibiotic. The positive control will have TGY + bacteria. The purpose of a negative control is that there shouldn’t be any growth. The positive control is there to see how the bacteria should grow. If something goes wrong with these controls then the rest of the results might not be as valid. 

1. Autoclave 8 test tubes of TGY with 0.5% agar to sterilize the mixture and the tubes

1. 13 x 100 mm test tubes with metal caps

2. 2.5 mL was pipetted into each tube 

2. Remove appropriate amount of TGY 

1. Amounts shown in table below 

3. Add in appropriate amount of antibiotic 

1. Amounts shown in table below 

4. Vortex on level 6 for 6 seconds

5. Pour the soft agar onto plates 

1. Pour onto plate that corresponds to the tetracycline tetracycline concentration 

6. Cover plate and move around to spread the soft agar all over the plate

1. We made duplicates of each concentration

7. Let agar set

8. Put into 30°C incubator

1. Should see results in about 24 hours 

Concentration	Antibiotic Added
3000 ng/mL	3.75 μL
2000 ng/mL	2.5 μL
1000 ng/mL	1.25 μL

Continuing MIC Testing on D. roseus 

1. 21 test tubes were prepared 

1. 2 mL of TGY was pipetted into each tube and autoclaved in order to sterilize the tube and the TGY 

2. Take OD600 value of D. roseus 

1. The value was 1.76

3. Dilute the bacteria to get it within the range of 0.80 - 1.00 

1. 1100 μL of TGY was mixed with 800 μL of D. roseus 

4. Remove TGY 

1. Amounts shown in table below 

5. Add antibiotic 

1. Amounts show in in table below  

6. Remove 50 μL of TGY + Antibiotic from each tube

7. Add 50 μL of D. roseus to each tube

8. Vortex on level 6 for 6 seconds 

9. Place in 30°C incubator 

Concentrations	TGY Removed/Methicillin & Kanamycin	TGY Removed/Ofloxacin	TGY+Antibiotic Removed/Bacteria Added
100 μg/mL	250 μL	500 μL	50 μL
50 μg/mL	125 μL	250 μL	50 μL
25 μg/mL	62.5 μL	125 μL	50 μL
12.5 μg/mL	31.25 μL	62.5 μL	50 μL
6.25 μg/mL	15.6 μL	31.25 μL	50 μL
3.125 μg/mL	7.8125 μL	15.625 μL	50 μL
1.56 μg/mL	3.91 μL	7.8 μL	50 μL

Results of D. aquaticus being tested with Tetracycline 

The plates showed no growth in the higher concentrations of 2,000 ng/mL and 3,000 ng/mL. The 1,000 ng/mL plate was covered in growth. This indicates that the MIC could be 2,000 ng/mL since the duplicates showed the same results. It was all consistent but we cannot say that 2,000 ng/mL is the MIC until we see if the concentration killed the bacteria or simply inhibited the growth. For this we would need to inoculate a sample from both the 2,000 ng/mL plate and the 3,000 ng/mL plate. We could potentially do this next week to see if this is the case. 

Results of D. roseus + Kanamycin, Methicillin, and Ofloxacin 

D. roseus Absorbance Values

Concentration	Kanamycin	Methicillin	Ofloxacin
100 μg/mL	0.11	0.00	0.02
50 μg/mL	0.02	0.07	0.09
25 μg/mL	0.01	0.03	0.06
12.5 μg/mL	0.02	0.00	0.03
6.25 μg/mL	0.03	0.11	0.05
3.125 μg/mL	0.01	0.05	1.76
1.56 μg/mL	0.41	0.21	0.08

Absorbance values of the antibiotic testing on D. roseus 

It seems as though  the bacteria responded to all three of the antibiotics. In order for us to continue our testing, we will have to retest the ofloxacin tube at concentration 1.56 μg/mL because there was a mistake when we were going through our protocol. Bacteria was not added into the tube so this makes the value that we got for that tube invalid. We will be redoing it to see what the actual result of it is.