Week 3 (9/19/22 - 9/23/22): MIC Test 2!

Introduction:

All antibiotics belong to one of the five mechanisms of action: cell wall synthesis inhibitor, protein synthesis inhibitor, nucleic acid synthesis inhibitor, cell membrane disruption, and cellular respiration inhibitor. The antibiotic of choice for Deinococcus aquaticus, kanamycin, is a protein synthesis inhibitor that belongs to the antibiotic class called the aminoglycosides. This antibiotic binds to the 30S subunit of the ribosome, causing misalignment between the ribosome and the messenger RNA. This leads to the mRNA being misread and incorrect amino acid production, making it impossible for proteins to be synthesized. Kanamycin typically treats gram-negative bacteria, with the main bacteria of target being Mycobacterium tuberculosis.

The Minimum Inhibitory Concentration (MIC) test run on 09/15 was performed using 3 different antibiotics: kanamycin, tetracycline, and chloramphenicol. Kanamycin and chloramphenicol were tested with the same concentrations of antibiotic, while tetracycline was tested with a much smaller concentration. This was done at smaller concentrations due to the lack of D. aquaticus' response to tetracycline at concentrations between 100 ug/ml and 1.56 ug/ml. After 48 hours of incubation, the only test tube that showed growth was K7, containing kanamycin at a concentration of 1.56 ug/ml. These results support the MIC being between 3.125 ug/ml and 1.56 ug/ml, as well as kanamycin being the antibiotic of choice for D. aquaticus. To find a more precise MIC value on kanamycin, another test will be done on 6 different antibiotic concentrations with values between 3.525 ug/ml and 1.56 ug/ml.

 

Methods: 

 

D. aquaticus has been inoculated and stored in a 100 ml flask in the 30^o C refrigerator. OD600 value for this D. aquaticus sample is 2.37.

 

250 ml of TGY media was prepared on 09/20. The TGY media was added to each of the test tubes prior to starting the experiment. The specific amounts of media added to each tube are shown in figure 1. Both the 250 ml and the test tubes full of TGY media were autoclaved overnight on 9/20.

 

The 2 mg/ml stock solution of Kanamycin was prepared by Chad on 09/15 and stored in the 30^o C refrigerator.

 

On 09/21, the autoclaved test tubes were lined up to prep for the MIC to be run. Kanamycin was retrieved from the refrigerator and was added to each of the test tubes using the P20 and fisher-brand micropipette. Amount of antibiotic added to each test tube is shown in figure 1. Each test tube was mixed 10 times using the P1000 pipette. After mixing, 50 ul was removed from each test tube and deposited in a waste beaker. 50 ul of D. aquaticus was added to each test tube, and each test tube was then flicked 10 times to mix.  50 ul of D. aquaticus was also added into the positive control tube.  All 14 test tubes were then placed in the shaking incubator at 30^o C for 48 hours to ensure a proper growth curve. At the 24- and 48-hour mark, all the test tubes were removed from the incubator and read on the spectrophotometer at 600 nm to find the absorbance values. The spectrophotometer was blanked with the negative control, which only contains TGY media. Before reading the test tubes on the spectrophotometer, each tube was placed on the vortex mixer for 10 seconds at level 4.

 

 

Run	Volume of Antibiotic Added (ul)	Volume of Media Added (ml)	Concentration of Antibiotic (ug/ml)
1	8.812	4.992	3.525
1 A	7.812	4.992	3.125
2	6.833	4.993	2.733
3	5.85	4.994	2.34
4	4.875	4.995	1.95
5	3.9	4.996	1.56

Figure 1: Amounts of antibiotic and media added into each tube to get the final antibiotic concentration. 

 

Results: 

 

The absorbance values in all six runs displayed zero bacterial growth after 24 hours of incubation. The absorbance values after 48 hours were all low values, with the most significant absorbance readings being 0.37 and 0.34.

 

	Contents	Absorbance After 48 Hours
Positive Control	5 ml TGY media	1.94
Negative Control	4.950 ml TGY media, 50 ul D. aquaticus	0.23

Figure 2: Absorbance of each of the controls used in the MIC experiment, along with the contents of each control.

 

Run	Concentration of Antibiotic (ug/ml)	Trial 1 Absorbance After 24 Hours	Trial 2 Absorbance After 24 Hours	Trial 1 Absorbance After 48 Hours	Trial 2 Absorbance After 48 Hours
1	3.525	0.01	0.02	-0.12	-0.12
1 A	3.125	0.01	0.00	0.37	-0.12
2	2.733	0.02	0.02	-0.01	-0.04
3	2.34	0.03	0.04	-0.02	-0.45
4	1.95	0.05	0.03	0.17	-0.09
5	1.56	0.02	0.02	0.34	0.19

Figure 3: Absorbance of Kanamycin at each antibiotic concentration after 24 and 48 hours of incubation.

Figure 4: Graph of the relationship between the concentration of kanamycin and the absorbance.

Discussion:

 

Absorbance values were initially taken after tubes were incubated for 24 hours, and all the results from trial 1 and 2 displayed that there was no growth this early on. The only tube that showed growth was the positive control (containing bacteria and TGY) with an absorbance value of 1.21. After 48 hours of incubation, the test tubes were removed from the shaking incubator and read on the spectrophotometer again, resulting in low readings. Trial 5 was the only absorbance value that showed any growth. The absorbance value at an antibiotic concentration of 1.56 ug/ml taken during the previous week was 0.59, only 0.25 larger than the absorbance taken this week at the same concentration. This is not a significant enough value to determine the MIC of D. aquaticus using kanamycin, therefore, further MIC testing will be done.

 

The results for 1 A during trial run do not line up with the other absorbance values; 0.37 is not a large value but it is much higher than -0.12. This is likely due to contamination by an unknown bacterium that grows quicker than D. aquaticus. The absorbance values during run 2 are expected to be negative, making trial 1 A a non-accurate result.

 

When comparing these results to the MIC test run in the previous week, it leads me to believe that 48 hours may not be long enough for D. aquaticus to grow. The MIC results from last week changed after the 48-hour mark, so the absorbance values will be measured again at 120 hours and compared with the data already collected.