Week 2 (9/12/22 - 9/16/22): Minimum Inhibitory Concentration

Introduction:

D. aquaticus is an aerobic mesophile that is best described as rod-shaped. It stains pink on a gram stain, making it gram- negative with a thin layer of peptidoglycan in the cell wall. The Deinococcus genus is well studied, however, there is still much to learn about D. aquaticus and the antibiotic that it responds to. To analyze this, a test called the Minimum Inhibitory Concentration (MIC) is performed. The MIC test determines the lowest concentration of antibiotic that can be added to a solution to inhibit bacterial growth. This differs from the Minimum Bactericidal Concentration because the goal for the MIC is not to kill the bacteria, but to prevent bacteria from growing. MIC testing for D. aquaticus includes creating a dilution range of the chosen antibiotic and incubating at 30 degrees C.

In this MIC test, we used three different antibiotics that all act as protein synthesis inhibitors. Chloramphenicol (CM) is an antibiotic of its own class that inhibits the 50S ribosomal subunit from forming a peptide bond, preventing protein synthesis. CM was selected for the MIC test because it was the antibiotic used in the transformation of D. radiodurans, another member of the Deinococcus species. Kanamycin is an aminoglycoside that alters the shape of the 30S ribosomal subunit, causing the ribosome to read the mRNA code incorrectly. Finally, tetracycline is an antibiotic of its own class; it binds to the 30S ribosomal subunit preventing the tRNA from binding to mRNA.

To meet our goal of having a low concentration of antibiotic, each antibiotic solution was made into a 2mg/ml stock solution by Chad and stored in 30^o C.

Antibiotic	Diluent	Stock concentration
Chloramphenicol	Alcohol	34 mg/ml
Kanamycin	Water	10 mg/ml
Tetracycline	Alcohol	5 mg/ml

Figure 1: Stock concentrations of each of the three antibiotics being tested with their associated diluents.

 

Methods: 

D. aquaticus was inoculated into liquid media and incubated at 30^o C for 24 hours. A gram stain was taken to prove the sample was not contaminated, and OD600 was obtained to determine the amount of cell growth. TGY broth was also prepared and autoclaved overnight on 09/07. TGY media was added to test tubes on 09/12 and autoclaved to ensure there was no contamination to the media, specific amounts of media added is shown in figure 2. 

To meet our goal of having a low concentration of antibiotic, each antibiotic solution was made into a 2mg/ml stock solution by Chad and stored in 30^o C.

The first MIC test done was performed on 09/07, testing only tetracycline with antibiotic concentrations of 100 ug/ml, 50 ug/ml, 25 ug/ml, 12.5 ug/ml, 6.25 ug/ml, 3.125 ug/ml, and 1.56 ug/ml. This MIC test resulted in absorbance values that were too low, meaning that the concentration of Tetracycline was too high in all 7 test tubes, inhibiting bacterial growth. This led to a redirection of our experiment: MIC was performed on Tetracycline with antibiotic concentrations of 3.125 ug/ml, 1.56 ug/ml, 0.78 ug/ml, 0.39 ug/ml, 0.195 ug/ml, and 0.0975 ug/ml, and Kanamycin and Chloramphenicol were done using our original spread of antibiotic concentrations varying from 100 ug/ml to 1.56 ug/ml, shown in figure 2. 

The tubes of media were lined up and grouped by the different types of antibiotics. The desired amount of antibiotic was then added to each test tube, specific amounts are listed in figure 2. Each test tube was then mixed using a P1000 pipette. After mixing, 50 ul was removed from each tube and placed in the contamination beaker. Then 50 ul of D. aquaticus was added to each test tube, and the tubes were mixed again using a P100 pipette. Test tubes were then placed in the shaking incubator for 48 hours to ensure there is a proper growth curve. After 48 hours, test tubes were removed from incubator and read on the spectrophotometer at 600 nm.

 

Antibiotic	Tube number	Volume of media added	Volume of antibiotic added	Concentration of antibiotic
Chloramphenicol	1	4.75 ml	250 ul	100 ug/ml
	2	4.875 ml	125 ul	50 ug/ml
	3	4.937 ml	62.5 ul	25 ug/ml
	4	4.968 ml	31.25 ul	12.5 ug/ml
	5	4.984 ml	15.625 ul	6.25 ug/ml
	6	4.992 ml	7.8125 ul	3.125 ug/ml
	7	4.996 ml	3.91 ul	1.56 ug/ml
Kanamycin	1	4.75 ml	250 ul	100 ug/ml
	2	4.875 ml	125 ul	50 ug/ml
	3	4.937 ml	62.5 ul	25 ug/ml
	4	4.968 ml	31.25 ul	12.5 ug/ml
	5	4.984 ml	15.625 ul	6.25 ug/ml
	6	4.992 ml	7.8125 ul	3.125 ug/ml
	7	4.996 ml	3.91 ul	1.56 ug/ml
Tetracycline	1	4.992 ml	7.8125 ul	3.125 ug/ml
	2	4.996 ml	3.91 ul	1.56 ug/ml
	3	4.998 ml	1.95 ul	0.78 ug/ml
	4	4.999 ml	0.975 ul	0.39 ug/ml
	5	5 ml	0.488 ul	0.195 ug/ml
	6	5 ml	0.244 ul	0.0975 ug/ml

Figure 2: Desired concentration of antibiotics, with associated media and antibiotic amounts that are added into each solution.

 

Results: 

The nanodrop readings obtained prior to the experiment showed sufficient growth of D. aquaticus. The MIC results for both chloramphenicol and tetracycline showed no growth in any of the tubes. The MIC results for kanamycin were more conclusive; there was growth seen in test tube 7 that had a antibiotic concentration of 1.56 ug/ml. These results were supported by the positive and negative control, which produced desirable results: positive control had an absorbance of 0.95, while the negative control had an absorbance of 0.23.

 

A600	Correction	λ	A(λ)
2.37	0.10	600	2.37

Figure 3: OD 600 results on D. aquaticus.

 

Antibiotic	Tube number	Concentration of antibiotic	Absorbance
Chloramphenicol	1	100 ug/ml	0.01
	2	50 ug/ml	0.06
	3	25 ug/ml	0.00
	4	12.5 ug/ml	-0.01
	5	6.25 ug/ml	-0.01
	6	3.125 ug/ml	0.03
	7	1.56 ug/ml	0.04
Kanamycin	1	100 ug/ml	-0.01
	2	50 ug/ml	-0.01
	3	25 ug/ml	-0.00
	4	12.5 ug/ml	-0.01
	5	6.25 ug/ml	-0.01
	6	3.125 ug/ml	-0.01
	7	1.56 ug/ml	0.59
Tetracycline	1	3.125 ug/ml	0.00
	2	1.56 ug/ml	-0.01
	3	0.78 ug/ml	0.01
	4	0.39 ug/ml	0.08
	5	0.195 ug/ml	0.04
	6	0.0975 ug/ml	0.02

Figure 4: Absorbance readings for each antibiotic concentration of chloramphenicol, kanamycin, and tetracycline.

 

Control	Contents	Contents	Absorbance
Negative control	Media only	5 ml media	0.23
Positive control	Media and bacteria	4.950 ml media, 50 ul D. aquaticus	0.95

Figure 5: Absorbance readings for negative control and positive control.

 

Discussion: 

The results for CM and tetracycline lead to the conclusion that D. aquaticus does not respond to these antibiotics at any given concentration. Additionally, the result on tetracycline is supported by the MIC run on 09/07 that showed zero growth at any concentration. The positive and negative control indicate the likelihood the experiment was done accurately. The positive control showed growth: the bacteria was able to grow well without antibiotics. The negative did not show growth: this shows that contamination did not happen with the TGY agar. 

Kanamycin was the only antibiotic that D. aquaticus responded to, making this aminoglycoside the only type of protein synthesis inhibitor to stop bacterial growth. There was growth in tube 7 and zero growth in tube 6, leading us to believe that the MIC for Kanamycin between concentrations 3.125 ug/ml and 1.56 ug/ml. For more accuracy, an additional MIC test will be run on kanamycin: there will be 6 test tubes with kanamycin concentrations of 3.125 ug/ml, 2.733 ug/ml, 2.34 ug/ml, 1.95 ug/ml, and 1.56 ug/ml.