Week 19 (02/27/23 - 03/03/23): Continuation of MIC/Antibiotic Testing

 Introduction: 

The goal for this week is to complete our protocol for the MIC testing with D. aquaticus and start a new procedure with D. roseus to identify if it will respond to methicillin, kanamycin, or ofloxacin. We are repeating the same procedures we have done in the previous weeks with both of our tests performed. The main outcome we are hoping for is to identify the MIC for D. aquaticus, and to see what antibiotics work with D. roseus to see if there are similarities with the previous Deinococcus species that underwent antibiotic testing: D. aquaticus and D. grandis.

 

Methods: 

 

MIC testing with D. aquaticus:

8 test tubes containing TGY with 0.5% agar were autoclaved and set out to cool. The 8 test tubes consisted of: 1 negative control tube, 1 positive control tube with no antibiotic added, 2 3000 ng/ml tubes, 2 2000 ng/ul tubes, and 2 1000 ng/ul tubes. The 2 mg/ml stock solution of tetracycline was vortexed on level 6 for 6 seconds, then added to all tubes besides the negative and positive controls. Exact amount of antibiotic added is shown in figure 1. The tubes were vortexed on level 6 for 6 seconds and poured over their designated plate with the same tetracycline concentration, prepared in the previous week.

Tetracycline concentration	Amount of antibiotic added
3000 ng/ml	3.75 ul
2000 ng/ml	2.5 ul
1000 ng/ml	1.25 ul
0 ng/ml (negative control)	0 ul
0 ng/ml (positive control)	0 ul

Figure 1: Amount of tetracycline added to each test tube to achieve the desired tetracycline concentration shown in the column on the left.
 
5 ml of TGY was added into 21 test tubes, and the tubes were autoclaved for 30 minutes. Following the autoclave, TGY was removed from each tube. This was done so that the final volume in each test tube after the antibiotic was added was 5 ml. Specific amounts of TGY that were in each tube are shown in figure 1. Before moving on, the sample of D. roseus needed to be diluted because the original OD600 value of our sample was 1.76. This was done by adding 800 ul of D. roseus to 1100 ul of TGY. The OD600 reading after this dilution was 0.9.

To start the procedure, antibiotic was added to each tube. Specific amounts of antibiotic added to each test tube are shown in figure 2. The test tubes were placed on the vortexer on level 6 for 6 seconds. Then 50 ul was removed from each tube using a P200 pipette. 50 ul of D. roseus was added to each test tube, and the test tubes were placed on the vortexer at level 6 for 6 seconds. The tubes were placed in the shaking incubator at 30^o C for 48 hours. After incubation, the tubes were read on a spec200 at a wavelength of 600 nm. Results of antibiotic testing are shown in figure 2.    
 

Figure 2: Outline of antibiotic testing on D. roseus. Amount of antibiotic added to each tube is marked over the arrows pointing to the tubes at each antibiotic concentration. Because the stock concentration of oflaxacin is different from the stock concentration of kanamycin and methicillin, ofloxacin amounts are written in purple ink instead of red.

Results: 
 
MIC testing with D. aquaticus:

Figure 3: Results of MIC testing on D. aquaticus with tetracycline. The antibiotic concentrations decrease from left to right: 1000 ng/ml, 2000 ng/ml, and 3000 ng/ml.
 

Antibiotic testing with D. roseus:

Antibiotic concentration	Absorbance with methicillin	Absorbance with kanamycin	Absorbance with ofloxacin
100 ug/ml	0.00	0.11	0.02
50 ug/ml	0.07	0.02	0.09
25 ug/ml	0.03	0.01	0.06
12.5 ug/ml	0.00	0.02	0.03
6.25 ug/ml	0.11	0.03	0.05
3.125 ug/ml	0.05	0.01	1.76
1.56 ug/ml	0.21	0.41	0.08*

Figure 4: Absorbances of D. roseus after being treated with either methicillin, kanamycin, or ofloxacin. The antibiotic concentrations were performed in the same range (100 ug/ml – 1.56 ug/ml) for each of the 3 antibiotics used. Absorbances were read on the spectrophotometer at 600 nm.
 

 

Discussion: 
 
The MIC testing on D. aquaticus appears to show that the MIC could potentially 2000 ng/ml. This will need to be confirmed next week by inoculating both 2000 ng/ml and 3000 ng/ml samples onto TGY plates to see if there is growth. If there is only growth from the 2000 ng/ml plates, that would mean that this is the MIC because the bacterial growth was inhibited on the plates because of this tetracycline concentration,  but it was still able to grow on a new plate without any antibiotics present. If there is growth on both the plates containing D. aquaticus that was grown at tetracycline concentrations of 2000 ng/ml and 3000 ng/ml, this could mean that the MIC is between these two values. If both of these values work to inhibit growth, I don’t see why either would work when it comes to future testing with D. aquaticus, but it may be beneficial to test concentrations between the two values for a more precise MIC value. 

 
In the antibiotic testing on D. roseus, it seems that the bacteria responded to all three antibiotics. There was an error made on the ofloxacin tube at 1.56 ug/ml concentration where bacteria was not added to the tube, so this may alter the results. To identify the absorbance at this concentration, the same test will be repeated with ofloxacin at concentrations of1.56 ug/ml, 3.125 ug/ml, and 6.25 ug/ml to properly identify that there was growth at the lowest antibiotic concentration. When it comes to all of the results that did not grow, we will inoculate the samples where growth stopped for each antibiotic and the samples at the next increasing concentration to see if the bacteria was either inhibited or killed. To provide more exact results, all of the antibiotics will be tested with D. roseus and the bacteria previously tested. This will be performed using a microplate and a microplate reader, so we can visualize the growth curves and accurately determine the MIC.