Week 18 (2/20/23 - 2/24/23): Beginning Antibiotic Testing on D. grandis

Introduction:

 

Antibiotic testing with D. grandis is the main goal for this week. This will be helpful moving forward because it will give us an idea if any Deinococcus species respond to these 3 types of antibiotics, and at what concentration it will inhibit growth at.

 

 

Methods:

 

Plates were made from our samples of 25 ml of 1.5% TGY that were in 10 different 125 ml flasks. The flasks were individually heated over a hot plate and removed when all the media was liquified. After the samples were cooled enough to pass the baby bottle test, TGY was removed from each flask so that the final volume in each flask after the tetracycline is added is 25 ml. Tetracycline was then added into each flask and it was swirled to mix. Exact amounts of TGY removed and antibiotic added to each flask  are shown in figure 1. Then the flasks were each poured over a plate and left out overnight so the media could fully solidify. 2 of these samples were prepared for another student using chloramphenicol at a concentration of 3 ug/ml.

 

Tetracycline concentration	TGY removed from each flask	Tetracycline added to each flask
3000 ng/ml	1.5 ml	1.5 ml
2000 ng/ml	1 ml	1 ml
1000 ng/ml	0.5 ml	0.5 ml
0 ng/ml	0 ml	0 ml

Figure 1: Amount of TGY removed and tetracycline added to each flask, depending on the antibiotic concentration.

 

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 2. Before moving on, the sample of D. grandis needed to be diluted because the original OD600 value of our sample was 1.87. This was done by adding 900 ul of D. grandis to 1100 ul of TGY. The OD600 reading after this dilution was 0.75. The antibiotic samples of kanamycin and methicillin needed to be diluted from 10 mg/ml to 2 mg/ml, so 4 ml of each sample was added to 6 ml of autoclaved water. Oflaxacin was already at a low concentration of 1 mg/ml, so the amount of media and antibiotic in these samples differed from the other 2 antibiotics.

 

Antibiotic concentration	TGY in methicillin and kanamycin samples	TGY in ofloxacin samples	Amount of antibiotic added to methicillin kanamycin samples	Amount of antibiotic added to oflaxacin samples
100 ug/ml	4.75 ml	4.5 ml	250 ul	500 ul
50 ug/ml	4.875 ml	4.75 ml	125 ul	250 ul
25 ug/ml	4.937 ml	4.875 ml	62.5 ul	125 ul
12.5 ug/ml	4.968 ml	4.938 ml	31.25 ul	62.5 ul
6.25 ug/ml	4.984 ml	4.969 ml	15.625 ul	31.25 ul
3.125 ug/ml	4.992 ml	4.984 ml	7.8125 ul	15.625 ul
1.56 ug/ml	4.996 ml	4.992 ml	3.91 ul	7.8125 ul

Figure 2: Amount of antibiotic and TGY in each test tube. Amounts are dependent on the antibiotic concentration.

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. grandis 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 4.    

Figure 3: Outline of antibiotic testing on D. grandis. 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:

 

Antibiotic concentration	Absorbance with methicillin	Absorbance with kanamycin	Absorbance with ofloxacin
100 ug/ml	0.316	0.592	0.435
50 ug/ml	0.662	0.847	0.447
25 ug/ml	0.774	0.707	0.576
12.5 ug/ml	0.582	1.57	0.497
6.25 ug/ml	1.21	1.68	0.929
3.125 ug/ml	1.37	1.36	1.04
1.56 ug/ml	1.39	1.44	1.17

Figure 4: Absorbances of D. grandis 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.

 

 

Discussion: 

 

The results from this test are not as conclusive as I would have hoped. My goal for this experiment was to have  the test tubes at the lowest antibiotic concentration having no growth, giving a range for what we should look for when we move forward to the minimum inhibitory concentration testing. We may want to repeat this with D. grandis at even smaller antibiotic concentrations, or move forward with different antibiotics all-together. 

 

The results do support that the procedure was performed correctly, because for each of the three antibiotics used, the absorbance increased s the antibiotic concentration decreased.