Week 20 (03/06/23 - 03/10/23): New Plan For Working With D. grandis

Introduction: 

 

Because our D. grandis results from 2 weeks ago showed that this bacterium was not resistant at antibiotic concentrations between 1.56 ug/ml and 100 ug/ml for all 3 antibiotics, we are shifting our focus to trying to classify the MIC for the 3 antibiotics we are testing on with an additional 3 antibiotics we have not worked on yet. The goal is to see if D. grandis has a higher MIC for all the antibiotics on the board in comparison to the other Deinococcus species.

 

Methods:

 

400 ml of TGY was prepared following the 3:3:1 method and was autoclaved overnight.

 

Gram staining was performed on 3/8 on select species shown in figure 3, and OD values were taken on 6 different species of Deinococcus on 3/10.

 

18 test tubes were filled with TGY and autoclaved for 30 minutes. The next day, TGY was removed from each tube to equate to the amount listed next to each test tube in red and purple ink in figure 1. Antibiotic was then added to each test tube, exact amounts added are shown in figure 1. Then each tube was placed on the vortexer level 6 for 6 seconds, and 50 ul was removed from each tube and deposited in the waste beaker. The D. grandis sample had an OD that was too high (1.5) so we diluted it with TGY to have a final OD value of 0.91. Then 50 ul of D. grandis was added to each tube. Each tube was vortexed on level 6 for 6 seconds, and the tubes were placed in the shaking incubator at 30^o C. 

Figure 1: Outline of antibiotic testing on D. grandis. 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 ofloxacin is different from the stock concentration of kanamycin and methicillin, ofloxacin amounts are written in purple ink instead of red.

 

 

Results:

 

Species	A600	Correction	λ	A(λ)
D. roseus	0.22	0.10	600	0.22
D. canei	0.16	0.10	600	0.16
D. aquatilis	0.42	0.10	600	0.42
D. grandis	1.71	0.10	600	1.71
D. gobi	0.23	0.10	600	0.23
D. indicus	0.24	0.10	600	0.24

 Figure 2: OD600 values of the 6 species of Deinococcus listed.

 

Figure 3: Gram stains performed on D. grandis (left), D. aquatilis (center), and D. roseus (right). 

 

 

Antibiotic concentration	Absorbance with methicillin	Absorbance with kanamycin	Absorbance with ofloxacin
300 ug/ml	-0.02	0.06	0.01
250 ug/ml	-0.03	-0.01	-0.03
200 ug/ml	0.00	0.01	-0.02
150 ug/ml	0.02	-0.01	-0.03
100 ug/ml	-0.03	0.00	0.01
50 ug/ml	0.08	0.11	-0.03

Figure 4: Antibiotic testing results with D. grandis with all 3 types of antibiotics we are working with.

 

Discussion: 

 

Based on the antibiotic testing results, I would believe the MIC for D. grandis to be between 50 ug/ml and 100 ug/ml for kanamycin and methicillin. The MIC with ofloxacin would be much lower due to our negative absorbance reading. I am unsure why our results do not match up with the results from the previous week, but I did not personally take those absorbance values so I cannot speak on how correct they are. Repeating the procedure with values from 150 ug/ml to 12.5 ug/ml will be necessary to fully identify where bacterial growth was inhibited.