Week 18: February 27, 2023 - March 3, 2023

 Monday, February 27, 2023 

MIC Testing with D. aquaticus 

To continue our MIC testing on D. aquaticus, we prepared 8 test tubes with metal caps with 2.5 mL of TGY with 0.5% agar. They were autoclaved for 30 minutes in order to be ready to be used for soft agar plates. These soft agar plates will be used to test the higher concentrations of kanamycin on D. aquaticus. The concentrations will be 1000 ng/mL, 2000 ng/mL, and 3000 ng/mL, and our negative control which will not include kanamycin. Each concentration will have a duplicate which brings our total plate count to eight. 

Inoculating D. indicus

We prepared to begin working with D. indicus. Since we plan on working with all of the Deinococcus species that we have available in the lab, we decided to continue our MIC testing with D. indicus. We took the OD600 value of our culture that we had growing in broth from 2/9/2023. A range that we aim for when it comes to OD600 values is anywhere between 0.80 - 1.00. The value for this sample was 0.63. It had been growing in the broth for several weeks and the value seemed low so we decided to inoculate a new sample. An OD600 value of D. grandis was also taken and it had a value of 1.80. 

MIC testing on D. grandis 

Last week, on one of the days that I was not present in lab, my lab partners performed our MIC protocol on D. grandis using three different antibiotics. They decided to work with D. grandis instead of D. indicus because it had significantly more growth. They diluted a little bit of the sample of D. grandis to get it within our desired range of 0.80 - 1.00. This included pipetting 5 ml of TGY into 13 x 100 mm test tubes with metal caps, autoclaving the TGY, removing TGY, adding in antibiotic, and adding in bacteria. The tubes were placed in the incubator at 30°C for 48 hours. Then, a spectrophotometer reading was taken for the different concentrations of D. grandis. 


Concentration 

Kanamycin

Methicillin

Ofloxacin

1.56 μg/ml

1.44

1.39

1.17

3.125 μg/ml

1.36

1.37

1.04

6.25 μg/ml

1.68

1.21

0.929

12.5 μg/ml

1.57

0.58

0.497

25 μg/ml

0.707

0.774

0.576

50 μg/ml

0.847

0.662

0.447

100 μg/ml

0.592

0.316

0.435


February 28, 2023 

It had now been 5 days since the MIC protocol was done on D. grandis using the antibiotics Kanamycin, Methicillin, and Ofloxacin. Another spectrophotometer reading was done to see if the results had changed since we last did a reading. We vortexed the tubes on level 4 for 6 seconds. This time, we did not vortex on level 6 because then we would risk the liquid spilling out of the tube. 


Concentration 

Kanamycin 

Methicillin 

Ofloxacin 

1.56 μg/ml

2.24

1.80

2.36

3.125 μg/ml

2.50

1.76

2.06

6.25 μg/ml

2.36

1.41

1.84

12.5 μg/ml

1.99

0.07

0.07

25 μg/ml

2.24

0.03

0.13

50 μg/ml

1.11

0.04

0.07

100 μg/ml

0.04

0.09

0.08


                                                                D. aquaticus MIC testing tubes 

                                                                           D. aquaticus MIC testing tubes 

OD600 Values of New Species

Earlier on in the semester, Jonathan had inoculated new species for us from freeze bacs. He had inoculated D. indicus, D. grandis, and D. roseus. We had them growing in broth so that we could begin working on them to find their MIC. They had each been growing in TGY for a while so we decided to check up on them and take their OD value along with D. aquaticus. 


Species 

OD600 Value 

D. aquaticus 

1.19

NEW D. indicus 

0.07

OLD D. indicus

0.61

D. roseus

0.84

D. grandis 

1.66


*Since we have two samples of D. indicus, we took the OD600 value of both of them* 

Inoculation of Four New Species

On this day, Jonathan also went ahead and inoculated four new species of Deinococcus from freeze bacs so that they could begin growing. We were told they were slower growers than the other species so it would be okay to begin growing them rather than waiting until we wrapped up our MIC testing with D. aquaticus, D. indicus, D. roseus, and D. grandis. 

New species inoculated onto TGY agar plates:

  • D. caeni

  • D. aquatilis

  • D. gobiensis 

  • D. deserti


                                                        New species inoculated onto agar plates





References 

Podstawka, A. (n.d.-a). Deinococcus aquaticus PB314 | Type strain | DSM 18376, KCTC 12552, NBRC 101311 | BacDiveID:3882. Bacdive.dsmz.de. https://bacdive.dsmz.de/strain/3882

Podstawka, A. (n.d.-b). Deinococcus aquatilis MIFE 35041/06 | Type strain | DSM 23025, CCM 7524, CCUG 53370 | BacDiveID:3891. Bacdive.dsmz.de. Retrieved April 25, 2023, from https://bacdive.dsmz.de/strain/3891#:~:text=Deinococcus%20aquatilis%20MIFE%2035041%2F06%20is%20an%20aerobe%2C%20mesophilic%2C

Podstawka, A. (n.d.-c). Deinococcus caeni Ho-08 | Type strain | DSM 18373, KCTC 12553, NBRC 101312 | BacDiveID:3881. Bacdive.dsmz.de. Retrieved April 25, 2023, from https://bacdive.dsmz.de/strain/3881

Podstawka, A. (n.d.-d). Deinococcus deserti VCD115 | Type strain | DSM 17065, CIP 109153, LMG 22923 | BacDiveID:3863. Bacdive.dsmz.de. Retrieved April 25, 2023, from https://bacdive.dsmz.de/strain/3863

Podstawka, A. (n.d.-e). Deinococcus gobiensis I-0 | Type strain | DSM 21396, CGMCC 1.7299, JCM 16679 | BacDiveID:3893. Bacdive.dsmz.de. Retrieved April 25, 2023, from https://bacdive.dsmz.de/strain/3893#:~:text=Deinococcus%20gobiensis%20I%2D0%20is

Podstawka, A. (n.d.-f). Deinococcus grandis KS 0485 | Type strain | DSM 3963, ATCC 43672, IAM 13005 | BacDiveID:3847. Bacdive.dsmz.de. https://bacdive.dsmz.de/strain/3847

Podstawka, A. (n.d.-g). Deinococcus indicus Wt/1a | Type strain | DSM 15307, MTCC 4913 | BacDiveID:3862. Bacdive.dsmz.de. Retrieved April 25, 2023, from https://bacdive.dsmz.de/strain/3862

Podstawka, A. (n.d.-h). Deinococcus roseus TDMA-uv51 | Type strain | DSM 22367, CCUG 53611, JCM 14370, NBRC 102117 | BacDiveID:3890. Bacdive.dsmz.de. Retrieved April 25, 2023, from https://bacdive.dsmz.de/strain/3890

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