Week 15 (01/23/23 - 01/27/23) Final MIC testing on D. aquaticus

 Introduction:

Before performing a procedure like transformation for a specific organism, it is essential to know what antibiotic that organism responds to, and the concentration of that antibiotic needed. When working with D. aquaticus, it was determined last semester that kanamycin is the best antibiotic to use to inhibit bacterial growth. Even though kanamycin is the ideal choice, we run into an issue when it comes to the plasmids that will be used in transformation. I believe we are working with the plasmid pwtCas9, which has an ampicillin resistance gene and a tetracycline resistance gene. Plasmid map for pwtCas9 is shown in figure 1. Because it does not have a kanamycin resistance gene, it is best to move forward with an antibiotic that the plasmid contains a resistance gene for. In this case, for our project we will be moving forward with tetracycline.


 Figure 1: Plasmid map for pwtCas9.


The minimum inhibitory concentration (MIC) is the minimum amount of antibiotic needed to inhibit bacterial growth. This differs from the minimum bactericidal concentration (MBC), which is the minimum amount of antibiotic needed to kill all bacterial cells. We are working with the MIC to determine the correct amount of antibiotic to add to our plates during a transformation, so it will only allow bacterial cells that are transformed to grow. Only transformed cells will grow because they correctly received the plasmid that contains the antibiotic resistance gene. We performed MIC testing on D. aquaticus with tetracycline at the end of last semester, but due to a contamination issue, our results were not sufficient enough to prove the MIC. The goal for this week and the next is to finish our procedure so we can accurately determine the MIC using tetracycline.

 

Methods:

 

Gram staining on D. aquaticus was done on 01/25 with lab partner  Jayce Thompson and viewed on the oil immersion lens.

 

200 ml of TGY was prepared using the 3:3:1 method; 0.6 g of tryptone, 0.6 g of yeast and 0.3 g of dextrose were measured in a weigh boat and poured into a 250 ml cylinder. Parafilm was placed on top and the cylinder was shaken to mix all of its contents. Then 150 ml was added from the cylinder to a 500 ml flask. The remaining 50 ml from the cylinder was added to a 250 ml flask. Then 2.25 g of agar was added to the 500 ml flask to make 1.5% agar, and 0.25 g of agar was added to the 250 ml flask to make 0.5% agar. Both flasks were placed in the autoclave for 30 minutes. After autoclaving, we started with the 1.5% agar first that contains 150 ml of TGY. 25 ml of TGY was added to 4 separate test tubes. Moving on to the 0.5% agar that contains 50 ml of TGY, 2.5 ml of TGY was added to 4 separate test tubes. All of the test tubes were placed in the autoclave again for 30 minutes. 

 

After determining the quality of each gram stain, sample 1 was used to make a new inoculation of D. aquaticus. The bacterial sample was inoculated into a 125 ml flask containing 10 ml of TGY. The inoculation was performed using an inoculating loop and a Bunsen burner. The new sample was then placed in the 30o C incubator to allow for growth. 

 

Planning for the MIC was done to prepare for next week’s work. The experimental design is shown in figure 2.



Figure 2: MIC planning for working with tetracycline and D. aquaticus.



Results:

Figure 3: Gram staining results done on D. aquaticus samples that were inoculated on 12/09, viewed on the oil immersion lens. Sample 1 is pictured on the left, sample 2 is pictured on the right.

 

 

Discussion: 

 

Based on the gram stains, sample 1 is the one that we decided go forward in using for our MIC experiment. This is because the cells on sample 1 seem to be the same morphology, despite the cells appearing to be gram positive and negative. Because D. aquaticus stains gram positive and negative, we can conclude that the color of the cells does not determine that the sample is contaminated. Sample 2 has a similar appearance with coloration, however there is a bright pink “spot” near the center of the gram stain. Because we cannot identify what it is, it’s safe to assume the sample is contaminated and should not be used in our MIC testing.

 

Because we started on our project in the middle of the week, most of the work done in lab was setting up the experiment and preparing TGY. This means that we do not have any other results for this week. However, we are finished with all the autoclaving of TGY so on Monday we can start right up with making plates and preparing the soft agar. The goal is to have the plates made and inoculated with soft agar by Wednesday, so Friday we will be able to determine the MIC and move forward with MIC testing with the other species of Deinococcus. 

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