Week 8 (10/24/22 – 10/28/22): Continuing the Growth

 This week was focused on preparing our bacteria so that we can have our bacteria undergo transformation once more -- hopefully this time with results! One thing that is interesting about doing research in a classroom setting, especially one where materials are shared by larger amounts of people, is that every once in a while, I will need to improvise, whether with scheduling or procedures that are still accurate and get the job done. 

One instance of this happened this past week. We had used a freezeback of E. coli that held the smaller plasmid that we will be using for transformation (this one, pRad1, is approximately 6,000 base pairs larger rather than the larger plasmids that are closer to 10,000 that we've been using) to incubate and have our own plates of the same bacteria. By inoculating (and hopefully having growth), we would use these plates to have E. coli (with the pRad1 plasmid) for future use. 

Now, when we inoculated the bacteria last week, we knew that we weren't going to be able to check on their growth until this past week, so we ended up inoculating into the 2X LB solution in a freezer that had a lower temperature. By doing this, growth of the E. coli would happen more slowly (this way, we wouldn't have overgrowth and too much on a plate to work with). Because of this, we did have the amount of growth that we were looking for. With this, we needed to ensure that we did have the correct bacteria, so we had to do a gram stain. However, something interesting needed to be done: Two of the bars that we used to set our plates on were missing (they may have been in another classroom that needed them for the same type of procedure), so I found a test tube rack that I cleaned to prepare for our gram stains and used as a makeshift holder. 


This setup did work in the end; We were able to do our gram stains and check to see if we had the correct bacteria. What was interesting to learn was that when I checked the first slide (slide A), I saw that the bacteria were purple, which indicates gram positive bacteria. 



However, E. coli are gram negative and are supposed to show up as pink, so I checked the other slides -- they were pink (as was expected): 





I also did notice that, in looking at Slide C that there was purple toward the edge, which helped me to realize that I might have not done the gram stain procedure correctly for slide A. Because of this, I rechecked Slide A and did see plenty of E. coli that had the correct pink coloring (indicating gram negative bacteria): 



In the end, we had successful growth and could proceed to our next steps (to be announced)!

Comments

  1. EricaNovember 4, 2022 at 8:56 AM

    Yay! A good week in the lab is always encouraging! Hope it continues.

    ReplyDelete

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