New Phase of My Project

In the beginning of this week, I started to transform my bacteria (E.Coli and Agrobacteria) again because I was not able to transform my Agrobacteria last week. On Monday, I did transform bacteria and follow the same procedures as I did last week which I couldn’t transform to Agrobacteria again. On Tuesday, Josh told me to transform my Agrobacteria again, but this time I should place my microtubes which contained the + pGLO and - pGLO tubes to a 95 degree C water bath for exactly 10 minutes instead of 42 degree C water bath for exactly 50 seconds. Then, I returned the tubes to a container of crushed ice and cultured my bacteria in agars, to later incubate them ina temperature of 37 degree C. On Wednesday, the first thing I did was to observe my Agrobacteria which I cultured on Tuesday and Monday. When I checked my agars from Monday with a UV light, nothing glowed, but transforming of E.coli was successful and it glowed and I exposed my petri dishes. The worst part is nothing did grow in my agars when I transformed them on Tuesday, but I put them back to the incubator. Today, after almost 48 hours I check my petri dishes. Unfortunately, petri dishes were clear and nothing did grow except that I saw two tiny colonies of bacteria on - pGLO LB/amp agar and I decided to keep it to grow more bacteria because I want to identify it with gram staining method next week. 

 According to the evidence, probably when I put my microtubes to a 95 degree C water bath for exactly 10 minutes. I did kill all of my Agrobacteria but we (Josh and I) decided to put some Agrobacteria to a 95 degree C water bath for 10 minutes again. Then, extract DNA to see that any Agrobacteria remain. In this way I’ll learn how to extract DNA and with the result of Bacterial DNA Extraction, we can tell why Agrobacteria didn’t grow. 

The good thing that happened today was the Vibrio Fischeri bacteria that josh ordered for my project arrived. I was so excited and started working with bioluminescence bacteria right away and leave DNA Extraction for next week. 

 

At first, I checked my Vibrio Fischeri tube in total darkness for 10 minutes to observe bioluminescence. Then, I started to transfer the culture that I did in the Biological cabinet, because this culturing should be done in a disinfected place. I cultured Vibrio (photobacterium) Fischeri into the two photobscterium agars that the company sent to us, and one luminescent agar which I made two months ago. At the end, I placed my tubes in the incubator at 23 degree Celsius.