Sp'23: Week 3 (1/30/23 - 2/3/23): Growth & Extraction

This week's focus was on making sure growth was obtained with E. coli that has the pRad1 plasmid so that I could do extractions. It started out with the help of a lab-mate who, on Monday, was able to inoculate this particular E. coli from my plates into the 2X LB that I made last week (and since I had incorrectly inoculated E. coli with the pRadZ1 plasmid onto the plates initially, he inoculated these E. coli into two sets of 2XLB as well). On Tuesday, he transferred the four sets of E. coli from the 2X LB into regular [1X] LB (resulting in four total 50mL centrifuge tubes: two of which held E. coli with the pRad1 plasmid, and two of which held the E. coli with the pRadZ1 plasmid). 

However, when I did a gram stain on each of the four LB tubes on Wednesday, I found contamination in all of them: 

*From top to bottom are the clearly-contaminated E. Coli tubes with pRad1 (Tube A), pRad1 (Tube B), pRadZ1 (Tube A), and pRadZ1 (Tube B). 

E. coli is gram negative and should be pink after being gram stained. This is because gram staining results in organisms that are colored either pink or purple/blue, due to the process of gram staining equivocating with the thickness of the bacteria's peptidoglycan layer (bacteria with a thick peptidoglycan layer will be stained blue/purple, while bacteria with a thin peptidoglycan layer will be stained pink). E. coli, with its thin peptidoglycan layer, thus should show up pink. Another thing I noticed is that the morphology of these assumed contaminants did not have the correct morphology that E. coli should have (which should be long [pink] rods). A sample photo of what gram stained E. coli should look like was pulled from the internet and is shown below: 

Since there was contamination in each of the four tubes, I hypothesized that the 2XLB, which was used to inoculate into the regular LB tubes, was contaminated, as contamination with 2XLB happened last semester (and especially because 2XLB contains ample concentrations of media that can allow other sets of microorganisms to grow). So, I took a gram stain of the 2XLB that I had, and it did seem that this broth was contaminated as there were microorganisms that stained gram-positive and gram negative, where the 2XLB broth should have been clean and shown no growth at all: 

Even though it was more likely to me that the 2XLB was the contaminant culprit (due to its doubled concentration of media that allows for growth), I should have gram stained my regular LB broth, too. 

Now, at the suggestion of my lab-mate, rather than doing a plasmid extraction from the contaminated tubes, I went ahead and did a new inoculation straight from the original two LB + Amp (mic concentration of 50 ug/mL) plates that held E. coli with pRad1 into two brand new tubes with regular LB broth (without the mic concentration of 50 ug/mL of ampicillin). The thought (or hope) was that this might allow just the E. coli with pRad1 that were in the contaminated tubes to grow: 

*Seen above are the original two LB + Amp (mic concentration of 50 ug/mL) plates that I inoculated from Freezeback (with E. coli + pRad1) along with the new tubes of [assumed] clean LB broth I inoculated into. 

He also suggested I take one of the two tubes and inoculate onto a clean LB Agar plate, so I used Tube 1A to inoculate its E. coli with pRad1 onto a new LB Agar plate. 

The next day, my lab-mate ended up doing a nanodrop reading on these new two LB broth tubes that held the E. coli with pRad1, this way when it was time for me to do hopefully do a plasmid extraction the next day (assuming the tubes were not contaminated, which was not actually the case), I knew what OD600 readings / bacterial concentrations I was working with: 

The next day, after being shown the OD600 readings from my two newly-inoculated tubes, I did a gram stain on them along with a gram stain from the newly-inoculated LB plate. What was odd was that the two tubes that had been inoculated from the original freezeback plates were contaminated (which leads me to think that perhaps my regular LB is contaminated, which I should check for), but that the plate, which had the contaminated E. coli + pRad1 inoculated onto, showed clean E. coli: 

*The two plates above show my newly-inoculated LB tubes with E. coli + pRad1, both of which show contamination. 

*The plate above shows the E. coli (hopefully one that still has pRad1) that was grown on a new LB plate. This is what non-contaminated E. coli (and what all of my other gram stains) should look like. 

Now, without having gram stained my regular LB broth, it is otherwise difficult to say why all of the E. coli gram stained thus far from LB broth was contaminated while the E. coli grown on an LB plate was clean. Yet, I moved forward with the process of plasmid extraction on both of my new tubes by using the ZymoPURE kit, just to see if it still held the E. coli with pRad1 (thus attempting to extract pRad1 from these contaminated tubes). I had to do extraction on each of the two tubes twice, as I messed up the first time (forgetting toward the end to discard the flow-through, which caused the Washes to sit in microcentrifuge tubes that were too full, and which I'm sure affected when they were centrifuged in tubes that were too full). I did end up making a mistake the second time by not allowing the elution buffer to incubate for 2 minutes at room temperature before centrifuging (in reality, they probably only sat about 30 seconds to a minute at most). In the end, I had four different tubes that had plasmid extractions done: 

My next steps are doing a nanodrop reading (dsDNA) on the four microcentrifuge tubes, along with a plasmid digest and a gel run, to see if I did extract pRad1 still. Then, I will move forward from there.