Monday, December 5, 2016

CFU Assay #3

This time around we started our liquid culture with LB Broth. Slowly but surely we got to log phase (O.D. of  0.6nm - 0.8nm) which is the point where we want to induce our bacteria with IPTG so they will begin to produce our IL-8 protein. Also we didn't have any contaminated plates but we had two contaminated bottles of LB Broth that had to be discarded. We concluded as a class that the contamination came from the water bath where  we stored the bottles overnight. Even though we got to log phase something was still off about our data. Along with the O.D. numbers being off and not going up like we expected, a lot of the time point plates looked very weird and not normal. After talking to Dr. Matt Koci, and Mrs. Rizwana from NCSU Biotechnology Lab we realized we were using too much Kanamycin (KAN)  in the two assays before. This time we added 100ul of KAN per every 100ml of LB Broth. The concentration of KAN is 100mg/ml.

DATA HERE for Assay # 3 !!!

The samples we took from the liquid culture and put into the spectrophotometer was diluted by 5 fold. We had 800 ul of LB Broth into a cuvette and 200ul from the sample of the liquid culture. Meaning the time points 6- 24 that say <UNDILUTED did not have any LB Broth in them, it was all bacteria. To see all the time points in the undiluted form you just multiply them by 5.

Tuesday, November 29, 2016

CFU "Buffer" Assay

If you paid any attention to the name of this blog you can figure we had a little mix up at the beginning of the Assay. When we were creating our liquid culture we were using SDS BUFFER instead of LB/Broth. SDS is a Sodium Diliatial Sulfate one of the main ingredients of Dish Detergent. When we did the liquid culture the bacteria died, which is what we ended up putting in the spectrophotometer and spreading on the plates. You can't get colonies with dead bacteria then it wouldn't be a Colony Forming Unit Assay.

We didn't realize that we had the wrong liquid until we got to time point 6.5. Sadly, we had already streaked the plates and made the serial dilutions for the experiment. Although this was an epic fail we still got a good test run of all the new techniques we thought of. We ended up making a new starter culture the next day and still put the O.D. numbers into the spreadsheet.

Attached HERE is the link to our redo of the liquid cultures O.D. numbers
Sheet 1 contains our O.D. numbers and the timepoints which we took them at
We started the experiment on November 9, 2016 at 8:00 am with time point 0 and it ended at November 10, 2016 at 8:00 am at time point 24 (meaning 24 hours)

Thursday, October 27, 2016

Results from CFU Assay

Recently we've did the CFU Assay, which is the Colony Forming Unit Assay which lets us know how much bacteria we have over a time period. We ran into a problem when pouring the plates we weren't  pouring the plates fast enough so the agar was cooling and solidifying. Also we had about 33 fungus contaminated plates out of almost 200 plates, meaning we didn't have triplicates for each tube, and we didn't get the best data. We had extra plates left over so we had to use them to make up for the plates loss. we still did not have enough plates the triplicates. After collecting the data for the spec and time points, we put it in a chart and we counted colonies. Looking at the plates we realized with the earlier time points the less diluted tube plates had more colonies and as we came towards the ending time points the more diluted tube plates had more colonies. Once we got all of our data into the spreadsheet we saw that we had a lot of outliers, and our O.D. numbers never reached Log phase nor did we see it on the chart. We decided as a class to do the CFU Assay over again but this time with more consistency in pipetting and pouring the plates. As a whole we would have to make rules to such as Pipette soon as the shaking incubator is opened and go to the bottom, make a 4 second time when flaming the hockey stick to spread the bacteria, label all plates ahead of time. We came up with a few ideas on why we didn't have as many bacteria as we expected:
- Too much KAN in the plates and flask/ or the KAN is old.
- Fungus could have make another antibiotic that the bacteria isn't resistant to.
- Media could have been old we used Millers
- We didn't have a new colony to start the culture.
- Did we have the right bacteria? In the process of streaking plates (to keep fresh bacteria) did another type of bacteria start to grow on the plates?

We also poured just regular LB/KAN plates and left them under the flow hood over the weekend. When we came back today to check on them they also had fungus on them so, we decided to make a solution of bleach and water to use to clean the flow hoods. We sprayed the solution like we would do the ethanol when preparing the flow hoods to be used and we let it sit for 5 mins. We plan on doing this once a week hoping the results get better and when we run the CFU Assay again we have less or even no contaminated plates.

Sunday, August 14, 2016

First Week of Class 8/8/16 - 8/12/16

This week, we worked on making and sterilizing KAN. Kanamycin (KAN) is an antibiotic that kills bacteria but the bacteria we have been working with (E.coli ) is KAN- resistant. Our TA's have been teaching us about all the machines in the lab and how to use them. Most of our work is done under the flow hood and that just keeps our bacteria or whatever we are working on from being contaminated. Earlier in the week we worked on our plate streaking techniques. You can't just streak all over the plates, if you do then you won't be able to get an isolated colony like you really want and need to experiment with. Last year, when I was a freshman, in Biology we did this experiment where we streaked bacteria onto a plate, each plate was different so we picked one plate, then we had to choose a temperature. The outcome was the bacteria was supposed to glow in the dark if put in the right conditions. Our TA's gave us a preview of the pGLO Transformation, Now we have to do the same thing but just on our own in groups of 4 without anybody telling us exactly what to do.

Sunday, August 7, 2016

Trip to NCSU for Biotech

On July 25, 2016 some of the Falcon Biomanufacturing team traveled to NCSU and we stayed at University Towers for a whole week. While at NCSU we worked in the BTEC Lab, basically having a crash course of what we will be doing in our Biotechnology class and the experiences. We toured Novozymes Inc., BRI Inc., the NCSU campus, and Hunt Library. Several mentors talked to us in small groups, we had time to ask questions about college life along with any other questions we had. Actually I would say we got a little taste of the college life sitting though lectures, doing labs, taking notes, walking around and catching the bus. Now that we are back in school we will start from the very beginning of the turkey IL-8 Protein we were working on.

-Due Aug. 7, 2016 by midnight