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.