S-STEM Scholar Jessica Sabel's Blog

Introduction:    This semester I have been working on determining the Minimum Inhibitory Concentration (MIC) in  D. aquaticus . It has been determined that out of the antibiotics tested, kanamycin is the best choice to use for this bacterium. The goal is to determine the exact concentration of antibiotic added so it inhibits growth without killing all of the cells.    This is significant when performing transformation; transformed cells should contain an antibiotic resistance gene so that when the sample is plated, only transformed cells will be able to grow. If the concentration of antibiotic is too high, it could prevent cells from growing. If the concentration of antibiotic is too low, non-transformed cells would be able to grow and we would not be able to tell the difference between transformed and non-transformed cells. Based on the MIC results from the previous week shown in figure 2, we determined that concentrations of 1.5 ug/ml and 1.75 ug/ml are potent...

  Introduction:  This week we attempted to move forward with our MIC test. In the previous week our results showed contamination, so we reflected on our technique and came up with ways to prevent it from happening again. The same antibiotic concentrations are being tested, however, mixing in the wells will be avoided this time because we believe it altered the results.    Methods:    Gram staining was done by lab partner Anh on 10/31 on our  D. aquaticus  samples in liquid media from 8/16/22 and 9/03/22. OD600 values were also taken on these two samples prior to starting the MIC procedure.   Before running the experiment, the UV light was turned on in the hood for 15 minutes. The blower was also turned on and the glass was lifted slightly.  All equipment, gloves, and surfaces under the hood were wiped down using Kim wipes with alcohol.   A sterile 96-well plate was used. 6 reps of different concentrations were done, with 3 runs...

  Introduction:  Minimum Inhibitory Concentration (MIC) is evaluated to determine the amount of antibiotic (kanamycin) to add to a bacterial sample to inhibit growth. This is important in the transformation experiment to determine if the cells are transformed or not. Plating transformed cells on media that contains an antibiotic at its MIC allows for only transformed cells to grow. This is due to the antibiotic of choice kanamycin, inhibiting growth in all cells that do not contain the kanamycin resistance gene. The kanamycin resistance gene will be added to all D. aquaticus cells during the transformation procedure, so growth leads to the conclusion that the bacterial cells are transformed and contain this gene. Methods:  OD600 of D. aquaticus was taken on the nanodrop prior to starting the MIC procedure. Protocol for MIC is the same as the previous week: Before running the experiment, the UV light was turned on in the hood for 15 minutes. The blower was also turned on...

Introduction: This week involved evaluating MIC results from the previous week, as well as preparation/planning for a more definitive result next week. Because the results from our MIC were finalized on a day we do not come in to the lab, an additional test needs to be performed to insure we are working with D. aquaticus at the time that the growth curve should be sufficient (at 48 hours). Methods: TGY (3:3:1 ratio) was made on 10/18 and autoclaved overnight.  D. aquaticus sample from 09/06/22 was inoculated onto a plate, creating 2 plates with a normal streaking pattern, and 2 plates done as a 4 way streaking pattern. Plates were stored in incubator.  The 96-well plate was removed from the microplate reader on 10/18, and the results were analyzed. Figure 1: Concentrations of kanamycin in each well, in ug/ml. Note that 96-well plate is upside down. Rows F-H have the same concentrations. Row C contains the negative controls (containing 250 ul TGY),    and row C con...

Introduction:  Over the last 5 lab reports, I have been working on different types of Minimum Inhibitory Concentration tests. The tests were first evaluated using a spectrophotometer, and as the results improved the tests were then moved to the microplate reader. Results from the MIC run done on 10/06 are shown in figure 1.    Based on the graphs created after 48 hours of incubation, the MIC should fall around column 7, which had a kanamycin concentration of 1.5 ug/ml. There was a pipetting error somewhere in between columns 7-9, which affects the results of this test. While trying to keep track of pipetting, I believe that I did not add antibiotic into row 8, and I added it into row 9 instead. This would explain why column 8 has exponential growth that matches closely with the positive controls, and why column 9 has little-to-no bacterial growth that corresponds with a higher concentration of antibiotic being in those wells. Because of this, another MIC test was performe...