S-STEM Scholar Jessica Sabel's Blog

  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...

  Introduction:  This week we were finally able to perform a true Minimum Inhibitory Concentration (MIC) test using a micro-plate reader. The results from last week pointed towards the MIC being around 1.75-2 ug/ml. This gives a general idea of where the MIC is, however, reading the absorbance in a spectrophotometer increases the risk of an inaccurate result. The spectrophotometer picks up any bacterial cells(dead or alive) and includes that in the absorbance reading. Our spectrophotometer readings could have been picking up dead cells and reading that as a positive result, which is why additional testing is useful. This week, the micro-plate reader allows us to get a more accurate result.  Methods:  TGY media was prepared on 10/03 using the 3:3:1 method (3g tryptone, 3g yeast extract, 1g dextrose). The TGY was autoclaved overnight on 10/3. The 1 mg/ml stock solution was prepared last week, combining 500 ul PCR water and 500 ul of 2mg/ml Kanamycin. Stock solution has...