Week 6 (10/10/22 - 10/14/22): Almost at the Finish Line!

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 performed to look at the smaller range of kanamycin concentrations shown in columns 6-10 (in rows A, B, and C only). Assuming this test is run correctly without contamination, the MIC will be determined and we will be able to move on with our transformation project.

Figure 1: Minimum Inhibitory Concentration results of kanamycin with D. aquaticus after 48 hours, resulted on 10/08. Row A has antibiotic concentrations decreasing from 3 ug/ml to 0.75 ug/ml. Rows B and C are duplicates of row A, making there 3 runs total. All samples in row F are positive controls, containing TGY and D. aquaticus. All Samples in row H are negative controls, containing only TGY.

Methods:

Gram staining was performed on 10/12, with 2 different stains performed on a sample of D. aquaticus inoculated on 09/06. TGY was prepared and autoclaved in the previous week using the 3:3:1 method and was stored at room temperature. The 1mg/ml stock solution was stored in the refrigerator at 30 degrees C. 

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 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. 5 reps of different concentrations were done, with 3 runs for each rep. Specific concentrations and amounts of TGY/kanamycin added are shown in figure 2. TGY was added to the wells first, using a P100, P20, and P2 pipette. Kanamycin was added next using a P2 pipette. Each well was mixed 6 times using a P1000 pipette, then 52.5 ul of liquid was removed from each well and deposited in the waste beaker. 2.5 ul of D. aquaticus was added to each well using a P20 pipette, making the total volume in each well 250 ul. The 96-well plate was placed in the micro-plate reader for 48 hours. Everything under the hood was wiped down with Kim wipes an alcohol, and the UV light was turned back on for 15 minutes to sterilize the area. 

Figure 2: Amount of antibiotic and TGY media added into each tube to get the final antibiotic concentration.

Figure 3: Outline of MIC procedure.

Figure 4: 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 contains the positive controls (containing 2.5 ul of D. aquaticus and 247.5 ul of TGY).

Results: 

Figure 5: Gram staining done on 10/12 on D. aquaticus. Each stain was viewed on a different microscope: the stain on the left was viewed on a newer microscope, while the stain on the right was viewed on an older one. Both stains are viewed with oil immersion drops on the 100x lens. 

Results from MIC run performed on 10/13 will be available after 48 hours. These results will be analyzed on 10/17. 

Discussion: 

The 2 gram stains appeared to have similar morphology: all of the cells present were rods. There was some variability in the gram stain viewed on the newer microscope. Some of the cells appeared pink, while some of the cells appeared purple. I’ve run into this problem when gram staining D. aquaticus before, which leads me to believe it has something to do with the Deinococcus species itself. Deinococcus are known to have thicker cell walls which can stain positive, but they are more closely related to gram negative bacteria because they have an outer membrane-like structure (Libretexts, 2021).

The microplate results from the previous week gave an indication of where the MIC is, however, the experiment performed on 10/13 should produce results that give a more accurate result. There were also significantly less wells used in this experiment than before, giving less opportunity for pipetting errors to happen again. MIC from this week was accidentally done upside-down on the 96-well plate, and the settings in the microplate reader were adjusted to reflect this.

References:

Libretexts. (2021, January 3). 8.12b: Deinococcus and Thermus. Biology LibreTexts. Retrieved October 14, 2022, from https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/8%3A_Microbial_Evolution_Phylogeny_and_Diversity/8.12%3A_Thermophiles/8.12B%3A_Deinococcus_and_Thermus