Week 5 (10/3/22 - 10/7/22): The big MIC Test

 

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 been stored in the refrigerator at 30o C.


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. 10 runs of different concentrations were done, with 3 trials for each run. Specific concentrations and amounts of TGY/kanamycin added are shown in figure 1. 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. Then 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.


Figure 1: Amount of antibiotic and media added into each tube to get the final antibiotic concentration.


Figure 2: Outline of MIC procedure. 



Figure 3:  Concentrations of kanamycin in each well, in ug/ml. A-C have the same concentrations. Row H contain the negative controls (containing 250 ul TGY),  row F 1-3 are blanks, row F 4-10 are positive controls (containing 2.5 ul of D. aquaticus and 247.5 ul of TGY).



Results:


Results were not obtained this week. Prepping and planning for the MIC run was done at the beginning of the week. MIC run was done on 10/07 with focus on helping the new TRAIN students in our transformation group learn how to work under the hood and with pipettes. 



Discussion: 


This MIC run was the first time that 4 of the people in our transformation  group worked under the hood. There was also only 1.5 hours to complete the entire procedure including the wait time for the UV to sterilize the area, which put a lot of added pressure onto our group. These two factors made it incredibly difficult to keep track of all the pipetting that was being done. I believe there is some risk for error because there were a few moments where we were confused over which well we were working on. If the results from the micro-plate reader display incorrect pipetting, the procedure may need to be repeated next week. In this circumstance, next week we can work with a smaller range of concentrations so there can be less runs, decreasing the chances of human error. 


If we are to repeat this procedure next week, the method for mixing will be changed. I noticed while mixing with the pipette that there were a lot of bubbles forming. There was a chance of the bubbles moving out of their well and into another one, messing up the result. Next time we will have to either decrease the total volume in the wells prior to mixing, or mix everything together in a separate test tube and then add the 250 ul to the 96-well plate at the end. It is unclear if we will be able to decrease the volumes of the well to 250 ul because if we are working with concentrations like 0.75 ug/ml,  the amount of kanamycin that would be added to keep the ratio is smaller than any pipette can measure out. In this case we will mix everything together in test tubes before adding the 250 ul to each well.


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