Week 10 (11/07/22 - 11/11/22) Plating With Different Kanamycin Concentrations - MIC Testing

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 potentially the right concentrations for D. aquaticus. To evaluate this further, we moved on to plating the bacterial cells at each concentration.

 

 

Methods: 

 

Two 250 ml flasks of TGY with agar were prepared using 3:3:1 method with Anh Nguyen on 11/08. The TGY was autoclaved for 30 minutes. TGY was heated on a hot plate. Amount of antibiotic added to each sample of TGY was calculated: the TGY containing a kanamycin concentration of 1.5 ug/ml needed 187.5 ul of antibiotic added, while the TGY containing a kanamycin concentration of 1.75 ug/ml needed 218.75 ul of antibiotic added. Once the antibiotic was added, the TGY was mixed thoroughly. The TGY was then poured onto plates; there are 5 plates with a kanamycin concentration of 1.5 ug/ml and 5 plates with a kanamycin concentration of 1.75 ug/ml. Plates were covered and left out to dry overnight. 

 

In the morning on 11/10, plates were flipped over, wrapped with parafilm, and placed in the refrigerator that contains all of the media. Later that day the plates were removed and an inoculation procedure was set up. Prior to inoculation, we determined that the D. aquaticus sample from 10/27 was the best to use based off of the OD values shown in figure 3 and the gram stain from the previous week done by Anh Nguyen. This bacterial sample was mixed throughly and set aside for inoculation. 

 

Inoculation was performed by myself, Anh Nguyen, Kaylee, Jayce Thompson, and Christine. First, 100 ul of D. aquaticus was added to each plate using a P200 pipette. The inoculating loop was placed over the flame to sterilize it, and once cooled down, we used the loop to spread the bacterial cells across the entire plate. Pipette tips were not reused, and the inoculating loop was sterilized between each plate inoculation. Once the inoculation was finished, the plates were left face-up for 10 minutes to allow the bacterial cells to sink into the media. Finally, all 10 plates were placed in the incubator at 30^o Celsius. Results should be available after 48 hours, on 11/12.

Figure 1: Concentrations of kanamycin in each well, in ug/ml. Rows A-C have the same concentrations. Row H, 1-3 contain the blanks (containing 250 ul of TGY), and H 5-10 contain the negative controls (containing TGY and kanamycin). Row F contains the positive controls (containing D.aquaticus and TGY).

Results: 

Figure 2: MIC plating results from test run on 11/03. Rows A-C have the same concentrations. Row H, 1-3 contain the blanks (containing 250 ul of TGY), and H 5-10 contain the negative controls (containing TGY and kanamycin). Row F contains the positive controls (containing D.aquaticus and TGY). D. aquaticus was taken from our 10/27 sample. 

 

 

Date sample was inoculated	A600	Correction	λ	A(λ)
9/6	0.99	0.10	600	0.99
10/27, taken from 8/16 sample	0.94	0.10	600	0.94

Figure 3: OD600 values taken on two different samples of D. aquaticus. Because the A600 values are so similar with each sample, the gram stains were considered in choosing the best sample to use on our MIC test.

 

 

Discussion: 

 

The bacterial sample from 10/27 was used when plating because it had the best gram stain result (see week 9 report for gram stain). The plating will not result this week, as the inoculation took place on a Thursday. We are hoping to see zero growth on either the 1.5 ug/ml plate or the 1.75 ug/ml plate. Zero growth shows that the D. aquaticus responded to that concentration, and bacterial growth was inhibited. If we see growth on both plates, that either means that there as either contamination, or something else went wrong in the procedure that will need to be evaluated next week. 

 

If the procedure went correctly and one set of plates has zero growth, this MIC project will be finished and I will be able to move on to working on transformation. This procedure may need to be repeated at least 1 time because the optimal growth time for D. aquaticus is 48 hours. Since our plating done on 11/11 will reach 48 hours over the weekend, the plates will not be assessed in time. The plates will be evaluated on 11/14 at ~ 92 hours, almost double the ideal growth time. If the plates show growth on 11/14 it could be due to the incubation time, so plates will be remade at the same concentrations and will be incubated for exactly 48 hours.