Week 9 (10/31/22 - 11/04/22) Starting Over With a New Procedure

 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 for each rep. Eppendorf tubes were used this time instead of adding the contents directly into the wells. TGY was first added to all of the eppendorf tubes, the exact amounts added are shown in figure 1. TGY was added using the P100, P20, and P2 pipettes. Kanamycin was added to the eppendorf tubes next using P20 and P2 pipettes, exact amounts shown in figure 1. 15.15 ul of D. aquaticus was added each of the testing tubes, as well as to the positive control. Once all of the contents were added to the eppendorf tubes, the experiment was moved under the hood to decrease chances of contamination. Before moving on, each eppendorf tube was flicked 6 times to “vortex” the mixture of TGY, antibiotic, and bacteria. When each solution was mixed, 250 ul was taken out of each eppendorf tube and added to the respective well. Specific concentrations and placement of the controls are shown in figure 3. The 96-well plate was placed in the micro-plate reader for 48 hours. Everything under the hood was wiped down with Kim wipes and alcohol, and the UV light was turned back on for 15 minutes to sterilize the area. 

 

Rep

Volume of Media (ul)

Volume of Kanamycin (ul)

Concentration of Antibiotic (ug/ml)

1

1497

3

2

2

1497

2.625

1.75

3

1498

2.25

1.5

4

1498

1.875

1.25

5

1498

1.5

1

6

1499

1.125

0.75

Figure 1: Amount of antibiotic and TGY 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. 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 4: Gram staining done on D. aquaticus by Anh Nguyen on 10/31. The stain on the left is from our bacterial sample from 8/16/22, the stain on the right is from our bacterial sample from 9/03/22.

 

 

Sample

A600

Correction

λ

A(λ)

8/16/22

0.90

0.10

600

0.90

9/03/22

0.88

0.10

600

0.88

Figure 5: OD600 values on our D. aquaticus samples. 

 

 

Discussion: 

 

The gram stain and the OD600 values both looked better from the 8/16/22 sample, so this was the sample specifically used in this MIC experiment. I personally believe mixing in the eppendorf tubes prior to adding the contents into the wells was a much better procedure. It was easier to work with the larger volumes and then add to the wells, since they only can hold about 250 ul each. Because the MIC test was done on Thursday, 11/03, the results will not be available until Saturday afternoon. It is likely that the results will point us in a new direction for next week: either we stop working with the eppendorf tubes and return to our original procedure, or we will continue on with what we are doing and potential experiment with other antibiotics that are available in the biotech lab.   

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