2/5-2/11 Week 5: Repeat of the Tetracycline Efflux Pump vs. caeni

Introduction

    In lab this week, my group and I will repeat the tetracycline and caeni experience once more. However, since we learned that both caeni and d.aquaticus showed that it is resistant to tetracycline in no more than the concentration of 10ug/ml, we will be redoing that experiment with caeni but now with only the concentrations of 0ug/ml, 1ug/ml, 2ug/ml, and 6ug/ml. Going into this, we want to expect at least growth on all plates especially between the concentrations 0ug/ml to 2 ug/ml. 

Procedure

    The prepping for week is different than last weeks because instead of working with soft agar and mixing it in with the tetracycline, we will be working with normal agar.

To prep, we made four 25ml flasks of normal TGY agar. The reason for this is because we want to ensure that each TGY plates we will be pouring is evenly 25ml. We then recieved our 2.5 ug/ml tetracycline stock from Chad. We also grew our caeni in a 10ml TGY flask. 
To keep in mind, after 2 days of growing our caeni, the OD value only measures to 0.04 which is quite low for a sample that is usually grown after 2-3 days. However, after talking to Dr. Tuohy, he recommends proceeding with the protocol because it will be growing on plates. Due to this, we did not need to further dilute our culture. 

1. We label each of four 25ml flasks of TGY agar their desired tetracycline concentrations and also labeled four empty plates the corresponding concentrations to the flasks. 

               Flask #1 and Plate #1: 0ug/ml 
                Flask #2 and Plate #2: 1ug/ml
                Flask #3 and Plate #3: 2 ug/ml 
                Flask #4 and Plate #4: 6 ug/ml 

2. We heat up the flask one at a time and wait until the TGY agar melted and waited until the flask is LUKEWARM (not baby bottle test because that can still denature our tetracycline due to the extreme heat) to pipette out the excess amount and add in the designated tetracycline amount in ul. See below for amount and concentrations

3. We then vortex the flask for 6-10 seconds to ensure that the tetracycline and media is evenly mixed together. 

4. We gently pour the flask into the corresponding empty plate.

5. While the plate did not solidify yet, we quickly pipette 250 ul of our caeni sample into the plate and using a sterilized loop, we spread the caeni around and we finally wait for the plate to solidify before placing it in the 27 degree Celsius incubator. 

6. Repeat steps 2-5 for the rest of the plates. 

Concentrations and Amounts

0ug/ml: 0 ul
1ug/ml: 10 ul
2ug/ml: 20 ul
6ug/ml: 60 ul

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