MIC of Vancomycin with K56-2, E.coli B/r, and K.aerogenes

Today I am testing the resistance to the antibiotic vancomycin by 3 different bacteria strains. The purpose of this is to help understand results of upcoming roach survival assays with this antibiotic added to the three strains. Burkholderia cepacia K56-2, Escherichia coli B/r, Klebsiella aerogenes, and Esherichia coli DH5alpha (from a different source other than the one used before) will be used. Overnight liquid cultures were made in 10mL LB in 50mL centrifuge tubes from plates made from freezer stocks. The strains will be tested using the protocol used before and shown below.

Minimal Inhibitory Concentration Determination Protocol

Materials

• Bacterial cultures on plates
• Sterile 5mL Culture tubes
• 2 sterile 96-wells plates
• Sterile water
• Tryptic soy broth (60mL)
• 2X tryptic soy broth (120mL)
• Working stock of 300 and 400μg/ml Vancomycin
• Sterile inoculating loops
• Multichannel p200 pipette with sterile tips
• p20 pipette with sterile tips
• Sterile 50mL reservoirs
• 37°C shaking incubator
• 37°C stationary incubator
• Sterile 10mL pipettes with pipette aid

Procedure

1. From overnight cultures, make 1:100 dilutions by inoculating 60μL culture into 6mL of fresh 2X TSB using 15mL conical centrifuge tubes.
2. Create a microtiter plate with 12 different dilutions of vancomycin.
1. Create 300 and 400μg/mL working stock
2. Add 200μL of 400μg/mL to the wells that will have the highest vanco concentration
3. Add 100μL of 300μg/mL to the wells that will have a final concentration of 150μg/mL
4. Add 100μL of sterile water to all other wells.
5. Serial dilute 1:2 subsequent wells by transferring 100μL and discard final 100μL so all wells have a final volume of 100μL
6. Concentrations of vancomycin should be 0, 0.78, 1.562 , 3.125, 6.25, 12.5, 25, 50, 100, 200, 300 , 400 μg/ml.
3. Add 100μL of each diluted bacterial culture to 4 sets of dilutions.
4. The final concentrations of vancomycin should be 0, 0.39, 0.78, 1.562, 3.125, 6.25, 12.5, 25, 50, 100, 150, and 200 μg/ml. 
5. All wells should have a volume of 200μL
6. Place 96 well plates in 37°C incubator overnight.
7. After 24 hours, check for the minimal inhibitory concentration of vancomycin for each strain.

Results

E.coli DH5alpha    50μg/mL

E.coli B/r               50μg/mL

K. aerogenes         >2000μg/mL

B. cepacia K56-2  >200μg/mL

//BEE