Minimal Inhibitory Concentrations of Vancomycin on S. maltophilia

Yesterday I read the 96-well plates that were used in the following protocol to determine the MIC of vacomycin on S.maltophilia. 

Minimal Inhibitory Concentration Determination Protocol

Materials

• Cultures of 20 Stenotrophomonas maltophilia isolates on plates
• 20 sterile 15mL centrifuge tubes
• 20 sterile 5mL Culture tubes
• 10 sterile 96-wells plates
• Sterile water (50mL)
• Tryptic soy broth (60mL)
• 2X tryptic soy broth (120mL)
• Working stock of 300 and 400μg/ml Vancomycin (16 mL each)
• 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. Make overnight liquid cultures from fresh plates in 3 mL TSB broth in 15 mL centrifuge tubes. Place in shaking 37°C incubator.
2. From overnight cultures, make 1:100 dilutions by inoculating 30μL culture into 6mL of fresh 2X TSB using 15mL conical centrifuge tubes.
3. 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.
4. Add 100μL of each diluted bacterial culture to 4 sets of dilutions.
5. 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. 
6. All wells should have a volume of 200μL
7. Place 96 well plates in 37°C incubator overnight.
8. After 24 hours, check OD600 with spectrometer for the minimal inhibitory concentration of vancomycin for Stenotrophomonas maltophilia.

Results
Isolate           MIC (μg/mL)
700                  100
BAA 2423       100
BAA 84           200
D                     200
E                       50
F                       50
G                     100
H                      25
I                      >200
J                       25
K                     150
L                      50
M                     50
N                    100
O                    >200
13637              50
17666             100
13270              150
51                   >200
49                    150

//BEE