Materials and Reagents

A. baumannii (ATCC, catalog number: 17978 )
Escherichia coli (E. coli) (ATCC, catalog number: 25922 )
Mueller Hinton broth (Sigma-Aldrich, catalog number: 70192 )
Tigecycline (Wyeth, catalog number. 0220620-09-7 )
NaCl (MDBio, catalog number: 101-1647-14-5 )
KCl (Sigma-Aldrich, catalog number: P1147 )
Na₂HPO₄ (J.T.Baker^®, catalog number: 3828-01 )
KH₂PO₄ (J.T.Baker^®, catalog number: 4921-07 )
HCl (J.T.Baker^®, catalog number: 9535-03 )
Tryptone (Pronadisa, catalog number: 1612 )
Yeast extract (Pronadisa, catalog number: 1702 )
Cation-adjusted Mueller-Hinton broth (CAMHB) (see Recipes)
PBS (1 L) (see Recipes)
Lysogeny broth (LB) (see Recipes)

Equipment

50 ml polystyrene culture tubes (sterile)
Spectrophotometer to measure absorbance of cell culture (OD₆₀₀)
37 °C shaking and static incubators
Multichannel pipette (volume ranges 10 μl-1,000 μl)
1.5 ml Eppendorf tube
A centrifuge machine
1 ml cuvette

Procedure

Preparation of antibiotic stock solution and dilution range
1. Obtain antibiotic powder from the pharmaceutical company and make a note of the relevant information, including expiry date, potency, stability and solubility.
2. Prepare 1 ml 10 mg/ml tigecycline stock solution.
3. Choose a suitable range of antibiotic concentrations to be tested for A. baumannii if available. If the range is not available, maximal concentration 512 µg/ml and serial diluted concentrations with CAMHB solution are used. The lowest dilution concentration is depended on the possible minimal inhibition concentration. 0.125 µg/ml is the lowest possible dilution concentration.
4. To get different tested concentrations, solution of 10-time maximal concentration is prepared by dispensing the appropriate amount of stock solutions with micropipette and diluting with CAMHB solution.
  For example, to get 1 ml 5,120 µg/ml solution, dispense 0.512 ml stock solution and dilute with 0.488 ml CAMHB solution.
Preparation of inoculum
1. Dissolve a single colony of A. baumannii, which is picked from a LB streak plate, in 3 ml LB broth and incubate overnight at 37 °C, 220 rpm.
2. Check OD₆₀₀ (1 OD₆₀₀= 10⁹ CFU/ml) with a spectrophotometer.
3. Dilute the bacterial solution with LB broth to get 0.1 OD₆₀₀ suspension and incubate at 37 °C, 220 rpm till mid-log phase (~2 h).
4. Put 1 ml mid-log phase bacterial solution in 1.5 ml Eppendorf tube, centrifuge at 6,000 rpm for five min, and wash with 1 ml PBS solution. Repeat the washing procedure twice.
5. Dissolve the bacterial pellet with 1 ml CAMHB solution.
6. Get 100 µl the above bacterial solution and mix it with 900 µl PBS, then check OD₆₀₀ with a spectrophotometer. The bacterial concentration can be deduced from the measured value x 10.
7. Adjust the bacterial concentration to 1 x 10⁷ CFU/ml with CAMHB solution (1 OD₆₀₀ ~10⁹ CFU/ml).
Inoculation and incubation
1. Mix 50 µl adjusted A. baumannii bacterial solution (1x10⁷ CFU/ml), 850 µl CAMHB and 100 µl solutions of 10-time serial tested antibiotic concentration. Use the E. coli ATCC25922 bacterial solution as a control.
2. Use 900 µl CAMHB and 100 µl solutions of 10-time serial tested antibiotic concentration for OD₆₀₀ measurement comparison as a negative control.
3. Incubate at 37 °C, 220 rpm for 20-24 h.
Reading and interpretation
1. Check OD₆₀₀ with a spectrophotometer.
2. Read the MIC endpoint as the lowest concentration of antibiotic at which there is no visible growth of bacteria (no solution turbidity on naked eyes), and the difference of measured and background OD₆₀₀ is less than 0.01.

Recipes

CAMHB
Dissolve 23 g Mueller Hinton broth in 0.9 L of distilled water
Adjust pH to 7.2 using HCl
Then fill up to 1,000 ml with distilled water
Sterilized by autoclaving at 121 °C for 15 min
And added
2 ml 10 g/L Ca²⁺ (8.36 g MgCl₂^․2H₂O in 100 ml ddH₂O)
1 ml 10 g/L Mg²⁺ (3.68 g CaCl₂^․6H₂O in 100 ml ddH₂O)
Stored at 4 °C
PBS (1 L)
8 g NaCl
0.2 g KCl
1.44 g Na₂HPO₄
0.24 g KH₂PO₄
Dissolve in 900 ml ddH₂O
Adjust pH to 7.2 using HCl
Sterilized by autoclaving at 121 °C for 15 min
LB
10 g tryptone
5 g yeast extract
5 g NaCl
Fill to 1 L with ddH₂O
Sterilized by autoclaving at 121 °C for 15 min

Acknowledgments

The development of this protocol was funded by a grant from the National Taiwan University Hospital, Chu-Tung Branch.

References

Lin, M. F., Lin, Y. Y., Yeh, H. W. and Lan, C. Y. (2014). Role of the BaeSR two-component system in the regulation of Acinetobacter baumannii adeAB genes and its correlation with tigecycline susceptibility. BMC Microbiol 14: 119.