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Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. MIC determination is used as not only a diagnostic tool in treating bacterial infections for clinicians but also a research method in evaluating the efficacy of an antimicrobial. Multidrug resistance Acinetobacter baumannii (A. baumannii) has emerged in recent years. Accurate determination of resistance by MIC assay is important in coping with this superbug. Here we described a protocol for determining MIC for A. baumannii in hope of assisting researchers and physicians in confirming resistance of clinical isolates correctly.

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Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii

Microbiology > Antimicrobial assay > Antibacterial assay
Authors: Ming-Feng Lin
Ming-Feng LinAffiliation 1: Department of Medicine, National Taiwan University Hospital Chu-Tung Branch, Taiwan
Affiliation 2: Institute of Molecular and Cellular Biology, National Tsing Hua University, Taiwan
Bio-protocol author page: a1836
Yun-You Lin
Yun-You LinAffiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Taiwan
Bio-protocol author page: a1837
 and Chung-Yu Lan
Chung-Yu LanAffiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Taiwan
For correspondence: cylan@life.nthu.edu.tw
Bio-protocol author page: a1838
Vol 4, Iss 23, 12/5/2014, 4002 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1308

[Abstract] Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. MIC determination is used as not only a diagnostic tool in treating bacterial infections for clinicians but also a research method in evaluating the efficacy of an antimicrobial. Multidrug resistance Acinetobacter baumannii (A. baumannii) has emerged in recent years. Accurate determination of resistance by MIC assay is important in coping with this superbug. Here we described a protocol for determining MIC for A. baumannii in hope of assisting researchers and physicians in confirming resistance of clinical isolates correctly.

Keywords: Acinetobacter baumannii, Minimal Inhibitory concentration, Drug resistance

Materials and Reagents

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

Equipment

  1. 50 ml polystyrene culture tubes (sterile)
  2. Spectrophotometer to measure absorbance of cell culture (OD600)
  3. 37 °C shaking and static incubators
  4. Multichannel pipette (volume ranges 10 μl-1,000 μl)
  5. 1.5 ml Eppendorf tube
  6. A centrifuge machine
  7. 1 ml cuvette

Procedure

  1. 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.

  2. 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).

  3. 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.

  4. 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

  1. 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 MgCl22H2O in 100 ml ddH2O)
    1 ml 10 g/L Mg2+ (3.68 g CaCl26H2O in 100 ml ddH2O)
    Stored at 4 °C
  2. 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
  3. 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

  1. 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.


How to cite: Lin, M., Lin, Y. and Lan, C. (2014). Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii. Bio-protocol 4(23): e1308. DOI: 10.21769/BioProtoc.1308; Full Text



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4/5/2016 9:42:01 AM  

Fahmeeda Mojan
Universiti Malaysia Terengganu

How do you obtain the volume of bacterial solution needed to get 1 x 10*7 CFU/mL (adjusted concentration) from OD600?

4/8/2016 8:54:24 PM  

Ming-Feng Lin (Author)
Department of Medicine,National Taiwan University Hospital Chu-Tung Branch

Answer: At first, the amount of bacteria in 1 OD600 (checked with a spectrophotometer) solution was determined. To get the result, 0.1 mL 1 OD600 bacterial solution was diluted with 10-time serial dilution for 6-7 times. Then the serial diluted bacterial solutions were inoculated and the colonies were measured after overnight culture. Next, the original amount of bacteria in these diluted solutions could be deduced by the colony number and dilution fold. Finally, we found that 1 OD600= 10*9 CFU/ml. Therefore, 0.01 OD600 = 10*7 CFU/ml.

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