Determination of Minimum Inhibitory Concentration (MIC)

["Collaborating Expert | Dr. Wang Zhang", "Infectious Diseases Zhejiang University"], ["Reviewer | Dr. Leyi Zhang", "Clinical Medicine Zhejiang University"]

Summary

Minimum Inhibitory Concentration (MIC) refers to the lowest concentration of a drug that can inhibit the growth and reproduction of microorganisms.There are many different methods for testing MIC, and three common methods are as follows: microdilution method, agar dilution method, and E-test method.

Principle

The growth of microorganisms at different concentrations of antimicrobials is determined by incubating the antimicrobial drug with the microorganisms to be tested over a range of dilutions, usually at a concentration of 1 million colony forming units (CFUs) per milliliter (mL) in suspension. A test system without antimicrobial activity will appear turbid due to the presence of microorganisms, while the absence of turbidity indicates that the growth of the microorganisms to be tested is inhibited.

Appliance

The minimum inhibitory concentration of the drug was determined to evaluate the antimicrobial activity and bacteriostatic capacity of the drug.

Operation method

Minimum Inhibitory Concentration (MIC) determination - microdilution method

Principle

Materials and Instruments

Sterilized cationic conditioning medium Mueller-Hinton Broth (CAMHB)

Drug powder to be tested, strain to be tested, sterilized saline

Sterilized distilled water, solid medium

Sterile V-/U-bottomed 96-well culture plates

McDonald's turbidimeter, sterilized plastic turbidimetric test tubes

Multi-channel pipettes

Move

1. Inoculate the strains to be tested on the corresponding solid culture plate in advance, and incubate in the bacterial incubator at 37 ℃ overnight. 2.

2. Weigh the appropriate amount of antimicrobial powder to be tested, add sterilized double-distilled water to fully dissolve, and prepare the storage solution for spare. 3.

3. According to the experimental requirements, use CAMHB liquid medium to dilute the storage solution to the highest concentration of the drug to be tested, take a sterile 96-well plate, and carry out drug dilution in a biological safety cabinet, the specific operation is as follows:

The first well (A1) add 200 μL of the highest concentration of the drug to be tested, A2 ~ A12 wells add 100 μL of CAMHB liquid medium, then 100 μL from the A1 wells pipetted into the A2 wells, mixing, then pipetted 100 μL from the A2 wells and added to the A3 wells, and so on, gradient dilution to the A12 wells, and discard the last 100 μL of the diluted liquid. 4.

4. Add 1 mL of sterilized saline into a transparent plastic test tube; put it on the turbidimeter to adjust the zero, then pick the strain to be tested and fully dissolve it in the saline, shake and mix well, adjust the turbidity between 0.4 and 0.6 McF, and then continue to dilute it 20 times with sterilized saline.

5. Add 10 μL of the diluted bacterial suspension into the wells (A1~A12) of each concentration of the drug in turn; place the 96-well plate in a bacterial incubator at 37 ℃ and incubate for 16~18 h. 6. Read the results of no bacterial growth.

6. Read the lowest concentration of the drug with no bacterial growth, which is the minimum inhibitory concentration (MIC) of the drug for the bacteria.

7. Use standard strains of Escherichia coli ATCC25922 as quality control strains for drug sensitivity test, and refer to CLSI and EUCAST guidelines for drug sensitivity judgment.

Caveat

1. The standard strains of Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Enterococcus faecalis ATCC29212 and Pseudomonas aeruginosa ATCC27853 should be selected for each experiment according to the different bacteria to be tested, and the MIC of the commonly used antimicrobials against these standard strains should be within the expected range.

Table 1 MIC range of quality control strains (mg/L)

2. The culture medium is suitable for the growth of microorganisms, some microorganisms have special requirements for the composition of the culture medium, and special culture medium should be used to carry out the drug sensitivity test: for example, HTM agar should be used for the drug sensitivity test of Haemophilus influenzae; MH agar with 5% of goat's blood should be added to the drug sensitivity test of gonococci; MH with 5% of goat's blood should be added to the drug sensitivity test of Streptococcus pneumoniae.

3. The stability of different antibiotics is different, antibiotic storage solution should be used now or kept at low temperature, and should be reconfigured if there are obvious changes in the state of antibiotics.

4, some special drugs need to be according to the nature of the drug pretreatment medium to eliminate antagonistic components; such as cefdinir sensitivity test using the removal of iron ions CAMHB.

Common Problems

The MIC of the quality control group is not within the expected range is called out of control, common factors affecting the results of MIC measurement are as follows:

(1) Culture medium: The pH, osmolality and electrolytes of the culture medium have an effect on the MIC;

(2) Antimicrobial drugs: Antimicrobial drugs must be standardized powder. The prepared drug stock solution should be used within the expiration date;

(3) Observation time of results: most microorganisms should be observed in 12-18 hours, and some pathogenic microorganisms should be observed in 20-24 hours. If the incubation time is too long, some pathogenic bacteria that have been mildly inhibited may start to grow again; in addition, because some antibacterial drugs are not stable enough, the incubation time is too long, so that their antibacterial activity is reduced, or even disappeared, thus increasing the MIC value.

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Tags:

Microbiology experiment