MIC Testing

Antibiotic minimum inhibitory concentration for pathogenic bacteria

1. MIC Testing Dr. Samira Fattah PhD in Medical Bacteriology College of Health Sciences-HMU

2. MIC Testing The lowest concentration of an antibiotic that will inhibit the growth of the organism being tested is known as the minimal inhibitory concentration (MIC). The MIC may assist a physician in deciding the concentration of the antibiotic needed to inhibit the pathogen. MIC = The minimum concentration leads to inhibition of bacteria under testing . MBC = The minimum concentration that kill the bacteria under testing.

3. MIC Testing Dilution method It is a quantitative method, depend on preparation of series of gradually duplicate concentration of antibiotic in a suitable medium for growth, then adding limited number of bacteria and checking the ability of antibiotic to inhibit or kill the bacteria under testing.

4. Methods of MIC determination Agar based dilution Agar dilution method Well diffusion method Broth based dilution Macrodilution method Microdilution method

5. Macrodilution Method The macrodilution method was among the first to be developed and still serves as a reference method.

6. Macrodilution Method Preparation of antibiotic stock solution A stock solution is a concentrated solution that will be diluted to some lower concentration for actual use. Stock solutions are used to save preparation time, conserve materials, reduce storage space, and improve the accuracy with which working lower concentration solutions are prepared.

7. Macrodilution Method Preparation of antibiotic stock solution The antibiotic stock solutions should be made to a final concentration of 10 mg/mL or 10 times the highest concentration to be tested and then diluted to an appropriate concentration in broth. Sterile water is generally used in the preparation of the antibiotic solutions as solvent and diluent. Antibiotic powder obtained from the manufacturer is not always 100% pure. Therefore, prior to making a stock solution, it is important to ensure the potency of each antibiotic. The following formula may be used to determine the amount of antimicrobial needed for the desired volume: volume (mL) × desired concentration ( µg/mL) Weight (mg) = antibiotic potency ( µg/mg)

8. Macrodilution Method Therefore, for 3 mL of 10 mg/mL solution of drug X with 99.4% potency (994 μ g/mg) volume (mL) × desired concentration ( µg/mL) Weight (mg) = antibiotic potency ( µg/mg) 1mg=1000 µg 99.4% = 99.4mg/100mg=0.994mg/mg 10mg=10,000 µg 0.994 ⸼ 1000=994 µg/mg 3 ml ˟ 10,000 µg/ml X = 994 µg/mg X = 30.18 mg (≈ 30.2 mg) of antibiotic powder dissolved in 3ml of sterile distilled water

9. Macrodilution Method The stock solutions can be further diluted by following the formula: C1 V1 = C2 V2 Suppose a working solution of 256μg/mL is desired in 5 mL. 10000 µg/ml ˟ V1 = 256 µg/ml ˟ 5ml V1 = 0.128 ml ( 128 µl) of the 10,000 µg/ml stock solution should be suspended in 4.872ml of sterile distilled water.

10. Macrodilution Method Small volumes of the stock solution can be stored at –70°C in freezer vials, without the loss of any activity. Solutions may be stored for up to 6 months unless otherwise indicated in the manufacturer’s package insert. Frozen stocks should be thawed the day of use, and any left over is generally discarded.

11. Macrodilution Method Preparation of inoculum Colony suspension method 4–5 isolated colonies from an overnight culture are touched with a loop and dilute in sterile broth or saline to a turbidity comparable to that of a 0.5 McFarland standard (1.5×10 8 cfu /mL). This suspension is further diluted 1:100 with sterile water, 0.85% saline, or broth to give a final organism density of 5×10 5 cfu /mL(range 3–7×10 5 cfu /mL) by transferring 0.1mL of organism suspension to a tube containing 9.9 mL of broth, this give an inoculum density of 1×10 6 cfu /mL which, when mixed with an equal volume of antimicrobial solution in tubes will result in a final inoculum of 5×10 5 cfu / mL.

12. Macrodilution Method Procedure Label sterile capped test tubes 1 through 11 Pipette 0.5 mL of Mueller-Hinton broth into tubes 1–11. Pipette 0.5 mL of antibiotic solution into tubes 1. Transfer 0.5 mL from tube 1 to tube 2 and continue through tube 9. Be certain to change pipettes between tubes to prevent carryover of antibiotic. Discard 0.5 mL from tube 9. The tenth tube, which serves as a control, receives no antibiotic. add 0.5 mL of bacterial broth suspension to each tube except the eleventh (last) tube, which is the broth control tube. incubated overnight at 35–37°C.

13. Macrodilution Method Result interpretation The lowest concentration of the antimicrobial agent that will inhibit the growth of the microorganism being tested as detected by lack of visual turbidity, matching with a negative control included with the test, is known as MIC. To determine MIC : select the 1st clear tube (no bacterial growth) ranked at serial turbidity tubes, this tube contains MIC of antibiotic. To determine MBC : take 0.1 ml from clear tubes and transfer to petri dishes containing Mueller Hinton agar and spread on the surface of agar then incubate at 37°C for 24-48 hrs and then check the growth of colonies in each plate ;the 1st plate that dose not show any colony represents the concentration of antibiotic for clear tube and is considered MBC.