The document describes the macrodilution method for determining the minimal inhibitory concentration (MIC) of antibiotics. Key steps include: 1. Preparing stock solutions of antibiotics at high concentrations like 10 mg/mL and diluting them to make testing concentrations. 2. Creating a standardized bacterial inoculum of around 5x105 CFU/mL. 3. Diluting the antibiotic in a series of tubes containing broth and adding the bacterial inoculum. 4. Incubating the tubes overnight and finding the lowest antibiotic concentration tube that shows no visible growth, which is the MIC. The MBC can also be determined by culturing samples from clear tubes.

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
1. Agar based dilution
i. Agar dilution method
ii. Well diffusion method
2. Broth based dilution
i. Macrodilution method
ii. 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=1000µg 99.4% = 99.4mg/100mg=0.994mg/mg
10mg=10,000µg 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
1. 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 (1.5×108 cfu/mL).
2. This suspension is further diluted 1:100 with sterile water, 0.85% saline, or
broth to give a final organism density of 5×105 cfu/mL(range 3–7×105 cfu/mL)
by transferring 0.1mL of organism suspension to a tube containing 9.9 mL of
broth, this give an inoculum density of 1×106 cfu/mL which, when mixed with
an equal volume of antimicrobial solution in tubes will result in a final inoculum
of 5×105 cfu/mL.

12. Macrodilution Method
Procedure
1. Label sterile capped test tubes 1 through 11
2. Pipette 0.5 mL of Mueller-Hinton broth into tubes 1–11.
3. Pipette 0.5 mL of antibiotic solution into tubes 1.
4. 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.
5. Discard 0.5 mL from tube 9. The tenth tube, which serves as a control,
receives no antibiotic.
6. add 0.5 mL of bacterial broth suspension to each tube except the eleventh
(last) tube, which is the broth control tube.
7. 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.