Standard Operating Procedure for Determination of the Minimum Inhibitory Concentration (MIC) of Different Antimicrobial Agents Against Different Bacteria

Set the electronic balance to mg unit

Place an empty sterilized 1.5 Eppendorf tube onto the balance and zero it.

Add the desired amount of the antimicrobial powder into the Eppendorf tube.

Add 1mL of the recommended solvent (depending on the antimicrobial compound) in the same tube.

Then, give it a quick spin down.

Make 100µL aliquots and store them at -20°C.

For example: if you want to prepare 20mg/mL of ampicillin, weigh 20mg of ampicillin in a 1.5 Eppendorf tube and then add 1mL of H₂O (the solvent).

To begin with, using sterilized pipette tip or sterilized loop, inoculate small amount of the bacterial stock (stored at -80°C) into MH broth medium in 1.5 Eppendorf tube. Incubate it at 37°C shaking 3h 0m 0s.

Next Day, prepare 1:50 or 1:100 dilution from the bacterial inoculum in fresh MH medium and incubate it for 2h 0m 0s- 3h 0m 0s until the OD₆₀₀ reach (0.3 – 0.7).

For example, if you want to make 3mL of the 1:100 dilution, divide the final volume by the dilution factor (e.g., 3ml (3000ul)/100) = 30ul.

Then, transfer 30µL of the bacterial culture into a 15ml tube containing 3mL of fresh MH broth and incubate for 2h 0m 0s - 3h 0m 0s at 37°C.

Prepare the 96-well plate while waiting for the optimal growth of bacteria at OD6₀₀0.3 – 0.7.

Label the plate and its lid with the name of the bacteria, antimicrobial agents, date and the name of the person performing the MIC experiment.

Using a multi-channel pipette, dispense 50µL of MH broth into each well of the 96-wells (except for column number 12 you need to dispense 100µL), use the same tip for one plate.

Add two-fold the required concentrations of the antimicrobial agents to the column no. 12.

For example, if highest desired concentration is 256μg/ml, add to 512μg/ml.

Use the following equation to calculate the needed volume of the antimicrobial agent:

                             **C1xV1 = C2xV2** <sub>1</sub>xV<sub>1</sub> = C<sub>2</sub>xV<sub>2</sub>

Where

C₁= The antimicrobial stock concentration

V₁= The volume that is needed to be taken from the antimicrobial stock concentration to achieve the required antimicrobial concentration in column no.12

C₂= The required antimicrobial concentration in column no.12

V₂= The total volume of the medium in column no.12

Follow this example to calculate the V₂, if the antimicrobial agent stock concentration is 20mg/mL and the highest desired concentration is 256μg/ml.

Then,

C1xV1 = C2xV2 ₁xV₁ = C₂xV₂

20mg/ml x V₁= 512ug/ml x 100ul

V₁= 0.512mg/ml x 100/ 20mg/ml = 2.56μl.

A volume of 2.56µL of antimicrobial agent will be added to the column no. 12, which contain 100µL MH broth, and mix by pipetting up and down 3 times.

Next, using a multi-channel pipette set at 50µL, mix antimicrobial agent in the wells in column no. 12 by pipetting up and down 3 times without splashing and introducing air bubbles.

Withdraw 50µL from column no.12 and add them to column no. 11 (This makes column no. 11 a two-fold dilution of column no. 12, with antimicrobial agent concentration at column no. 11 of 128μg/ml). Mix up and down 3 times.

Repeat the same procedure in step 13 by taking 50µL from the corresponding column and add it to the preceding column until column no. 2.

Discard the 50µL from column no.2.

Column no. 1 should not contain any antimicrobial agent (just the 50µL of MH broth you added from the starting of the MIC experiment).

After 2h 0m 0s - 3h 0m 0s of bacterial incubation at 37°C, measure OD₆₀₀using the following steps: Turn on the spectrophotometer and wait until the automatic calibration is finished, make sure that the absorbance is set at 600nm.

Add 1mL of MH medium only into a cuvette as a blank, then place the cuvette into the spec. and close the lid.

Press blank. The screen will give you a reading of the absorbance equals to 0.000.

Remove the cuvette and discard the MH medium from it.

Add 1mL of the growing culture to the same cuvette and place back into the spectrophotometer. Press measure and record your reading.

After achieving the desired OD₆₀₀of 0.3-0.7, dilute the bacterial culture OD₆₀₀to 0.004, using the following equation:

                            **C1xV1 = C2xV2** <sub>1</sub>xV<sub>1</sub> = C<sub>2</sub>xV<sub>2</sub>

Where

C₁= The measured bacterial OD₆₀₀

V₁= The volume that is needed to be taken from bacterial culture to achieve the required bacterial OD₆₀₀of 0.004

C₂= The required bacterial OD₆₀₀ of 0.004

V₂= The required volume of the bacterial culture of OD₆₀₀ of 0.004

For example, if the bacterial OD₆₀₀is 0.5

        **0.5 x V1 = 0.004 x 5 ml** <sub>1</sub>= 0.004 x 5 ml 

We selected V₂= 5 ml because the amount of bacterial culture for one plate is around 5 ml.

As 96 wells x 50 ul = 4800 μl ≈ 5000 μl (5 ml).

Therefore, V₁= 40 ul.

Next, transfer the calculated 40µL of the growing culture to 4960µL of fresh MH broth.

Mix well or vortex.

Then, quick spin down.

Divide the diluted bacteria into 8 Eppendorf tubes each with 625µL. Therefore, each tube will be used for one row of the 96-well plate (to avoid cross contamination).

Dispense 50µL of the bacterial cultural of the OD₆₀₀of 0.004 to all the wells.

Mix the solution 1-2 times after each addition of the bacterial culture.

Cover the plate and place it in a wet and closed plastic box then incubate it for 18h 0m 0s at 37°C 18h 0m 0s.

Next day, column no. 1 should show bacterial growth as a control (presence of turbidity).

Determine the MIC for each antimicrobial agent by finding the lowest concentration of the antimicrobial in each row that inhibit the visible bacterial growth (clear broth with no turbidity) and record the results.