TITLE: Standard Operating Procedure for Determination of the Minimum Inhibitory Concentration (MIC) of Different Antimicrobial Agents Against Different Bacteria
AUTHORS: Alanoud T Aljasham, Mashal M. Almutairi

[DESCRIPTION]
This standard operating procedure (SOP) describes the standardized laboratory procedure for performing the MIC experiment.

[BEFORE_START]
Before starting, the work area should be sterilized with 70% ethanol, and all the instruments that will be used during the experiment should be sterilized.

[GUIDELINES]

Scope:

This procedure applies to all antimicrobial agents and bacteria when performing MIC experiment.


Definitions and Acronyms:

 


[STEPS]
SECTION: Antimicrobial Agents Stock Solution Preparation
1. Set the electronic balance to mg unit
SECTION: Antimicrobial Agents Stock Solution Preparation
3. Add the desired amount of the antimicrobial powder into the Eppendorf tube.
SECTION: Antimicrobial Agents Stock Solution Preparation
4. Add 1 mL of the recommended solvent (depending on the antimicrobial compound) in the same tube. 
 


SECTION: Antimicrobial Agents Stock Solution Preparation
5. Then, give it a quick spin down.
SECTION: Antimicrobial Agents Stock Solution Preparation
6. Make 100 µL aliquots and store them at -20 °C.


SECTION: Bacterial subculturing from glycerol stock
7. 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  180 min.

SECTION: Bacterial subculturing from glycerol stock
8. Next Day, prepare 1:50 or 1:100 dilution from the bacterial inoculum in fresh MH medium and incubate it for 120 min- 180 min until the OD600 reach (0.3 – 0.7). 

SECTION: 96-Well Plate Preparation:
9. 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.
SECTION: Bacterial subculturing from glycerol stock
8.1. For example, if you want to make 3 mL of the 1:100 dilution, divide the final volume by the dilution factor (e.g., 3ml (3000ul)/100) = 30ul.

SECTION: Bacterial subculturing from glycerol stock
8.2. Then, transfer 30 µL of the   bacterial culture into a 15ml tube containing 3 mL of fresh MH broth and incubate for 120 min - 180 min at  37 °C. 

SECTION: Bacterial subculturing from glycerol stock
8.3.  Prepare the 96-well plate while waiting for the optimal growth of bacteria at OD600  0.3 – 0.7.
SECTION: Antimicrobial Agents Stock Solution Preparation
6.1. For example: if you want to prepare 20 mg/mL of ampicillin, weigh 20 mg of ampicillin in a 1.5 Eppendorf tube and then add 1 mL of H2O (the solvent).

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

SECTION: 96-Well Plate Preparation:
11. Add two-fold the required concentrations of the antimicrobial agents to the column no. 12. 
SECTION: 96-Well Plate Preparation:
11.1. For example, if highest desired concentration is 256 μg/ml, add to 512 μg/ml. 

SECTION: 96-Well Plate Preparation:
11.2. Use the following equation to calculate the needed volume of the antimicrobial agent: 


                                   C1xV1 = C2xV2

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
SECTION: 96-Well Plate Preparation:
12. Next, using a multi-channel pipette set at 50 µL, mix antimicrobial agent in the wells in column no. 12 by pipetting up and down 3 times without splashing and introducing air bubbles.

SECTION: 96-Well Plate Preparation:
11.3. Follow this example to calculate the V2, if the antimicrobial agent stock concentration is 20 mg/mL and the highest desired concentration is 256 μg/ml. 

SECTION: 96-Well Plate Preparation:
11.4. Then, 

             C1xV1 = C2xV2 

20mg/ml x V1= 512ug/ml x 100ul 
V1= 0.512mg/ml x 100/ 20mg/ml = 2.56μl. 
SECTION: 96-Well Plate Preparation:
11.5. A volume of 2.56 µL of antimicrobial agent will be added to the column no. 12, which contain 100 µL MH broth, and mix by pipetting up and down 3 times.
 



SECTION: 96-Well Plate Preparation:
13. Withdraw 50 µL 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 128 μg/ml). Mix up and down 3 times.

SECTION: 96-Well Plate Preparation:
14. Repeat the same procedure in step 13 by taking 50 µL from the corresponding column and add it to the preceding column until column no. 2. 

SECTION: 96-Well Plate Preparation:
14.1. Discard the 50 µL from column no.2.

SECTION: 96-Well Plate Preparation:
14.2. Column no. 1 should not contain any antimicrobial agent (just the 50 µL of MH broth you added from the starting of the MIC experiment).

SECTION: Bacterial Preparation for MIC: Measuring OD600
15. After 120 min - 180 min of bacterial incubation at 37 °C, measure OD600 using the following steps:  Turn on the spectrophotometer and wait until the automatic calibration is finished, make sure that the absorbance is set at 600nm.

SECTION: Bacterial Preparation for MIC: Measuring OD600
16. Add 1 mL of MH medium only into a cuvette as a blank, then place the cuvette into the spec. and close the lid. 

SECTION: Bacterial Preparation for MIC: Measuring OD600
16.1. Press blank. The screen will give you a reading of the absorbance equals to 0.000. 
SECTION: Bacterial Preparation for MIC: Measuring OD600
16.2. Remove the cuvette and discard the MH medium from it.
SECTION: Bacterial Preparation for MIC: Measuring OD600
17. Add 1 mL of the growing culture to the same cuvette and place back into the spectrophotometer. Press measure and record your reading.

SECTION: Bacterial Preparation for MIC: Bacterial Addition
18. 
After achieving the desired OD600 of 0.3-0.7, dilute the bacterial culture OD600 to 0.004, using the following equation: 

                                 C1xV1 = C2xV2

Where 

C1= The measured bacterial OD600
V1= The volume that is needed to be taken from bacterial culture to achieve the required bacterial OD600 of 0.004
C2= The required bacterial OD600 of 0.004
V2= The required volume of the bacterial culture of OD600 of 0.004
SECTION: Bacterial Preparation for MIC: Bacterial Addition
19. Divide the diluted bacteria into 8 Eppendorf tubes each with 625 µL. Therefore, each tube will be used for one row of the 96-well plate (to avoid cross contamination).

SECTION: Bacterial Preparation for MIC: Bacterial Addition
18.1. 
For example, if the bacterial OD600 is 0.5

           0.5 x V1 = 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. 
SECTION: Bacterial Preparation for MIC: Bacterial Addition
18.2. Next, transfer the calculated 40 µL of the growing culture to 4960 µL of fresh MH broth.

SECTION: Bacterial Preparation for MIC: Bacterial Addition
18.3. Mix well or vortex. 
SECTION: Bacterial Preparation for MIC: Bacterial Addition
18.4. Then, quick spin down.
SECTION: Bacterial Preparation for MIC: Bacterial Addition
20. Dispense 50 µL of the bacterial cultural of the OD600 of 0.004 to all the wells.
 


SECTION: Bacterial Preparation for MIC: Bacterial Addition
21. Cover the plate and place it in a wet and closed plastic box then incubate it for 1080 min at 37 °C 1080 min.

SECTION: Bacterial Preparation for MIC: Bacterial Addition
20.1. Mix the solution 1-2 times after each addition of the bacterial culture.
SECTION: Bacterial Preparation for MIC: Bacterial Addition
22. Next day, column no. 1 should show bacterial growth as a control (presence of turbidity).
SECTION: Bacterial Preparation for MIC: Bacterial Addition
23. 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.
SECTION: Antimicrobial Agents Stock Solution Preparation
2. Place an empty sterilized 1.5 Eppendorf tube onto the balance and zero it.