Oct 26, 2022
Standard Operating Procedure for Determination of the Minimum Inhibitory Concentration (MIC) of Different Antimicrobial Agents Against Different Bacteria
- Alanoud T Aljasham1,
- Mashal M. Almutairi1,2
- 1Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
- 2The Chair of Vaccines Research for Infectious Diseases, King Saud University, Riyadh 11495, Saudi Arabia
Protocol Citation: Alanoud T Aljasham, Mashal M. Almutairi 2022. Standard Operating Procedure for Determination of the Minimum Inhibitory Concentration (MIC) of Different Antimicrobial Agents Against Different Bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw4owl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 07, 2022
Last Modified: October 26, 2022
Protocol Integer ID: 70997
Keywords: Standard Operating Procedure, Antimicrobial Agents, Minimum Inhibitory Concentration, MIC
Abstract
This standard operating procedure (SOP) describes the standardized laboratory procedure for performing the MIC experiment.
Guidelines
Scope:
This procedure applies to all antimicrobial agents and bacteria when performing MIC experiment.
Definitions and Acronyms:
| A | B | |
| Term/Acronym | Definition | |
| SOP | Standard Operating Procedure | |
| MH | Mueller Hinton | |
| OD 600 | Optical density at a wavelength of 600 nm |
Materials
Solutions and Materials:
- MH broth media
- Antimicrobials
- 96 Well Round Bottom Non-treated Plate SterileCell Treat Scientific ProductsCatalog #229590
- Medium Reservoir
- 1.5 ml Eppendorf tubes.
- 15 ml tubes
- Ice
- Spectrophotometer cuvettes
Instrument:
- Spectrophotometer
- Incubator
- Electronic balance
- Vortexer
- Minicentrifuge
- Plastic box
- Different pipettes
Safety warnings
Safety Precautions:
- All the MIC procedures should be taking place in a designated area at the lab.
- Before and after processing of the samples, the workstation should be disinfectant with 70% ethanol or 10 % bleach, and all the tools used during the processing must be sterilized.
- A properly fastened laboratory coat, long pants/skirt, closed toed shoes and gloves are required when working with clinical isolates.
- After handling any contaminated samples, gloves must be removed, and hands must be washed properly.
- All contaminated materials and gloves must be discarded in the biohazard containers.
- Prior to disposal, decontaminate all waste with 70% ethanol or 10% bleach.
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.
Antimicrobial Agents Stock Solution Preparation
Antimicrobial Agents Stock Solution Preparation
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 1 mL of the recommended solvent (depending on the antimicrobial compound) in the same tube.
Note
Vortex until the antimicrobial powder completely dissolved in the solvent.
Then, give it a quick spin down.
Make 100 µL aliquots and store them at -20 °C .
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).
Bacterial subculturing from glycerol stock
Bacterial subculturing from glycerol stock
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 Overnight .
3h
Next Day, prepare 1:50 or 1:100 dilution from the bacterial inoculum in fresh MH medium and incubate it for 02:00:00 - 03:00:00 until the OD600 reach (0.3 – 0.7).
5h
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.
Then, transfer 30 µL of the Overnight bacterial culture into a 15ml tube containing 3 mL of fresh MH broth and incubate for 02:00:00 - 03:00:00 at 37 °C .
5h
Prepare the 96-well plate while waiting for the optimal growth of bacteria at OD600 0.3 – 0.7.
96-Well Plate Preparation:
96-Well Plate Preparation:
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 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.
Add two-fold the required concentrations of the antimicrobial agents to the column no. 12.
For example, if highest desired concentration is 256 μg/ml , add to 512 μg/ml .
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
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 .
Then,
C1xV1 = C2xV2
20mg/ml x V1= 512ug/ml x 100ul
V1= 0.512mg/ml x 100/ 20mg/ml = 2.56μl.
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.
Note
The concentration of antimicrobial agent in column no. 12 will be 256 μg/ml .
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.
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.
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.
Discard the 50 µL from column no.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).
Bacterial Preparation for MIC: Measuring OD600
Bacterial Preparation for MIC: Measuring OD600
After 02:00:00 - 03:00:00 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.
5h
Add 1 mL 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 1 mL of the growing culture to the same cuvette and place back into the spectrophotometer. Press measure and record your reading.
Bacterial Preparation for MIC: Bacterial Addition
Bacterial Preparation for MIC: Bacterial Addition
1d 12h
1d 12h
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
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.
Next, transfer the calculated 40 µL of the growing culture to 4960 µL of fresh MH broth.
Mix well or vortex.
Then, quick spin down.
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).
Dispense 50 µL of the bacterial cultural of the OD600 of 0.004 to all the wells.
Note
Use one Eppendorf tube and one tip for each row, start adding the bacterial culture from column no. 1 until column no. 12 (from zero to the highest concentration of antimicrobial to prevent the contamination of the wells by the highest concentrations of antimicrobial).
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 18:00:00 at 37 °C Overnight .
1d 12h
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.