TITLE: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assays Using Broth Microdilution Method
AUTHORS: Tomasz Swebocki, Alexandre Barras, Aleksandra Maria Kocot, magdalena.plotka, rabah.boukherroub

[DESCRIPTION]
The presented protocol outlines a comprehensive assessment of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for bacterial cell cultures. These experiments are vital for screening bacterial susceptibility to antibiotics and substances with potential antibacterial properties. The protocol not only covers the necessary preparatory steps but also introduces the application of the widely recognized Gompertz model. The protocol ensures a smooth execution of the assessment through thorough preparation and step-by-step instructions. The user-friendly instructions provided enable researchers to easily follow the protocol, facilitating the implementation of the assessment. By adhering to the outlined procedures, researchers can acquire a deeper understanding of bacterial susceptibility, evaluate the efficacy of antimicrobial agents through MIC and MBC values, and contribute to the advancement of antibacterial strategies.

[BEFORE_START]
Be sure to read the protocol fully, before starting any manipulation. Be sure that all the equipment needed is working properly and you have enough time to do all the manipulations. If it is your first time doing this, be sure to plan more time, as usually first times take longer. Be sure that both you and environment is well protected and your laboratory regulations allow for manipulation with bacteria.

For a single test, that consist of a triplicate you will need around 4 mL of MHB, however, it is recommended to have much more prepared (preferably around 20 mL) in case any mishaps occur. You have to have an inoculated medium on a Petri dish with discrete colonies already prepared.

[GUIDELINES]
The following protocol is optimised for substances that are soluble in water. However, we recommend to prepare a small portion of the solution of the investigated substance and mix it with three potions of Mueller-Hinton Broth medium to see if any precipitation or liquid-liquid phase separation occurs. If that is the case – addition of small amount of DMSO (up to 1%) may help to solubilise the substance. If this won't help, we recommend using different technique to assess MIC and MBC values.

The following media have been used in the protocol:
   – MHB
   – MHA

The protocol was optimised and tested using following strains:
S. aureus 43300 (ATCC)
P. aeruginosa 15442 (ATCC)
E. coli K-12 (ATCC)

This protocol can be easily adapted to other strains of bacteria, however, make sure to use suitable media, if the investigated strain is not growing on the MHA/MHB. Bare in mind, that use of MHA/MHB is in accordance with CLSI guidelines; hence, any changes have to be indicated when publishing the results.

Gompertz model file was adapted from OriginPro forums and was originally posted by the user YimingChen.

[STEPS]
SECTION: Pre-preparation of stock colony
1. Take the inoculated Petri dish and transfer one colony to a tube with approx.3 mL of fresh MHB.
SECTION: Pre-preparation of stock colony
2. Incubate the tube for around 180 min (or more, depending on the strain and the size and age of the colony used) at 37 °C.
 


SECTION: Preparation of diluted standardized inoculum
3. 
 
Prepare standardized inoculum by first measuring the OD600 of your stock colony. If the value of the OD600:
Is greater than 0.1, dilute the culture with MHB to reach a value of 0.1,
Is between 0.09 and 0.1 you can move to a next step,
is below 0.09 put the culture back and incubate it for 15-30 minutes more.
SECTION: Preparation of diluted standardized inoculum
4. Pour 10 mL of MHB into a trough and add 100 µL of the standardized inoculum. Mix it by flushing couple of times with a pipette.
 


SECTION: Preparation of 96-well microplate
6. Pour around 10 mL of MHB to the trough and:
Add 50 µL of MHB to each well in columns 1-10 (growth control, GC (1)+ serial dilution (2-10)),
Leave wells in column 11 empty (highest concentration of serial dilution),
Add 100 µL to the wells in column 12 (sterility control, SC).

  

SECTION: Preparation of 96-well microplate
5. Dissolve tested substance (X) in MHB at twice the maximum concentration for the test.

 

SECTION: Preparation of 96-well microplate
7. Add 100 µL of the just prepared solution of X diluted in MHB to the wells in column 11 and remove and pass 50 µL to next well until reaching wells in column 2,
 

  

SECTION: Preparation of 96-well microplate
8. Using multichannel pipette add 50 µL of diluted standardized inoculum to each well of columns 1-11. For more information    .
 

 


SECTION: Preparation of 96-well microplate
9. Put a protective film on that-prepared well plate. Cover it with a lid. Put a name and/or other details on the side of the plate. Avoid leaving any marks on the lid.
SECTION: Preparation of 96-well microplate
10. Incubate the plate at 37 °C 15 min .

SECTION: Minimal inhibitory concentration (MIC) assessment
11. After the incubation take the microplate out of the incubator. Remove the lid from the plate and protective film if applied and let it cool down in Room temperature for 15 min.
SECTION: Minimal inhibitory concentration (MIC) assessment
12. 
After that time put the microplate into the plate reader and read OD600 values of the wells' content.

SECTION: Minimal inhibitory concentration (MIC) assessment
13. Gather the data, and using plotting program such as Prism, OriginPro, Excel or Kaleida plot the data and fit it using modified Gompertz model.
 

    

SECTION: Minimal bactericidal concentration (MBC) assessment
14. Transfer 100 µL of the content of the wells were no turbidity was observed to a separate 96-well microplate and dilute it ten fold serially with the solution of NaCl 9 g/L

SECTION: Minimal bactericidal concentration (MBC) assessment
15. Plate 20 µL of the content of the wells directly onto square Petri dish/dishes with MHA and incubate   
  

SECTION: Minimal bactericidal concentration (MBC) assessment
16. Next morning count the colonies on the plates.