Broth Dilution Method for MIC Determination

The lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration (MIC).

Minimum inhibitory concentration (MIC) is determined when a patient does not respond to treatment thought to be adequate, relapses while being treated or when there is immunosuppression.

Dilution methods can be carried out in 2 ways; broth dilution and agar dilution.

Broth Dilution Method

Broth dilution testing allows providing both quantitative (MIC) and qualitative (category interpretation) results. MIC can help establish the level of resistance of a particular bacterial strain and can substantially affect the decision to use certain antimicrobial agents.

Broth dilution can again be performed in 2 ways.

  1. Macro dilution:  Uses broth volume of 1 ml in standard test tubes.
  2. Microdilution: Uses about 0.05 to 0.1 ml total broth volume and can be performed in a microtiter plate or tray.

The procedure for macro and microdilution is the same except for the volume of the broth. MIC of an antibiotic using the broth dilution method is determined by using the following procedure 

  1. Preparation of antibiotic stock solution
  2. Preparation of antibiotic dilution range
  3. Preparation of agar dilution plates
  4. Preparation of inoculum
  5. Inoculation
  6. Incubation
  7. Reading and interpreting results

Preparation of antibiotic stock solution

Antibiotic stock solution can be prepared by commercially available antimicrobial powders (with given potency). The amount needed and the diluents in which it can be dissolved can be calculated by using either of the following formulas to determine the amount of antimicrobial powder (1) or diluent (2) needed for a standard solution:

microdilution formula

Prepare antimicrobial agent stock solutions at concentrations of at least 1000 μg/mL (example: 1280 μg/mL) or 10 times the highest concentration to be tested, whichever is greater.

Because microbial contamination is extremely rare, solutions prepared aseptically but not filter-sterilized are generally acceptable. If desired, however, solutions may be sterilized by membrane filtration. Dispense small volumes of the sterile stock solutions into a sterile glass, polypropylene, polystyrene, or polyethylene vials; carefully seal; and store (preferably at −60 °C or below, but never at a temperature warmer than −20 °C and never in a self-defrosting freezer). Vials may be thawed as needed and used the same day.

Preparation of antibiotic dilution range

  • Use sterile 13- x 100-mm test tubes to conduct the test. If the tubes are to be saved for later use, be sure they can be frozen.
  • Close the tubes with loose screw caps, plastic or metal closure caps, or cotton plugs.
  • Prepare the final two-fold (or other) dilutions of antimicrobial agent volumetrically in the broth. A minimum final volume of 1 mL of each dilution is needed for the test.

Note: For microdilution, only 0.1 ml is dispensed into every 96 wells of a standard tray.

 Preparation of inoculum

  1. Prepare the inoculum by making a direct broth suspension of isolated colonies selected from an 18- to 24-hour agar plate (use a non-selective medium, such as blood agar).
  2. Adjust the suspension to achieve turbidity equivalent to a 0.5 McFarland turbidity standard. This results in a suspension containing approximately 1 to 2 x 10^8 colony forming units (CFU)/mL for Escherichia coli ATCC®a 25922.
  3. Compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines.
  4. Optimally within 15 minutes of preparation, dilute the adjusted inoculum suspension in broth so, after inoculation, each tube contains approximately 5 x 10^5 CFU/mL. Note: This can be accomplished by diluting the 0.5 McFarland suspension 1:150, resulting in a tube containing approximately 1 x 10^6 CFU/mL. The subsequent 1:2 dilution in step 3 brings the final inoculum to 5 x 10^5 CFU/mL.
Broth dilution method for measuring minimum inhibitory concentration of antibiotics. (image
Broth dilution method for measuring the minimum inhibitory concentration of antibiotics. (image


Within 15 minutes after the inoculum has been standardized as described above, add 1 mL of the adjusted inoculum to each tube containing 1 mL of antimicrobial agent in the dilution series (and a positive control tube containing only broth), and mix.

This results in a 1:2 dilution of each antimicrobial concentration and a 1:2 dilution of the inoculums.


Incubate the inoculated tubes at 35 ± 2 ºC for 16 to 20 hours in an ambient air incubator. To maintain the same incubation temperature for all cultures, do not stack microdilution trays more than four high.


Compare the amount of growth in the wells or tubes containing the antimicrobial agent with the growth in the growth-control wells or tubes (no antimicrobial agent) used in each set of tests when determining the growth endpoints. For a test to be considered valid, acceptable growth (≥ 2 mm button or definite turbidity) must occur in the growth-control well.

Quality Control

Use reference bacterial strains that are genetically stable and have well-defined MICs in the middle range of the expected MICs of each antimicrobial agent to be tested. A dilution series should include at least two concentration increments above and below the previously established MIC for the reference organisms.

CLSI has established QC limits for dilution susceptibility tests; an unacceptable QC result falls outside these published limits. Reference strains recommended by the CLSI for

QC of dilution tests for aerobic bacteria are

  • E. coli ATCC 25922,
  • E. faecalis ATCC 29212,
  • P. aeruginosa ATCC 27853, and
  • S. aureus ATCC 29213.

Troubleshooting Microdilution Assays

  1. Inappropriate MICs report:
    • When MICs are lower than expected—the inoculum may be too light.
    • When MICs are higher than expected—the inoculum may be too heavy.
      In such conditions, repeat testing using McFarland 0.5 turbidity standard or standardizing device. Check steps in the inoculum preparation and inoculation procedure.
  2. When MICs are either higher or lower than expected—the composition of the cation-adjusted Müeller–Hinton broth may not be optimal. Check the pH and calcium concentration of in-house prepared media. Use an alternative commercial lot of media or an alternative lot of commercial panels.
  3. When there are skipped wells—it may be caused by several problems:
    1. Check for contamination.
    2. The panel may have been inadequately inoculated, or the inoculum may have been inadequately mixed.
    3. Drug concentration in the wells may be inaccurate.
    4. The volume of broth in the wells may be inaccurate.
  1. When several MICs are too high or too low—which may indicate a possible reading/transcription error. Recheck all of the readings and repeat testing using an alternative lot.

References and further readings

  1. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

21 thoughts on “Broth Dilution Method for MIC Determination

    1. Dear Chinonye
      In our laboratory we use visual inspection while categorizing tube as turbid or clear. You can compare it with control tubes too. Sometime it might have slight confusions, regarding which tube (tire) to pick out of two consecutive tube as MIC, at that time your experience will guide you or you can consult with senior Microbiologist of you laboratory.

  1. If microbroth dilution technique was used, on a 96-well plate how to read the interpretation based on the OD value? what is the acceptable OD value compared to the control? Thank you very much

  2. sir, which book should I consider for the topic – Antifungal activity of neem leaves extract I am totally confused about the method for leaves extraction and the mic DISC DIFFUSION & BROTH DILUTIONS that could be considered please help me as soon as possible

  3. Pls explain whole process step by step…because i don’t have any idea about it……I want to do this process in my project work. Plz help me

    1. Dear Shivani, thank you for the comment. I will try to write about this in future, when time favors.

  4. Hello sir can you write about 0.5 Macferland standard, i want to make 0.5 macferland suspension of ecoli but i have no idea if you have time please write step by step. Thanks

  5. Please I want to understand something. I’m not working with antibiotics but with a certain leaf extract. How do I go about the MIC as most write-ups are about antibiotics. Thanks I need this info for my project.

  6. when u add diluent ,what should be type of that diluent …in isenberg it is written that tween 80

  7. I’d like to know if how much of the nutrient rich broth will be added together with the antibiotic and the test organism?

  8. I would like to request you to write an article on MIC determination using Micro plate method.

  9. Why is it mentioned that the stock solution should be prepared in 1000 ug/ml of concentration. I have read the same in many research articles, but there is no specific reason given anywhere, as to what would happen if its taken below 1000. I just want to know if I could make stock solution having concentration less than 1000 ug/ml ( like 320 ug/ml …could you please help me with this query

  10. Why is it mentioned that the stock solution should have at least 1000 ug/ml of concentration. I have read the same in many research articles, but they have not given reasons as to why is that so. I want to know what would happen if the stock solution has concentration below 1000 (say 320 ug/ml). If you could help me with this query…please

    1. Because antibiotic powder is quite dangerous to handle all the time (and bleddy annoying), so it is recommended to make a stock solution of high concentrations of the antibiotic, divide it into say.. 1ml tubes, freeze them at -20C and then whenever you need to use the antibiotic, you can just take one tube out of the freezer, and use it to make whatever concentrations you want.

  11. if we want to do mic of two different bacterial strains for the checking of combine effect at the same time so how we can perform our experiment ?

  12. hello sir i read your blog sir i request you i want to know about MIC test in test tubes for the fungus please tell me the procedure

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