About the MIC Calculator

The Minimum Inhibitory Concentration (MIC) is a cornerstone of microbiology, representing the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism. This MIC Calculator is designed to simplify the two most critical preparatory steps in antimicrobial susceptibility testing (AST): preparing an accurate stock solution and calculating the volumes needed for a serial dilution series.

What This Calculator Does

This tool provides clear, actionable instructions for laboratory use, divided into two distinct functions:

• Stock Preparation: Calculates the precise mass of a compound needed to create a stock solution of a desired concentration and volume. It accommodates both mass-based (e.g., mg/mL) and molar-based (e.g., mM) concentrations, requiring the compound's molecular weight for the latter.
• Serial Dilution: Generates a step-by-step pipetting protocol for creating a series of decreasing antimicrobial concentrations in a microtiter plate. It provides a detailed table, total volume requirements, and a visual layout of the final concentrations per well.

When to Use It

This calculator is intended for researchers, technicians, and students in microbiology, pharmacology, or clinical laboratory settings. Use it when:

• Preparing a new antimicrobial stock solution from a powdered compound.
• Setting up a broth microdilution or agar dilution assay to determine the MIC of a compound against a specific microorganism.
• Needing to quickly generate a reliable pipetting plan for an experiment with multiple replicates.
• Training new laboratory personnel on the principles and practice of serial dilutions.

Inputs Explained

Serial Dilution Tab

• Stock Solution Concentration: The concentration of your starting, concentrated solution of the antimicrobial agent.
• Highest Final Concentration: The highest concentration you want to test in your first well. This must be lower than your stock concentration.
• Dilution Factor: The factor by which the concentration decreases between wells. A value of '2' represents a standard two-fold serial dilution.
• Number of Dilutions: The total number of wells in your dilution series (e.g., 8, 10, or 12).
• Final Volume per Well (µL): The total volume in each well after all components (diluent, drug, inoculum) are added. The calculator plans for the pre-inoculum volume.
• Number of Replicates: The number of identical dilution series you plan to run, used to calculate total reagent volumes.

Stock Preparation Tab

• Compound Molecular Weight (g/mol): The molar mass of your compound. This is only required if you are working with molar concentrations (M, mM, µM).
• Desired Stock Concentration: The target concentration for your stock solution.
• Desired Final Volume: The total volume of stock solution you wish to prepare.

Results Explained

After calculation, the tool provides a comprehensive output designed for direct use at the lab bench:

• Pipetting Table: A clear, well-by-well guide showing the volume of stock solution, diluent (e.g., broth), and transfer liquid required for each step.
• Volume Summary: The total amount of stock solution and diluent needed for your entire experiment, including a 10% excess to account for pipetting losses.
• Step-by-Step Protocol: A plain-language guide detailing the entire dilution process, from initial reagent preparation to the final serial transfer.
• Stock Preparation Instructions: For the stock tab, it provides a simple, direct instruction: "Weigh out X mg of compound and dissolve in Y mL of solvent."

Formula / Method

Stock Preparation

The calculation is based on the fundamental concentration formula. For mass-based units:

Mass (mg) = Concentration (mg/mL) × Volume (mL)

For molar concentrations, it incorporates molecular weight (MW):

Mass (g) = Molarity (mol/L) × Volume (L) × MW (g/mol)

Serial Dilution

The calculator uses the dilution equation C₁V₁ = C₂V₂ to determine the volume of stock needed for the first well:

• C₁ = Concentration of Stock Solution
• V₁ = Volume of Stock Solution to add (this is what's calculated)
• C₂ = Desired Highest Final Concentration (in well 1)
• V₂ = Final Volume per Well

For subsequent wells, the transfer volume is calculated based on the dilution factor:

Transfer Volume (µL) = Final Volume (µL) / Dilution Factor

Step-by-Step Example

Let's calculate a standard two-fold serial dilution for a single replicate.

• Stock Solution: 1024 µg/mL
• Desired Highest Concentration: 128 µg/mL
• Dilution Factor: 2
• Number of Dilutions: 8
• Final Volume per Well: 200 µL

1. Well 1 Preparation (using C₁V₁ = C₂V₂):
   • V₁ = (128 µg/mL × 200 µL) / 1024 µg/mL = 25 µL of stock
   • Volume of Diluent = 200 µL - 25 µL = 175 µL of diluent
2. Subsequent Wells (2 through 8):
   • Transfer Volume = 200 µL / 2 = 100 µL
   • Volume of Diluent = 200 µL - 100 µL = 100 µL

Add 25 µL of stock and 175 µL of diluent to Well 1. Add 100 µL of diluent to Wells 2-8. Then, transfer 100 µL from Well 1 to Well 2, mix, then from Well 2 to Well 3, and so on, discarding 100 µL from the final well.

Tips + Common Errors

• Inoculum Volume: Standard protocols (e.g., CLSI) often specify a final inoculum volume. This calculator plans for pre-inoculum volumes. Ensure your "Final Volume" input accounts for any subsequent additions.
• Check Solubility: Before preparing a large volume, ensure your compound is fully soluble in the chosen solvent at the target concentration. Use of a vortex or sonicator may be necessary.
• Pipetting Technique: Use calibrated pipettes and fresh tips for each transfer to prevent carryover contamination and ensure accuracy. Pre-wetting tips can improve precision.
• Impractical Volumes: If the calculator reports an error about pipetting volumes being too small (<1 µL), it means your stock concentration is too high relative to your target. Prepare an intermediate dilution of your stock and use that as your starting material.
• Concentration Mismatch: A common error is setting the "Highest Final Concentration" to be equal to or greater than the "Stock Solution Concentration." This is mathematically impossible for a dilution.

Frequently Asked Questions

Why is my calculated pipetting volume for the first well different from the transfer volume?

The first well is a direct dilution from your main stock solution to achieve the highest target concentration. Subsequent wells are part of a serial dilution chain, where a fixed volume is transferred from the previous, more concentrated well. These are two different dilution methods, hence the different volumes.

What is a typical dilution factor for MIC assays?

A factor of 2 (two-fold dilution) is the most common and is recommended by standard bodies like CLSI and EUCAST. This creates concentrations such as 128, 64, 32, 16 µg/mL, etc.

Does this calculator account for the volume of the bacterial inoculum?

No. The "Final Volume per Well" should be the volume *before* adding the inoculum. For example, if you need a 200 µL final volume and plan to add 10 µL of inoculum, you should set the calculator's final volume to 190 µL. Always consult your specific laboratory protocol.

Why does the calculator add a 10% excess to the total required volumes?

This is a standard laboratory practice to account for small losses during pipetting (e.g., solution adhering to reservoir walls or pipette tips) and to ensure you have enough reagent to complete all replicates without running short.

Can I calculate dilutions for molar concentrations (e.g., mM)?

The serial dilution tab works with any unit as long as the stock and highest concentration units are the same (e.g., µM). However, the output concentrations will be in µg/mL, as this is the standard reporting unit for clinical MICs. First, use the Stock Preparation tab to determine the mass-based concentration (mg/mL or µg/mL) of your molar stock.

What is a growth control well?

A growth control well contains only the broth and the microorganism, with no antimicrobial agent. It is essential to confirm that the organism can grow under the assay conditions. This calculator does not explicitly plan this well, but you should always include one in your plate setup.

What is a sterility control well?

A sterility control well contains only the broth medium to check for contamination. It should show no growth. This should also be included in your experimental setup.

What if my compound is dissolved in a solvent like DMSO?

You must ensure the final concentration of the solvent in each well is low enough to not affect microbial growth. This often requires running a separate control plate with just the solvent at the same concentrations.

References

1. Wiegand, I., Hilpert, K., & Hancock, R. E. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163–175. doi.org/10.1038/nprot.2007.521
2. Clinical and Laboratory Standards Institute (CLSI). (2018). M07: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 11th Edition. CLSI guideline.
3. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). (2023). Routine and extended internal quality control for MIC determination and disk diffusion as recommended by EUCAST. Version 13.0. eucast.org
4. Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6(2), 71–79. doi.org/10.1016/j.jpha.2015.11.005

Disclaimer

This tool is for informational and educational purposes only. It is not a substitute for professional clinical judgment, validated laboratory protocols, or regulatory guidelines (e.g., CLSI, EUCAST). All calculations should be independently verified before use in a research or clinical setting. The user assumes all responsibility for the application of the information generated by this tool.