To calculate the Minimum Inhibitory Concentration (MIC) of the probiotic using the microdilution method, you need to determine the lowest concentration of the probiotic at which there is no visible growth of the target microorganism. Here's the correct protocol to calculate the MIC:

Prepare the probiotic dilutions: Start by preparing a series of dilutions of the probiotic in the 96-well microdilution plate. Each well will contain a different concentration of the probiotic, usually ranging from highest to lowest concentration.

Add the target microorganism: Inoculate each well of the microdilution plate with the target microorganism.

Incubation: Allow the microdilution plate to incubate at the appropriate temperature for a sufficient period. The incubation time and temperature depend on the microorganism and are usually specified in your experimental protocol.

Visual inspection: After incubation, visually inspect each well for growth of the target microorganism. Growth may be indicated by turbidity or visible colonies.

Determine the MIC: The MIC is the lowest concentration of the probiotic at which no visible growth of the target microorganism is observed. It is the highest dilution that shows no turbidity or visible growth. The corresponding concentration in that well is your MIC value.

Controls: Always include positive and negative controls. A positive control should contain the target microorganism without any probiotic treatment to ensure growth occurs in the absence of inhibition. A negative control should contain only the growth medium to verify there is no contamination.

Replicates: It's essential to perform replicates for each dilution of the probiotic to ensure the accuracy of your results.

Interpretation: Based on the MIC value, you can categorize the sensitivity of the target microorganism to the probiotic. A lower MIC value indicates greater inhibitory activity against the microorganism.

This is a difficult methodologic concept to realize. You can't consider a probiotic as you would a chemical reagent.

Both the probiotic and target microbe are dynamic quantatively and qualitatively in any assay. The intial probiotic titer will not be maintained during the test so the intial titer can only be treated as threshold not absolute Further, the unstated presumption that efficacy is a direct and singular function of cell number is questionable. Certainly metabolites of the probiotic have an impact and you have no asssurance that these would be produced in vitro as in vivo. or that preobiotic cellk numb er is a valid indicator,

Hello,

The 96-well microdilution method is ideal for determining MICs. However, in your case, your probiotic is not in dry weight; instead, it remains qualitative. If I were in your position, I would freeze-dry the probiotic, weigh it to obtain the dry weight, and then prepare a precise probiotic suspension to determine the MICs.

Best regards

@Rima thanks 👍