Question

How do you determine the MIC value using the broth dilution method?

Answer 1

MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. MIC is defined as the lowest concentration of the antimicrobial agent that prevents visible growth of a microorganism under defined conditions, Broth dilution uses liquid growth medium containing geometrically increasing concentrations (typically a twofold dilution series) of the antimicrobial agent, which is inoculated with a defined number of bacterial cells. The final volume of the test defines whether the method is termed macrodilution, when using a total volume of 2 ml, or microdilution, if performed in microtiter plates using r500 ml per well. After incubation, the presence of turbidity or a sediment indicates growth of the organism.

Broth dilution is a technique in which containers holding identical volumes of broth with antimicrobial solution in incrementally (usually geometrically) increasing concentrations are inoculated with a known number of bacteria.

Broth microdilution denotes the performance of the broth dilution test in microdilution plates with a capacity of ≤ 500 µL per well.

NOTE : This protocol colud be helpful to determine the MIC through broth dilution. you Can use accordingly the testubes for dilution and different concentration of your choice.

PROTOCOL:

	Grow the test strains in the chosen medium to the right A600
	Using the multipipettor, dispense 100ul of medium into all wells of a microtitre plate
	Pipette 100ul of appropriate 2x antibiotic solutions into the wells in column 1
	Using the multipipettor set at 100ul, mix the antibiotics into the wells in column 1 by sucking up and down 6-8 times.
	Withdraw 100ul from column 1 and add this to column 2. This makes column 2 a twofold dilution of column 1, e.g. for the example above this would be 64ug/ml. Mix up and down 6-8 times. Transfer 100ul to column 3. Repeat the procedure down to column 10 only. The same set of tips can be used for the entire dilution series. Discard 100ul from column 10 rather than putting it in column 11.
	Pour bacteria of the right A600 into a sterile petri dish. The bacteria may be diluted first depending on the desired inoculum. The appropriate inoculum size for standard MIC is 104 to 105 CFU/ml.
	With the smaller multipipettor set to 5ul, dispense bacteria into wells in columns 11 to 1 in that order. Do not add bacteria to column 12 (sterility control and blank for the plate scanner).
	Incubate the plates at 37oC or other desired temperature.
	When satisfactory growth is obtained (18-36 hours) scan the plates with an ELISA reader (e.g. Levy’s lab). Use column 12 as the blank
	MIC can be taken as the lowest concentration of drug that reduces, by more than 50% or 90% for MIC50 or MIC90 respectively.