MIC DETERMINATION BY MICROTITRE BROTH DILUTION METHOD
liquid cultures of bacteria at suitable growth phase
sterile petri dishes
sterile 96-well microtitre plates (round-bottom wells are best)
filter sterilized antibiotics
- Grow the test strains in the chosen medium to the right A600. Have antibiotic solutions and plates ready before the cultures reach the desired growth phase.
- Thaw and weigh the antibiotics. Take a note of the purity at this stage, e.g. gentamicin, 577ug/mg solid. Dissolve the antibiotics (solvent depends on the compound), then dilute in the test medium to 2x the top concentration desired in the test, e.g. if highest desired concentration is 128ug/ml, dilute to 256ug/ml. Keep on ice until use.
- Using the multipipettor, dispense 100ul of medium into all wells of a microtitre plate. Label the plate and lid.
- Pipette 100ul of appropriate 2x antibiotic solutions into the wells in column 1 (far left of plate).
- Using the multipipettor set at 100ul, mix the antibiotics into the wells in column 1 by sucking up and down 6-8 times. Do not splash.
- 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.
- Streak the bacterial cultures on plates to check their purity.
- 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 (this means putting the plate in back-to-front).
- MIC can be taken as the lowest concentration of drug that reduces, by more than 50% or 90% for MIC50 or MIC90 respectively.
NOTE: Put different drugs on different rows of the same plate, but try to avoid putting bacteria together, to prevent cross-contamination.