MIC DETERMINATION BY MICROTITRE
BROTH DILUTION METHOD
(NB. For cationic antimicrobial
peptides use the modified MIC method)
REFERENCE: Amsterdam, D.
1996. Susceptibility testing of antimicrobials in liquid media, p.52-111.
Loman, V., ed. Antibiotics in laboratory medicine, 4th ed. Williams and
Wilkins, Baltimore, MD.
liquid cultures of bacteria at suitable growth phase
sterile petri dishes
sterile 96-well microtitre plates (round-bottom wells are
filter sterilized antibiotics
NOTE: Put different drugs on different rows of the
same plate, but try to avoid putting bacteria together, to prevent cross-contamination.
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, 577 µg/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 128 µg/ml, dilute to 256 µg/ml. Keep on ice until use.
Using the multipipettor, dispense 100 µl of medium
into all wells of a microtitre plate. Label the plate and lid.
Pipette 100 µl of appropriate 2x antibiotic solutions
into the wells in column 1 (far left of plate).
Using the multipipettor set at 100 µl, 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 64 µg/ml. Mix up and down 6-8 times. Transfer 100 µl
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 100 µl 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 2 x 104
to 105 CFU/ml.
With the smaller multipipettor set to 5 µl, 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 (or read by eye). 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
Updated 01 December 2000.