NCCLS的微量稀释法的PROPOTAL

National Committee for Clinical Laboratory Standards. 2003.
Methods for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically,5th ed. Approved standard M7�A6.
National Committee for Clinical Laboratory Standards, Wayne, Pa.

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. In Loman, V., ed. Antibiotics in laboratory medicine, 4th ed. Williams and Wilkins, Baltimore, MD.

MATERIALS:
•  liquid cultures of bacteria at suitable growth phase
•  sterile petri dishes
•  sterile 96-well microtitre plates (round-bottom wells are best)
•  filter sterilized antibiotics
•  sterile diluents
•  test tubes.

METHOD:
1.  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.
2.  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.
3.  Using the multipipettor, dispense 100 µl of medium into all wells of a microtitre plate. Label the plate and lid.
4.  Pipette 100 µl of appropriate 2x antibiotic solutions into the wells in column 1 (far left of plate).
5.  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.
6.  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.
7.  Discard 100 µl from column 10 rather than putting it in column 11.
8.  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.
9.  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).
10.  Incubate the plates at 37oC or other desired temperature.
11.  Streak the bacterial cultures on plates to check their purity.
12.  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).
13.  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