MIC values out of range

Hi all,

please I really need your help. I am doing some MIC with Amikacin and the values I obtained so far for the reference controls are totally out of range and higher than expected. Can you tell me if the procedure I am following for the antibiotic diluition is correct? Just to be sure that it does not depend on me.

I prepare an amikacin stock solution of 5 mg in 500 ul.

I prepare my antibiotic dilutions that are in the range : 128-0.125 ug/ml. The final volume in each eppendorf (I have one for each antibiotic dilution) should be 1.4 ml and the dilution should be made two-fold . So what I do is to prepare the solution of antibiotic most concentrated (128 ug/ml) with this calculation:

I apply the formula CiVi=CfVf

Initial volume to be taken from the stock solution is = 128 x 1.4/ 5000/500 .This is 17.9 ul of stock

So the Volume of diluent is 1400 ul- 17.9 ul= 1382ul

From this dilution I take 700 ul and I pour it in the lower dilution (64 ug/ml) where already 700 ul of diluent are. And so on to halve the concentration everytime.

Is it correct?

Thank you very much in advance.

Silvia