The size of inoculum does affect the MIC, but the effect depends on the antibiotic and its mechanism of action. There are two main reasons behind this: 

1- Increased cell number result in decreased effective number of antibiotic molecules/cell (you may say per target molecule). Depending on the mechanism of action it may change kinetics of killing or prevent killing altogether. 

2- Increased inoculum results in fewer cell generations before culture reaches saturation. Some antibiotics require active growth for killing (e.g. beta-lactams) and it takes few generations to accumulate a defect which would result in cell death. Hence, if you start with the dense culture you may not have enough time (number of cell divisions) to observe the killing.  

Unfortunately, the MIC change isn't just simply proportional to the cell number, and there is no way of calculating it. That's why the MIC determination protocols are standardized for 10^5 cell/ml, so it can be directly comparable. 

In our observations either you keep the concentation 100 or 10000/ ml, MIC show no appreciable change but when it is above 1000000/ ml then absorbance test often gives variable results. 

The MIC should remain the same regardless of cell number. What happens at higher cell densities is that you are unable to accurately measure the effect of the inhibitor, so for example if you have a cell density at a high enough number to measure visible turbidity, the inhibitor might stop further growth but the existing cells will contribute to the turbidity even if now dead. 

Thanks that was quite helpful 

The size of inoculum does affect the MIC, but the effect depends on the antibiotic and its mechanism of action. There are two main reasons behind this: 

1- Increased cell number result in decreased effective number of antibiotic molecules/cell (you may say per target molecule). Depending on the mechanism of action it may change kinetics of killing or prevent killing altogether. 

2- Increased inoculum results in fewer cell generations before culture reaches saturation. Some antibiotics require active growth for killing (e.g. beta-lactams) and it takes few generations to accumulate a defect which would result in cell death. Hence, if you start with the dense culture you may not have enough time (number of cell divisions) to observe the killing.  

Unfortunately, the MIC change isn't just simply proportional to the cell number, and there is no way of calculating it. That's why the MIC determination protocols are standardized for 10^5 cell/ml, so it can be directly comparable. 

I agree with Anna Konovalova. We did some growth curve experiments with different inocula, keeping the previous determined MIC the same. So while for 10^5 growth occured after a specific lag-time, which was around 72 hours, no growth was appeared for 10^2 in that period of time.

So I would advise you to stick to e.g. the guidelines of EUCAST. 

Hi. I agree with Anna Konovalova, too. The MIC depends on the inoculum you use to set up the experiment, together with other variables, such as the culture media you used, that can under/overestimate the value. You can see these effect in the attached paper. You can try to use standard inoculum as suggested, and if you want you can determine the MBC (minimum bactericidal concentration) the lowest concentration of an antibacterial agent required to kill a particular microorganism.

Have a nice day

Alessandro

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