Hi,

I need to perform a microdilution agar assay to determine MIC values for some essential oils according to the method described by Golus Et al., 2016 (DOI: 10.1111/jam.13253) which is a modified method of CLSI (2015). They suggest netting 100 µL consisting of approximately 10 µL of essential oil and 90 µL of molten agar media and below 2% DMSO and then filling each well of the microplate with 100 µL of the mixture. Then each well would be inoculated with 10^4 CFU. I guess they have used nearly 200 µL of essential oil + medium in the first well, right? Or else how is it possible to use an initial 100 µL and be able to dilute it in wells while having a final volume of 100 µL per well?

1. If I use less medium, say 40 µL of medium with 10 µL of essential oil to obtain near 20% concentration then inoculate with 10^4 CFU, will the growth of microbes alter significantly? If yes, does it even notably affect the results of MIC?

2. What about using a more medium to essential oil ratio?

3. Can I reduce the amount of inoculum the same ratio as the medium volume has been reduced in respect of the reference method to prevent any adverse effects?

My main purpose is to use less essential oil and also to obtain a higher initial concentration for MIC assay.

I appreciate any suggestions or answers.

Sincerely, Nafis