I have 30 different samples, each of which have undergone different treatments and thus have water holding capacities (WHCs) that vary from 14%-39%, and I would like to measure the respiration rates of these samples on the same plate and compare across treatments.

The MicroRespTM protocol instructs the user to first adjust the moisture content of each sample to 30-60% (40% is recommended) of WHC and then, after incubating for 3-5 days, to add 30mg of substrate per gram of soil water in one 25uL aliquot per well.

It is unclear to me how to apply these instructions to multiple samples with different WHCs.

Question A) For multiple samples, is it best to adjust the moisture content of each sample to a specific percentage of WHC across all samples, i.e. 40%? Or is it sufficient to adjust each sample's moisture content to lie within the range 30-60% of WHC, i.e. sample 1 has a MC equal to 30% of its WHC while sample 2's MC is 45% of its WHC, etc and the moisture content of all samples lies within the range 30-60% WHC.

Question B) If the moisture content (g water/well) differs across samples, is there a disadvantage to adjusting the amount of stock substrate solution added to each well to ensure 30mg substrate/g soil water, i.e. 20uL to well 1 and 27uL to well 2, etc. Or is it necessary to make up multiple stock substrate solutions to ensure adding the same volume of solution (water) to each well?

Thank you.