We are measuring soil nitrification potential in soil using the Griess-Ilosvay's method, according to Raglan et al., 2022 (see extract below). We have in total 360 soil samples which were stored at -20°C immediately after the harvest. In the protocol below, the rate is calculated from comparing each sample incubated at RT with a sample incubated at -20°C.
Since I work with already frozen soil samples, I am wondering how would you minimize the effect of freeze-thawing processes on the two samples?
Thank you
Method from:
Article Long Term Influence of Fertility and Rotation on Soil Nitrif...
"Briefly, 5 g of homogenized field soil in 50 mL Falcon tubes was shaken for 5 h at room temperatures after adding 1 mM (NH4)2SO4, and 1.5 M sodium chlorate. Each sample had a corresponding control sample which was treated identically, but frozen at −20°C for the 5-h incubation. 2M KCl was added after the incubation, and the tubes were manually shaken, then centrifuged for 2 min at 2,000 RPM. The supernatant was filtered using Whatman 42 filter papers. NO2-N was measured using a Genesys 20 spectrophometer (Thermo Scientific, Rochester, NY) after adding Griess-Ilosavay reagent (sulfanilamide and N-napthylethyldiamine) at 520 nm. NO2-N concentration was quantified against a NaNO2-N standard curve. Potential nitrification rates were measured as the change in NO2-N concentration between the aerated and frozen samples, by the soil gram dry weight (% dry matter) per hour."
Not directly related to nitrification potential but similar issues also present for enzyme activities. I think most important thing is to make sure every sample is treated in the same way for all. You can take them out of freezer one by one and keep them on ice while weighing them. It's important you try to follow the same procedure for each sample.
Some further reading might help you figure out also some other things for yourself;
Article Considerations in the storage of soil samples for enzyme act...
https://soil.copernicus.org/articles/7/95/2021/soil-7-95-2021.pdf
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