The MicroResp™ system requires calibration for individual laboratories to take into account different spectrophotometers that are used, different types of soil or other environmental samples, and incubation conditions. The conversion of Absorbance to %CO2 is a non-linear relationship and the best fitted curve (regression analysis) is used to obtain the formula and parameters.
There are various ways to carry out the calibration. The Detection gel is incubated with varying CO2 concentrations for a period of time and then measured on a microplate spectrophotometer reader. The incubation time and temperature should be the same as that of your experiment. In our case MicroResp™ was carried out on soils incubated at 25oC over 6 hours.
Microplate strips (MicroStrip)
Assembled 1 x 8 well breakable strips in a microplate frame are filled with the detection gel. These can be broken (usually into a strip of 4 wells) and placed into a sealed jar or suitable container for the measurement of CO2.
Duran® bottles (or similar) containing various volumes of soil (or a variety of soils with different respiration rates), with a Microstrip of 4 wells (and detection gel) are sealed with a rubber bung for a set time, and incubated at a constant temperature. After incubation a sample of CO2 is removed and measured on a GC. The bung is then removed and the strip of wells inserted into the carriage plate and read on the spectrophotometer.
To obtain a range of %CO2 and absorbance values it is advisable to use a range of samples with varying respiration rates and/or differing amounts of the sample. Containers filled with different concentrations of %CO2 could also be used to calibrate the system.
Insert a strip of 4 wells from the breakable microplate, containing the detection gel, into several glass vials (with septa). Fill a Tedlar® gas sampling bag with a standard CO2 gas, e.g. 5% CO2 in N2. From each vial, remove a known quantity of air using a gas tight syringe and replace with the corresponding quantity of the standard CO2. Aim to have a range of at least seven different volumes for the calibration curve. After incubation, open the vials and read the strip wells on the spectrophotometer. The theoretical concentration of CO2 and the absorbance reading from the spectrophotometer are used to obtain the calibration curve.