Microbial respiration of soil has received considerable attention because it can be used as a soil quality indicator and it is one important variable to quantify soil microbial activity.
— Brendecke, J et al., 1993 and Alef, K., 1995 Alef, K.

 

MicroRes® Preparatory steps

Reagents:

A. Prepare the 60-ml blue (alkaline - ALK) working solution from the stock solution: 

  1. Fill the 60-ml square bottle with DI or quality tap water to approximately the 20-mL mark.
  2. Open a blue stock solution capsule and empty the content into the 60-ml square bottle.
  3. Fill the bottle to the 60-ml mark with more water.
  4. Cap the square bottle tightly with the septum cap and mix the solution thoroughly.
  5. From this point on, the solution can only be withdrawn by syringe. 

NOTE: FROM THIS POINT ON, keep CAP closed since SOLUTION IS SENSITIVE TO CO2 IN THE AIR.


Prep Syringes A and B:

B. Prepare a 1-mL syringe for each of two solutions: Syringe A for the standardized acid (HCL) solution and Syringe B for the blue (ALK) working solution:

1. Label one 1-ml syringe “Syringe A” = A(cid) and fill with the 2 mM HCI acid solution exactly to the 1-ml mark.

You can open the cap of acid solution because it is not sensitive to the air. Dispel all the bubbles in the syringe by tapping it with the needle side up and eject the bubbles out of the syringe. 

                             Syringe A - Acid Solution

                        Syringe A - Acid Solution

Note: You may prepare and fill multiple Syringes for handling multiple samples.

2. Label the second 1-ml syringe “Syringe B” = B(lue). Withdraw the blue (ALK) working solution through the septum cap of the square bottle and fill exactly to the 1-ml mark.

Dispel all the bubbles in the syringe by tapping it with the needle side up to eject bubbles.

               Syringe B - Blue Stock Solution

        Syringe B - Blue Stock Solution

NOTE: YOU MAY PREPARE AND FILL MULTIPLE SYRINGES FOR HANDLING MULTIPLE SAMPLES.


Assembling MR - soil samples (S) and blank (B)

A. Assembling the sample MRs and loading the soil samples:

  1. Insert the reaction chamber into the chamber seat.
     
                   Reaction Chamber in Chamber Seat

            Reaction Chamber in Chamber Seat

     2. Insert a small septum into the small hole on the reaction chamber. Use thumb nail to twist          and push septum into hole.    

      Septum in Reaction Chamber

   Septum in Reaction Chamber

      3. Insert the cylinder plug into the bottom (straight wall) of the sample cylinder. 

        Cylinder plug in sample cylinder           

     Cylinder plug in sample cylinder
       


       


Note: Now you need to turn on a fan (not provided) blowing towards you to homogenize the uneven CO2 in the air caused by operator's breath. hold the MicroRes away from face towards the blowing fan when coupling the reaction chamber to the sample cylinder.

          Small tabletop fan is sufficient

       Small tabletop fan is sufficient


B. Loading soil samples and coupling the sample cylinder with the reaction chamber

  1. Pre-weigh 2-g (dry weight basis) of each soil sample in separated weighing boats.
  2. Load each 2-g soil sample into a bottom-plugged sample cylinder. 

  3. Tap the sample cylinder on a flat surface to level the soil surface.

                                                                                            Pre-weigh 2-gram sample and load in sample cylinder

                                                                                         Pre-weigh 2-gram sample and load in sample cylinder

Note: The following steps 4, 5 and 6 need to be done under a blowing fan               directed towards the operator: 


    4. Put a syringe needle through the small septum (to vent air pressure when coupling) and couple           the reaction chamber (with the seat) to the top of the sample cylinder. 

   Insert needle in septum and couple sample             cylinder with reaction chamber

Insert needle in septum and couple sample             cylinder with reaction chamber


     5. Minimize the head-space of the sample cylinder by pushing the cylinder plug up leaving only a gap      approximately 1/8 inch between the soil surface and the reaction chamber seat. 

     6. Pull the syringe needle out of the small septum to form a closed headspace in the MR. This also          marks the start of the time of measurement duration of the MR. 
 

   Push up cylinder plug and        remove syringe needle

Push up cylinder plug and        remove syringe needle

C. Assembling the Blank MR:

    1. Prepare a blank MR the same way as you did for the sample MR, except leaving an empty space of     approximately 4.5 ml in the sample cylinder. This can be easily done by pre-marking a line on the           blank sample cylinder 1 inch below the cylinder collar and pushing the bottom plug so that its top is in     line with the mark.
 

         Prepare Blank MR and push up cylinder plug                          to line marked on sample cylinder

      Prepare Blank MR and push up cylinder plug                          to line marked on sample cylinder


Procedure (Adding blue reagent, pre-assay shaking and titration)

A. Using Syringe B to add Blue (ALK) Solution and Pre-Assay Shaking:

    1. Using Syringe B, inject exactly 0.5 ml blue working solution into the reaction chamber through the small septum to each of sample MRs and the blank MR .

   

    2. Shake all the MRs (2 Samples and 1 Blank) in the Orbital Mini-Shaker at a constant rate around 400 rpm for 60 minutes.

    3. At the end of the 60-minute shaking period, stop the shaker and remove the MRs.

Note: One mini shaker can accommodate 4 MRs: one control (Blank) and 3 samples.

B. Using Syringe A to add HCL for titration purposes:

  1. Using Syringe A (2 mM HCI), titrate the blue working solution (inside the reaction chamber) by inserting the needle through the small septum and doing the following:
    a. Slowly inject Syringe A acid solution into the MR until the blue solution turns to almost colorless. 
    b. Record the volume of the acid solution used in syringe A.
    c. Do the same titration for all sample and blank MRs.

Note: Soil respiration continues after the HEAD-SPACE of a MR is closed. The duration of a MR measurement starts after you close the HEAD-SPACE (When you pull the syringe needle). You should be able to close three or more sample MRs within 3 minutes. If not, you should record the initial time of each MR and use that to correct for the final result. 


Calculation of Soil Respiration Rate

Calculate the soil respiration rate (SRR) by the difference of the amount of acid used between the control (B)and the sample (S) in mL using the following formula:

Equation:

SRR (µmol CO2/g/h) = (B-S)*M*30/(t- t0), or

SSR (µL CO2/g/h) = (B-S)*M*1465/(t – t0) at 25o C

where M is the concentration of the acid in mM, t is the time when the shaking stops and t0 is the initial time of the MicroRes in minutes.

Note: If the initial times among sample MRs are different within five minutes, (t- t0) can be considered a constant for the batch. If the blue solution color changed before 60 minutes of shaking ends, record that time as t for the MR.


Reusable TEST KIT COMPONENTS:

The principal component of the Real Time Soil CO2 Respiration Test Kit is the Microrespirometer (MR) which consists of four components: Reaction Chamber, Chamber Seat, Sample Container and Cylinder Bottom Plug:

Most of these components are reusable except for the the small septum (small white plug inserted into the syringe hole of a reaction chamber, 50/pkg). The reaction chamber can be reused three times following cleaning instructions in the manual.  The remaining components (chamber seat, sample container and cylinder bottom plug) can be cleaned just by rinsing and reusing again.

REFURBISHED REACTION CHAMBER:

The used reaction chamber can be returned for a $3.00 credit, if not damaged, towards the next purchase of new reaction chambers.