Methods for the Pig Project Activities

Lianne Kurina

KCl extracts

1) Initial pool size

  1. take 10 cm soil cores
  2. sieve soil with 2 mm sieve (if dry enough), or if by hand, using a long plastic bag and pulling out rocks and roots
  3. weigh out 10 g (field weight) into a solo cup
  4. add 50 ml KCl (2 molar) and shake for one minute (also put 50 ml in a cup with no soil for a blank)
  5. let sit for 24 hours
  6. set up a metal rack with filter papers in funnels
  7. leach each filter paper with 90 ml of KCl (fill to the top of the funnel 3 times) into used solo cups
  8. pour a few ml of your soil solution supernatant into the appropriate funnel to get rid of the pure 2 M NaCl
  9. set up the rack over clean solo cups and pour the supernatant of the soil solution into the filter paper
  10. transfer the solution from solo cups to autoanalyzer vials (fill 2 autoanalyzer vials 2/3rds full to allow room for expansion when the solution freezes and keep one set in the freezer at Magma Lab in case the other set gets lost). Put them into the freezer.

2) Mineralization/Nitrification

  1. follow steps a-c above
  2. put the solo cups into the dark and let them sit for 10 days
  3. follow steps d-k above
  4. net mineralization = (nh4 + no3 at time 10) - (nh4 + no3 at time 0)
  5. net nitrification = (no3 at time 10) - (no3 at time 0)

3) Making the KCl

  1. ultimately, we're after 2 molar KCl, which is 2.98 kg KCl / 20L water.
  2. fill big carboy with 10 L of DI water
  3. add the KCl (2.98 kg) and get it into solution
  4. bring the carboy up to volume (20 L)
  5. try to use only one batch of KCl per set of soils
  6. if you finish (or come close to finishing) a batch of KCl - be sure to make a new batch.

4) Soil dry weight

  1. Put some soil into a metal tin (keep track of which soil is going into which labelled tin - Write this information down in the rite-in-the-rain book). Weigh the wet soil.
  2. Put in the 100 deg oven for 72 hours.
  3. Weigh the soils for dry weight.
  4. Grind the soils in the wiley mill, put some into a coin envelope and ship this to Doug At Stanford for carbon and nitrogen analyses (make sure the envelope is clearly labelled).

Resin bags

1) Making bags

Cut out strips of material that are 6cm x 10cm. Fold the material in half and sew the bag so that you have a 6cm x 5cm bag with one side open. Put 3.0 g (get it accurate to 3.01) of either cation or anion exchange resin into the bag and submerge the bottom part of the bag in DI until you're ready to sew the last side. Sew up the top with either red (anion bags) or white (cation bags) thread. Also, sew on a red or white leash (30-40cm) to each bag for easy recovery. The thread for the sides of the bag should be all purpose cotton/polyester thread. The thread for the leash should be strong "Button/carpet" thread. Put the uncharged bags into ziplocs with some DI water in them so that the bags stay moist. Pop them into the fridge.

2) Charging the bags

  1. Submerge them in the square tupperware in either 2 M NaCl (for anion bags) or 2 M HCl (for cation bags).
  2. Shake the tupperwares by hand for a while (10 minutes) then let them sit for 24 hours.
  3. Rinse all the bags with DI and put them in ziplocs and send them out into the field.

3) Placing the bags

  1. Use a trowel to make two slits next to each other, about 4 to 6 cm deep, disturbing the soil As little as possible.
  2. Lift the soil mat and place one of your resin bags into the horizontal space. Pat the soil down On top of it and tie the leash to the flag.
  3. Do steps a and b again for the other resin bag, not placing it too close to the first.

4) Collect bags and put them into a labelled ziploc (anions and cations separate).

5) Extracting the bags

  1. Rinse the bags thoroughly in DI to get dirt off and shake well to get the excess water off.
  2. Put each bag into its own 50ml culture tube (the ones with blue caps sitting in styrofoam containers on our shelves). Label the culture tubes clearly.
  3. Add 20 ml of 0.5 M NaCl to the anion bags and 20 ml of 0.5 M HCl to the cation bags. Put the tubes into the cups on the two shaker plates (4 tubes should be able to fit in each cup). Turn the shakers on and let the resin bags shake for 6 hours.
  4. Take the resin bag out, trying not to remove any of the liquid. Rinse the resin bags well in DI. Put them in a ziploc (with some DI - remember to always keep them wet) and pop them in the fridge.
  5. Transfer the extract from the 50 ml tubes to clearly-labelled scintillation vials.
  6. Cap tightly and freeze.

6) Making solutions.

  1. 0.5 M NaCl = 29.225 g NaCl/1000 ml solution (in DI H2O)
  2. 0.5 M HCl = 41.5 ml concentrated HCl/1000 ml solution (in DI H2O)
  3. 2.0 M NaCl = 116.9 g NaCl / 1000 ml solution (in DI H2O)
  4. 2.0 M HCl = 166 ml concentrated HCl / 1000 ml solution (in DI H2O)

(remember to be careful with the concentrated HCl - work in the hood when you're using it)