Transcript
WSH June 3, 2009
NutNet Resin Bag Protocol Modified by S Harpole from Hobbie Lab Protocol by Adelia Falk and Chinelo Njaka (5 April 2001), which was based on Giblin et al. (Giblin, A. E., J. A. Laundre, K. J. Nadelhoffer, and G. R. Shaver. 1994. Measuring nutrient availability in arctic soils using ion exchange resins: a field test. Soil Science Society of America Journal. 58: 1154-1162). Fabric and extraction modifications adapted from K. Suding lab and S. Allison lab protocols.
Bag Preparation Materials: • Gloves, latex or alternative • Nylon/Lycra Swimsuit liner material • Measuring scoop, 1 Tablespoon (15mL) • Plastic zip ties / cable ties (at least 7 in) • Mixed Bed Ion Exchange Resin: Sigma Aldrich Dowex Marathon MR-3 hydrogen and hydroxide form, Catalog 428736 - 1kg. • Fabric scissors or rotary cutter and cutting mat (only use on fabric to keep them sharp). • Prepare 10% (=1.2M) HCl (For 1 L, add 103 ml of 37% HCl to bottle filled with 800 ml Nanopure deionized water; fill bottle to 1 L with additional DI water). ADD ACID TO WATER! USE FUME HOOD AND SAFE LAB PRACTICE • Tubs for acid and DI water rinsing • Ziploc bags (1 gallon) Construction: 1. Cut material into 5 inch squares. 2. Measure the resin by volume; scoop a leveled tablespoon of resin onto each square (1 Tbsp = 15 ml). 3. Bundle up corners and edges of fabric and tie off with a zip tie. Use 4 inch zip ties that you have started to close to form a loop. Tie a 1 m piece of nylon twine to the loop. 4. Once the zip tie is over the fabric edges and not squeezing the resin excessively, pull it tight with your fingers or pliers. Trim off excess fabric. 5. Bags are ready for acid wash: *
Put on gloves—you should not touch bags with bare hands once they have been washed *
6. Soak a set of 20-30 resin bags in a bath of 10% HCl solution for 45 minutes to 1 hour. 7. Rinse multiple times in de-ionized water, 10-15 minutes each rinse, until litmus paper indicates the rinse water is neutral or at least the same pH as the DI water supply. 8. Store bags in ziploc bags in the refrigerator until you are ready either to deploy them in the field.
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WSH June 3, 2009 Resin Bag Deployment and Retrieval: Materials: • Gloves, latex or alternative • String, nylon • Hori-Hori / Soil Knife (or garden trowel) → • Labeled ziploc bags (sandwich or snack sized, one per plot) label: site name, plot#, resin bag#, date in, date out • Cooler with ice (for retrieved bags) Deployment: 1. Put on gloves—you should not touch bags with bare hands once they have been washed. 2. Tie 2 bags on the string (about 2m total length), about 60 cm apart, leaving about 10 cm to attache to center post. (See resin bag location example diagram) 3. In the “Site” subplot, use the soil knife to cut a slit at a 45° angle in the soil to the hilt of the soil knife and pry up wedge of soil. 4. Gently push bag into the slit to a depth of 10 cm (bottom of bag), and push down on the soil surface to close the opening. 5. Leave the tail of the cable tie and string sticking out of the ground. 6. Optionally, mark the location so you can find it again (e.g., pin flag). 7. Attach the string to the marker. 8. Leave bags out for an appropriate period of time (1 year). Retrieval: 1. Using twine attached to marker and exposed zip tie end of bag as a guides, GENTLY excavate around bag location to loosen soil until the bag is exposed and free. Brush off excess soil. 2. DO NOT PULL BAG DIRECTLY FROM SOIL USING THE TWINE OR ZIP TIE because they may pull free of the bag and you will lose the resin! 3. Place each resin bag into its appropriately labeled (site name, plot#, resin bag#, date in, date out) ziploc bag and into the cooler and take back to the lab to mail or extract. NutNet Resin Bag Locations (actual location of the “Site” subplot is random for each plot)
Future
Site Resin Bags
(2, or 3 if deploying optional 1-month bag)
String
10 cm Future
Core Center post
2 Corner “ID” post
WSH June 3, 2009 Extracting the Resin Bags (DRAFT-Still in revision) Materials: • Gloves, latex or alternative • Extracting solution: (2M NaCl). • 250 mL Nalgene bottles, acid washed, numbered, one per resin bag and blanks • Shaker table • 50 ml centrifuge tubes (one per bag plus blanks) • Vacuum filter holders, vacuum pump and manifold (Or centrifuge them?) • Filter paper Whatman xxx, Dia=xx, Fisher #XXXX • Filter forceps • Squirt bottle with extractant • Zip lock bags (1 gallon) Make extractant (To make 14 L, enough to extract approximately 100 resin bags): 1. Fill a clean carboy (14 L or larger) with 10 L of Nanopure deionized water. 2. Add 121 ml of 37% HCl to carboy (8.6 ml/L). ALWAYS ADD ACID TO WATER! 3. Add 1636.3 grams of NaCl to carboy (116.8g/L). 4. Fill carboy with another 4 L of Nanopure deionized water. 5. Stir with stir bar or let sit until all of the NaCl is dissolved. Extract Bags: 1. Put on gloves—you should not touch bags with bare hands once they have been washed. 2. Rinse each resin bag using Nanopure deionized water to remove soil/litter/debris from the outside of the bag. 3. (Use the specially prepared funnel to rinse resins if the bag has broken—allow resin to air dry and record dry weight.) 4. Label a set of 50ml centrifuge tubes, one for each resin bag. 5. With tweezers, place filter in the bottom of each filter holder. 6. Squirt 10-20 ml of extractant onto each filter to rinse it, discarding the collected liquid and return the centrifuge tube underneath the filter holder. 7. Place resin bag into a acid-washed numbered 250 mL bottle that has been previously filled with 100 mL of extractant. 8. Shake bottles for an hour at medium speed. 9. After the bags are done shaking, let them sit for an hour 10. Filter the solution into labeled centrifuge tubes. Only about 20-30 ml should be transferred; Fill tubes to about half full. 11. Close the tubes and then store in freezer until they are analyzed. The tubes can be collected in a large labeled Ziploc TM freezer bag. 12. The remainder of the liquid can be discarded.
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WSH June 3, 2009 Analyzing the extracts TBD
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