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Microbial_Biomass_sequential_extraction.md

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Sequential Extraction For Peat Microbial Biomass Carbon and Nitrogen Analyses

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  1. Preparations (at least 2 days before extraction!!):

    1. Make 0.5M K2SO4 solution
      1. Hard to dissolve!
      2. Make 2L or 4L at once in a big erlenmeyer flask
      3. Dissolve by applying low heat (Do not boil!)
      4. Use a 2L volumetric flask to bring the solution to final volume quantitatively
    2. Pre-leach filter papers
      1. Whatman #42: 2.5µm, 11cm diameter
      2. Use a Buchner funnel and side-armed flask vacuume set-up
      3. Stack 10 filter papers in the funnel (lay flat), pump ~20ml K2SO4 onto the stack, then turn on the vacuume to suck the salt solution through
      4. Repeat the above step for each stack of 10 three times
      5. Spread web filter paper to dry in the fume hood over night
        1. Just to speed up the process...

  2. 1st extraction:

    1. Add 24ml 0.5 M K2SO4 directly into the subsampled soil containing tubes
      1. 1:3 soil weight/K2SO4 volume
    2. Place on shaker for 2 hours at 200 rpm at room temp (~25ºC)
    3. Balance tubes and centrifuge at 4000 rpm for 15 min
    4. Gravity filter the supernatant through K2SO4 leached filter papers
      2. Into pre-labeled 25ml scintillation vials
      3. Pour carefully to avoid splashes
      1. In-growth peat is really fine and may fall out as a big chunk when pouring
      2. If any solids were observed in the filtrate, re-filter and save the filter paper
    5. Add 2 drops of concentrated phosphoric acid to each filtrate
    6. Store the filtrate in -20ºC
      1. Make sure there are room in the vials for frozen liquid to expand.
      2. Too much filtrate will crack the glass vial.
    7. Put the filter back into the pellet containing 50ml conical tube
      1. To preserve the biomass
    8. Continue the process for fumigation and second extraction

  3. In-tube Chloroform Fumigation:

    1. In the fume hood, add 2ml chloroform to each filter paper containing peat pellet
      1. 1.5ml for 13g (Prakis & Hedin, 2001)
      2. Use vortexer to make sure everything is mixed well
        1. Or hand shake/flick to make sure the pellet and filter paper mix
    2. Keep the chloroform containing tubes in dark for ~36 hours.
      1. No shaking needed
    3. Open the tube in the fume hood and let them air out for at least 17 hours (over night).
      1. Use vacuum pump to evacuate excess chloroform fume for 5 X 1min.

  4. 2nd extraction:

    1. Extract aired pellets with 0.5M K2SO4
      1. Repeat 1st extraction step 2 (1st extraction)