<commit_before>Sequential Extraction For Peat Microbial Biomass Carbon and Nitrogen Analyses
==============
Adopted from 
------------

+ **Subsample peat (stored at +4ºC until all subsampled)**
        1. One subsample for determining gravimetric soil moisture 
                a. \>15 g/sample in pre-weighted aluminum trays
                        i. the more sample the better
                        ii. peat (especially fresh peat) contains ~90% of water
                b. Dried peat can be stored in co:in bags 
                c.Part of the dried peats can be used to determine soil pH (per location, per geography)
        2. One subsample for sequential extraction
                a. Directly subsample into 50ml conical tubes
                b. 8.1 ± 0.1 g 

+ **Sequential Extraction**
        1. 1st extraction:
                a. Add 24ml 0.5 M K2SO4 directly into the subsampled soil containing tubes
                        i. 1:3 soil weight/K2SO4 volume
                b. Place on shaker for 2 hours at 200 rpm at room temp (~23ºC)
                c. Balance tubes and centrifuge at 4000 rpm for 20 min 
                d. Gravity filter the supernatant through K2SO4 leached filter papers
                        i. Whatman #42: 2.5μm
                        ii. Into pre-labeled 25ml scintillation vials
                        iii. 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
                e. Add 2 drops of concentrated phosphoric acid to each filtrate
                f. Store the filtrate in -20ºC
                        i. Make sure there are room in the vials for frozen liquid to expand.
                        ii. Too much filtrate will crack the glass vial. 
                g. Put the filter back into the pellet containing 50ml conical tube
                        i. To preserve the biomass
                h. Continue the process for fumigation and second extraction
        2. In-tube Chloroform Fumigation:
                a. In the fume hood, add 2ml chloroform to each filter paper containing peat pellet 
                        i. 1.5ml for 13g (Prakis & Hedin, 2001)
                        ii. Use vortexer to make sure everything is mixed well
                                1. Or hand shake/flick to make sure the pellet and filter paper mix
                b. Keep the chloroform containing tubes in dark for 24 hours. 
                        i. No shaking needed
                c. Open the tube in the fume hood and let them air out for at least 17 hours (over night). 
                        i. Use vacuum pump to evacuate excess chloroform fume for 5X1min. 
        3. 2nd extraction:
                a. Extract aired pellets with 0.5M K2SO4
                        i. Repeat 1st extraction step a). through d). 
<commit_msg>fixing the line breaks<commit_after>Sequential Extraction For Peat Microbial Biomass Carbon and Nitrogen Analyses
==============
Adopted from 
------------

+ **Subsample peat (stored at +4ºC until all subsampled)**  
        1. One subsample for determining gravimetric soil moisture  
                a. \>15 g/sample in pre-weighted aluminum trays   
                        i. the more sample the better   
                        ii. peat (especially fresh peat) contains ~90% of water   
                b. Dried peat can be stored in co:in bags   
                c.Part of the dried peats can be used to determine soil pH (per location, per geography)   
        2. One subsample for sequential extraction   
                a. Directly subsample into 50ml conical tubes
                b. 8.1 ± 0.1 g    

+ **Sequential Extraction**    
        1. 1st extraction:   
                a. Add 24ml 0.5 M K2SO4 directly into the subsampled soil containing tubes   
                        i. 1:3 soil weight/K2SO4 volume   
                b. Place on shaker for 2 hours at 200 rpm at room temp (~23ºC)   
                c. Balance tubes and centrifuge at 4000 rpm for 20 min   
                d. Gravity filter the supernatant through K2SO4 leached filter papers   
                        i. Whatman #42: 2.5μm   
                        ii. Into pre-labeled 25ml scintillation vials   
                        iii. 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   
                e. Add 2 drops of concentrated phosphoric acid to each filtrate   
                f. Store the filtrate in -20ºC   
                        i. Make sure there are room in the vials for frozen liquid to expand.  
                        ii. Too much filtrate will crack the glass vial.   
                g. Put the filter back into the pellet containing 50ml conical tube   
                        i. To preserve the biomass   
                h. Continue the process for fumigation and second extraction   
        2. In-tube Chloroform Fumigation:   
                a. In the fume hood, add 2ml chloroform to each filter paper containing peat pellet    
                        i. 1.5ml for 13g (Prakis & Hedin, 2001)   
                        ii. Use vortexer to make sure everything is mixed well   
                                1. Or hand shake/flick to make sure the pellet and filter paper mix   
                b. Keep the chloroform containing tubes in dark for 24 hours.   
                        i. No shaking needed  
                c. Open the tube in the fume hood and let them air out for at least 17 hours (over night).    
                        i. Use vacuum pump to evacuate excess chloroform fume for 5X1min.   
        3. 2nd extraction:   
                a. Extract aired pellets with 0.5M K2SO4   
                        i. Repeat 1st extraction step a). through d).   

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