TITLE: Sequential Microbial Biomass and Nitrogen Extraction
AUTHORS: maggie.bowman

[DESCRIPTION]
The method is modified from the Hofmockel lab at PNNL, modified from Suding Lab protocol, modified from S. E. Hobbie, 5 May 1998. 

[STEPS]
SECTION: Subsample soil
1. Subsample (~8 g) place in V- bottomed conical tube labeled with Cat# and weight. Label caps with Cat#

SECTION: Subsample soil
2. Gravimetric water content will also be essential for final calculations on a dry weight basis.
SECTION: Data to Collect
3. Data to Collect
SECTION: Data to Collect
3.1. Weight of soil and Cat# for each sample on the data sheet
SECTION: Data to Collect
3.2. Time onto and off the shaker 1st extraction (by batch)
SECTION: Data to Collect
3.3. Time of chloroform addition (by batch)
SECTION: Data to Collect
3.4. Time of chloroform evacuation (by batch)
SECTION: Data to Collect
3.5. Time onto and off the shaker 2nd extraction (by batch)
SECTION: Rinsing Filters
4. Gather the vacuum pump, Erlenmeyer filter flask, number of filters needed, K2SO4, Nano pure water, Buchner funnel, and tweezers.
SECTION: Rinsing Filters
5. Set up a tray with bench paper. Wipe the plastic side of the bench paper with ethanol.
SECTION: Rinsing Filters
6. Set up vacuum, Erlenmeyer flask, and Buchner funnel.
SECTION: Rinsing Filters
7. Place a stack of 5-8 Filters in the funnel.
SECTION: Rinsing Filters
8. to cover the filters. Turn on the vacuum pump. 
SECTION: Rinsing Filters
9. Once the K2SO4 has been pulled through, repeat with K2SO4 two more times.
SECTION: Rinsing Filters
10. Wait a few seconds to ensure all K2SO4 is pulled thought (you will hear the vacuum noise change) Then add ultrapure water until filters are completely covered.
SECTION: Rinsing Filters
11. Wait a few seconds to vacuum off as much liquid as possible then turn off the vacuum and use tweezers to remove the filters.
SECTION: Rinsing Filters
12. Spread the filters out on the tray with the clean bench paper. Place the tray in the hood overnight to dry.
SECTION: Extraction
13. Work in batches (when possible randomized experimental treatments among batches, but do not mix isotopically labelled and natural abundance samples in order to prevent cross contamination) 
SECTION: Extraction
14. Include multiple (minimum 3 per day) empty 50 mL conical tubes. Carry these through the entire process as blanks.

SECTION: Extraction
15. Add 24 mL K2SO4 to non-fumigated subsample using repeat dispenser. Cap well. 

SECTION: Extraction
16. Place on shaker for   at room temp (20 °C)

SECTION: Extraction
17. Balance samples and centrifuge @ 4000 rpm, 20 min.

SECTION: Extraction
18. Place funnels in support racks and insert a pre-leached filter in each funnel.
SECTION: Extraction
19. Remove sample from centrifuge and place scintillation vial with matching Cat# below funnel. 
SECTION: Extraction
20. Pour supernatant into funnel, take care to check that filtrate is dripping directly into the vial. 
SECTION: Extraction
21. Leave at least 1cm head space in vials to prevent bursting (collect excess in waste container)
SECTION: Extraction
22. Using ethanol clean forceps transfer filter paper back into conical tube with soil pellet.
SECTION: Extraction
23. In the fume hood- add 2 drops of phosphoric acid to the extract (for preservation for TOC analysis).
SECTION: Extraction
24. Store extracts at-20 °C. 

SECTION: Extraction
25. Using ethanol clean forceps transfer filter paper back into conical tube with soil pellet.
SECTION: Extraction
26. Rinse and acid wash funnels for next extraction. 
SECTION: FUMIGATION
27. FUMIGATIONS MUST BE DONE IN THE FUME HOOD 

*CHLOROFORM IS A KNOWN CARCINOGEN*

Wear Viton/Butyl gloves with disposable nitrile gloves inside during steps 28-31. Always wear a lab coat and safety glasses with side guards when working with chloroform.
SECTION: FUMIGATION
28. Add 2 mL chloroform to soil pellet, plus filter.

SECTION: FUMIGATION
29. Add additional labeling to tube and cap if needed, chloroform will cause ink to run if it comes in contact with ink
SECTION: FUMIGATION
30. Let sit in the dark in the hood for 1440 min: cover the tube racks with a black garbage bag (darkness prevents the chloroform from breaking down). 

SECTION: FUMIGATION
31. Remove garbage bags and caps and allow to vent 2880 min. Be sure to keep track of which cap goes to each tube.

SECTION: FUMIGATION
32. Extract the sample as above: as per steps #13-24.
SECTION: FUMIGATION
33. As an added precaution against any residual chloroform, perform the K2SO4 addition, filtration, and phosphoric acid addition steps inside the hood
SECTION: FUMIGATION
34. The filter from the second extraction can be discarded. 
SECTION: FUMIGATION
31.1. Note: Be sure to place notice on the fume hood that chloroform containing samples are inside
SECTION: FUMIGATION
34.1. For all experiments leveraging isotopic labeling the leftover soil pellets should be archived at 20 °C. This includes 1abeled samples and the unlabeled controls.

SECTION: FUMIGATION
34.2. For experiments not requiring the pellet: At the end of the extraction open all tubes inside the fume hood and leave to dry completely to remove any trace of chloroform before disposing of tubes containing filter and soil.