Sequential Microbial Biomass and Nitrogen Extraction

Subsample (~8g) place in V- bottomed conical tube labeled with Cat# and weight. Label caps with Cat#

Gravimetric water content will also be essential for final calculations on a dry weight basis.

Data to Collect

Weight of soil and Cat# for each sample on the data sheet

Time onto and off the shaker 1st extraction (by batch)

Time of chloroform addition (by batch)

Time of chloroform evacuation (by batch)

Time onto and off the shaker 2nd extraction (by batch)

Gather the vacuum pump, Erlenmeyer filter flask, number of filters needed, K2SO4, Nano pure water, Buchner funnel, and tweezers.

Set up a tray with bench paper. Wipe the plastic side of the bench paper with ethanol.

Set up vacuum, Erlenmeyer flask, and Buchner funnel.

Place a stack of 5-8 Filters in the funnel.

to cover the filters. Turn on the vacuum pump.

Once the K2SO4 has been pulled through, repeat with K2SO4 two more times.

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.

Wait a few seconds to vacuum off as much liquid as possible then turn off the vacuum and use tweezers to remove the filters.

Spread the filters out on the tray with the clean bench paper. Place the tray in the hood overnight to dry.

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)

Include multiple (minimum 3 per day) empty 50mL conical tubes. Carry these through the entire process as blanks.

Add 24mL K2SO4 to non-fumigated subsample using repeat dispenser. Cap well. 

Place on shaker for 200rpm at room temp (20°C)

Balance samples and centrifuge @ 4000rpm.

Place funnels in support racks and insert a pre-leached filter in each funnel.

Remove sample from centrifuge and place scintillation vial with matching Cat# below funnel. 

Pour supernatant into funnel, take care to check that filtrate is dripping directly into the vial. 

Leave at least 1cm head space in vials to prevent bursting (collect excess in waste container)

Using ethanol clean forceps transfer filter paper back into conical tube with soil pellet.

In the fume hood- add 2 drops of phosphoric acid to the extract (for preservation for TOC analysis).

Store extracts at-20°C.

Using ethanol clean forceps transfer filter paper back into conical tube with soil pellet.

Rinse and acid wash funnels for next extraction. 

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.

Add 2mL chloroform to soil pellet, plus filter.

Add additional labeling to tube and cap if needed, chloroform will cause ink to run if it comes in contact with ink

Let sit in the dark in the hood for 24h 0m 0s: cover the tube racks with a black garbage bag (darkness prevents the chloroform from breaking down). 

Remove garbage bags and caps and allow to vent 48h 0m 0s. Be sure to keep track of which cap goes to each tube.

Note: Be sure to place notice on the fume hood that chloroform containing samples are inside

Extract the sample as above: as per steps #13-24.

As an added precaution against any residual chloroform, perform the K2SO4 addition, filtration, and phosphoric acid addition steps inside the hood

The filter from the second extraction can be discarded.

For all experiments leveraging isotopic labeling the leftover soil pellets should be archived at 20°C. This includes 1abeled samples and the unlabeled controls.

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.