Modified 25 April 2014
Ecoss Lab
Chloroform Fumigation Protocol for Microbial Biomass C and N
Modified from Hofmockel Lab protocol, modified from Suding Lab, modified from Hobbie Lab
Materials Needed:
- 0.05M K2SO4 (8.713g K2SO4 per L RO/DI water), with 50 mL re-pipetter
- Chloroform
- Alumina
- Boiling chips
- Specimen cups (2 x # samples for Step 2, and 2 x # samples for Step 3)
- V-bottom 50mL Falcon tubes (2 x # samples)
- 50mL glass beakers (1 x # samples)
- 2 - 250mL glass beakers for chloroform
- Whatman #1 filter paper (2 x # samples)
- Aluminum tins for gravimetric soil moisture content (1 x # samples)
Step 1: Subsample Soil Sample
- Weigh one sample (~10g) into a soil tin for gravimetric water content (see gravimetric water
content protocol for details)
- Weigh one non-fumigated sample (~15-20g) into specimen cup for immediate extraction with
K2SO4 for extractable C and N
- Weigh one fumigated sample (~15-20g) into 50mL glass beaker to be extracted after chloroform
incubation for extractable C and N + microbial C and N
Step 2: Non-Fumigated Sample Extraction
- Add 50mL 0.05M K2SO4 to specimen cup using a re-pipetter
- Put on lid and shake by hand to mix
- Place samples on shaker table (in 104A, cell culture room) @ ~ 125 rpm for 1 hour
(move to Step 3 while these samples are shaking)
-
Let samples sit for a few minutes to let soil particles settle
Transfer supernatant to a 50mL Falcon tube
Centrifuge for 2 minutes @ 2500 rpm
Gravity filter supernatant on pre-leached Watman filter into clean specimen cup
Place filtered extract in oven @ 60°C for ~ 48 hours – until extract is dry and crystal have formed
Extracts can be stored at room temp after completely dried.
Step 3: Fumigated Samples – do next steps in Fume Hood! Chloroform is a known carcinogen!
- In one 250mL glass beaker, pour in enough alumina to cover the bottom of the beaker
- Pour chloroform over alumina (~100mL) and let sit in hood for a few minutes to get rid of the
ethanol
- In the second, clean 250mL beaker, put ~ 1 spatula of boiling chips.
Modified 25 April 2014
-
Pour chloroform into the 250mL beaker with boiling chips, carefully avoiding the alumina settled
at the bottom
Place chloroform beaker into the vacuum desiccator with samples in it.
Evacuate the desiccator until chloroform boils (~2-5min). Open desiccator. Repeat evacuation and
opening 2 more time, NOT opening after the last evacuation.
Cover desiccator with black garbage bag because chloroform breaks down in the light.
Incubate for 5 days.
After incubation, open the desiccator, remove chloroform, and place in the ‘ethanol free used
chloroform’ bottle.
Re-evacuate the desiccator (~1min) with only the soil samples in, and open desiccator. Repeat 4
more times.
In the hood, transfer the soils in the glass beakers to specimen cups.
Add 50mL 0.05M K2SO4 and follow directions from Step 2 (shake, centrifuge, filter, dry)
Step 4: Salt prep for C and N analysis
- Grind salts with a mortar and pestle
- Store salts in glass scintillation vials
- Weigh salts in tin capsules
o Talk to Melanie in CPSIL about how much to weigh