Extraction Preparations & Procedures
*Performed on freshly sampled soils within 24 hours of collection; keep soil samples refrigerated.
*Include duplicates and blanks of your samples for each extraction procedure.
*For maximum efficiency, weigh soil samples out for all procedures (T0, PMN, MB, gravimetric moisture) at the same.
Basic Extraction Procedure
1. Line wooden racks with funnels that contain folded Whatman filter paper.
2. Decant supernatant in spec cup (after 1 hour of low-speed shaking) through filters into labeled scintillation vials
(spec cup will not be emptied; recap and save remainder for wet sieving).
3. Fill scintillation vials (leaving some room at the top for expansion). Refrigerate or freeze extractants immediately.
Where to use Extractions
Analyze T0 and PMN extractions for NH4+ and NO3- on BioTek spectrophotometer.
Analyze T0 and MB extractions on TOC.
Time Zero (T0)
a. Label spec cup for each sample/lab #.
b. Weigh out 10-11 g of moist soil into cup (tare cup).
c. Add 50 mL of 0.5 M K2SO4.
d. Cover tightly with lid and put on shaker (low speed) for 1 hour, then extract.
Gravimetric Moisture
a. Label weighing tin for each sample/lab #.
b. Record empty tin weight.
c. Weigh out 10-11 g of moist soil onto tin and record weight (tare tin).
d. Dry at 105˚C for 24 hours.
e. Record tin+ soil weight.
Potentially Mineralizable Nitrogen (PMN)
a. Label spec cup for each sample/lab #.
b. Weigh out ~22 g (21.9-22.1 g range) of moist soil and record weight (tare cup).
c. Cover tightly with lid and refrigerate until the next day.
d. Assemble enough wide-mouth Mason jars and lids with rubber septa for each sample. Add 1 mL of DI to each
Mason jar.
e. After gravimetric moisture is calculated, adjust each PMN sample with DI water based on 23% field capacity
determinations.
f. Place PMN spec cup into Mason jar and seal tightly. Place jars in dark area (e.g. cupboard) at room temperature;
record time going into cupboard.
g. After 24 hours (record start time for each block of jars), withdraw 30 cc of gas from each jar using labeled syringes
(needle passes through septa).
h. After gas withdrawal from each block of jars, open lids and use vacuum hose to vent remaining gas (but not soil).
Use the same time frame/procedure for each jar. Record end time and place block of jars back into cupboard.
i. Repeat above gas withdrawal procedure for each reading (usually Day 01, Day 07 and Day 14). **Start/stop at
the same time for each block of jars for each Day reading.
j. Record [CO2] (i.e. labile carbon) using LiCor 820.
k. After Day 14, remove 10-11 g of moist soil to weighing tin (record tin only and tin + soil weights). Oven dry tin +
soil at 105˚C for 24 hours. Record dry weight.
l. Add 50 mL K2SO4 to the remaining soil in the spec cup, and then shake for 1 hour (low speed) and extract.
Microbial Biomass (MB) (Horwath and Paul, 1994)
a. Label beaker (30-50 mL, should use same size for group of samples) for each sample/lab #.
b. Weigh out 11 g soil (10.9-11.1 g range) and record weight (tare beaker).
c. Place in fumigation chamber.
d. Warm a larger beaker (~150-200 mL size) with DI water on a stir plate.
e. While DI water is warming, add 50 mL (less may be needed with fewer samples) chloroform (under the fume
hood) to a smaller beaker (~100 mL size) that contains boiling chips.
f. Place chloroform beaker into DI water beaker (DI water MUST NOT overflow into chloroform beaker!) and place in
bottom of chamber.
g. Seal door. Open chamber vent and pull vacuum until chloroform boils, then vent chamber to hood.
h. Repeat vacuum/venting two more times, but DO NOT vent chamber to hood on third vacuum. Instead, leave
chamber sealed with chloroform boiling for 48 hours.
i. After 48 hours fumigation, vent chamber to hood. Repeat vacuum and venting two more times.
j. Empty each beaker’s sample into a labeled spec cup and add 50 mL K2SO4.
k. Put on shaker for 1 hour (low speed), then extract.
References
Horwath, W.R., and E.A. Paul. 1994. Microbial Biomass, p. 753-773, In R. W. Weaver, et al., (eds.) Methods of soil
analysis Part 2 Microbiological and Biochemical Properties. ed. SSSA Book Ser. 5. Soil Science Society of America,
Madison, WI.