Updated 09/03/13
Optimized Lab Protocols for Testing Soils
By Jared L. DeForest
Department of Environmental and Plant Biology
Ohio University, Athens, OH 45701
Phone: 740-593-0742
email: [email protected]
Disclaimer: Most of these methods are modified for testing acidic, fine-texture forest
soils commonly found in Eastern Deciduous forests. Some methods may not directly
apply to other soils (e.g. agricultural, calcareous, or sandy soils). Adjustments in the
amount of soil (g) used and standard range might be necessary.
Sampling & Preparing
Soil Physical Properties
Texture
General Soil Chemistry
Soil pH
Lime Requirement (SMP)
Organic Carbon (wet combustion)
Total C and N (elementar)
CN Standard & Sample Range
Exchangeable Acidity & Aluminum
Extracting for Base Cations - Volumetric
Extracting for Bioavailable Base Cations - Gravimetric
Phenolics (microplate)
Ammonium Analysis (microplate)
Nitrate Analysis (microplate)
Soil Phosphorus
Ascorbic Acid Method
Ascorbic Acid Method - For Anion Exchange Membranes (microplate)
Modified Ascorbic Acid Method
The advantage of the modified ascorbic acid method is it does not include
orthophosphate derived from the hydrolysis of polyphosphates and/or acid-labile
organic P. If the research question involves just general determination of P, then both
methods are fine or use an ICP. However, if the research question involves determining
specifically inorganic ortho-P (e.g. resin P) then the modified method is recommended.
Phosphorus Fractions (Hedley Fractionation)
Available Phosphorus (Bicarb Extraction, Total and Inorganic)
Phosphorus Sorption
Microbial Phosphorus
Microbial Fuction & Community
Phospholipid Fatty Acid (PLFA) Analysis
Hydrolytic Enzyme Activities
Nitrogen Mineralization
Litter
Litter Fractionations
Major Analytical Equiptment:
HP 6890 GC-FID w/ autosampler
Synergy HT Multi-Detection Microplate reader
DeForest Lab
Ohio University
Sampling & Preparing for Analysis
Recommended diameter of soil core
Physical: 6 cm to 10 cm
Chemical: 6 cm
Biological: 2 cm to 6 cm
Fine Roots: > 6 cm
Depth of soil core
Typically between 5 cm and 20 cm
For biological (5 cm) or chemical sampling (10 cm), try to keep within the A horizon. The cores
should be 15 to 20 cm deep if you are interested in determining basic physical properties or near
total nutrient pools.
IMPORTANT - Always carefully scrape away the organic horizon before you take a mineral
soil sample. Become familiar with the differences between the Oe/a and A horizons.
Procedure - Sampling
When sampling, it is important to have several (3 to 12) soil cores within a sample bag. This
will account for natural variation found within the plot and minimize outliers. Also, you will
have plenty of soil for analysis and archiving.
Procedure - Preparing
It is important to completely homogenize soil samples prior to analysis. Any rocks, roots, or
pieces of organic matter should be removed. Furthermore, large aggregates should be reduced in
size to less than 2 mm in diameter. Soil should be passed through a 2 mm sieve.
Store at 4oC for biological activity or labile P analysis
It is important to measure soil biological activity (e.g. mineralization & enzymes) and
liable nutrients as soon as possible (within a week).
Air dry for basic physical (e.g. texture) or chemical analysis (e.g. total C, N, CEC, Exc. Al, etc.)
To quickly air dry, place sieved soil in clearly labeled paper lunch bags in the forced-air
oven with the fan on, but heater off (~45oC) for a few days. Shake at least once a day.
Because most report soil in units per kg of oven-dried soil, you must determine the moisture
content of the soil. Weight around 10 to 20 g of fresh soil (or air-dry) on a tin weight boat and
oven-dry at 105oC for at least 48 hrs. Find the ratio of oven-dried to fresh soil.
You can also measure gravimetric water content (g): g = (g moist - g dry ) / (g dry ).
With bulk density you can determine volumetric water content (v): v = g * bulk
density (g cm-3)
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Created: 12/19/2008
Revised: 11/01/2011
DeForest Lab
Ohio University
Soil Texture - Hydrometer method
Materials – Dispersing agent
In 1 l flask (enough for 10 samples)
50 g sodium hexametaphosphate
Bring to approximate volume with distilled water
250 ml Erlenmeyer flask
Electric blender (milkshake machine) with metal cups
Glass sedimentation/graduated cylinder (1 liter)
Stopwatch/timer
Soil hydrometer (ASTM 152H)
Amyl alcohol
Procedure
Add the following to 250 ml Erlenmeyer flask
40 g
Oven-dried equivalent air dried, sieved soil
100 ml
Dispersing agent
Place on orbital shaker (150 rpm) overnight.
Swirl mixture then quickly transfer into metal blender cup, using tap water from a squirt bottle to
rise out all the soil particles. Add tap water to the blender cup to 2/3 full and blend for 5 minutes
on high speed.
Transfer the soil slurry into cylinder and use squirt bottle to rinse out ALL soil particles. Fill the
cylinder to the 1 liter mark with tap water. Allow the suspension to equilibrate to room
temperature.
Set up a blank cylinder with just tap water with 100 ml of the dispersing agent to the 1 liter mark.
This blank reading (RL) will be used to correct your hydrometer readings.
Thoroughly mix the suspension using the metal plunder for 30 sec. Be careful when doing this to
avoid splashing any of the solution and soil particles out the top! Remove the plunger and
immediately note the time.
Gently ease the hydrometer into the cylinder and after exactly 45 seconds (R45s), take the
hydrometer reading. If foam obscures the reading, add 1-2 drops of amyl alcohol. After taking
the 30 second reading, do not move or disturb the suspension.
An hour and a half after stirring carefully insert the hydrometer and take a reading (R1.5h).
At least 24 hours after stirring, carefully insert the hydrometer and take a reading (R24h).
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Created: 01/28/11
Revised: 10/10/11
DeForest Lab
Ohio University
Soil pH - glass electrode method
Standards
Buffer solution 4.00 (Fisher # SB101-500)
Buffer solution 5.00 (Fisher # SB102-500)
Buffer solution 7.00 (Fisher # SB108-500)
Deionized (DI) water (503 Porter)
Materials
pH meter with glass electrode
125 ml sample cup
Graduated cylinder (25 ml) or repipettor
Orbital shaker
Procedure
Add the following to 125 ml sample cup
10 g
Oven-dried equivalent, sieved soil (can be air dried or field fresh)
20 ml
Deionized (DI) water
A 1:1 dilution (10 g soil to 10 ml DI water) is also acceptable. Do not exceed a 1:5
dilution.
Place on orbital shaker (150 rpm) for 30 minutes.
Let stand for 10 min to equilibrate with atmospheric CO2 and then swirl gently.
Calibrate pH meter with pH 4.0, 5.0, and 7.0 buffer solutions.
Note that buffer solutions are standardized to specific temperatures. You may have to
adjust buffer pH when room temperature differs greatly from 25oC.
Insert pH electrode into soil slurry and read the pH after display has stabilized on a calibrated pH
meter.
Rinse pH electrode with DI water to remove any soil or organic matter before moving onto next
sample. Please do not blot with a tissue (i.e., Kimwipe).
Check calibration after every 15 samples to nearest pH buffer of soil pH. Recalibrate if
necessary.
What to report
Soil slurry dilution (1:2)
Water extracted (as opposed to 0.01 CaCl2 or 1 M KCl solution)
Air dried or field fresh soil
pH units to at least 0.1
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Created: 01/27/09
Revised: 10/10/11
DeForest Lab
Ohio University
Lime Requirement
Slightly Modified Shoemaker-McLean-Pratt (SMP) Single Buffer Method
Reagents
SMP Buffer – Part A
In 1 liter flask
Fill one-third the flask with DI water
1.8 g
p-nitrophenol (HO · C6H4 · NO2)
3.0 g
potassium chromate (K2CrO4)
53.1 g
calcium chloride dihydrate (CaCl2 · 2H2O)
Shake vigorously
SMP Buffer – Part B
In 250 ml flask
100 ml
DI water
2.0 g
Calcium acetate (Ca(OAc)2)
Mix until fully dissolved
Add part B to part A in the 1 liter flask and shake vigorously when they are combined.
Place on an orbital shaker for 2 hrs.
SMP Buffer – Part C
In the same 1 liter flask from above
2.5 ml
triethanolamine (TEA)
Shake mixture periodically until it is completely dissolved.
Adjust to pH 7.50 ± 0.02 with 4 M NaOH or 4 M HCl
Transfer mixture into a 1 liter volumetric flask
Bring to volume with DI water
Purge the solution with N2 and store in a dissector. CO2 or water vapor will contaminate the
buffer.
Procedure
In a 125 ml sample cup
5.2 g
Fresh sieved soil
10 ml
DI water
10 ml
SMP buffer
Place on orbital shaker (250 rpm) for 10 minutes
Let sit for 30 minutes
The times of shaking and standing are of prime importance. Therefore, stagger samples, at most,
in sets of six to keep close to these times as possible.
Gently swirl the solution before measuring pH. Record to nearest 0.01 pH unit as soil-buffer pH.
Adapted from Sim (1996) Chapter 17 “Lime Requirement”
SSSA Methods of Soil Analysis. Part 3. Chemical Methods
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Created: 09/15/10
DeForest Lab
Ohio University
Organic Carbon Determination
Analysis of Soil Organic Matter by Wet Combustion
Reagents
In 1 L volumetric flask
49.04 g
Potassium Dichromate (K2Cr2O7)
Bring to volume with DI water
36 N Sulfuric Acid (H2SO4)
Standard
In 1 L volumetric flask
9.5 g Sucrose
Bring to volume with DI water
Add 1, 2, 4, 6, and 8 ml of the 4 mg C ml-1 sucrose standard solution into five 250 ml
Erlenmeyer flasks.
Add 10 mL Potassium Dichromate, swirl
Add 20 mL Sulfuric Acid
Add 100, 99, 98, 96, 94, and 92 ml of H2O to the 0, 4, 8, 16, 24, and 32 mg C standards
Standards: 0, 4, 8, 16, 24, and 32 mg C
Procedure
Add the following to 250 mL Erlenmeyer flask
1.0 g
Soil (0.5 g if samples is dark)
10 mL
Potassium Dichromate
Swirl (make sure soil is completely covered)
20 mL
Concentrated H2SO4
Swirl & let sit for 10 minutes
100 mL
DI water
Centrifuge for 5 min at 2000 rpm
Using a pipette, transfer supernatant 200 l into each well in one column on a clear well plate
Measure absorbance at 620 nm
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Created: 6/25/07
DeForest Lab
Total Carbon and Nitrogen – (Analyzer Set Up)
1) Turn on computer and wait until it finishes loading.
2) Remove black carousel from top of C/N analyzer.
3) Turn on (green switch) C/N analyzer and allow 2 minutes for initialization.
a. If initialization is successful, two carousel alignment pins will line up on top.
4) Return black carousel to top of C/N analyzer. Make sure the two receiving holes are aligned.
5) Load Vario EL software
a. Note furnace temperature on lower gray status bar, if it doesn’t read ambient (e.g., 23oC) then
there is no PC-C/N communication.
b. Only one copy of the software can be open at one time.
6) Fully open valves on gas tanks. Note tank pressure, it must be above 200 psi.
7) Check flow on C/N analyzer:
a. Left flow meter should read ~1.0 bar; O2: 2.5 bar (blue tank).
b. Right flow meter should read ~3.0 bars; He: 29 psi (orange tank).
c. Round pressure gauge should read 1.25 bars.
8) Always perform a leak check if system was repacked or pressure gauge is < 1.0 bars.
a. Lower the He pressure to 18 psi.
b. Plug white hoses in back of C/N analyzer with fittings.
c. In the software go to options, misc., leak check, to start auto leak check.
d. If you pass the test with < 80%, then contact Dr. DeForest.
e. Remember to reset He pressure to 29 psi.
9) Within the software, go to task bar and select Options, Parameter, (no password, click ok), enter
values for furnace temperatures and click ok.
a. Furnace 1 = 950oC
b. Furnace 2 = 500oC
10) Confirm that ovens are warming:
a. Lower right status bar should read “heating up”.
b. Lower left status bar, furnace temperatures should be rising.
11) System is operational when furnaces attain target temperatures for at least 1 hour. It will take ~ 2
hours after turning on the furnace. For best results, let it run overnight.
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Created: 07/01/09
Revised 11/21/12
DeForest Lab
Total Carbon and Nitrogen – (Sample entry & Standards)
Carousel tray should be set up with:
2 empty (blank) tins
2 run-in samples (conditioner, 1-3 mg of Acetanilide)
3 standards (see reference standard section)
Oxygen setting for soil and litter should be 1, wood should be 3, and blank is 2.
Set up Vario-EL spreadsheet prior to run (double click on sample lines to access input). The following
is an example for soil (2-5% carbon) on the large carousel:
Sample
1
2
3
4
5
6
7
8 - 23
24
25-40
41
42-57
58
59-74
75
76-80
Name
Blank
Blank
Run-in
Run-in
Atro
Atro
Atro
[Sample 1-15]
Atro
[Samples 16-31]
Atro
[Samples 31-46]
Atro
[Samples 47-62]
Atro
[Samples 62-66]
Wt. (mg)
1.0
1.0
~2.5
~1.5
~1.0
~0.6
~2.0
[balance]
~1.2
[balance]
~0.8
[balance]
~2.5
[balance]
~0.7
[balance]
O2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
If the analyzer has been inactive for several months, it is ok to start a carousel with a run-in to purge the
system.
Reference standard (font color will be blue)
(Atropine – 70.56% C & 4.84% N) – Name must be ‘Atro’: Best for soil/litter/wood
or
(Acetanilide - 71.09% C & 10.36% N) – Name must be ‘Ace’: Best for biomass
For most samples (atropine standard):
SOIL - Weigh out the standards between 0.6 mg and 3.0 mg.
Half of the standards should be between 0.6 mg and 1.25 mg
LITTER - Weigh out the standards between 1.25 mg and 4.5 mg.
Half of the standards should be between 1.75 mg and 3.00 mg
Make sure each standard weight is at least 0.2 mg apart from the other standard weights.
Refer to the table on the side of the fume hood for details.
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Created: 07/01/09
Revised 11/21/12
DeForest Lab
Total Carbon and Nitrogen (Sample Prep)
Procedure – Pulverize soils
Use the 8000M ball mill (Room 415); ask Dr. DeForest for permission before using.
1)
2)
3)
4)
5)
Add enough air-dry, sieved soil to fill the chamber half way (~15 g).
Add several pulverizing balls.
Pulverize for 1 min.
Place pulverized soil in a labeled small vial.
Store in a desiccator away from light.
Procedure – Encapsulating samples
Do not touch the tin with your hands, use forceps
1) Place a blank tin on the microbalance
2) Tare balance so it reads 0.000.
3) Remove tin and place within a 96-well plate.
4) Add pulverized soil to tin using a micro scoop or spatula.
Do not add soil or litter to the tin while on the balance!
5) Place back on scale.
If weight is off target, add or remove soil and re-weigh.
Encapsulated weight for typical forest soils is between 25 and 30 mg.
Organic soils/compost- 10-15 mg
Subsoil/mineland - 45-50 mg
Encapsulated weight for litter is between 3 and 4 mg.
If weight is on target, carefully fold tin over and form a plug with the press.
It is very important that the encapsulated sample form a ball or thick disk.
6) Place encapsulated sample back on scale.
7) Press the yellow button on the microbalance to record weight in Vario EL software. (Optional)
8) Always record weight of the soil plug in your lab notebook.
9) Place sample in a 96-well plate, record position (e.g., A1, A2, A3…..H10, H11, H12). Store the 96well plate in a desiccator until analysis.
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Created: 07/01/09
Revised 11/21/12
DeForest Lab
Total Carbon and Nitrogen – (Analyzing Samples)
YOU MUST FOLLOW THESE METHODS FOR PUBLISHABLE RESULTS.
REMEMBER TO ALWAYS BE PATIENT AND TAKE YOUR TIME.
FALIURE TO CALIBRATE THE ANALYZER WILL YIELD INACCURATE RESULTS!
•
C/N analyzer should be at operating temperature for at least three (3) hours before running blanks!
You should wait overnight if you repacked the reduction tube. Best method: let the system sit
overnight to fully purge and condition the analyzer after maintenance. This should make it
easier to calibrate the C/N analyzer.
•
To initiate run sequence, click on Auto Start button on command line.
•
DO NOT LEAVE THE C/N ANALYZER UNTIL THE END OF THE FIRST SAMPLE! This
will take at least 70 minutes. Load your samples on the carousel after the blanks, run-ins, and
two of the standards.
•
Blanks are used to confirm the system is working well. Second blank should stabilize carbon and
nitrogen peaks (< 50). DO NOT CONTINUE TO RUN-INS UNLESS BLANK PEAKS ARE
BELOW 50! Repeat blanks until peaks are below 50 or purge the system with a run-in and
run another blank.
•
Run-ins are used to condition machine for the following standards. Run-ins are not part of the
standard curve. Values should be consistent with little variation (i.e., < ± 0.3% for C & N). DO
NOT CONTINUE TO STANDARDS UNLESS RUN-INS ARE SIMILAR. Repeat run-ins
until % C & N are similar.
•
It is very important to have the correct sample weight. See sample prep page for details.
•
Each sample requires ~8-10 minutes, so it is around 12 hrs for the large carousel (80 slots).
•
ALWAYS FILL OUT THE MAINTENANCE LOG ON THE C/N ANALYZER! See
maintenance page for details.
•
Always record the data (% C and % N) in your lab notebook ASAP after the run. Save the run with
either your name or the project followed by the date. (e.g. PAX_092709).
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Created: 07/01/09
Revised 11/21/12
DeForest Lab
Total Carbon and Nitrogen – (Maintenance)
Please record the number of samples in your analyzing session on the maintenance
log. The log is attached to the C/N analyzer. You must record the following:
Date
Full name and project
Type of sample (e.g., soil, leaves, litter)
The number of runs (this includes blanks, run-ins, standards, and samples)
The pressure in psi of the He tank and the start and end of a session
The score (%) on the leak check test
Put a check mark if the ash trap, H2O absorption tube, combustion (oxidation) or reduction tubes were
changed after your session.
Contact Dr. DeForest if maintenance is required. Only a fully trained person may
independently service the analyzer.
Maintenance will be required shortly if maintenance display at bottom left corner is blinking.
• Ash trap
o Every 80 soil samples if tin wt. is < 50 mg
o Every 60 soil samples if tin wt. is between 50 - 60 mg
o Every 50 soil samples if tin wt. is between 60 - 80 mg
•
H2O – Absorption-U-Tube (i.e., H2O scrubber)
o As needed (~200 soil sample or 150 litter samples)
o Fully spent Sicapent is blue, partially spent Sicapent is green
•
Reduction tube = every 500 runs or until only 50 cm of the Cu is still copper in color
•
Combustion tube = every large carousel (~80 samples)
Shut down
• Within the software, go to task bar and select Options, Parameter, (no password, click ok), enter
values for furnace temperatures and click ok.
o Furnace 1 = 0oC
o Furnace 2 = 0oC
• Shut off gases at the tank. Turn valve clock-wise to close.
•
Let stand overnight
o Do not turn off analyzer until furnaces are below 100oC.
•
Turn off (green switch) C/N analyzer
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Created: 07/01/09
Revised 11/21/12
DeForest Lab
Soil Samples
The actual amount of C and N in mg within a sample tin based on sample weight and % C
and likely % N (C:N ratio = 15). Shaded values indicates outside the detection range.
C%
0.5%
1%
2%
3%
4%
5%
6%
7%
8%
9%
10%
15
0.08
0.15
0.30
0.45
0.60
0.75
0.90
1.05
1.20
1.35
1.50
mg of sample
25
0.13
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
30
0.15
0.30
0.60
0.90
1.20
1.50
1.80
2.10
2.40
2.70
3.00
40
0.20
0.40
0.80
1.20
1.60
2.00
2.40
2.80
3.20
3.60
4.00
N%
0.03%
0.07%
0.13%
0.20%
0.27%
0.33%
0.40%
0.47%
0.53%
0.60%
0.67%
15
0.005
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
mg of sample
25
0.008
0.017
0.033
0.050
0.067
0.083
0.100
0.117
0.133
0.150
0.167
30
0.010
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0.160
0.180
0.200
40
0.013
0.027
0.053
0.080
0.107
0.133
0.160
0.187
0.213
0.240
0.267
Leaf & Litter Samples
The actual amount of C and N in mg within a sample tin based on sample weight and % C
and % N (C:N ratio = 30).
C%
40%
42%
44%
46%
48%
50%
52%
54%
56%
58%
60%
4
1.6
1.7
1.8
1.8
1.9
2.0
2.1
2.2
2.2
2.3
2.4
mg of sample
5
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.0
6
2.4
2.5
2.6
2.8
2.9
3.0
3.1
3.2
3.4
3.5
3.6
7
2.8
2.9
3.1
3.2
3.4
3.5
3.6
3.8
3.9
4.1
4.2
N%
1.33%
1.40%
1.47%
1.53%
1.60%
1.67%
1.73%
1.80%
1.87%
1.93%
2.00%
4
0.053
0.056
0.059
0.061
0.064
0.067
0.069
0.072
0.075
0.077
0.080
mg of sample
5
0.067
0.070
0.073
0.077
0.080
0.083
0.087
0.090
0.093
0.097
0.100
6
0.080
0.084
0.088
0.092
0.096
0.100
0.104
0.108
0.112
0.116
0.120
7
0.093
0.098
0.103
0.107
0.112
0.117
0.121
0.126
0.131
0.135
0.140
Atropine Standard (70.56% C, 4.84% N)
Atropine Carbon Nitrogen (mg)
(mg)
(mg)
0.60
0.75
1.00
1.25
1.50
1.75
0.423
0.529
0.706
0.882
1.058
1.235
0.029
0.036
0.048
0.061
0.073
0.085
Atropine Carbon Nitrogen (mg)
(mg)
(mg)
1.75
2.00
2.25
2.50
2.75
3.00
1.235
1.411
1.588
1.764
1.940
2.117
0.085
0.097
0.109
0.121
0.133
0.145
Atropine Carbon Nitrogen (mg)
(mg)
(mg)
3.00
3.50
4.00
4.50
5.00
5.50
2.117
2.470
2.822
3.175
3.528
3.881
0.145
0.169
0.194
0.218
0.242
0.266
Revised: 07/01/2009
DeForest Lab
Ohio University
Exchangeable Acidity & Aluminum
Reagents
In 10 l carboy (enough for 65 samples)
745.9 g
Potassium Chloride (1 M KCl)
Bring to volume with DI water
In 1 l volumetric flask (enough for 100 samples)
58.1 g Potassium Fluoride (1 M KF)
Bring to volume with DI water
Phenolpthalein indicator
Sodium Hydroxide Standard (1N NaOH)
Hydrochloric acid Standard (1N HCl)
Procedure
Add the following to 125 ml sample cup
10 g
Oven-dried equivalent, sieved soil (can be field fresh)
75 ml
Potassium Chloride (1 M KCl)
Place on orbital shaker (200 rpm) for 30 minutes
Filter suspension (coarse filter paper) in a plastic funnel into a 250 ml Erlenmeyer flask
Wash soil THREE successive 25 ml aliquots of KCl for a total of 150 ml per sample
Add five drops of Phenolpthalein indicator
Stir constantly with a magnetic stir plate
Titrate the solution with 1 N NaOH (or 0.1 N) using a titrating electronic pipette (accuracy 5 l)
Note the volume to a permanent pink end-point for KCl-exchangeable total acidity
Repeat this procedure with 150 ml of 1M KCl (accounts for acidity in KCl)
Add 10 ml of 1 M KF & swirl to mix (solution should become dark pink)
Titrate the solution with 1 N HCl (or 0.1 N) using a titrating electronic pipette (accuracy 5 l)
Note the volume to a permanent clear end-point for KCl-exchangeable Al3+
Adapted from Sims JT. (1996) Lime Requirement. Methods of soil Analysis. Part 3. Chemical Methods-SSSA Book
Series no. 5.
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Created: 08/01/07
Revised: 10/10/11
DeForest Lab
Ohio University
Extracting for Base Cations
Reagents
“FREELY EXCHANGEABLE AND BIOAVAILABLE”
In 10 l carboy (enough for 40 samples)
770.8g
Ammonium Acetate (1 M NH4OAc)
Adjust to pH 7.0 with acetic acid (CH3COOH)
Bring to volume with DI water
“BOUND ELEMENTS”
Mehlich III Stock
In 1 l volumetric flask
600 ml
DI water
56.1 g
Ammonium Fluoride (1.5 M NH4F)
29.23
EthyleneDiamineTetraAcetic acid (0.1 M EDTA)
Bring to volume with DI water
Mehlich III Working Solution
In 10 L carboy (enough for 40 samples)
8l
DI Water
Mehlich III Stock
100 ml
115 ml
Acetic acid (CH3COOH)
82 ml
10% Nitric acid (HNO3)
Bring to volume (10 liters) with DI water
Procedure
Add the following to 125 ml Erlenmeyer flask
10 g
Oven-dried equivalent sieved soil (field fresh or air-dried)
~75 ml
Ammonium Acetate (1 M NH4OAc)
or
~75 ml
Mehlich III working solution
Place on orbital shaker (200 rpm) for 30 minutes
Filter suspension with coarse filter paper in a plastic funnel in a 250 ml volumetric flask
Wash soil THREE successive ~50 ml aliquots of NH4OAc or Mehlich III
Be careful not to pour any sediment into the funnel
Bring to 250 ml volume
Pour ~10 ml of the mixed extract into a 15 ml centrifuge tube with proper identification &
extraction type. Centrifuge for 5 minutes @ 300 rpm. Using certified standards, analyze on the
ICP-OES within a week stored at 4oC or freeze at -20oC.
Note: The use of NH4OAc may overestimate the in situ exchangeable cations and CEC in acidic soils. This method
is to estimate “freely bioavailable” cations and CEC, not the effective CEC under “field” conditions.
[email protected]
Created: 09/11/07
Revised: 10/10/11
DeForest Lab
Ohio University
Bioavailable Base Cations (gravimetrically)
This method is preferable over the volumetric method if sample size is high (>36) due to limited
number of 250 ml volumetric flasks.
Reagents
In 10 l carboy (enough for 80 samples)
770.8g
Ammonium Acetate (1 M NH4OAc; density = 1.0778 g/ml)
Adjust to pH 7.0
Bring to volume with DI water. Store at 4oC.
Ammonium Acetate has a limited shelf-life.
Procedure
Add the following to 125 ml sample cup
Oven-dried equivalent sieved soil (field fresh or air-dried)
10 g
50 ml
Ammonium Acetate (1 M NH4OAc)
Place on orbital shaker (200 rpm) for 30 minutes
Filter suspension with coarse filter paper in a plastic funnel in a 125 ml flask
Wash soil TWO successive ~25 ml aliquots of NH4OAc
Be careful not to pour any sediment into the funnel
Pour into a 125 ml sample cup that is already tarred on a balance
Add NH4OAc until weight is 80.0 g (i.e., 74.225 ml)
Pour ~10 ml of the mixed extract into a 15 ml centrifuge tube with proper identification &
extraction type. Centrifuge for 5 minutes @ 3000 rpm. Using certified standards, analyze on the
ICP-OES within a week stored at 4oC or freeze at -20oC.
Note: The use of NH4OAc may overestimate the in situ exchangeable cations and CEC in acidic soils. This method
is to estimate “freely bioavailable” cations and CEC, not the effective CEC under “field” conditions.
[email protected]
Created: 08/23/10
Revised: 10/10/11
DeForest Lab
Ohio University
Methods in Determining Soluble Soil Phenolic Content
Standard
In 1000 ml volumetric flask
50.0 mg
Syringic acid
Bring to volume
Mix concentrations: 100%, 75%, 50%, 25%, 10%, and 0%
Soluble Soil Phenolic Extraction
In a 50 ml centrifuge tube
5g
Soil sample
25 ml
Distilled Water
Agitate on an orbital-action shaker for 18 h
Centrifuge 15 m @ 900 rpm
Filter with 0.45 m nylon filters
Folin-Ciocalteu Method
In a 15 ml test tube
5.0 ml Sample
0.75 ml
Na2CO3 (1.9 M; 201.38 g L-1)
0.25 ml
Folin-Ciocalteu reagent
After 1 h @ 25oC in the dark
Record the absorbance at 750 nm
[email protected]
Created 7/20/2007
DeForest Lab
Ohio University
Ammonium Analysis
Reagents
Sodium Salicylate solution
In a 100 ml volumetric flask of DI water mix
6.8 g sodium salicylate
5.0 g sodium citrate
5.0 g sodium tartrate
25 mg sodium nitroprusside
NaOH Solution
In a 100 ml volumetric flask
6g
sodium hydroxide
Bring to volume
Bleach Solution
Make fresh each day in 50 ml volumetric flask
1 ml bleach
Bring to volume with NaOH Solution
Matrix
In a 1 liter volumetric flask
74.59 g KCl (1M KCl) & Bring to volume with DI water
Standard (25 ppm NH4)
In 100 ml volumetric flask
10 ml NH4 standard (Certified 250 ppm NH4)
Bring to volume with matrix
Mix standards in 15 ml centrifuge tubes using dilutions from the
adjacent table.
Procedure
In a clear 96-wellplate
Assay the standards in duplicate (Col. 1 & 2) - 50 μl standard
Assay the samples in quadruple – add 50 μl of sample to each
well. A 96-wellplate can hold 20 samples with standards.
Standard Dilutions
Concentration
(ppm )
0
Standard
(ml)
0
1 M KCl
(ml)
10
0.5
0.2
9.8
1
0.4
9.6
2.5
1
9
5
2
8
10
4
6
15
6
4
25
10
0
Add 150 μl salicylate solution to each well using the multi-channel pipette
Add 150 μl bleach solution to each well using the multi-channel pipette
Tap edge of plat to mix well and incubate 50 min. Read plate at 650 nm.
Method detection limit: 0.1 - 50 ppm
[email protected]
Created 7/20/07
Revised 10/10/11
Nutrient Analyses on Microplate Reader
DeForest Lab
Ohio University
Nitrate & Nitrite Analysis
Reagents
Vanadium solution (VCl3)
In 100 ml volumetric flask
0.80 g vanadium (III) chloride in 50 ml of 1 M HCl
CAUTION: Work quickly because Cl3V powder will react with air!
Flush with N2, store in the dark at 4oC, or freeze, should be blue.
2.0 % (w/v) Sulfanilamide solution (SULF)
In 100 ml volumetric flask
2g
Sulfanilamide
Bring to volume with 5% (v/v) HCl
Flush with N2, may be stored in the dark at 4oC for several months, and discard if colored
0.1% (w/v) NEDD solution
In 100 ml volumetric flask
100 mg
N-(1-naphthyl)-ethylenediamine dihydrochloride
Bring to volume with DI water
Flush with N2, may be stored in the dark at 4oC for several months, and discard if colored
Premix solutions just prior to analysis to a ratio of 2:1:1 (20 ml VCl3 : 10 ml SULF : 10 ml NEDD)
Standard (40 ppm NO3)
In 100 ml volumetric flask
4 ml 1,000 ppm nitrate (NO3) standard
Bring to volume in 1 M KCl
Procedure
Soil solution extraction
10 g
Oven-dried equivalent field fresh, sieved soil
1M KCl
20 ml
Shake for 1 hr and centrifuge (10 min @ 3000 rpm)
Standard Dilutions
Concentration
Standard
1 M KCl
(ppm )
(ml)
(ml)
0
0
10
1
0.25
9.75
2
0.5
9.5
4
1.0
9.0
8
2.0
8.0
16
4.0
6.0
24
6.0
4.0
Ambient (initial):
Add 100 l of reagent to 100 l of sample, or standard in
40
10
0.0
clear 96-wellplate.
Incubated (or farm soils):
Add 50 l KCl and 100 l of reagent to 50 l of sample, or standard in clear 96-wellplate.
Incubate at 37oC and measure when color develops. Measure absorbance at 540 nm. Samples should
turn pink, if dark magenta (OVRFLW), redo by adding only 10 l of sample with 90 l of KCl and
100 l of reagent.
Adapted from Miranda et al., 2001, Nitric Oxide-Biology and Chemistry
[email protected]
Created: 03/06/07
Revised: 10/10/11
DeForest Lab
Ohio University
Inorganic Phosphorus (Ascorbic Acid Method)
Adapted from Kuo S. (1996) Phosphorus. Methods of soil Analysis. Part 3. Chemical Methods-SSSA Book Series no. 5.
Reagents
Solution A
In 500 ml volumetric flask
250 ml
DI water
70 ml
Sulfuric acid (18M H2SO4)
Mix well & bring to volume with DI water
Solution B
In 500 ml volumetric flask
250 ml DI water
20 g
Ammonium molybdate [(NH4)6Mo7O24 · 4H2O]
Mix well & then bring to volume with DI water
Solution C
In 100 ml volumetric flask
50 ml
DI water
0.2728 g
Antimony potassium tartrate [K(SbO) · C4H4O6 · ½ H2O]
Bring to volume with DI water
Working Solution - Use within 8 hours (enough for 50 samples + standards)
In 500 ml volumetric flask
250 ml
Solution A
75 ml
Solution B
2.64 g
Ascorbic acid (1 M C6H8O6)
25 ml
Solution C
Bring to volume with DI water
Standards
In 100 ml volumetric flask
50 ml
Extraction solution (e.g. 0.5M NaHCO3)
Certified standard P solution (1,000 ppm)
2.0 ml
2 drops
0.25% p-nitrophenol
If acidic, then add 5 M NaOH dropwise until yellow, then add 0.25 M
H2SO4 until clear. If alkaline, add acid until clear. Beware, it might
strongly effervesce.
Bring to volume to make a 20 ppm working standard. Follow table for
standard dilutions.
P ppm
0.0
0.5
1
2
5
10
15
20
20 ppm
Stock (ml)
0.0
0.25
0.5
1.0
2.5
5.0
7.5
10.0
Extract
(ml)
10.0
9.75
9.5
9.0
7.5
5.0
2.5
0.0
Suggested Procedure
In 15 or 50 ml centrifuge tube
Transfer an aliquot of sample solution that contains 2 to 40 g P.
Dilute with DI water, if necessary, but record dilution for later calculation
It is recommended to use a 6.25:1 ratio between sample/standard and the working solution.
Mix well, incubate for exactly 10 min, dispense 300 l, 4 times into clear microplate and read at 880
nm. Twenty samples can fit on one microplate. Run standards in duplicate. It is recommended to
have sample absorbance between 0.5 and 3.0. If below 0.5, consider using the Spec-20 w/ cuvettes,
which has a longer path length. This procedure is sensitive to 0.5 g P ml-1.
[email protected]
Created: 07/02/10
DeForest Lab
Ohio University
Inorganic Phosphorus (Ascorbic Acid Method)
For Anion Exchange Membranes (i.e., resin strips)
96-well plate method
Reagents
Solution A (2.5 M H2SO4)
In 250 ml volumetric flask
Fill flask half-way with DI water
35 ml
Sulfuric acid (18M H2SO4)
Mix well, bring to volume with DI water
Solution B
In 250 ml volumetric flask
Fill flask half-way with DI water
10 g
Ammonium molybdate
Mix well, bring to volume with DI water
Solution C
In 100 ml volumetric flask
50 ml
DI water
0.2728 g
Antimony potassium tartrate
Bring to volume with DI water
Working Solution - Use within a day or flush with N2 for cold storage
In 50 ml volumetric flask
Solution A
25 ml
7.5 ml
Solution B
0.264 g
Ascorbic acid (1 M C6H8O6)
Solution C
2.5 ml
Bring to volume with DI water
Working Standard
In 100 ml volumetric flask
50 ml
0.5 M HCl (i.e. matrix)
2.5 ml
Certified standard P solution (1,000 ppm)
Bring to volume to make a 25 ppm working standard.
Standard Dilutions
In 15 ml centrifuge tube
1 ppm
0.4 ml standard, 9.6 ml matrix
5 ppm
2.0 ml standard, 8.0 ml matrix
10 ppm
4.0 ml standard, 6.0 ml matrix
Procedure
In a clear 96-well plate (2:1 dilution)
200 l
Solution (in 0.5 M HCl) using the single channel electronic pipette into 4 wells.
100 l
Using the 8-channel pipette, add working solution directly into the well to mix the
solution.
Using the same procedure as above, add the three (3) standards to the last three columns in the
plate. Incubate between 18 and 24 hours (Make sure standards are linear). Read absorbance at
880 nm.
[email protected]
Created: 04/26/11
Revised: 10/10/11
DeForest Lab
Ohio University
Charging AEM (Anion Exchange Membranes)
In 250 ml Flask
0.5 M NaHCO3
150 ml
AEM strips
Rapidly shake for 15 minutes
Remove strips and place AEM in another flask
Rinse AEM with DI water three (3) times.
Immediately use or store at 5 oC
In situ soil phosphorus (Field deployment)
Prep work
Use a large syringe needle to poke a hole in a corner
Thread Firewire finishing line and tie. Have at least 20 cm of loose line.
Place AEM inside a plastic bag with the treads taped on the outside of the bag
Keep moist and cool
In the field
Remove O horizon by hand
Using a 1” putty knife, insert into the soil at an angle to a vertical depth of 5 cm
Slide the AEM into the hole and press the soil back to make contact
Tie the end of the line to a pin flag
Keep in the field between one (1) and two (2) weeks
Place each individual AEM, or grouped within a plot (3), into a small bottle or plastic bag.
Keep moist with DI water & remove the thread
Extraction
Clean off any loose debris with DI water (squirt bottle)
In 150 ml Flask
Place each AEM (or group of AEM) in the flask
25 ml
0.5 M HCl
Shake for four (4) hours
Analyze extract for anions (i.e. Phosphate) – See above
Readily Available Soil Phosphorus (Resin P)
In 50 ml centrifuge tube
Field fresh soil
10 g
25 ml
DI water
1
Charged AEM strip
Shake for four (4) hours
Take out strip and rise to remove debris, and place into another 50 ml centrifuge tube
Follow extraction procedure
[email protected]
Created: 04/26/11
Revised: 10/10/11
DeForest Lab
Ohio University
Inorganic Phosphorus (Modified Ascorbic Acid Method)
Adapted from Kuo S. (1996) Phosphorus. Methods of soil Analysis. Part 3. Chemical Methods-SSSA Book Series no. 5.
Reagents
Reagent A
In 500 ml volumetric flask
400 ml
DI water
8.8 g
ascorbic acid (0.1 M C6H8O6)
40.9 g
trichloroacetic acid (0.5 M C2HCl3O2)
Bring to volume with DI water
Reagent B – Make daily
In 100 ml volumetric flask
Fill one-third with DI water
1.24 g
Ammonium molybdate (0.01 M (NH4)6Mo7O24 · 4H2O)
Bring to volume with DI water
Reagent C
In 1 liter volumetric flask
Fill one-half with DI water
29.4 g
Sodium Citrate (0.01 M Na3C6H5O7 · 2H2O)
26.0 g
Sodium Arsenite (0.1 M NaAsO2)
50 mL
99% Acetic Acid (5% NaAsO2)
Bring to volume with DI water
Standards
In 100 ml volumetric flask
50 ml
Extraction solution (e.g. 0.5M NaHCO3)
2.0 ml
Certified standard P solution (1,000 ppm)
2 drops
0.25% p-nitrophenol
If acidic, then add 5 M NaOH dropwise until yellow, then add 0.25 M
H2SO4 until clear. If alkaline, add acid until clear. Beware, it might
strongly effervesce.
Bring to volume to make a 20 ppm working standard. Follow table for
standard dilutions.
P ppm
0.0
0.5
1
2
5
10
15
20
20 ppm
Stock (ml)
0.0
0.25
0.5
1.0
2.5
5.0
7.5
10.0
Extract
(ml)
10.0
9.75
9.5
9.0
7.5
5.0
2.5
0.0
Procedure
Add the following in 15 ml centrifuge tube
5 ml
Reagent A
2.5 ml
Circumneutral pH (pH 5 – 7.5) aliquot of sample or standard
Add immediately
1 ml
Reagent B
2.5 ml
Reagent C
Mix well, incubate for 10 min, centrifuge, dispense 250 l, 4 times into clear microplate and read at 700
nm. Twenty samples can fit on one microplate. Run standards in duplicate. It is recommended to have
sample absorbance between 0.5 and 3.0.
[email protected]
Created: 8/19/09
Revised: 10/10/11
DeForest Lab
Ohio University
[email protected]
Created: 8/19/09
Revised: 10/10/11
DeForest Lab
Ohio University
Soil P Hedley Fractionation
Reagents - enough for 40 samples
0.5M NaHCO3
1liter volumetric flask
42 g Sodium Bicarbonate
Bring to volume with DI water
Adjust to pH 8.5
0.1 M NaOH
1 liter volumetric flask
4 g Sodium Hydroxide
Bring to volume with DI water
1.0 M HCl
1liter volumetric flask
83.3 ml Hydrochloric Acid (12 M)
Bring to volume with DI water
0.5 M HCl
1liter volumetric flask
41.7 ml Hydrochloric Acid (12 M)
Bring to volume with DI water
Sequential Extraction Procedure
In a 50 ml centrifuge tube
10 g Field fresh, sieved soil
Resin P
1
Bicarb charged resin strip (2 x 6 cm; Charging: shake strips 10 min in 100 ml 0.5 M
NaHCO3, rinse 3 times with DI water, shake dry)
25 ml DI water
Shake for 4 hrs, centrifuge, and discard supernatant
In a 250 ml flask
Extract resin with 25 ml of 0.5 M HCl for 1 hr on a shaker
NaHCO3 P
25 ml 0.5M NaHCO3
Vortex briefly to break up soil plug
Shake for 18 hrs, centrifuge, and store supernatant at 4oC.
NaOH P
25 ml 0.1M NaOH
Vortex briefly to break up soil plug
Shake for 18 hrs, centrifuge, and store supernatant at 4oC
Sonicate NaOH P
25 ml 0.1M NaOH
Vortex briefly to break up soil plug and Sonicate for 2 min
Shake for 18 hrs, centrifuge, and store supernatant at 4oC
HCl P
25 ml 1.0M HCl
Vortex briefly to break up soil plug
Shake for 18 hrs, centrifuge, and store supernatant at 4oC
[email protected]
Revised: 06/30/09
Revised: 10/10/11
DeForest Lab
Ohio University
P Residue
In a 60 ml heat treated test tube
Add soil from HCl P fraction with a minimal amount of DI water
5 ml Sulfuric acid
Heat slowly to evaporate water (block digester) & allow cooling to hand-warm
0.5 ml 30% H2O2 (Careful, it may overflow)
Reheat for 30 min
Repeat H2O2 additions until liquid is clear (usually 10 times)
Cool, make to volume (i.e., 25 ml) and analyze on the ICP
Standards
In a 50 ml volumetric flask
5 ml of a 1000 ppm certified stand P solution
Bring to volume with DI water to make a 100 ppm standard
Mix standards with the extracting solution (matrix)
Resin (5 to 10 ppm)
In a 15 ml disposable centrifuge tube
1.5 ml
100 ppm solution
13.5 ml
0.5 M HCl (matrix)
Will make a 10 ppm working solution
Follow dilution table
Working
standard Matrix
(ml)
(ml)
Dilution
0%
0.0
5.0
1%
0.1
9.9
10%
1.0
9.0
25%
2.5
7.5
50%
3.0
3.0
100%
6.0
0.0
NaHCO3 (5 to 20 ppm)
In a 15 ml disposable centrifuge tube
100 ppm solution
3 ml
12 ml
0.5M NaHCO3 (matrix)
Will make a 20 ppm working solution
Follow dilution table
NaOH (50 ppm)
In a 15 ml disposable centrifuge tube
7.5 ml
100 ppm solution
7.5 ml
0.1M NaOH (matrix)
Will make a 50 ppm working solution
Follow dilution table
HCl P (10 to 20 ppm)
In a 15 ml disposable centrifuge tube
3 ml
100 ppm solution
12 ml
1.0M HCl (matrix)
Will make a 50 ppm working solution
Follow dilution table
P Residue (50 to 100 ppm)
Follow dilution table with DI water
[email protected]
Revised: 06/30/09
Revised: 10/10/11
DeForest Lab
Ohio University
Bicarbonate Phosphorus Extraction
Inorganic & Organic
Reagents
0.5M NaHCO3 (enough for 50 samples)
In 2 l volumetric flask
84 g
Sodium Bicarbonate
Adjust pH to 8.5
Bring to volume with DI water
Keep refrigerated for a month
Procedure - Total bicarb P
Soil solution extraction (1:20 dilution)
In a 50 ml Oakridge centrifuge tube
2g
Oven-dried equivalent field fresh, sieved soil
40 ml
0.5M NaHCO3
Shake for 18 hrs on an angle
Centrifuge @ 2500 rpm for 5 min
Decant into 15 ml centrifuge tube
Analyzing for P on ICP-OES for total bicarb P
Procedure - Inorganic bicarb P
In a 15 ml centrifuge tube
Solution (draw off before analyzing for total bicarb P)
5 ml
Slowly add 2.5 M H2SO4 (expect effervescences) to neutralize the solution. Record the amount
added to determine dilution. Test with pH paper.
Vortex after ~2 min to de-gas the sample.
If necessary, centrifuge @ 2500 rpm for 5 min.
Analyze for inorganic P using the ascorbic acid method.
Organic bicarb P = Total bicarb P - Inorganic bicarb P
[email protected]
Created: 12/17/08
Revised: 10/10/11
DeForest Lab
Ohio University
Phosphorus Sorption
Reagents
In a 10 liter carboy
7.46 g
Potassium Chloride (0.01 M KCl)
Bring to volume with DI water
Stock solution (1000 µg P/ml)
In 1 liter volumetric flask
4.39 g
Oven-dried KH2PO4
Bring to volume with DI water
Store in bottle at 4oC with 2 drops of chloroform
Follow the adjacent table for P working solution (enough for 32
samples)
Dilute daily from stock solution
µg P/ml
(ppm)
0
10
25
50
100
150
200
Stock
(ml)
0
10
25
50
100
150
200
0.01 M KCl
(ml)
1000
990
975
950
900
850
800
Procedure
In a 50 ml Oakridge centrifuge tube
3g
Oven-dried equivalent sieved soil (field fresh)
P working standards at each concentration
30 ml
1 drop
Chloroform
Shake for 2 days at 150 rpm and then centrifuge for 1 hour.
Repeat the procedure, but add 2 bicarb charged resin strips
Analyze solutions for ortho-P using the ascorbic acid method.
Calculations
Xs = [(s – c) × F] - E
Where:
Xs = sorbed P at working solution concentrations (µg P/g soil)
s = µg P/ml of original working solution
c = µg P/ml of working solution after shaking (i.e., equilibrium)
F = ml working solution/g dry soil (e.g. 10 ml/g = 30 ml/3 g dry soil)
E = resin-extractable P (µg P/g soil)
Adapted from Tiessen et al (1991) Geoderma and Lajtha K. et al. (1999) Soil Phosphorus. Standard soil methods
for long-term ecological research.
[email protected]
Created: 08/19/09
DeForest Lab
Ohio University
Microbial Phosphorus - Chloroform Extraction Method
Reagents
0.5M NaHCO3 (enough for 50 samples)
In 2 l volumetric flask
84 g
Sodium Bicarbonate
Adjust pH to 8.5
Bring to volume with DI water
Keep refrigerated for a month
0.5 M HCl
In 2 liter volumetric flask
85.8 ml Hydrochloric Acid
Bring to volume with DI water
Procedure
In 50 ml Oakridge centrifuge tubes
In duplicate
2g
Field fresh, sieved soil
40 ml
DI water
2
Bicarb charged resin strip (2 x 6 cm; Charging: shake strips 10 min in 100 ml 0.5 M
NaHCO3, rinse 3 times with DI water, shake dry)
Shake @ 150 rpm for 4 hours
Remove resin strips and wash with minimal amount of DI water back into the sample cup
The purpose for the resin strips is to remove inorganic ortho-P
Centrifuge at high speed for 45 minutes, discard supernatant without removing the soil.
Fumigation
Add 1 ml chloroform (CHCl3) to one tube labeled “F” for fumigated
Shake @ 250 rpm for 1 hour
Allow chloroform to evaporate overnight in the fume hood
Store the other tube labeled “C” for control (i.e., unfumigated) at 4oC.
Add 30 ml of 0.5 M NaHCO3 to F and C labeled tubes
Shake @ 150 rpm for 16 hrs, centrifuge at high speed for 30 minutes.
Pour supernatant into 15 ml tubes and centrifuge again at high speed for 10 min.
Analyze for total P on the ICP
Calculations
µg P/g in microbial biomass = [(Total P in „F‟ soil) – (Total P in „C‟ soil)]/kp
Where:
kp = extraction efficiency (0.4) or sorption efficiency for each soil (see P sorption
methods)
Lajtha K. et al. (1999) Soil Phosphorus. Standard soil methods for long-term ecological research.
[email protected]
Created: 8/19/09
Revised: 10/10/11
Phase I: Extraction of Soil Lipids
DeForest Lab
Preparation
Calibrate the methanol repipetter to dispense 10 ml
Calibrate the chloroform repipetter to dispense 5 ml
Consumable supplies and glassware
All glass must be very clean (i.e., 4 hrs @ 450 oC)
2 beakers (150 ml)
2 beakers (50 ml)
Each sample needs:
2 large (50 ml) test tubes – blue-labeled
1 small (15 ml) test tube – green-labeled
2 Pasteur pipettes
4 ml of phosphate buffer
5 ml nanopure water
250 l internal standard
Procedure
1.
Add 5 g of freeze-dried soil to large test tube and label with blue tape.
2.
Add 4 ml phosphate buffer.
3.
With the repipetter, add 10 ml methanol followed by 5 ml chloroform.
4.
Add 250 µl internal standard 19:0.
5.
Vortex each sample briefly and vent.
6.
Sonicate samples for two minutes, and then set for three hours.
7.
Centrifuge samples for 10 minutes @ 3000 rpm.
8.
Label second set of large test tubes with blue tape.
9.
Pour clear supernatant into second set of large test tubes.
10. Add 5 ml chloroform to first test tube, vortex, & centrifuge for 10 min @ 3000 rpm.
11. Decant supernatant into second set of large test tubes.
12. Add 5 ml nanopure water to second set of large test tubes.
13. Vortex each sample briefly and vent.
14. Allow mixture to sit overnight (best) or proceed to step 15.
Day Two: Turn on water bath (37oC) for the N-EVAP. Make sure the bath is nearly full with
distilled water.
15. Centrifuge samples for 10 minutes @ 3000 rpm.
16. With the pasture pipette, transfer the bottom organic phase for each sample into small test
tube. To prevent uptake of unwanted material, blow small bubbles through the pipette
while passing through the top phase.
17. Evaporate the solvent in the N-EVAP until standing liquid is gone, but still looks wet.
Process will take ~30 minutes.
18. Store samples at -20oC.
Created: 10/29/08
Revised: 10/10/11
Phase II: Silicic Acid Chromatography
DeForest Lab
Preparation
Calibrate the chloroform and methanol repipetter to dispense 2.5 ml
Turn on water bath (37oC) for the N-EVAP. Make sure the bath is full with distilled water.
Consumable supplies & glassware
A set (i.e., 6) waste test tubes for unwanted lipid fraction
1 beaker (50 ml)
Each sample:
1 SPE chromatography column
1small (15 ml) test tubes – yellow-labeled
1 pasture pipette
Procedure
Note: In order for the chromatography columns to work, they must not dry out. Keep the columns in contact
with an organic solvent at all times.
1.
Remove samples from freezer and while still cold, add ~150 l chloroform.
2.
Place SPE columns on vacuum manifold.
3.
Condition each column with ~10 ml acetone then with ~10 ml chloroform.
4.
Load the column with a Pasteur pipette. Drip the sample directly into the center of the
column.
5.
Add another 150 l of chloroform to the green-labeled test tube and load that into the
column.
6.
Add another 150 l of chloroform to the green-labeled test tube and swirl the chloroform
to get lipids from the side of the test tube. Load that into the column.
7.
Elute column with 2.5 ml chloroform twice to remove the neutral lipid fraction.
8.
Elute column with 2.5 ml acetone twice to remove the glycolipid fraction.
9.
Switch to the yellow-labeled test tubes and elute with 2.5 ml methanol four times.
10.
Evaporate the solvent for the polar lipids in the N-EVAP until standing liquid is gone, but
still looks wet. Process will take 2 hours.
11.
Store samples at -20oC.
Created: 10/29/08
Revised: 10/10/11
Phase III: Methylation of Polar Lipids
DeForest Lab
Preparation
Turn on water bath (60oC). Make sure the bath is full.
Consumable supplies & glassware
3 beakers (100 ml) - methanolic KOH, hexane, water
5 beakers (50 ml) - methanol, chloroform, acetic acid, cleaning, hexane-MTBE
Each sample:
1small (15 ml) test tubes – orange-labeled
4 pasture pipette
Reagents
0.2 M methanolic KOH
10 ml of methanol in a 100 ml beaker
Quickly weigh 2-3 pellets of KOH and record weight
Immediately add pellets to the methanol
Add enough methanol to achieve the correct concentration (0.28 g KOH : 25 ml methanol)
Sonicate to dissolve. Shelf life is 6 months, so make enough for the whole session.
Procedure
1.
Remove samples from freezer and while still cold, add 500 l chloroform and 500 l
methanol.
2.
Add 1 ml methanolic KOH, then vortex briefly.
3.
Place samples in the 60oC water bath for 30 minutes; allow samples to cool before the
next step.
4.
Add 2 ml hexane and vortex briefly.
5.
Add 200 l 1N acetic acid and swirl to mix.
6.
Add 2 ml nanopure water to break phase.
7.
Vortex samples for 30 seconds.
8.
Centrifuge samples for 5 minutes @ 3000 rpm.
9.
With a Pasteur pipette, transfer the top phase into orange-labeled test tubes.
10. Add 2 ml hexane to the yellow-labeled test tube and vortex for 30 seconds.
11. Centrifuge samples for 5 minutes @ 3000 rpm.
12. With a Pasteur pipette, transfer the top phase into orange-labeled test tubes.
13. Perform steps 10 and 12 ONE more time.
14. Check to see if bubbles (i.e., water) appear at the bottom of the orange-labeled test tubes.
Remove bubbles with a Pasteur pipette.
15. Evaporate the solvent in the N-EVAP until standing liquid is gone, but still looks wet.
Process will take ~15 minutes.
16. Store samples at -20oC or add 300 l hexane-MTBE and transfer into GC vial for analysis.
Created: 10/29/08
Revised: 10/10/11
DeForest Lab
Ohio University
Hydrolytic Enzyme Analysis
Buffer
Acetate buffer (Stock Solution; 1 M)
In 500 ml Beaker
Fill two-thirds with DI water
68.304 g
Sodium acetate trihydrate
Using a stir bar, bring to pH 5.5 by adding, with a pasture pipette, concentrated HCl
Transfer to 500 ml volumetric flask and bring to volume with DI water
Store in an opaque bottle at 4oC and use within 7 days
Acetate buffer (Working Solution; 50 mM)
In 2 L volumetric flask
100 ml
Acetate buffer stock solution
Bring to volume with DI water
Two liters is enough for 15 samples. Put in a carboy w/spout for easy dispensing.
Standards
Methylumbelliferyl (MUB; 10 µM)
In a 500 ml opaque bottle
500 g
DI water (i.e, 500 ml)
0.881 mg
4-Methylumbelliferone
Mix very well.
Amino-Methylcoumarin (AMC; 10 µM)
In a 500 ml opaque bottle
500 g
DI water (i.e, 500 ml)
0.876 mg
7-amino-4-methylcoumarin
Mix very well
Substrates (200 µM)
In a 50 ml opaque bottle
Weigh on a microbalance in tin capsules
Substrates
4-MUB β-D-glucopyranoside
4-MUB beta-D-cellobioside
4-MUB N-acetyl-β-D-glucosaminide
4-MUB phosphate
bis-(4-MUB) phosphate
L-Leucine-7-AMC
Enzyme
β-1,4-glucosidase
β-D-1,4-cellobiosidase
β-N-acetylglucosaminidase
Phosphomonoesterase
Phosphodiesterase
Leucine amino peptidase
ID
BG
BC
NAG
PM
PD
LAP
mg /
50 ml
3.383
5.005
3.794
2.562
4.143
3.248
Assay
(hours)
3 to 6
3 to 6
0.5 to 3
0.5 to 3
3 to 6
6 to 24
Add the correct about of DI water to achieve correct concentration, gravimetrically. Mix well
and store at 4oC. Discard after 3 days. Assay each enzyme at least four times and at least 30
minutes apart for PM & NAG or 1 hour apart for BG, BC, PD, and LAP.
[email protected]
Created: 08/19/2010
Revised: 07/09/12
DeForest Lab
Ohio University
Nitrogen and Phosphorus Mineralization
Procedure
Initial Nitrogen (Day 0)
In a 50 ml centrifuge tube
10 g
Field fresh soil
20 ml
1 M KCl (74.59 g KCl/liter)
Place on orbital shaker for one (1) hour
Centrifuge and decant into a 15 ml centrifuge tube
Measure NH4+ and NO3- using colorimetric methods within a week
Initial Phosphorus (Day 0)
In a 50 ml centrifuge tube (if available, Oakridge tubes)
Field fresh soil
10 g
1
Bicarb charged AEM resin strip (Charging: shake strips 10 min in 100 ml 0.5 M
NaHCO3, rinse 3 times with DI water, shake dry)
20 ml
DI water
Shake for 4 hrs, centrifuge, and discard supernatant
In a 250 ml flask
AEM strip
1
Extract resin with 25 ml of 0.5 M HCl (41.7 ml 12M HCl/liter) for 18 hr on a shaker
Measure Resin P using the ascorbic acid colorimetric method within a day
Incubated N and P (Day 14)
In a 125 ml sample cup
20 g
Field fresh soil
Loosely put on the lid
Place in the incubator at 25oC for around two (2) weeks
Check moisture every week. If dry, then spray once with distilled water
In 14 days (two weeks)
In a 50 ml centrifuge tube (if available, Oakridge tubes)
10 g
Soil for P analysis from the incubated soil
Label “incubated”
Repeat procedure P procedure from above
In the incubated 125 ml sample cup
Remaining soil for N analysis
10 g
20 ml
1 M KCl
Label “incubated”
Repeat procedure N procedure from above
[email protected]
Created: 02/02/11
Litter Fractionation
DeForest Lab
Ohio University
Adapted from Moorhead & Reynolds (1993) Am. Midl. Nat. 130:83-89.
This method separates C compounds into three parts: (1) Soluble in ethanol and water, (2) sulfuric acid
soluble and (3) sulfuric acid insoluble.
Equipment:
Sonicating water bath, High-speed centrifuge, muffle furnace, 50-ml centrifuge tube (preweighed),
pasture pipette, 15-ml centrifuge tubes, 72% H2SO4 (sulfuric acid), 125 ml flask, pre-weighed Pall
50mm glass fiber filter, Corning Mini-Miser Filter
Sample Preparation
1)
Oven dry litter at 65oC
2)
Grind litter sample into a fine powder
3)
Place 0.25g of sample into preweighed 50-ml centrifuge tube
Soluble Fraction (Soluble in water and ethanol)
1)
Add 25 ml of ethanol to each tube
2)
Place into 60oC water bath for 30 m
3)
Centrifuge at 1,000 rpm for 15 min
Suction off supernatant (try to keep polar lipids on top)
4)
5)
Repeat steps 1-4 two more times
6)
Add 25 ml of water to each tube
7)
Repeat steps 1-4 two more times, but with water
8)
Oven dry samples at 60oC for 24 h
9)
Weigh residue
Soluble content was estimated as the difference between original and residual weight
Hemicellulose (Acid soluble) & Lignin (Acid insoluble)
1)
For the same centrifuge tube, add 2 ml of 72% sulfuric acid
2)
Incubate for 1 h at 30oC
3)
Add 30 ml of distilled water to transfer sample to 125 ml flask
4)
Autoclave sample for 1 h at 120oC
5)
Pass sample through a pre-weighed Millipore filter.
6)
Oven-dry filter at 60oC for 24 h
Weigh filter
7)
Hemicellulose content calculated by difference between pre- and post acid digested dry
sample weight. The residue is lignin content (acid insoluble fraction plus the mineral
fraction).
Mineral Fraction (Ash)
1)
Place residue from acid digested in crucible
2)
Muffle furnace at 500oC for 24 h
3)
Weigh ash
Subtract the ash value from the acid insoluble fraction.
[email protected]
Created: 10/05/06
Revised: 10/10/11