Dairy OneDairy One
HomeLinksAbout Us



u  Procedures
Dairy One Analytical Procedures - Forage, Manure, Water
November 2011
I.
 Dry Matter
A.
 Oven – 135ºC for 2 hours - AOAC 930.15.
1.
 Goering, H.K. and P.J. Van Soest. 1970. Forage Fiber Analyses (apparatus,reagents, procedures, and some applications). ARS/USDA Handbook No. 379, Superintendent of Documents, US Government Printing Office, Washington, D.C. 20402. p15.
2.
 NFTA Method 2.2.1.1 - Partial Dry Matter using Forced-air Drying Ovens.
B.
 Oven – 135ºC for 2 hours - AOAC 930.15 - Loss on Drying (Moisture) for Feeds.
C.
 Oven – 105ºC for 3 hours - NFTA Method 2.2.2.5 - Dry Matter by Oven Drying for 3hr at 105C.
D.
 Near Infrared Reflectance Spectroscopy (NIRS) – AOAC 991.01 - Moisture in Forage.
II.
 NIR
AOAC 989.03 - Fiber (Acid Detergent) and Protein (Crude) in Forages.

Foss NIRSystems Model 6500 with Win ISI II v1.5. Components analyzed by NIR include: DM, CP, SP, RDP, ADI-CP, NDI-CP, ADF, NDF, lignin, starch, WSC, ESC, fat, ash, Ca, P, Mg, K, S, Cl, NDFD 24, NDFD 30, NDFD 48. Silages also receive lactic acid, acetic acid, and ammonia CPE.

Near infrared reflectance spectroscopy (NIR) is an instrumental method for rapidly and reproducibly measuring the chemical composition of forage and feed samples. It is based on the fact that each of the major chemical components of a sample has near infrared absorption properties that can be used to 1) differentiate one component from another and 2) determine nutrient concentration.

NIR is a calibration based technology, meaning that analysis is limited to only feeds and nutrients for which calibrations have been developed. Dairy One has built broad based calibrations by incorporating samples collected over several decades. Calibrations are based on reference chemistry using traditional procedures. Visit this link for more info http://www.dairyone.com/Forage/services/Forage/forage_NIR_service.htm.

Foss NIRSystems, 7703 Montpelier Road, Suite 1, Laurel, MD 20723 www.foss.us
III.
 Protein
A.
 Crude Protein (CP) and Nitrogen (N)
1.
 Leco FP-528 Nitrogen/Protein Analyzer
a.
 AOAC 990.03 - Protein (Crude) in Animal Feed
b.
 AOAC 992.15 - Crude Protein in Meat and Meat Products including Pet Foods
c.
 AOAC 992.23 - Crude Protein in Cereal Grain and Oilseeds
d.
 Leco Application Note "Nitrogen/Protein in Animal Feeds" Form 203-821-146, 07/03 Rev1
Leco Corporation, 300 Lakeview Avenue, St. Joseph, MI 49085 www.leco.com
2.
 Leco TruMac N Macro Determinator
a.
 AOAC 990.03 - Protein (Crude) in Animal Feed
b.
 AOAC 992.15 - Crude Protein in Meat and Meat Products including Pet Foods
c.
 AOAC 992.23 - Crude Protein in Cereal Grain and Oilseeds
d.
 Leco Application Note "Nitrogen/Protein in Feeds, Grains, and Oil Seeds" Form 203-821-392, 09/10-Rev0
e.
 Leco Application Note "Nitrogen in Soil and Plant Tissue" Form 203-821-394, 09/10-Rev0.
Leco Corporation, 300 Lakeview Avenue, St. Joseph, MI 49085 www.leco.com
3.
 NIRS - Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03
B.
 Soluble Protein (SP)
1.
 Cornell Sodium Borate-Sodium Phosphate Buffer Procedure. Soy products incubated at 39°C. All other samples incubated at ambient temperature. Residues analyzed for crude protein using Leco TruMac N Macro Determinator. Cornell Nutrition Conference Proceedings, 1990, pp. 85-86.
2.
 NIRS - Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
C.
 Degradable Protein (Rumen Degradable Protein (RDP))
1.
 Cornell Streptomyces griseus (SGP) enzymatic digestion. Enzyme concentration held constant. Residues analyzed for crude protein using Leco TruMac N Macro Determinator.

a.

a. Concentrates incubated for 18 hrs. Cornell Nutrition Conference Proceedings, 1990. pp. 81-88.
b.
 Forage samples incubated for 2 hrs at higher SGP concentration. J. Dairy Sci. 1999. 82: 343-354.
2.
 NIRS - Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
D. Acid Detergent or Neutral Detergent Insoluble Crude Protein (ADI-CP, NDI-CP).
1.
 ADF or NDF residue is analyzed using a Leco TruMac N Macro Determinator to determine the protein fraction bound to the fiber. Sodium Sulfite not used for NDI-CP.
2.
 NIRS - Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
E.
 Non Protein Nitrogen (NPN; reported as Crude Protein Equivalent – CPE)
1.
 Ammonia and Urea - Timberline TL-2800 Analyzer

Extraction - Samples are extracted in deionized water using a single speed blender at 20,000 rpm for 2 minutes (50g/750ml) or a reciprocal shaker for 30 minutes at 280 epm (Forage - 5g/100ml wet or 1g/100ml dry; Manure - 10g/150ml). For urea, a prepared urease solution is added to a duplicate sample prior to shaking (5g/100ml wet or 1g/100ml dry). All extracts then centrifuged at 4000 rpm for 5 minutes, decanted into tubes, then analyzed.

Analysis - A peristaltic pump directs the sample, caustic, and absorbing solutions into a diffusion cell. Within the cell, the sample is mixed with the caustic solution, resulting in a pH of 11-13 which converts the ammonium ion present in the sample to dissolved ammonia gas. The sample/caustic solution flows past one side of a membrane that is permeable to gases but not to liquids or ionic species. The dissolved ammonia gas in the sample/caustic mixture diffuses across the membrane.

On the other side of the membrane, a buffered solution absorbs the diffused ammonia gas then flows through a low volume heat exchanger to establish thermal equilibrium then into the conductivity detector. The conductivity cell measures the change in electrical conductance of the absorbing solution. This change is proportional to the concentration of ammonium in the original sample.

Timberline Instruments, 1880 S. Flatiron Ct. Suite I, Boulder, CO 80301 www.timberlineinstruments.com

Extraction using reciprocating shaker - Kalra, Y.P. 1998. Determination of Ammonium-Nitrogen in Plant Tissue. Handbook of Reference Methods for Plant Analysis. 11:90.
2.
 Urea – AOAC 967.07 - Urea in Animal Feed, colorimetric method.
3.
 NIRS – Ammonia – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03. Analyzed on corn silage and fermented haycrop forages only.
IV.
 Fiber
A.
 Acid Detergent Fiber (ADF)
1.
 ANKOM Technology Method 5 - Acid Detergent Fiber in Feeds - Filter Bag Technique for A200 (4-13-11).

Solutions as in AOAC 973.18 - Fiber (Acid Detergent) and Lignin (H2SO4) in Animal Feed.

Samples individually weighed at 0.5g into filter bags and digested for 75 minutes as a group of 24 in 2L of ADF solution in ANKOM A200 Digestion Unit. Samples are rinsed three times with boiling water for 5 minutes in filter bags followed by a 3 minute acetone soak and drying at 100°C for 2 hours.

ANKOM Technology, 2052 O'Neil Road, Macedon, NY 14502 www.ankom.com
2.
 AOAC 973.18 (C). Liquid samples. Whatman 541 filter paper and buchner funnels.
3.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
B.
 Neutral Detergent Fiber (NDF)
1.
 ANKOM Technology Method 6 - Neutral Detergent Fiber in Feeds - Filter Bag Technique for A200 (4-13-11).

Solutions as in Van Soest, P.J., J.B. Robertson, and B.A. Lewis. 1991. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. J.Dairy Science 74:3583-3597.

Samples individually weighed at 0.5g into filter bags and digested for 75 minutes as a group of 24 in 2L of NDF solution in ANKOM A200 Digestion Unit. Four ml of Alpha Amylase and 20g sodium sulfite are added at the start of digestion. Samples are rinsed three times with boiling water for 5 minutes. Alpha Amylase is added to the first 2 rinses. Water rinses are followed by a 3 minute acetone soak and drying at 100ºC for 2 hours.
2.
 Journal of Dairy Science 74:3583 - 3597, 10/91. Liquid samples.
3.
 NIRS - Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
C.
 Crude Fiber
1.
 ANKOM Technology Method 1 - Crude Fiber Analysis in Feeds - Filter Bag Technique (FBT) - AOAC Approved Procedure Ba 6a-05 (Rev E 4-13-11).
Same digestion solutions as in AOAC 962.09 - Fiber (Crude) in Animal Feed and Pet Food. Acetone used for presoak fat extraction. 0.5g weighed for forages, dried manure, wood, and wood pellets. 1.0g weighed for all other sample types.
2.
 AOAC 962.09. Liquid samples. Samples pre-extracted for fat by rinsing three times with approximately 40ml acetone per rinse. Samples filtered through bingham linen after first boil and through Whatman AH-934 glass membrane filter paper after second boil.
D.
 Lignin
1.
 ANKOM Technology Method 9 - Method for Determining Acid Detergent Lignin in the DaisyII Incubator - 04/11.

Solution as in AOAC 973.18 - Fiber (Acid Detergent) and Lignin (H2SO4) in Animal Feed. ADF performed as in IV.A.1. ADF residue digested as a group of 24 in 72% w/w sulfuric acid for 3 hours in ANKOM DaisyII Incubator at ambient temperature.
2.
 AOAC 973.18(D). Liquid samples. No asbestos.
3.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
E.
 In-Vitro True Digestibility (IVTD / NDFD)
1.
 ANKOM Technology Method 3 - In Vitro True Digestibility using the DaisyII Incubator (08/05).

Reagents and solutions as in: Goering, H.K. and P.J. Van Soest. 1970. Forage Fiber Analyses (apparatus,reagents, procedures, and some applications). ARS/USDA Handbook No. 379, Superintendent of Documents, US Government Printing Office, Washington, D.C. 20402. p13-14.

Rumen fluid collected from TMR fed, high producing lactating cow. Dry, ground feed samples (0.25g; 1mm particle size) incubated in Van Soest buffer/rumen fluid mixture for 24, 30, or 48 hours under anaerobic conditions at 39ºC. After incubation, samples extracted using NDF procedure as in IV.B.1. to remove bacterial contamination. Residue is undigested fibrous material and is used to determine in-vitro true digestibility (IVTD) and neutral detergent fiber digestibility (NDFD).
2.
 NIRS - Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
V.
 Minerals
A.
 Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, Co, S, Al, B, Cr, Sr

Analyzed using a Thermo IRIS Advantage HX or Intrepid Inductively Coupled Plasma (ICP) Radial Spectrometer after microwave digestion.
1.
 CEM Microwave Accelerated Reaction System (MARS) with MarsXpress Temperature Control using 50ml calibrated Xpress Teflon PFA vessels with Kevlar/fiberglass insulating sleeves.

CEM Application Note for Acid Digestion - Feed Grain
CEM Application Note for Acid Digestion - Alfalfa

Sample weights - 0.5g for forages, ingredients, byproducts (1.0g for Co or Cr); 0.5g for grain mixes; 0.2g for mineral mixes.

Samples predigested at ambient temperature 15 minutes with 8ml nitric acid (HNO3) and 2ml hydrochloric acid (HCl) then ramped to 190ºC in 15 minutes and finally held at digestion temperature of 190ºC for 15 minutes at 1600W. Vessels brought to 50-ml volume, aliquot used for analysis.

CEM, 3100 Smith Farm Road, Matthews, NC 28106 www.cem.com
Thermo Fisher Scientific Inc., 81 Wyman Street, Waltham, MA 02454 www.thermo.com
2.
 NIRS – (Ca, P, Mg, K, S) – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
B.
 Chloride Ion (Cl-)
1.
 Brinkmann Metrohm 716 Titrino Titration Unit – 0.5g dried, ground sample extracted in 50ml 0.1N HNO3 followed by potentiometric titration with AgNO3 using Brinkmann Metrohm 716 Titrino Titration Unit with silver electrode.
a.
 Metrohm Application Bulletin No. 130 by Metrohm Ltd., C-H-9101 Herisau, Switzerland.

Metrohm USA, 6555 Pelican Creek Circle, Riverview Fl, 33578 www.methohmusa.com
b.
 The method by Metrohm is similar to the concepts found in: Cantliffe, D.J., MacDonald, G.E. and Peck, N.H. 1970. The potentiometric determination of nitrate and chloride in plant tissue. New York’s Food and Life Sciences Bulletin. No.3, September 1970. Plant Sciences. Vegetable Crops Geneva. No. 1: 5-7.
2.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
VI.
 Supplemental Services
A.
 Crude Fat
1.
 Ether Extraction – AOAC 2003.05. Crude Fat in Feeds, Cereal Grains, and Forages.

Extraction by Soxtec HT6 System using anhydrous diethyl ether. Crude fat residue determined gravimetrically after drying.
2.
 Acid Hydrolysis – AOAC 954.02 – Crude Fat in Pet Food.
3.
 Dried Milk – AOAC 932.06 A (b) and 932.06 B (Roese-Gottlieb Method).
4.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
B.
 Ash
1.
 AOAC Method 942.05 - Ash of Animal Feed.
2.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
C.
 Nitrates (%NO3 or ppm NO3-N)

RQflex® Reflectometer Method

Merck Millipore Application Note "Nitrates in Plant Sap", 2006-05-23; extraction modified.
(Similar to AOAC 998.11 - Screening Test for Nitrate in Forages by Test Strip)

EMD Chemicals Inc., One International Plaza, Suite 300, Philadelphia, PA, 19113 www.emdchemicals.com

1g of dried, ground sample is extracted in 50ml deionized water for 20 minutes by shaking at 280 oscillations/minute. Samples are filtered through Whatman 934-AH (1.5um) filter paper, then analyzed by RQflex® Reflectometer using Reflectoquant® Nitrate test strips.

When the Nitrate test strip is immersed in a sample, a reducing agent reduces nitrate ions to nitrite ions. In the presence of an acidic buffer, the nitrite ions react with an aromatic amine to form a diazonium salt. The salt reacts with N-(1-naphthyl)-ethyelene-diamine to form a red-violet azo dye that is measured reflectometrically. Nitrate concentration is proportional to the color reaction.

Each strip contains two reaction zones generating dual replicate analyses per sample. The RQflex® Reflectometer's double optic system measures the analyte concentration based on the light reflected from the dual reaction zones. Barcode-controlled software calculates the mean of those two measurements.
D.
 pH

15g wet sample placed into 250-ml beaker. 200ml deionized water added, stirred, and allowed to stabilize five minutes. Analyzed using Thermo Orion Posi-pHlo SympHony Electrode and Thermo Orion 410A meter.

Calibrated with buffers referenced to NIST SRMs. pH 4 buffer contains potassium hydrogen phthalate and pH 7 buffer contains sodium phosphate dibasic and potassium phosphate monobasic.

Thermo Fisher Scientific, 81 Wyman Street, Waltham, MA 02454 www.thermo.com
E.
 Starch
1.
 YSI 2700 SELECT Biochemistry Analyzer

YSI Incorporated Life Sciences, 1725 Brannum Lane, Yellow Springs, Ohio 45387 Application Note Number 319. www.ysilifescience.com

Samples are pre-extracted for sugar by incubation in 40ºC water bath and filtration on Whatman 41 filter paper. Residues are thermally solubilized using an autoclave, then incubated with glucoamylase enzyme to hydrolyze starch to produce dextrose (glucose).

Prepared samples injected into sample chamber of YSI Analyzer where dextrose diffuses into a membrane containing glucose oxidase. The dextrose is immediately oxidized to hydrogen peroxide and D-glucono-4-lactone. The hydrogen peroxide is detected amperometrically at the platinum electrode surface. The current flow at the electrode is directly proportional to the hydrogen peroxide concentration, and hence to the dextrose concentration. Starch is determined by multiplying dextrose by 0.9.
2.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
F. Water Soluble Carbohydrates (WSC)
1.
 West Virginia University Procedure by W.H. Hoover and T.K. Miller Webster. Determination of Nonstructural Carbohydrates.

Hall, M.B., W.H. Hoover, J.P. Jennings and T.K. Miller Webster. 1999. A method for partitioning neutral detergent soluble carbohydrates. J. Sci. Food Agric. 79: p.2081.

Samples incubated with water in a 40ºC bath extracting water soluble carbohydrates comprised of simple sugars and fructan. WSC determined after acid hydrolysis with sulfuric acid and colorimetric reaction with potassium ferricyanide.
2.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
G.
 Ethanol Soluble Carbohydrates (ESC)
1.
 Hall, M.B., W.H. Hoover, J.P. Jennings and T.K. Miller Webster. 1999. A method for partitioning neutral detergent soluble carbohydrates. J. Sci. Food Agric. 79: p.2079-2086.

Samples shaken with 80% ethanol to extract ethanol soluble carbohydrates comprised of simple sugars. ESC determined colorimetrically using a phenol-sulfuric acid reaction.
2.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
H.
 Fermentation Volatile Fatty Acids (VFA)
1.
 Gas Chromatography and Biochemistry Analyzer methods

Extraction – Samples blended for 2 min. in deionized water, filtered through cheesecloth, then filtered through disposable syringe filter. Adapted from Personal Communication, L.E. Chase, Ph.D., Cornell University.

Analysis –
a.
 Acetic, Propionic, Butyric, Iso-butyric acids

Aliquot of extract mixed 1:1 ratio with 0.06M oxalic acid containing 100ppm trimethylacetic acid (internal standard). Samples injected into a Perkin Elmer Autosystem XL Gas Chromatograph containing a Supelco packed column with the following specifications: 2m x 2mm Tightspec ID, 4% Carbowax 20M on 80/120 B-DA.

Procedure based upon:
  • "GC Separation of VFA C2-C5" Supelco GC Bulletin 749F, 1975.
  • "Analyzing Fatty Acids by Packed Column Gas Chromatography" Supelco GC Bulletin 856A, 1990.
  • "Volatile Fatty Acid SOP" W.H. Miner Institute, Chazy, NY.
Sigma Aldrich (Supelco), 3050 Spruce Street, St. Louis, MO 63103 www.sigmaaldrich.com

Perkin Elmer, 940 Winter Street, Waltham, MA 02451 www.perkinelmer.com
b.
 Lactic acid

Aliquot of extract analyzed for L-Lactate using a YSI 2700 SELECT Biochemistry Analyzer equipped with an L-Lactate membrane. YSI User’s Manual, page 4-7.

Samples injected into sample chamber of YSI Analyzer where L-Lactate diffuses into a membrane containing L-Lactate oxidase. The L-Lactate is immediately oxidized to hydrogen peroxide and pyruvate. The hydrogen peroxide is detected amperometrically at the platinum electrode surface. The current flow at the electrode is directly proportional to the hydrogen peroxide concentration, and hence to the L-Lactate concentration. Total lactic acid is determined by multiplying L-Lactate by 2.0.
2.
 NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.

Lactic and Acetic acids only. Analyzed on corn silage and fermented haycrop forages only.
I.
Gross Energy (GE)

Gross energy (gross calorific value) of solid and liquid materials expressed as calories per gram (cal/g) using an IKA C2000 basic Calorimeter System.

Instrument is set to IKA’s dynamic mode with an outer vessel temperature set at 25ºC. Analysis time is 7-12 minutes. Dried samples weighed into polyethylene bags or oil type samples weighed into gelatin capsules. Samples placed in a crucible, then ignited in an oxygen rich atmosphere in a sealed decomposition vessel where the increase in temperature of the system is measured.

The specific gross calorific value of the sample is calculated from:
  • The weight of the sample.
  • The heat capacity of the calorimeter system determined from benzoic acid calibration standards.
  • The increase in temperature of the water within the inner vessel of the measuring cell.
IKA Works, Inc., 2635 North Chase Pkwy SE, Wilmington, NC 28405-7419 www.ika.net
VII.
 Manure
 All results corrected for density except total solids.
A.
 Total Solids
1.
 Liquid or solid; no bedding
a.
 Oven - 105°C for 16 hours (gravity).
2.
 Liquid or solid with bedding
a.
 Oven - 60°C for 6-8 hours (forced air).
b.
 Near Infrared Reflectance Spectroscopy - AOAC 991.01.

Hoskins, B., A. Wolf, and N. Wolf. 2003. Dry matter analysis. Recommended methods of manure analysis. ed J. Peters, pp14-17. University of Wisconsin Extension Publication. A3769.
B.
 Nitrogen, Total (N)
1.
 Combustion by Leco TruMac N Macro Determinator.

Leco Corporation, 300 Lakeview Avenue, St. Joseph, MI 49085 www.leco.com

Watson, M., A. Wolf, and N. Wolf. 2003. Total nitrogen. Recommended methods of manure analysis. ed J. Peters, pp18, 23-24. University of Wisconsin Extension Publication. A3769.
C.
 Ammonia-Nitrogen (Amm-N)
1.
 Timberline TL-2800 Analyzer.

Extraction - 10g samples are extracted in 150ml deionized water using a reciprocal shaker for 30 minutes at 280 epm. For urea, a prepared urease solution is added to a duplicate sample prior to shaking. All extracts then centrifuged at 4000 rpm for 5 minutes, decanted into tubes, then analyzed.

Analysis - A peristaltic pump directs the sample, caustic, and absorbing solutions into a diffusion cell. Within the cell, the sample is mixed with the caustic solution, resulting in a pH of 11-13 which converts the ammonium ion present in the sample to dissolved ammonia gas. The sample/caustic solution flows past one side of a membrane that is permeable to gases but not to liquids or ionic species. The dissolved ammonia gas in the sample/caustic mixture diffuses across the membrane.

On the other side of the membrane, a buffered solution absorbs the diffused ammonia gas then flows through a low volume heat exchanger to establish thermal equilibrium then into the conductivity detector. The conductivity cell measures the change in electrical conductance of the absorbing solution. This change is proportional to the concentration of ammonium in the original sample.

Timberline Instruments, 1880 S. Flatiron Ct. Suite I, Boulder, CO 80301 www.timberlineinstruments.com
D.
 Organic Nitrogen (Org-N)
1.
 Calculated by difference (Total Nitrogen - Ammonia-Nitrogen).
E.
 Minerals
1.
 Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, Co, S, Al, B, Cr, Sr

CEM Microwave Accelerated Reaction System (MARS) with MarsXpress Temperature Control using 50ml calibrated Xpress Teflon PFA vessels with Kevlar/fiberglass insulating sleeves.

Sample weight - 0.5g dried, ground or 2-10g wet sample.

Samples predigested at ambient temperature 15 minutes with 8ml nitric acid (HNO3) and 2ml hydrochloric acid (HCl) then ramped to 190ºC in 15 minutes and finally held at digestion temperature of 190ºC for 15 minutes at 1600W. Vessels brought to 50-ml volume, aliquot used for analysis.

Wolf, Ann, M. Watson, and N. Wol- 2003. Digestion and dissolution methods for P, K, Ca, Mg and trace elements. Recommended methods of manure analysis. ed J. Peters, pp30, 32-35. University of Wisconsin Extension Publication. A3769.

CEM Application Note for Acid Digestion - Feed Grain
CEM Application Note for Acid Digestion - Alfalfa

CEM, 3100 Smith Farm Road, Matthews, NC 28106 www.cem.com
Thermo Fisher Scientific Inc., 81 Wyman Street, Waltham, MA 02454 www.thermo.com
2.
 Chloride (Cl)

Brinkmann Metrohm 716 Titrino Titration Unit - 0.5g dried, ground or 5g wet sample extracted in 50ml 0.1N HNO3 followed by potentiometric titration with AgNO3 using Brinkmann Metrohm 716 Titrino Titration Unit with silver electrode.
a.
 Metrohm Application Bulletin No. 130 by Metrohm Ltd., C-H-9101 Herisau, Switzerland.

Metrohm USA, 6555 Pelican Creek Circle, Riverview Fl, 33578 www.metrohmusa.com
b.
 The method by Metrohm is similar to the concepts found in:

Cantliffe, D.J., MacDonald, G.E. and Peck, N.H. 1970. The potentiometric determination of nitrate and chloride in plant tissue. New York's Food and Life Sciences Bulletin. No.3, September 1970. Plant Sciences. Vegetable Crops Geneva. No. 1: 5-7.
F.
 Ash
1.
 AOAC 942.05 - Ash of Animal Feed.
G.
 pH
1.
 AOAC 973.04 - pH of Peat. (Manure pH technique similar to pH of peat).

35ml liquid sample poured into 50ml beaker. 15g solid or semi-solid sample weighed into 200 ml deionized water, stirred, and allowed to stabilize five minutes. Analyzed using Thermo Orion Posi-pHlo SympHony Electrode and Thermo Orion 410 A meter.

Wolf, Nancy. 2003. Determination of manure pH. Recommended methods of manure analysis. ed J. Peters, pp48-49. University of Wisconsin Extension Publication. A3769.
H.
 Nitrates (%NO3 or ppm NO3-N)
1.
 RQflex Reflectometer Method.

Merck Millipore Application Note "Nitrates in Plant Sap", 2006-05-23; extraction modified.
Merck Millipore Application Note "Nitrates in Waste Water", 2006-05-24.

EMD Chemicals Inc., One International Plaza, Suite 300, Philadelphia, PA, 19113 www.emdchemicals.com

1g of dried, ground sample or 10g wet sample is extracted in 50ml deionized water for 20 minutes by shaking at 280 oscillations/minute. Samples are filtered through Whatman 934-AH (1.5um) filter paper, then analyzed by RQflex® Reflectometer using Reflectoquant® Nitrate test strips.

When the Nitrate test strip is immersed in a sample, a reducing agent reduces nitrate ions to nitrite ions. In the presence of an acidic buffer, the nitrite ions react with an aromatic amine to form a diazonium salt. The salt reacts with N-(1-naphthyl)-ethyelene-diamine to form a red-violet azo dye that is measured reflectometrically. Nitrate concentration is proportional to the color reaction.

Each strip contains two reaction zones generating dual replicate analyses per sample. The RQflex® Reflectometer's double optic system measures the analyte concentration based on the light reflected from the dual reaction zones. Barcode-controlled software calculates the mean of those two measurements.
I.
 Density
1.
 Standard Vial Method. 2002. Dairy One.

Samples weighed into fixed volume vessel. Density calculated and expressed in kg/l, lbs./ft3, and lbs./gal.
J.
 Gross Energy (GE)
Gross energy (gross calorific value) of solid and liquid materials expressed as calories per gram (cal/g) using an IKA C2000 basic Calorimeter System.

Instrument is set to IKA's dynamic mode with an outer vessel temperature set at 25°C. Analysis time is 7-12 minutes. Dried samples weighed into polyethylene bags or oil type samples weighed into gelatin capsules. Samples placed in a crucible, then ignited in an oxygen rich atmosphere in a sealed decomposition vessel where the increase in temperature of the system is measured.

The specific gross calorific value of the sample is calculated from:
- The weight of the sample.
- The heat capacity of the calorimeter system determined from benzoic acid calibration standards.
- The increase in temperature of the water within the inner vessel of the measuring cell.

IKA Works, Inc., 2635 North Chase Pkwy SE, Wilmington, NC 28405-7419 www.ika.net
K.
 Minerals
1.
 Fermentation and Volatile Fatty Acids (VFA)

Gas Chromatography and Biochemistry Analyzer methods

Extraction -

50g samples blended at 20000 rpm for 2 min. in 450ml deionized water, filtered through cheesecloth, then filtered through disposable syringe filter. Adapted from Personal Communication, L.E. Chase, Ph.D., Cornell University.

Analysis -
a.
 Acetic, Propionic, Butyric, Iso-butyric acids

Aliquot of extract mixed 1:1 ratio with 0.06M oxalic acid containing 100ppm trimethylacetic acid (internal standard). Samples injected into a Perkin Elmer Autosystem XL Gas Chromatograph containing a Supelco packed column with the following specifications: 2m x 2mm Tightspec ID, 4% Carbowax 20M phase on 80/120 Carbopack B-DA.

Procedure based upon:
- "GC Separation of VFA C2-C5" Supelco GC Bulletin 749F, 1975.
- "Analyzing Fatty Acids by Packed Column Gas Chromatography" Supelco GC Bulletin 856A, 1990.
- "Volatile Fatty Acid SOP" W.H. Miner Institute, Chazy, NY.

Sigma Aldrich (Supelco), 3050 Spruce Street, St. Louis, MO 63103 www.sigmaaldrich.com

Perkin Elmer, 940 Winter Street, Waltham, MA 02451 www.perkinelmer.com
b.
 Lactic acid

Aliquot of extract analyzed for L-Lactate using YSI 2700 SELECT Biochemistry Analyzer equipped with an L-Lactate membrane. YSI User's Manual, page 4-7.

Samples injected into sample chamber of YSI Analyzer where L-Lactate diffuses into a membrane containing L-Lactate oxidase. The L-Lactate is immediately oxidized to hydrogen peroxide and pyruvate. The hydrogen peroxide is detected amperometrically at the platinum electrode surface. The current flow at the electrode is directly proportional to the hydrogen peroxide concentration, and hence to the L-Lactate concentration. Total lactic acid is determined by multiplying L-Lactate by 2.0.
VIII.
 Water
A.
 Coliform and E. coli (presence/absence in 100ml)
Colilert® - IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092,

Colilert® uses the patented Defined Substrate Technology® (DST®) to simultaneously detect total coliforms and E. coli. Two nutrient-indicators, ONPG and MUG, are the major sources of carbon in Colilert® and can be metabolized by the coliform enzyme ß-galactosidase and the E. coli enzyme ß-glucuronidase, respectively.

As coliforms grow in Colilert®, they use ß-galactosidase to metabolize ONPG and change it from colorless to yellow to indicate presence.

E. coli use ß-glucuronidase to metabolize MUG and create fluorescence to indicate presence. Since most non-coliforms do not have these enzymes, they are unable to grow and interfere.

Colilert® is US FDA Approved for Dairy Waters.
  • Milk Laboratory Evaluation Form FDA 2400m (3/01)

Colilert® is also US EPA-approved for drinking water presence/absence(P/A) and Most Probable Number (MPN) and for source water. Pertinent references:
  • June 29, 1989 US EPA Federal Register Colilert® coliform approval
  • June 10, 1992 US EPA Federal Register Colilert® E. coli approval
Colilert® detects total coliforms and E. coli at 1 organism/100 ml.
B.
 Standard Plate Count (colonies per ml)
US FDA Milk Laboratory Evaluation Form FDA 2400a (1/01)

Petrifilm Aerobic Count Method - Deposit 1ml of sample onto petrifilm and cover. Distribute sample with spreader and allow gel to solidify for 1 minute. Incubate 48 hours at 32°C. Count colonies when incubation time is complete.
C.
 pH
AOAC 973.41 - pH of Water

Analyzed using Thermo Orion Posi-pHlo SympHony Electrode and Thermo Orion 410 A meter. Calibrated with buffers referenced to NIST SRMs. pH 4 buffer contains potassium hydrogen phthalate and pH 7 buffer contains sodium phosphate dibasic and potassium phosphate monobasic.
D.
 Nitrates (ppm NO3 and ppm NO3-N)
RQflex Reflectometer Method.

Merck Millipore Application Note "Nitrates in Waste Water", 2006-05-24.
EMD Chemicals Inc., One International Plaza, Suite 300, Philadelphia, PA, 19113 www.emdchemicals.com

When the Nitrate test strip is immersed in a sample, a reducing agent reduces nitrate ions to nitrite ions. In the presence of an acidic buffer, the nitrite ions react with an aromatic amine to form a diazonium salt. The salt reacts with N-(1-naphthyl)-ethyelene-diamine to form a red-violet azo dye that is measured reflectometrically. Nitrate concentration is proportional to the color reaction.

Each strip contains two reaction zones generating dual replicate analyses per sample. The RQflex® Reflectometer's double optic system measures the analyte concentration based on the light reflected from the dual reaction zones. Barcode-controlled software calculates the mean of those two measurements.

Nitrate-Nitrogen (NO3-N) calculated as Nitrates (NO3) divided by 4.427.
E.
 Sulfates (ppm SO4 and SO4-S)
Turbidimetric Method. 957-13-3. (Based upon principles in AOAC 973.57).
Orbeco Hellige, 6456 Parkland Drive, Sarasota, FL 34234, 1-941-756-6410 www.orbeco.com

BaCl2 tablet is added to the sample resulting in precipitation of sulfate as BaSO4. Suspension is measured photometrically at 420nm using an Orbeco-Hellige Series 942 Mini-Analyst to determine the sulfate concentration in mg/l (ppm).

Sulfate-sulfur (SO4-S) calculated as sulfates (SO4) divided by 2.996.
F.
 Total Dissolved Solids (ppm TDS)
Conductivity Method. ES&D Model 76 Conductivity meter.

The total quantity of free ions is determined by ability of the sample to conduct an electrical current. Electrode immersed in water while gently stirring. Temperature of water measured, temperature knob set, meter allowed to stabilize for 15 seconds, and then reading recorded.
G.
 Minerals
1.
 ppm Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo

Analyzed directly with no sample preparation by Thermo IRIS Advantage HX or Intrepid Inductively Coupled Plasma (ICP) Radial Spectrometer.

Thermo Fisher Scientific Inc., 81 Wyman Street, Waltham, MA 02454 www.thermo.com

ppm Hardness as CaCO3 equivalent calculated as (Ca x 2.5) + (Mg x 4.1).
2.
 ppm Chloride (Cl)

25ml 0.2N HNO3 added to 25ml of water followed by potentiometric titration with AgNO3 using Brinkmann Metrohm 716 Titrino Titration Unit with silver electrode.
a.
 Metrohm Application Bulletin No. 130 by Metrohm Ltd., C-H-9101 Herisau, Switzerland.

Metrohm USA, 6555 Pelican Creek Circle, Riverview Fl, 33578 www.metrohmusa.com
b.
 The method by Metrohm is similar to the concepts found in:

Cantliffe, D.J., MacDonald, G.E. and Peck, N.H. 1970. The potentiometric determination of nitrate and chloride in plant tissue. New York's Food and Life Sciences Bulletin. No.3, September 1970. Plant Sciences. Vegetable Crops Geneva. No. 1: 5-7.
IX.
 Websites of Interest
ANKOM Technology - www.ankom.com
AOAC International - www.aoac.org
CEM - www.cem.com
EMD Chemicals - www.eshop.emdchemicals.com
Foss North America - www.foss.us
IKA Works - www.ika.net
Leco Corporation - www.leco.com
Metrohm USA - www.metrohmusa.com
National Forage Testing Association (NFTA) - www.foragetesting.com
Perkin Elmer - www.perkinelmer.com
Supelco - www.sigmaaldrich.com
Thermo Scientific - www.thermo.com
YSI Incorporated - www.ysilifescience.com

Home I Publications I Contact Us I Links I About Us I Employment
Farm & DHIA Services I Forage Laboratory Services I Soils Laboratory Services default.htm" class="D1SubLinks">Soils
Dairy Management Resources I Milk Laboratory Services
Equi-Analytical Laboratory Services I Farmland Environmental I Current Events