Title Page 

Determination of the δ15N of 

Nitrate in Water; 

RSIL Lab Code 2899

Chapter 16 of

Section C, Stable Isotope-Ratio Methods

Book 10, Methods of the Reston Stable Isotope 

Laboratory 

 

 

 

 

 

 

 

 

U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 10–C16 
This page left blank intentionally.
Determination of the δ15N of

Nitrate in Water; 

RSIL Lab Code 2899

By Tyler B. Coplen, Haiping Qi, Kinga Révész, Karen Casciotti, and Janet E. 

Hannon 

Chapter 16 of

Section C, Stable Isotope-Ratio Methods 

Book 10, Methods of the Reston Stable Isotope 

Laboratory 

 

Edited by Kinga Révész and Tyler B. Coplen

U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 10–C16
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey 

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

Version 1.0, 2007 

Version 1.1, 2012 

For sale by U.S. Geological Survey, Information Services

Box 25286, Denver Federal Center

Denver, CO 80225

For more information about the USGS and its products:

Telephone: 1–888–ASK–USGS

World Wide Web: http://www.usgs.gov/

Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted material contained within this report.

Suggested citation:

Coplen, T.B., Qi, Haiping, Révész, Kinga, Casciotti, Karen, and Hannon, J.E., 2012, Determination of the δ15N of nitrate 

in water; RSIL lab code 2899, chap. 16 of Stable isotope-ratio methods, sec. C of Révész, Kinga, and Coplen, T.B. eds., 

Methods of the Reston Stable Isotope Laboratory (slightly revised from version 1.0 released in 2007): U.S. Geological 

Survey Techniques and Methods, book 10, 35 p., available only online at http://pubs.usgs.gov/tm/2006/tm10c16/. 

(Supersedes version 1.0 released in 2007.)
iii

Foreword 

The Reston Stable Isotope Laboratory (RSIL) provides stable isotope analyses on a routine basis 

for a large user community within the U.S. Geological Survey (USGS) and elsewhere. The RSIL 

also serves the USGS National Research Program (NRP) through its project on Stable Isotope 

Fractionation in Hydrologic Processes. The NRP conducts basic and problem-oriented hydrologic 

research in support of the mission of the USGS. The stable isotope project conducts research on 

the use of isotope-ratio measurements in studies of water resources and environmental quality. 

One objective of this project is to develop new techniques for isotopic analysis of hydrogen, 

nitrogen, oxygen, carbon, and sulfur in environmental samples. New analytical techniques 

expand the range of tools available for studying the movement of those elements in hydrologic 

and biogeochemical systems. Another objective of the project is to test new applications of 

isotope measurements in specific field settings. Field studies of isotope behavior have 

contributed to understanding water-supply sustainability, groundwater/surface-water 

interactions, paleoclimate history, biologic cycling of nutrients, groundwater contamination, and 

natural remediation. This project also contributes to the improvement of measurement science 

and the development of isotope databases.

Book 10 of the Techniques and Methods Series of the USGS contains detailed descriptions of 

methods of the RSIL, including routine analytical methods called standard operating procedures 

(SOPs), along with safety guidelines, maintenance procedures, and other information about the 

operation of the RSIL. Section C of book 10 contains the SOPs for a variety of methods to 

measure stable isotope ratios, each of which constitutes a chapter. Each chapter is limited to a 

narrow field of subject matter to permit flexibility in revision as the need arises.

Pierre Glynn

Chief, Branch of Regional Research, Eastern Region 
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v 

Contents 

Foreword............................................................................................................................................................ iii

Summary of Procedure.................................................................................................................................... 1

Reporting Units and Operational Range ...................................................................................................... 1

Reference Materials and Documentation ................................................................................................... 2

Reference Materials Used, Storage Requirements, and Shelf Life....................................... 2

Documentation.................................................................................................................................. 2

Labware, Instrumentation, and Reagents ................................................................................................... 3

Sample Collection, Preparation, Analysis, Retention Times, and Disposal.......................................... 4

Sample Containers, Preservation, and Handling Requirements............................................ 4

Sample Preparation and Time Requirements ............................................................................ 5

Performing the Analysis and Time Requirements..................................................................... 6

Problematic Samples....................................................................................................................... 7

Interferences..................................................................................................................................... 7

Troubleshooting and Bench Notes............................................................................................... 7

Maintenance and Maintenance Records................................................................................... 8

Sample Retention Time and Disposal .......................................................................................... 8

Data Acquisition, Processing, Evaluation, Quality Control, and Quality Assurance .......................... 8

Laboratory Information Management System for Light Stable Isotope (LIMS-LSI) .......... 8

Quality Control (QC) Samples......................................................................................................... 9

Acceptance Criteria for All QC Samples ..................................................................................... 9

Corrective Action Requirements................................................................................................... 9

Responsible Parties for All QA/QC Functions for Procedures Covered in RSIL SOPs .... 10

Data Management and Records................................................................................................. 10

Health, Safety, and Waste-Disposal Information..................................................................................... 10

Applicable Health and (or) Safety Issues.................................................................................. 10

Personal Protection ...................................................................................................... 10

Electrical Hazards.......................................................................................................... 10

Chemical Hazards.......................................................................................................... 10

Gas Cylinder Handling .................................................................................................. 10

Biological Hazards ........................................................................................................ 11

Specific Waste-Disposal Requirements ................................................................................... 11

Revision History............................................................................................................................................... 12

References Cited............................................................................................................................................. 14

Appendix A. Step-by-Step Procedure to Log-In Samples to LIMS-LSI............................................... 15

Appendix B. Step-by-Step Procedure to Print a Template and a Samples-to-Be-Analyzed 

List.............................................................................................................................................................. 16

Template........................................................................................................................................... 16

Appendix C. Step-by-Step Preparation Procedure for Nitrogen Isotopic Analysis of Nitrate 

by Bacterial Denitrification................................................................................................................... 18

Making the Plates .......................................................................................................................... 18

Preparing the Tryptic Soy Broth (TSB) ...................................................................................... 18

Testing Bacteria for Incomplete Conversion of Nitrate......................................................... 19

Harvesting the Bacteria—Concentrating Culture by Centrifugation.................................. 20

Adding Concentrated Cell Culture to Sample Vial and Purging Headspace and 

Adding Samples ....................................................................................................................... 20

Appendix D. Step-by-Step Procedure to Add Sample Information to Sequence Table.................. 31

Appendix E. Step-by-Step Procedure to Transfer Data to LIMS-LSI, to Transfer Data to 

Back-Up Computer, and to Reevaluate Old Data ............................................................................ 32

Appendix F. Step-by-Step Procedure to Determine and Apply Correction Factors and 

Evaluate Data .......................................................................................................................................... 33
vi

Appendix G. Daily Check List ........................................................................................................................34

Appendix H. Step-by-Step Procedure to Report Data.............................................................................35

Figures

1. Schematic of Modified Finnigan MAT (now Thermo Scientific) GasBench 2 

Inlet System. ................................................................................................................................. 3

2. Schematic of a continuous flow isotope-ratio mass spectrometer (CF-IRMS) 

(modified from Clark and Fritz, 1997)....................................................................................... 5

B–1. Sample list generated by LIMS...............................................................................................16

C–1. Tryptic soy broth (TSB), with KNO3

, prepared, mixed in 9-L bottle (yellow 

liquid), and divided into 500-g aliquots..................................................................................21

C–2. Autoclaved for 60 minutes at 120 o

C.......................................................................................21

C–3. Top view of 580-mL bottles with media ready to be autoclaved. ....................................22

C–4. Tryptic Soy broth (TSB) without KNO3

, prepared with 100-mL aliquots in 

125-mL serum bottles................................................................................................................22

C–5. Selecting a separate colony from the plate using a sterile flamed loop. ......................23

C–6. Selected colony is shown........................................................................................................23

C–7. Selected colony transferred to labeled tube containing 5 mL of TSB............................24

C–8. Shaker showing 5 mL of culture before injection and showing previously 

prepared 580-mL bottles injected with bacteria. ................................................................24

C–9. A bottle of nitrate-free bacterial culture ready for use.....................................................25

C–10. Centrifuge with four 250-mL bottles.......................................................................................25

C–11. Twenty-milliliter sample vial containing 3 mL of concentrated cell culture. ................26

C–12. Mechanical crimper..................................................................................................................26

C–13. PTFE/Aluminum-lined septa with aluminum crimps...........................................................27

C–14. Empty vial showing placement of vent needle....................................................................27

C–15. Manifold showing helium purging of 32 vials. .....................................................................28

C–16. Loading vial with sample..........................................................................................................28

C–17. Aluminum tray with inverted sample vials. ..........................................................................29

C–18. Finnigan MAT GasBench 2. .....................................................................................................29

C–19. Sample tray with 32 vials and a 1⁄4-inch-thick aluminum plate to secure vials.............30
vii

Conversion Factors 

Multiply By To Obtain 

Length

millimeter (mm) 0.03937 inch

centimeter (cm) 0.3937 inch

meter (m) 3.281 foot

Volume

microliter (μL) 0.0610210–3 cubic inch

milliliter (mL) 0.06102 cubic inch

cubic centimeter (cm3) 0.06102 cubic inch

Mass

nanogram (ng) = 10–3μg 3.527 × 10–11 ounce

microgram (μg) = 10–3mg 3.527 × 10–8ounce

milligram (mg) = 10–3 g 3.527 × 10–5 ounce

gram (g) = 10–3 (kg) 3.527 x 10–2 ounce

kilogram = 103g; 106mg; 109μg; 1012 ng 2.205 pound

Density

gram per cubic centimeter (g/cm3) 0.5780 ounce per cubic inch

Temperature

Celsius (°C) °F = 9/5 (°C) + 32 Fahrenheit (°F)
viii

Acronyms and Abbreviations 

ANSI American National Standards Institute 

CF-IRMS continuous flow isotope-ratio mass spectrometer

cm centimeter

DIW deionized water

g gram

h hour 

HDPE high-density polyethylene 

IRMS isotope-ratio mass spectrometer

LIMS-LSI Laboratory Information Management System for Light Stable Isotopes 

MSDS Material Safety Data Sheets

mg milligram

mg/L milligram per liter

min minute

mL milliliter

mL/min milliliter per minute

mm millimeter

mm/s millimeter per second

mM millimolar

nmol nanomole 

NED N-(1-haphthyl)ethylenediamine dihydrochloride)

NFPA National Fire Protection Association

NWQL National Water Quality Laboratory (USGS)

per mil, ‰ one part in one thousand parts, with value 10–3

pF picofarad

QA quality assurance

QC quality control

QC/QA quality control/quality assurance

RSIL Reston Stable Isotope Laboratory

SOP standard operating procedure

std international measurement standard

TSA tryptic soy agar

TSB tryptic soy broth

USGS U.S. Geological Survey 

μg microgram

μM micromolar

Symbols 

Ω ohm 

< less than

> greater than

≤ less than or equal 
1 

Determination of the δ15N of Nitrate in Water;

RSIL Lab Code 2899

By Tyler B. Coplen1, Haiping Qi1, Kinga Révész1, Karen Casciotti2

Summary of Procedure

, and Janet E. Hannon1

The purpose of the Reston Stable Isotope Laboratory (RSIL) lab code 2899 is to determine the δ

15N 

of nitrate (NO3

–

) in water. The δ

15N of the dissolved NO3

– is analyzed by conversion of the NO3– to 

nitrous oxide (N2

O), which serves as the analyte for mass spectrometry. A culture of denitrifying bacteria 

is used in the enzymatic conversion of the NO3

–

 to N2O, which follows the pathway shown in equation 1: 

NO3

–  NO2–  NO  1/2 N2

O (1) 

Because the bacteria Pseudomonas aureofaciens lack N2

O reductive activity, the reaction stops at 

N2

O, unlike the typical denitrification reaction that goes to N2. After several hours, the conversion is 

complete, and the N2

O is extracted from the vial, separated from volatile organic vapor and water vapor 

by an automated –65 O

C isopropanol-slush trap, a Nafion drier, a CO2 and water removal unit (Costech 

#021020 carbon dioxide absorbent with Mg(ClO4

)2), and trapped in a small-volume trap immersed in 

liquid nitrogen with a modified Finnigan MAT (now Thermo Scientific) GasBench 2 introduction system. 

After the N2

O is released, it is further purified by gas chromatography before introduction to the isotoperatio mass spectrometer (IRMS). The IRMS is a Thermo Scientific Delta V Plus continuous flow IRMS 

(CF-IRMS). It has a universal triple collector, consisting of two wide cups with a narrow cup in the 

middle; it is capable of simultaneously measuring mass/charge (m/z) of the N2

O molecule 44, 45, and 46. 

The ion beams from these m/z values are as follows: m/z = 44 = N2

O = 14N14N16O; m/z = 45 = N2O = 14N15N16O or 14N14N17O; m/z = 46 = N2

O = 14N14N18O. The 17O contributions to the m/z 44 and m/z 45 ion 

beams are accounted for before δ

15N values are reported.

Reporting Units and Operational Range

Variations in stable isotope ratios typically are small. Stable isotope ratios commonly are measured 

and expressed as the relative difference in the ratio of the number of the less abundant isotope (usually the 

heavy isotope) to the number of the more abundant isotope (usually the light isotope) of a sample with 

respect to the measurement standard, std (Coplen, 2011). This relative difference is designated δ

i

E, which 

is a shortened form of δ(

i

E/jE) or δ(

i

E), and is defined according to equation 2 (Coplen, 2011): 

( ) ( ) ( ) ( ) ( ) ( )

( ) ( ) NN

NN NN δ = δ = δ = i j

i j i j

i i ji

std std

P P std std

EE

EE EE EE E E − (2)

 

1

U.S. Geological Survey, Reston Stable Isotope Laboratory, Reston, Virginia 20192

2

Stanford University, Department of Environmental Earth System Science, 397 Panama Mall, California 94305-

2210
2 

where N(

i

E)P and N(

j

E)P are the numbers of the two isotopes iE and jE of element E in specimen P and 

equivalent parameters follow for the international measurement standard, “std.” A positive δ

i

E value 

indicates that the specimen is enriched in the heavy isotope, 

i

E, relative to the std. A negative δ

i

E value 

indicates that the specimen is depleted in the heavy isotope, 

i

E, relative to the std. For stable nitrogen 

isotope–amount ratios, δ

15N is defined as follows: 

( ) ( ) ( ) ( ) ( ) ( )

( ) ( )std

14

std

15

std

14

std

15

P

14

P

15

15 15 1415

N N

NN N N NNNN

N N

NNN N δ = δ = δ = − (3)

The primary reference material for relative nitrogen isotope-ratio measurements (δ

15N) is atmospheric 

nitrogen gas (N2

), which is widespread and homogeneous and, by convention, has a δ

15N consensus value 

of 0 (Mariotti, 1983). By interspersing internationally distributed isotopic reference materials with 

accepted δ values among unknown samples, the δ values can be determined on normalized δ

15N scales. 

The δ

15N scale is determined by analyzing reference materials IAEA-N-1 and USGS32, which have an 

assigned values of +0.43 and +180 ‰, respectively (Böhlke and Coplen, 1995). The δ

15N measurements 

are made with a CF-IRMS, which alternately measures the isotope-amount ratios of the sample N2

O gas

and one or more injections of the “working reference” N2

O gas. Values of δ

15N typically are reported in 

parts per thousand (‰). 

The system was tested by analyzing pure inorganic samples (potassium and sodium nitrates). The 

routine analysis requires 0.28 μg N; however, the system is capable of analyzing samples as small as 

0.03 microgram (μg) nitrogen in one sample aliquot. The δ

15N values of reference materials ranged from 

approximately –1.8 to +180 ‰, which covers much of the range of isotopic composition of natural 

samples. The precision and accuracy of the results were acceptable, and no memory effect was observed. 

The uncertainties of precision and accuracy were less than ±0.25 ‰ for the δ

15N measurement. 

Reference Materials and Documentation

Reference Materials Used, Storage Requirements, and Shelf Life

Internationally distributed reference materials used in the RSIL are solid nitrogen-bearing materials, 

which have wide range of isotopic composition. In the measurement of δ

15N, USGS34 potassium nitrate 

(KNO3

)with a δ

15N value of –1.8 ‰ (Böhlke and others, 2003) and a USGS32 KNO3 with a δ15N value of 

+180 ‰ (Böhlke and others, 1993) are used. 

All of these reference materials are stored in the RSIL at room temperature in glass bottles capped 

with Teflon-coated or cone-shaped caps to keep moisture out. Their shelf life is indefinite. 

Primary stocks of 20-millimolar (mM) reference nitrates are prepared by dissolving solid references, 

and they are stored in a refrigerator at 5 O

C. The 20-micromolar (μM) secondary working stock solutions 

are prepared every 1 to 2 months by diluting th primary stocks. These 20-μM working stocks are also 

stored in a refrigerator between uses and are analyzed along with the samples in amounts identical to that 

of the samples analyzed in a particular batch.

Documentation 

All calibration results are stored in the Laboratory Information Management System for Light Stable 

Isotopes (LIMS-LSI) (Coplen, 2000) under sample identifiers N-7 and N-34. All daily sample process 

templates are saved in a binder.
3 

Labware, Instrumentation, and Reagents

Preparatory labware and apparatus include beakers, watch glasses, 580-milliliters (mL) glass serum 

bottles, Petri dishes, flasks, a torch, sterile plastic test tubes, spreading loop, a laboratory shaker, freezers, 

a refrigerator, an autoclave, and a tabletop centrifuge. 

The analytical apparatus consists of three different units: (1) an automated sample extraction and 

analysis system, (2) IRMS, and (3) computer software. The automated sample extraction and analysis 

system is a modified and improved version (Coplen and others, 2004) of the Finnigan MAT GasBench 2 

(fig. 1), first described by Sigman and others (2001) and by Casciotti and others (2002). 

 

 

Figure 1. Schematic of Modified Finnigan MAT (now Thermo Scientific) GasBench 2 Inlet System.

The automated sample extraction and analysis unit is constructed to hold 32 samples and is furnished 

with a CTC Analytical CombiPAL robotic sampling arm by which the sample ultimately is sent to the 

IRMS through a volatile organic vapor and water removal unit (an automated –65 O

C isopropanol-slush 

trap), a water removal unit (Nafion drier), CO2

 and water removal unit [Costech #021020 carbon dioxide 

absorbent and Mg (ClO4

)2/Ascarite trap), a cryogenic trap, a GC column, another water removal unit 

(Nafion drier), and an open split. To eliminate the need for an automatic liquid-nitrogen-trap filler, the 

stroke of the trap of the Finnigan MAT GasBench 2 was increased from 25 centimeter (cm) to 40 cm by 
4 

replacing the SMC model C85KN20-250 piston with a model C85KN20-400 piston. The liquid nitrogen 

dewar of the Finnigan MAT GasBench 2 was replaced by one with an inner diameter of 9.5 cm and an 

inner depth of 49 cm, which held liquid nitrogen for 20 h. The CTC CombiPAL autosampler is fitted with 

a LEAP Technologies, Inc. headspace sampler (P/N PAL.VENTTOOL). Helium enters the sample vial 

through a 17-cm-long 22s-gauge side-hole inlet needle (Hamilton #17305). Helium and sample gas exit 

the sample vial through a 22-gauge sidehole outlet needle. Bending of the inlet needle during septum 

penetration is minimized by using a 22s-gauge needle and by reducing the Z-axis motor speed of the 

CombiPAL to 500 millimeters per second (mm/s). The helium inlet needle is held by a VICI bulkheadreducing union (ZBRU1.5) mounted in a 1-mL CombiPAL syringe holder. The lower needle guide of the 

CombiPAL was lowered 4 cm by adding a pair of 4-cm stainless steel extenders. The Finnigan MAT 

sample holder was replaced with a 32-hole VT32-20 CombiPAL sample holder, which was screwed to 

the base of the CombiPAL. Because the friction of the needles in the vial septum is sufficiently high to 

pull vials with samples out of the sample holder, a 32-hole (2.222-cm diameter holes), 6.3-millimeter 

(mm)-thick aluminum plate was positioned over the vials and held down by screws extending from the 

sample holder. The CTC Analytical CombiPAL autosampler allows separate helium inflow [25 milliliters 

per minute (mL/min))to purge the entire vial and a helium + N2

O outflow for sample collection. The 

purged sample flows through a Nafion water removal tube and is collected for 13 minutes (min) in the 

cryogenic trap (95 × 0.16 cm-outside-diameter, stainless steel) submerged in liquid N2

 (–192 oC). By 

removing the trap from liquid N2

, the sample warms to room temperature, and the sublimed sample is 

released to the GC (PoraPLOT Q CP7551, held at room temperature) under a helium flow rate of 

3 mL/min through a Nafion drier, an open split that limits the flow to 0.3 mL/min, and ultimately into the 

IRMS. To ensure sufficient separation of CO2 and N2

O, the original 25-m PoraPLOT Q CP7551 GC

column has been extended to 37.5 m. 

The IRMS is a Thermo Scientific Delta V Plus CF-IRMS (fig. 2). The fundamental principle of the 

CF-IRMS technique is that a carrier gas transports the analyte through an initial stage of online chemistry 

for conversion to a form acceptable by the IRMS (Brenna and others, 1997), in this case N2

O gas. This is 

an automated system generating online, high-precision δ values. In the IRMS, gas molecules are ionized 

in a source by electrons emitted from a hot filament. The ions are accelerated into an analyzer, forming an 

ion beam, are separated in a magnetic field, and then collected in Faraday-cup collectors. The ion-beam

intensities are measured with electrometers. This IRMS has a universal triple collector, consisting of two 

wide cups with a narrow cup in the middle; it is capable of simultaneously measuring m/z 44, 45, and 46. 

The resistor–capacitor combination on the electrometers used to measure these masses are as follows: 

3 × 108 ohms (Ω) and 680 picofarads (pF) for cup 1, 3 × 1010 Ω and 5 pF for cup 2, and 3 × 1011 Ω and 

2 pF for cup 3. 

The Finnigan ISODAT 2.0 software (1) controls the Finnigan MAT GasBench 2 preparation system, 

(2) operates the IRMS, (3) acquires data from the IRMS, and (4) calculates delta values.

Sample Collection, Preparation, Analysis, Retention Times, and 

Disposal

Sample Containers, Preservation, and Handling Requirements

Each sample is collected in a maximum size of 4 oz (125 mL) amber, high-density polyethylene 

(HDPE) (Q33FLD or Q405FLD in USGS’s One-Stop Shopping online store) bottle, untreated, shipped 

capped by a Polyseal cap, and stored frozen or at 4 °C to minimize microbiological activity. The bottles 

are labeled with isotopes whose concentrations are to be determined with the respective laboratory code 

or schedule number. The sample should be filtered through a 0.25-μ filter; the bottle should be filled with 

sample with the water level at a maximum of three quarters of the volume of the bottle to allow for 
5 

 

Figure 2. Schematic of a continuous flow isotope-ratio mass spectrometer (CF-IRMS) (modified from Clark 

and Fritz, 1997).

expansion during freezing. The sample size required will depend on NO3

–

concentration. A minimum total 

amount of 0.002 milligram (mg) of NO3

– as nitrogen is required; therefore, the minimum sample 

concentration should be 0.03 milligram per liter (mg/L) as N. The submitter must provide the 

concentration of NO3

– in the sample to the RSIL before the sample can be analyzed. Obtaining National 

Water Quality Laboratory (NWQL) analysis LC 1975 or LC 1979 and reporting concentration to the 

RSIL is fully satisfactory. 

Sample Preparation and Time Requirements

Sample preparation involves logging samples into the LIMS-LSI (see appendix A). The logging-in 

procedure involves logging samples in batches to LIMS-LSI that could contain from 1 to 100 samples per 

batch (also called a project), printing labels for each sample, and printing a summary project report. Each 

sample label contains the Field ID (the identifier by which the sample submitter knows each sample) and 
6 

the unique RSIL Lab ID assigned by the LIMS-LSI. The summary project report is inserted into a threering binder titled “Samples in Progress.” 

A sample work-order sheet is created by LIMS-LSI by printing a “Template” and a “Samples to be 

Analyzed” form; step-by-step procedures are given in appendix B. The time requirement for sample 

preparation is about 1 to 5 days. 

Performing the Analysis and Time Requirements

A denitrifier culture is prepared by cultivating the appropriate strain of Pseudomonas aureofaciens

for δ

15N determination. Using tryptic soy broth (TSB) medium amended with 10-mM NO3

–

 and 15-mM 

NH4

+

, the bacteria are first inoculated in a starter tube that contains 5 mL of culture medium.The bacteria 

are allowed to grow overnight, and then transferred into autoclaved 580-mL serum bottles containing 

500 mL of medium. 

The bacteria (5- to 10-day-old cultures) are harvested by centrifugation in four 200-mL centrifuge 

bottles and resuspended with a total of 100 mL of new medium that contains no NO3

–

 to achieve a 5-fold 

increase in concentration. Several drops of antifoaming agent are added to the 100-mL concentrated cells. 

The 3-mL concentrated cells are dispensed into thirty-two 20-mL headspace vials. The vials are sealed 

with a Teflon-aluminum-lined silicone septum and purged with helium for 1 h. At this point, the TSB in 

the vials should be NO3

– and nitrite (NO2–

) free, and the headspace helium should be N2O free. 

An amount between 1 and 10 mL of a sample containing dissolved NO3

–

 is added to the prepared 

vials to achieve a constant amount of analyte N2

O, typically 10 to 20 nanomoles (nmol). For this reason, 

the sample submitter must provide the NO3

– concentrations of each sample. After the samples are injected, 

the sample vials are stored upside down for at least 2 h to minimize leakage through the septum while 

NO3

–

 is converted to N2O. The step-by-step procedure is given in appendix C.

After a 2-h conversion, the 32 vials that typically have 20 unknown samples and 12 references, 

among which are 4 vials of N-7 (USGS32; KNO3

), 4 vials of N-34 (USGS34; KNO3), and 4 vials of N-35 

(USGS35), are analyzed. N-7 and N-34 are used to calibrate δ

15N values. The sample vials are loaded into 

a 32-position aluminum tray that is accessed by the autosampler. The process is automated and controlled 

by the ISODAT 2.0 mass spectrometer program; therefore, it requires entering sample identifications into 

the “Sequence Table” in ISODAT 2.0, choosing appropriate “Methods” (the method that controls the 

CTC Analytical CombiPAL system and the method that controls the analysis, both of which were set up 

previously in the system), and starting sequence acquisition. The step-by-step procedure is given in 

appendix D.

For each sample analysis, the vial is purged by the autosampler for 13 min using helium gas at a flow 

rate of 30 mL/min. The flow from the exit needle is directed through a –65 O

C isopropanol-slush trap, a 

Nafion drier, followed by Mg(ClO4

)2 and Costech #021020 carbon dioxide absorbent to remove volatile 

organic vapor, water, and CO2

 from the gas stream before trapping N2O under liquid nitrogen. The N2O is 

then released from the liquid nitrogen trap and is passed through a PoraPLOT Q CP7551 GC column and 

on to the IRMS. 

After the analyses are completed, the data are exported from the ISODAT 2.0 database to a 

removable disc or “memory stick” (see appendix E) and imported to LIMS-LSI, and transferred to a Data 

Back-Up Computer. Final daily correction factors are determined (see appendix F) based on the daily 

analyses of reference materials using equations 4 and 5, which are represented with numerical values: 

+180 ‰ = m × δ

15NUSGS32/ workingrefgas + b (4)

–1.8 ‰ = m × δ

15NUSGS34/ workingrefgas + b (5)

The values +180 ‰ and –1.8 ‰ are the assigned values of the two international reference materials, 

USGS32 and USGS34, respectively. The δ

15N values in equations 4 and 5 are the mean daily delta values 

of the respective international reference materials relative to the working reference gas; b is the additive 
7 

correction factor, and m is the expansion coefficient correction factor. When two or more reference 

materials with different isotopic compositions are analyzed, the correction for sample blank does not need 

to be applied if the magnitude of the blank in the samples and reference materials is identical. However, if 

only a single reference material is analyzed, only one-point calibration is possible; therefore, a blank 

correction must be applied.

If replicates do not agree within acceptable tolerances, they are analyzed until acceptable statistics are 

achieved. The computerized LIMS-LSI will not release data until the statistics are acceptable. The time 

requirement for performing the analyses described earlier is a minimum of 2 days if the bacterial culture 

is already prepared.

The systematic procedure is listed in the “Lab Procedures” binder. The supervisor should review 

suggested changes before they are adopted. 

Problematic Samples

Problematic samples include (1) samples that have concentrations less than 14 μg of nitrogen per 

liter, (2) samples that have a mixture of NO3

– and NO2–

, because this technique does not distinguish 

between these two forms of nitrogen, and (3) samples that have been poisoned with mercury chloride or 

other preservatives. Samples that have anomalous n(

17O)/n(16O) ratios can be problematic for δ15N analysis 

because the δ

15N calculation assumes that the fractionation of oxygen isotopes in the NO3

– is mass 

dependent, that is, that δ

17O = 0.52 × δ18O. For samples containing atmospheric NO3

– that have excess 17O, 

the bacterial method may overestimate the δ

15N values by as much as 1 to 2 ‰. Therefore, for samples 

that are expected to contain more than 20-percent atmospheric NO3

–

, users should contact the RSIL for 

methods to quantify δ

17O values and determine accurate δ15N values. 

Interferences 

Because CO2 has the same m/z values as N2O, it poses an analytical interference if it is not completely 

removed from the sample stream. Ammonia (NH4

+

) and dissolved organic nitrogen do not cause 

interference. To ensure a sufficient separation of CO2 from N2

O, the original 25-m PoraPLOT Q CP7551 

GC column has been extended to 37.5 m in length. 

When analyzing KOH- or NaOH-preserved samples with a typical pH of about 11, if weaker or older 

bacteria are used, the N2

O yield and isotopic results can be affected because very basic sample solutions 

can kill the denitrifier bacteria, especially for samples containing very small amounts of nitrate. In such a 

case, several milliliters of raw sample are needed. 

When analyzing very dirty samples (microbial rich), such as landfill samples, sewage contaminated 

samples, etc, if the samples are only filtered by 0.45-μm filters, the small microbes (<0.45 μM) can 

compete with denitrifier bacteria. Consequently, unreliable N2

O yield and isotopic results can be 

observed. 

Troubleshooting and Bench Notes

Bacterial growth during the initial culture phase is monitored by the amount of NO3

– remaining in the 

medium solution. In a healthy culture, NO2

– is completely consumed in 5 to 7 days. If NO3– is not 

completely consumed in 7 days, the culture is discarded. 
8 

Maintenance and Maintenance Records

Stocks of bacteria are in a –80 °C freezer. New plates are streaked from that stock and transferred on 

agar plates. After 3 to 5 days, the second plates are streaked by individual colonies from the first plates. 

Likewise, the third-generation plates can be streaked from the second plates. The working cultures are 

inoculated only from the second and third-generation plates. The history of all working cultures is kept in 

a culture log, with the bacterial strain, date of inoculation, source of inoculum, and date of harvest. 

The isopropanol-slush trap is replaced every few weeks. The Nafion driers require replacement every 

8 to 10 months. The Mg(ClO4

)2 and Costech #021020 carbon dioxide absorbent is repacked every 1 to 2 

months. The GC is baked out (150 °C overnight) as needed. 

The IRMS requires a daily check (see appendix G). A logbook is kept for each IRMS with notes 

concerning maintenance checks, normal settings, problems, and repairs. 

Routine maintenance is necessary for the upkeep of the IRMS vacuum system, including 

(1) checking the pump performance from time to time, (2) changing the pump oil, and (3) changing the 

oil cartridge in each turbomolecular pump at 6-month intervals. Pump conditions, such as date of oil 

changes, problems, and repairs, are listed in the pump database (file path C:\RSIL\vacuum pumps). 

Maintenance of ISODAT 2.0 includes importing/transferring results from the ISODAT 2.0 database 

to the LIMS-LSI computer hard disk and the Data Back-Up computer hard disk (appendix E).

Sample Retention Time and Disposal 

Samples are retained in the RSIL for at least 4 months after reporting data. Samples are then 

discarded unless the submitter has requested that the samples be returned. 

The ISODAT sample files from the IRMS computer are kept indefinitely on two different hard disks 

of the Data Back-Up computer. Paper reports are kept for a few weeks. Analytical results from the IRMS 

are exported to LIMS-LSI and kept indefinitely. No data are entered by hand, and no manual calculations 

are performed on the data.

Data Acquisition, Processing, Evaluation, Quality Control, and Quality 

Assurance

Laboratory Information Management System for Light Stable Isotope (LIMS-LSI)

In the RSIL, the LIMS-LSI (Coplen, 2000) is used for data processing and evaluation. This system is 

a database program capable of (1) storing information about samples, (2) storing the results of mass 

spectrometric delta values of samples after importing from the ISODAT database, (3) calculating 

analytical results using standardized algorithms stored in a database, (4) normalizing delta values using 

isotopic reference materials, and (5) generating templates to facilitate loading of samples. With this 

system, the following are ensured: (1) quality assurance (QA), (2) laboratory efficiency, (3) reduction of 

workload and errors owing to the elimination of retyping of data by laboratory personnel, and (4) a 

decrease of errors in data reported to sample submitters. This database provides a complete record of 

information on how laboratory reference materials have been analyzed and provides a record of what 

correction factors have been used as an audit trail for the RSIL. 
9 

Quality Control (QC) Samples

Samples are prepared and analyzed in batches—a maximum of 32 daily. Each batch contains 

approximately 12 vials of 3 reference materials; one pair depleted in 15N and 18O and another pair enriched 

in 15N and 18O.All of these reference materials serve as QC samples. 

Daily, the analyst (1) examines the computer printouts for indications of analytical problems, 

(2) determines the daily additive and expansion correction factors by the LIMS-LSI, and (3) applies 

correction factors to isotopic data. The values of these correction factors along with the date and range of 

analysis numbers are recorded manually in the laboratory “GasBench Book” even though the data are 

already in the LIMS-LSI. After printing the list of isotopic results from the “Table of Samples in 

Progress,” the analyst reviews the results, determines which samples need to be analyzed a third time to 

achieve acceptable results (i.e., ∆δ ≤ 0.25 ‰ for δ

15N) and puts them back to the template. The final 

average isotopic values for each QC sample, with dates and analysis numbers, are recorded manually in 

the laboratory “GasBench book” even though the data are already in LIMS-LSI.

Acceptance Criteria for All QC Samples

Acceptance criteria for QC samples are the same as acceptance criteria for the samples. The rules are 

as follows: 

• If standard deviation is less than or equal to 0.25 ‰ for δ

15N, use mean delta.

• If there are three or more analyses, delete the outlier and recalculate.

• If standard deviation of this recalculation is less than or equal to 0.25 ‰ for δ

15N, use the mean from 

this recalculation. 

• If none of the above is the case, the result is not acceptable and corrective action is required. 

The RSIL estimates the expanded uncertainty (U = 2μc

) of δ

15N measurement results. The expanded 

uncertainty provides an envelope that represents a 95-percent probability of encompassing the true value 

that has been determined from the aggregation of measurement results over a period of time. The 

expanded uncertainty can be determined using the guide to the expression of uncertainty (Joint 

Committee for Guides in Metrology, Working Group 1 (JCGM/WG1), 2010). The application of 

expanded uncertainty to the reporting of stable isotope measurements is discussed by Coplen and others 

(2006). The estimated expanded uncertainty of δ

15N measurement results for samples with nitrate 

concentrations of at least 0.06 mg/L as nitrogen is ±0.5 ‰ unless otherwise specified, and this value is 

conservative. If any given sample were resubmitted to the RSIL for nitrogen isotopic analysis, the 

measured values would fall within the uncertainty bounds of the previous results more than 95 percent of 

the time. The δ

15N uncertainty for nitrate samples with concentrations less than 0.06 milligrams per liter 

as nitrogen is ±1.0 ‰ unless otherwise specified. 

Corrective Action Requirements

If an analyst finds any problem with the daily reference-sample data, the analyst contacts the 

supervisor. The trouble-shooting process will require an evaluation and reanalysis of certain samples to 

identify the origin of the problem. 

If samples do not give satisfactory results after three or more separate analyses, the analyst averages 

all the data and reports the mean value. Such analytical results are indicated with a comment, and the 

customer will be advised by e-mail or other means. 
10

Responsible Parties for All QA/QC Functions for Procedures Covered in RSIL SOPs 

The analyst, with supervisor approval, is responsible for qualifying data and notifying customers. 

Data Management and Records

In addition to evaluating daily sample analyses, every week, an analyst evaluates the data project-byproject, reports results to the customers, and files final project data reports in the laboratory 

“Correspondence” binder (appendix H). 

Health, Safety, and Waste-Disposal Information

Applicable Health and (or) Safety Issues

Personal Protection 

Safety glasses and protective gloves are recommended whenever samples are handled, especially 

when the samples are of biological origin. For other precautions and safety procedures, consult the 

Material Safety Data Sheets (MSDS); they are on file in the laboratory and at the URL 

http://www.ilpi.com/msds/#Manufacturers. This URL provides links to the MSDSs of most chemical 

companies. 

Electrical Hazards 

Electrical systems must conform to the National Electric Code, National Fire Protection Association 

Code (NFPA 70–1971), and the American National Standards Institute (ANSI) Code (C1–1971). For 

more information, consult the U.S. Geological Survey’s Safety and Environmental Health Handbook 

(U.S. Geological Survey, 2002, sec. 4–4.1).

Shock hazards exist inside the instruments. Only an authorized service representative or an individual 

with training in electronic repair must remove panels or circuit boards where voltages are greater than 

20 volts. The instruments require a third-wire protective grounding conductor. Three-to-two wire adapters 

are unsafe for these instruments. 

Chemical Hazards 

Use protective gloves to handle all chemicals.

Gas Cylinder Handling 

Compressed gas cylinders must be handled and stored according to the U.S. Geological Survey’s

Safety and Environmental Health Handbook (U.S. Geological Survey, 2002, sec. 4–4.5.1). Each cylinder 

must be (1) carefully inspected when received; (2) securely fastened at all times with an approved chain 

assembly or belt; (3) capped at all times when not in use; (4) capped when transported; (5) transported 

only by a properly designed vehicle (hand truck); and (6) stored separately with other full, empty, 

flammable, or oxidizing tanks of gas, as appropriate. 
11

Biological Hazards 

The bacteria used in this method are not a significant health hazard. However, infection may occur if 

bacterial culture comes in contact with open wounds or if needles that have been used with the bacteria 

puncture the skin. If contact with bacterial culture occurs, wash the affected area thoroughly with water 

and use an alcohol swab (70-percent EtOH) to cleanse the affected area. The used or old bacterial cultures 

should be killed by autoclaving at 120 O

C for 30 min before sink disposal. 

Needles that have been used with the bacteria should be placed in a “Sharps” container, sealed, and 

disposed of when the container is full. The container should be labeled “biological hazard” and disposed 

of in the same manner in which hazardous chemicals are disposed. 

Specific Waste-Disposal Requirements

The used or old bacterial culture should be killed by autoclaving at 120 OC for 30 min before disposal 

in the sink. 
12

Revision History

Publication Series and Series Number: Techniques and Methods 10–C16 (Book 10, Section C, 

Chapter 16)

Publication Title: Determination of the δ

15N of Nitrate in Water; RSIL Lab Code 2899 

Publication Authorship: Coplen, Tyler B., Qi, Haiping, Révész, Kinga, Casciotti, Karen, and Hannon, 

Janet E. 

Version 1.0, released 2007 

Version 1.1, released 2012 

Summary of Revised Product Components 

Component Description 

Last Revised 

In Publication Version 

Date of 

Last Revision 

Title Replaced δ(

15N/14N) by δ15N to be in 

accordance with recommendations of the 

Commission on Isotopic Abundances and 

Atomic Weights of the International Union 

of Pure and Applied Chemistry. 

1.1 July 2012 

Authorship Tyler B. Coplen, and Haiping Qi, and Janet 

E. Hannon added as coauthors. 

1.1 July 2012 

Summary of 

procedure 

Updated text to reflect improved method of 

Coplen and others (2004) and new 

instrumentation.

1.1 July 2012 

Reporting units 

and operational 

range 

Updated text to reflect expression of delta 

values with number ratios. 

1.1 July 2012 

Reference 

materials and 

documentation 

Updated text to reflect improved method of 

Coplen and others (2004). 

1.1 July 2012 

Labware, 

instrumentation, 

and reagents 

Updated text to reflect improved method of 

Coplen and others (2004) and new 

instrumentation.

1.1 July 2012 

Sample 

collection, 

preparation, 

analysis, 

retention times, 

and disposal 

Updated text to reflect improved method of 

Coplen and others (2004) and to reflect that 

samples are no longer submitted through 

the National Water Quality Laboratory 

(NWQL). 

1.1 July 2012 
13

Data acquisition, 

processing, 

evaluation, 

quality control, 

and quality 

assurance

Updated text to reflect improved method of 

Coplen and others (2004) and to reflect that 

samples are no longer submitted through 

the National Water Quality Laboratory 

(NWQL). 

1.1 July 2012 

Health, safety, 

and wastedisposal 

information

Updated text to reflect improved method of 

Coplen and others (2004). 1.1 July 2012 

References cited Added four references. 1.1 July 2012 

Appendix A Updated text to reflect that samples are no 

longer submitted through the National 

Water Quality Laboratory (NWQL). 

1.1 July 2012 

Appendix B Updated text to reflect improved method of 

Coplen and others (2004). 

1.1 July 2012 

Appendix C Updated text to reflect improved method of 

Coplen and others (2004). 

1.1 July 2012 

Appendix H Updated text to reflect that samples are no 

longer submitted through the National 

Water Quality Laboratory (NWQL). 

1.1 July 2012 

Main text footer Removed from document. 1.1 July 2012 
14

References Cited

Böhlke, J.K., Gwinn, C.J., and Coplen, T.B., 1993, New reference materials for nitrogen-isotope-ratio 

measurements: Geostandards Newsletter, v. 17, p. 159–164. 

Böhlke, J.K., and Coplen, T.B., 1995, Interlaboratory comparison of reference materials for nitrogenisotope-ratio measurements, in Reference and Intercomparison Materials for Stable Isotopes of Light 

Elements: Vienna, Austria, International Atomic Energy Agency, IAEA–TECDOC–825, p. 51–66. 

Böhlke, J.K., Mroczkowski, S.J., and Coplen, T.B., 2003, Oxygen isotopes in nitrate: New reference 

materials for18O:17O:16O measurements and observations on nitrate-water equilibration: Rapid 

Communications in Mass Spectrometry, v. 17, p. 1835–1846. 

Brenna, J.T., Corso, T.N., Tobias, H.J., and Caimi, R.J., 1997, High-precision continuous-flow isotoperatio mass spectrometry: Mass Spectrometry Reviews, v. 16, p. 227–258. 

Casciotti, K.L., Sigman, D.M., Hastings, M., Böhlke, J.K., and Hilkert, Andreas, 2002, Measurement of 

the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method: 

Analytical Chemistry, v. 74, p. 4905–4912. 

Clark, Ian, and Fritz, Peter, 1997, Environmental isotopes in hydrogeology: Boca Raton, Fla., Lewis 

Publishers, 328 p. 

Coplen, T.B., 2000, A guide for the laboratory information management system (LIMS) for light stable 

isotopes—Version 7 and 8: U.S. Geological Survey Open-File Report 00–345, 121 p., accessed April 

27, 2012, at http://water.usgs.gov/software/code/geochemical/lims/doc/ofr00345.pdf. 

Coplen, T.B., 2011, Guidelines and recommended terms for expression of stable-isotope-ratio and gasratio measurement results: Rapid Communications in Mass Spectrometry, v. 25, p. 2538–2560, 

accessed April 27, 2012, at http://onlinelibrary.wiley.com/doi/10.1002/rcm.5129/abstract. 

Coplen, T.B., Böhlke, J.K., and Casciotti, K.L., 2004, Using dual-bacterial denitrification to improve δ

15N 

determinations of nitrates containing mass-independent 17O: Rapid Communications in Mass 

Spectrometry, v. 18, p. 245–250. 

Coplen, T.B., Brand, W.A., Gehre, Matthias, Gröning, Manfred, Meijer, H.A.J., Toman, Blaza, and 

Verkouteren, R.M., 2006, New guidelines for δ

13C measurements: Analytical Chemistry, v. 78, no. 7, 

p. 2439–2441. 

Joint Committee for Guides in Metrology, Working Group 1 (JCGM/WG1), 2010, Evaluation of 

measurement data—Guide to the expression of uncertainty in measurement (known as the GUM) 

(2010 corrected version of the first edition of 2008): Joint Committee for Guides in Metrology 

[publication] JCGM 100:2008, 120 p., accessed July 16, 2012, at 

http://www.bipm.org/en/publications/guides/.

Mariotti, A., 1983, Atmospheric nitrogen is a reliable standard for natural 15N abundance measurements: 

Nature, v. 303, p. 685–687. 

Sigman, D.M., Casciotti, K.L., Andreani, M., Barford, C., Galanter, M., and Böhlke, J.K., 2001, A 

bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater: Analytical 

Chemistry, v. 73, p. 4145–4153. 

U.S. Geological Survey, 2002, USGS handbook 445–3–H, Safety and environmental health handbook, 

435 p. 
15

Appendix A. Step-by-Step Procedure to Log-In Samples to LIMS-LSI 

1. For sample submitter:

a. Download “Standard Submission Excel Form” or the “QWDATA Compatible Submission Excel 

Form” from the RSIL Web site at http://isotopes.usgs.gov/. 

b. Fill out the requested sample information.

c. Send a diskette or CD and a hard copy along with the samples or e-mail the completed RSIL 

Excel worksheet to isotopes@usgs.gov. 

2. For RSIL personnel: 

a. Match up information on sample bottles with the submitted “Standard Submission Excel Form”

or the “QWDATA Compatible Submission Excel Form.” 

b. Enter all Standard Submission Excel Form or “QWDATA Compatible Submission Excel Form” 

information into LIMS-LSI by loading media. Submission date is the date samples are logged in. 

c. Use “Import a New Project From Excel Submission” in LIMS-LSI to assign G#s. 

d. Print out one project report and container labels (one for each sample). 

e. Put a label on each sample bottle and cross-check Field IDs between bottles and Excel worksheet 

forms.

f. Punch holes in the original Excel worksheet and all the project information you have and put in 

“Samples-in-Progress” binder.
16

Appendix B. Step-by-Step Procedure to Print a Template and a Samplesto-Be-Analyzed List 

Template 

1. Use “Create a Sample List -->” in LIMS-LSI (fig. B–1). 

2. Select appropriate template and mass spectrometer for GasBench, aureofaciens, and N&O.

3. Design analysis sequence with 32 ports. 

4. Click “Create Sample List for Mass Spec.” 

5. Add any sample information you want in column “Sample/Extraction Info.” 

6. Click “Save and Print.”

7. Insert diskette to receive sample headings.

8. Click “OK.”

9. Exit LIMS-LSI. 

10. Write sample amount (mL) needed to be loaded into each vial and write the date that samples should 

be analyzed on the paper template.

 

 

 

Figure B–1. Sample list generated by LIMS. 
17

Samples to be Analyzed List 10/20/2003 3:54:33 PM

[N-7, N-34, and N-35 are reference materials] 

Port Sample # Amount 

1 N-7 

2 N-34

3 G-70

4 G-71

5 G-72

6 G-73

7 G-74

8 G-75

9 N-34

10 N-7 

11 G-76

12 G-77

13 G-78

14 G-79

15 G-80

16 G-81

17 G-82

18 G-83

19 G-84

20 N-34 

21 G-85

22 G-86

23 G-87

24 G-88

25 G-89

26 G-90

27 G-91

28 G-92

29 G-93

30 G-94

31 N-34 

32 N-7 
18

Appendix C. Step-by-Step Preparation Procedure for Nitrogen Isotopic

Analysis of Nitrate by Bacterial Denitrification

Making the Plates

1. Preparing the plates: 

a. Mix together:

i. 23 grams (g) of tryptic soy agar (TSA). 

ii. 0.5 g of KNO3

. 

iii. 500 mL of E-pure or Milli-Q water. 

b. Autoclave for 30 min at 120 O

C. 

c. Cool ~15 min (until you can handle the flask with a gloved hand). 

d. Pour into sterile plastic Petri dishes, flaming the lip of the flask every 3 to 4 plates (500 mL of 

solution should make ~20 plates).

e. Allow the plates to solidify overnight. 

f. Place all plates in a plastic bag. Label the bag with a piece of tape; include type of plates (TSA, 

plus NO3

–

), today’s date, and initials of technician who made the plates. 

g. Store plates in the refrigerator until use.

2. Streaking the bacteria: 

a. Remove the small box that contains the small tube of frozen stocks of Pseudomonas aureofaciens

American, Type Culture Collection (ATCC)13985 from the –80 °C freezer. 

b. Take bacteria from the frozen stock: Do not wait for the vial to thaw—use a sterile toothpick to 

scrape a very small amount of bacterial slush from the tube. CLOSE the tube before doing

anything else. The frozen stocks are our most important resource. 

c. Lightly touch the surface of a plate with the toothpick to transfer the bacterial slush. It should 

form a small puddle. 

d. Using a flamed loop, spread the puddle by streaking back and forth through it. Flame the loop. 

Proceed with streaking by crossing trails twice with tracks of the first streak. Flame the loop. 

Cross trails twice with the second streak and proceed. This plate will be numbered as #1.

e. To streak new plates from earlier plates: Using a single colony from #1plate, streak, flame, streak, 

flame, streak (as above from frozen stocks). This plate will be numbered as #2.

f. Plate labeling: 

i. Strain (Pseudomonas aureofaciens). 

ii. Plate number. 

iii. Today’s date. 

iv. Source information in parentheses (“freezer” or date of earlier plate).

g. Plates take 3 to 5 days to mature so one will typically go through a round each week. 

h. Keep plate lineages together for ease of organization. Discard old plate lineages when a new #2 plate 

has matured and is ready to be used for inoculating. If streaking #3s also streak new #1s on the same 

day. 

Preparing the Tryptic Soy Broth (TSB)

1. Wash the 580-mL medium bottles (580-mL bottle with vial neck (23 × 75.5-mm diameter and height, 

20-mL volume)): 

a. Brush and wash inside with Liqui-Nox detergent. 

b. Rinse with warm water and deionized water (DIW). 

c. Bake in 200 °C oven overnight. 
19

2. Making the medium: (making 7000 mL of medium and distributing it into fourteen 580-mL serum 

bottles): 

a. Mix together (fig. C–1): 

i. 210 g of DIFCO Tryptic soy broth (TSB). 

ii. 7.0 g of KNO3

. 

iii. 3.5 g of (NH4

)2SO4. 

iv. 34.3 g of potassium phosphate. 

b. Add 7000 mL of DIW.

c. Transfer the well-homogenized, 500 mL of medium into a 580-mL bottle by peristaltic pump. 

d. Seal bottles with blue butyl stoppers and aluminum crimp-seals.

e. Autoclave for 60 min; the time starts after the autoclave reached 120 °C (figs. C–2 and C–3). 

3. Making the NO3

–

-free medium: (making 2000 mL of medium and transferring it into twenty 100-mL 

serum bottles):

a. Mix together:

i. 30 g of Tryptic soy broth. 

ii. 0.5 g of (NH4

)2SO4. 

iii. 4.9 g of KH2

PO4. 

b. Add 2000 mL of DIW.

c. Transfer the well-homogenized, 100-mL volume of NO3

–

-free medium into 160-mL serum bottles 

by peristaltic pump. 

d. Autoclave for 60 min; the time starts after the autoclave reaches 120 °C (fig. C–4). 

4. Incubating the bacteria: 

a. Measure 5 mL of autoclaved medium into a sterile plastic test tube.

b. Using a well-flamed loop, pick a colony from a #1 or #2 plate (figs. C–5 and C–6).

c. Open the tube and touch the end of the loop to the inside of the tube or swish the end of the loop 

in the broth to transfer the colony. Whichever you do, do it quickly. Get in and out of the bottle as 

quickly as possible (fig. C–7). 

d. Label tube with the following information: 

i. Bacteria strain. 

ii. Today’s date. 

iii. Source of the inoculum (plate number and date). 

e. Incubate the tube on a shaker overnight (fig. C–8). This is good for 1 or 2 days. 

f. Add 5 mL of starting solution to each 580-mL culture bottle and enter each inoculated bottle in 

the Culture Log, one line per bottle. 

g. Put the bottles on the shaker, and grow the bacteria for a week.

5. Generally, inoculations will be performed as often as needed to supply a sufficient supply for the 

following week. These cultures will grow for a week before they are harvested. 

Testing Bacteria for Incomplete Conversion of Nitrate 

1. Withdraw 1 mL of solution from each 580-mL culture to be harvested.

2. Add 40 μL of sulfanilamide to convert NO3

–

to NO2. 

3. Add 40 μL of N-(1-naphthyl) ethylenediamine dihydrochloride (NED), which turns to pink if any 

NO2

-

is present in the solution.

4. If it turns pink, it indicates that that the bacteria has not quantitatively converted NO3

– to N2

O, and the 

bottle should not be used until further testing shows no pink coloring. A cloudy culture should be 

visible after 5 days. When consumption of the NO3

– is complete, label the bottle as ready for use (fig. 

C–9). 
20

Harvesting the Bacteria—Concentrating Culture by Centrifugation

1. Evenly distribute 500 mL of culture into four 250-mL centrifuge bottles by weighing. 

2. Screw caps on tightly and put the bottles into centrifuge, making sure they balance each other

(fig. C–10). 

3. Centrifuge the bottles for 15 min at 18 °C and 3800 rpm. (Bacteria should be pelleted on the bottom 

of the bottle).

4. Discard clear medium supernatant, taking care not to disturb the ring of bacteria. 

5. Add 15 mL from a 100-mL, NO3

–

-free medium unit to each centrifuge bottle.

6. Swirl bottles to re-suspend the bacteria and transfer to a sterile 250-mL beaker.

7. Use remaining NO3

–

-free medium for rinsing all four centrifuge bottles, adding the rinse to the 

250-mL beaker. 

8. Add 10 drops antifoam B solution to the beaker. 

Adding Concentrated Cell Culture to Sample Vial and Purging Headspace and Adding Samples

1. Dispense 3 mL of concentrated cell culture into each sample vial (fig. C–11). 

2. Using a mechanical crimper (fig. C–12), seal vials with Teflon-aluminum-lined silicone septum 

inserted into aluminum crimps (fig. C–13). 

3. Insert a helium vent needle (1.5-in. long, 25 gauge) into vial (fig. C–14). 

4. Insert each vial upside down into helium purging manifold. 

5. Purge vials with 3 to 4 psi helium for 1 hour (h) (fig. C–15). 

6. Inject a sufficient amount of sample water to produce 5 to 20 nmol N2

O (1 to 10 mL) with an airtight 

syringe equipped with a 25-gauge needle (fig. C–16). If the amount of water needed is more than 

4 mL, insert a venting needle while injecting to avoid over pressurization. Mix well by inverting the 

vial several times.

7. Turn the vial upside down (fig. C–17) and incubate for 2 h. 

8. Samples now are ready to be loaded on the Finnigan MAT GasBench 2 (fig. C–18) for isotope 

analysis. Loaded samples are shown in fig. C–19. 
21

 

Figure C–1. Tryptic soy broth (TSB), with KNO3, prepared, mixed in 9-L bottle (yellow liquid), and divided into 500-g 

aliquots. 

Figure C–2. Autoclaved for 60 minutes at 120 oC. 
22

 

Figure C–3. Top view of 580-mL bottles with media ready to be autoclaved.

 

Figure C–4. Tryptic Soy broth (TSB) without KNO3, prepared with 100-mL aliquots in 125-mL serum bottles. 
23

 

Figure C–5. Selecting a separate colony from the plate using a sterile flamed loop.

 

 

Figure C–6. Selected colony is shown. 

 

 
24

 

Figure C–7. Selected colony transferred to labeled tube containing 5 mL of TSB.

 

 

Figure C–8. Shaker showing 5 mL of culture before injection and showing previously prepared 580-mL bottles injected with 

bacteria.
25

 

Figure C–9. A bottle of nitrate-free bacterial culture ready for use.

 

Figure C–10. Centrifuge with four 250-mL bottles. 
26

 

Figure C–11. Twenty-milliliter sample vial containing 3 mL of concentrated cell culture.

 

 

 

Figure C–12. Mechanical crimper. 
27

 

Figure C–13. PTFE/Aluminum-lined septa with aluminum crimps. 

 

 

 

Figure C–14. Empty vial showing placement of vent needle. 
28

 

Figure C–15. Manifold showing helium purging of 32 vials.

 

Figure C–16. Loading vial with sample. 
29

 

Figure C–17. Aluminum tray with inverted sample vials. 

 

 

Figure C–18. Finnigan MAT GasBench 2.
30

 

Figure C–19. Sample tray with 32 vials and a 1⁄4-inch-thick aluminum plate to secure vials. 
31

Appendix D. Step-by-Step Procedure to Add Sample Information to 

Sequence Table

1. Insert the diskette with sample headings generated by LIMS-LSI.

2. Open sequence table in Excel. 

3. Open “N2

O Sample Sequence File” under the GB configuration. 

4. Copy the sequence template from Excel and paste it to sequence table of the mass spectrometer’s data 

acquisition and control computer. 

5. Select the appropriate “method” for each sample. 

6. Define a reference line and blank line (as needed).

7. Start.

8. Give Folder Name: Comment.

a. Select pre: “Date.”

9. Give File Name: leave it empty. 

a. Select pre: “Analyzes #.”

b. Select post: “Identifier 1.”

c. Select: “Print Result.”

10. Click OK.

11. Wait for first sample to be analyzed.
32

Appendix E. Step-by-Step Procedure to Transfer Data to LIMS-LSI, to 

Transfer Data to Back-Up Computer, and to Reevaluate Old Data

1. Transfer data to LIMS-LSI:

a. Start LIMS.

b. Choose “Import Data From Instruments.”

c. Choose mass spectrometer, for example “H --> Hercules.” 

d. Click “Import.”

e. Select the file that will be imported.

f. Depending upon the mass spectrometer, the user may be given the opportunity to select the 

columns containing the isotope data. Enter column names as appropriate for the isotope or 

isotopes to be imported. 

g. Click “Import.” Note: Sample ID, sample weight, Peak Area, Analyses #s are automatically 

imported to LIMS-LSI. 

2. Transfer data to Data Back-Up computer: 

a. Go to Windows Explorer in the Data Back-Up computer.

b. Find the drive where your back ups are stored. 

c. Choose: “RSIL.” 

d. Choose: “Mass. Spec. Analysis Back Up.”

e. Choose the mass spectrometer. For example, “H,” which stands for “Hercules,” a Thermo 

Scientific Delta V Plus IRMS. 

f. Under that folder create a new folder. The name of the folder should be the date range of the 

analyses that one wishes to back up. 

g. Transfer data to that folder. 

h. Make back ups every 2 weeks or so, as needed. 

3. Reevaluate old data: 

a. Find your samples in the Data Back-Up computer.

b. Transfer data by a memory stick to the computer where a virtual version of ISODAT 2.0 is 

installed.

c. Reevaluate your data.
33

Appendix F. Step-by-Step Procedure to Determine and Apply Correction 

Factors and Evaluate Data

1. Open “Apply Data Normalization” in LIMS-LSI.

2. Select instrument and isotope, for example “H (Hercules) for 34S.” 

3. Select “Query.”

4. Double click on the last sample analyzed on that day.

5. Evaluate data of the reference materials.

6. Choose “Normalize with all References.”

7. Click “Apply Normalization -->.” 

8. Print out correction factor sheet.

9. Report daily reference values and correction factors along with date and range of analysis number to 

the “EA” binder. 

10. Go back to LIMS-LSI main menu by closing open windows. 

11. Choose “Print/Save Analyses in Progress”: 

a. Choose appropriate isotope. 

b. Choose appropriate prefix (W for water, N for nitrogen, S for sulfur). 

c. Enter sample ID range from “Samples-to-Be-Analyzed” sheet.

d. Click Print.

12. Review results and identify samples that need to be re-analyzed. 

13. Add the samples needing analyses to the Samples to Be Analyzed: 

a. Go back to LIMS-LSI main menu.

b. Open “Create a Sample List -->.” 

c. Select appropriate sample list for the mass spectrometer and the samples that are to be analyzed.

d. Find each sample in the “List of samples.”

e. Change entry in “Repeats” column from 0 to 1. 

f. Close Windows and exit LIMS-LSI.
34

Appendix G. Daily Check List

Analyst in charge: ______________________Date: ______________________ 

1. Change the working reference gas tank and helium gas tank if the pressure is <500 psi. Order new 

ones for spares; the reference gas is research grade, helium; the carrier gas is zero grade.

2. Change the Nafion every 10 months, and change the water and CO2

 trap every 1 to 2 months. 

3. Replace isopropanol-slush trap daily. Clean the used trap with ethanol and DIW, and dry it in a 

150 O

C oven. 

4. Clean the sample-transfer line (1/32-inch diameter stainless steel tubing) between the needle and the 

isopropanol-slush trap every 6 months by injecting ethanol and DIW. 

THESE ITEMS ARE TO BE CHECKED OFF AS YOU CHECK THEM DAILY!

1. Check the helium and reference gas flow. [ ]

2. Check the background masses. [ ]

Cups Mass Intensity (V) Mass Intensity (V) Resistor (Ω) Capacity (pF) 

1 28 0.007 0 3 × 108 680

2 29 0.003 18 1.256 3 x 1010 5 

3 30 0.083 18 6.818 3 × 1011 2 

3. Check the peak center. [ ] 

4. Analyze the Ref on/off method 10 times to stabilize the IRMS (STD should be < 0.1 ‰). [ ]

ConFlo II Pressure Setting 

N2O (bar) 

N2O signal (V) Mass 45 on 

Cup 2 

0.25 0.638

0.50 1.59

0.75 Optimal setting 2.653

1.00 3.921

1.25 5.315

1.50 7.059

1.75 8.778

2.00 >10.8

5. Record the analysis date, the project, the sample submitter, and the mass spectrometer analysis 

number in the electronic lab-log book (in xls format). Also record the instrument working status. 
35

Appendix H. Step-by-Step Procedure to Report Data

1. Open “Store Samples in Progress” in LIMS-LSI.

2. Choose the appropriate isotope. 

3. Choose sample ID range from “Sample in Progress” print out.

4. Store data.

5. Go back to the main menu of LIMS-LSI.

6. Open “Project” and find the appropriate project in the list. 

7. Select “Print Report” and check whether the project report contains all the results. If not, search for 

the missing results in the database. 

8. Select “Results,” transfer data in Excel format or (and) text format to a diskette, and report data to 

customer through e-mail.

9. Click “Print Report” to print a project report and put it in the “Correspondence” binder along with all 

the other documents in the “Samples In Progress” binder that are related to this project.