NSDB - Sampling - Analysis - Manual (Soil Sampling and Analysis Procedures Used For NSDB)

Soil Sampling and Analysis Procedures Used for
the National Soil Database (NSDB)

Gaelene Kramers
1
, Deirdre Fay
1
, David McGrath
1
, Chaosheng Zhang
2
,
Cora Carrigg
3
, Vincent OFlaherty
3
, Owen T. Carton
1
, Eamonn Grennan
4

1
Teagasc, Environmental Research Centre, J ohnstown Castle, Co. Wexford;
2
Department of Geography, National University of Ireland, Galway;
3
Department of
Microbiology and Environmental Change Institute, National University of Ireland,
Galway;
4
Institute of Technology, Sligo

PREFACE.................................................................................................................................... 3
1. SAMPLING PROCEDURE IN THE FIELD....................................................................... 1
1.1 LOCATION OF SAMPLING POINTS......................................................................................... 1
1.2 SOIL SAMPLING PROCEDURE................................................................................................ 1
1.3 MICROBIOLOGICAL SAMPLING PROCEDURE ...................................................................... 2
1.4 ARCHIVING OF SAMPLES....................................................................................................... 2
2. SAMPLE PREPARATION AND ANALYSIS..................................................................... 3
2.1. CHEMICAL ANALYSIS ........................................................................................................... 3
2.1.1 PREPARATION OF SOIL SAMPLES AFTER ARRIVAL .................................................................... 3
2.1.2 ANALYSIS OF SOIL SAMPLES FOR ACIDITY (PH) ....................................................................... 3
2.1.3 ANALYSIS OF SOIL SAMPLES FOR AVAILABLE ELEMENTS .......................................................... 3
2.1.4 ANALYSIS OF SOIL SAMPLES FOR SOIL ORGANIC CARBON AND TOTAL N................................... 4
2.1.5 ANALYSIS OF SOIL SAMPLES FOR ALL MEASURED ELEMENTS ................................................... 5
2.2 MICROBIOLOGICAL ANALYSIS ............................................................................................. 6
2.2.1 SAMPLING AND SAMPLE PRESERVATION ................................................................................. 6
2.2.2 CELL COUNTS AND DETERMINATION OF LYSIS EFFICIENCY ..................................................... 6
2.2.3 DNA EXTRACTION METHODS ................................................................................................. 6
2.2.4 QUANTIFICATION OF SOIL DNA YIELD................................................................................... 8
2.2.5 NUCLEIC ACID PURIFICATION................................................................................................ 8
2.2.6 DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)..................................................... 8
2.2.7 SEQUENCING AND PHYLOGENETIC ANALYSIS OF DGGE BANDS.............................................. 9
REFERENCES.......................................................................................................................... 10
APPENDIX 1 ............................................................................................................................. 11
PROTOCOL FOLLOWED BY THE SAMPLERS FOR THE NSDB ........................................................ 11
APPENDIX 2 ............................................................................................................................. 14
FIELD SHEET FOR THE NSDB...................................................................................................... 14

2
Preface

The National Soil Database (NSDB) project, jointly funded by the Environmental Protection
Agency and Teagasc, has produced for the first time a national baseline database of soil
geochemistry that includes data-point and spatial-distribution maps of major nutrients, major
elements, essential trace elements, trace elements of special interest and minor elements. In
addition, this study has generated a National Soil Archive, comprising both dried soil samples
and a nucleic-acids (DNA) archive in addition to sampling and location information for each
sampling point.

The report and archive will provide Ireland with a sound, well-structured baseline of soil
geochemical properties relevant to environmental, agronomic and health-related pressures and
against a background of increasing soil protection policies.

The NSDB has generated baseline soil geochemical maps (point and spatial distribution) of
Ireland, and has begun an interpretation of these in a pedological context. This study also
applied large-scale microbiological analysis of soils for the first time in Ireland and in doing so
also investigated microbial community structure in a range of soil types.

This report describes the soil sampling and analysis procedures used for the NSDB.

Soil Sampling and Analysis Procedures Used for the National Soil Database (NSDB)
1
1. Sampling Procedure in the Field
1.1 Location of Sampling Points
A total of 1365 sampling sites were predetermined as fixed locations on the National Grid.
During 1995 and 1996, 295 soil samples were collected in the south-eastern region of Ireland,
while an additional 1015 soil samples were collected when the study was extended to include
the remaining three-quarters of the country in 2002.

Two samples were taken from each 10 x 10 km segment of the National Grid. One location
corresponded to each intersection and one to each centre point. In order to avoid the problem
presented by map corners, each location was moved exactly 1 km to the north and 1 km to the
east of the defined position. In the event of it not being possible to take a sample at the
sampling position, a default procedure similar to that used in the Geochemical Survey of
England and Wales (McGrath and Loveland, 1992) was used. Sampling was next attempted at
a point 50 m north, then east, south and west in turn with the procedure being repeated at a
distance of 100 m, 200 m and 400 m until successful or until a distance of 800 m from the
original point was reached. Sites were located using 1:50,000 maps assisted by a global
positioning system (GPS).

1.2 Soil Sampling Procedure
At sampling sites, 25 soil cores were taken to a depth of 10 cm at 5 m intervals on a grid
measuring 20 m x 20 m and with the centre point of the grid at the sample location. This mini-
grid approach was chosen in order to minimise the variation at each site. A Dutch auger (5 cm
diameter; Eijkelkamp, The Netherlands) was used. Cores were combined and the resulting
composite sample weighed approximately 24 kg.

All field data were recorded by samplers on field sheets (see Appendix 2, Field Sheet for
Samplers). A protocol was written up to be followed by all samplers (see Appendix 1).
Essentially, the sampler on site determined where the sample was taken. Reasons for any
deviation from the sampling protocol were reported on the field sheet. The sample sheet also
included a section for land use which was filled in by the sampler. The land-use categories
comprised grassland, tillage, forestry, and peatland types. A number of land-use categories
were described as tillage, but the crop type was not recorded. All sheets were inspected on
receipt at the laboratory and data incorporated into a Microsoft Excel database.

The samples were placed in clear polythene bags, which were labelled clearly and delivered to
Teagasc, Johnstown Castle, Wexford within one week. Inspection of field sheets, Ordnance
Survey maps, and soil samples, at Teagasc indicated no lack of correspondence between what
might have been expected for soil and accompanying land information on the one hand and

field sheets and soil samples received on the other. Occasionally, clarification of recorded data
was sought.

1.3 Microbiological Sampling Procedure
The procedure followed for obtaining sub-samples for microbiological analyses is detailed in
Appendix 1. Briefly, samples for microbiological analyses were obtained as sub-samples from
the composite sample described in Section 2.1.1. As far as was physically possible the
composite sample was mixed thoroughly in a large plastic bag. Sub-samples, numbering 25
(approximately 1 g each), were then taken using a spatula. In incidences where it was not
possible to thoroughly mix the composite sample, sub-samples were taken from different parts
of the bag. Sub-samples were subsequently transferred to a vial containing 50 ml stabilisation
buffer (cetyltrimethylammonium bromide [CTAB] extraction buffer [Griffiths et al., 2000] + 2.5 M
guanidine isothiocyanate), ensuring that the soil was submerged in the buffer solution. After
transfer the vial was stoppered and shaken gently. Vials, numbered by site number, were stored
in a cool place and delivered to National University of Ireland (NUI), Galway within three to four
days. Upon receipt at NUI, Galway the soil samples were processed using the protocols
described in Section 2.2.

A total of 1310 authenticated soil samples were received at Teagasc, Johnstown Castle and
1005 soil samples were received by NUI, Galway. The difference in the number of samples
received by Teagasc and NUI, Galway is mainly accounted for by the 295 samples that were
collected as part of the south-eastern 1995/1996 study. The remaining 10 can be attributed to
the inherent difficulties often encountered in such large-scale sampling campaigns.

1.4 Archiving of Samples
A total of 1310 soils have been stored in 1-l glass jars at Teagasc, Johnstown Castle, while 100-
ml sub-samples have also been stored in small plastic containers. The soils have been labelled
clearly and are linked to the National Soil Database via unique identification codes. Ordnance
Survey maps and the original field sheets have been stored in addition to the standard
operating procedures for chemical and microbiological analyses undertaken.

A nucleic acids archive, comprising 1005 soil sub-samples, has been generated at NUI,
Galway. All soil samples were processed using the nucleic acids extraction protocol developed
by the project team (see Section 2.2.5). The nucleic acids are stored in 96-well micro-titre plates
at 80C in freezers with uninterrupted power sources. Three copies of the archive have been
produced and are stored in separate storage facilities at NUI, Galway. A total of 1005 soil sub-
samples, collected during the sampling campaign, are also stored in buffer solution in 200-ml
plastic containers.
2
2. Sample Preparation and Analysis

The National Soil Database study investigated 40 elements, including aluminium (Al),
arsenic (As), barium (Ba), calcium (Ca), cadmium (Cd), cerium (Ce), cobalt (Co),
chromium (Cr), copper (Cu), iron (Fe), gallium (Ga), germanium (Ge), mercury (Hg),
potassium (K), lanthanum (La), lithium (Li), magnesium (Mg), manganese (Mn),
molybdenum (Mo), sodium (Na), niobium (Nb), nickel (Ni), phosphorus (P), lead (Pb),
rubidium (Rb), sulphur (S), antimony (Sb), scandium (Sc), selenium (Se), tin (Sn),
strontium (Sr), tantalum (Ta), thorium (Th), titanium (Ti), thallium (Tl), uranium (U),
vanadium (V), tungsten (W), yttrium (Y), and zinc (Zn). In addition, soil samples were
analysed for soil acidity (pH), available nutrients (P, K and Mg) and soil organic carbon
(SOC).

2.1. Chemical Analysis
2.1.1 Preparation of soil samples after arrival
All soil samples were air-dried at ambient temperature. Soil samples were sieved to remove
stones and plant debris, and mixed thoroughly to obtain a representative sample. Soil samples
were rolled manually with a steel roller and then sieved through a mechanically vibrating 2-mm
stainless steel mesh. Both devices were cleaned after each sample had been processed to
avoid cross-contamination. As described in Section 1.3, soils were stored in 1-l glass jars at
Johnstown Castle, with 100-ml sub-samples also stored in small plastic containers.

2.1.2 Analysis of soil samples for acidity (pH)
Soil pH was determined for all samples by mixing 10 ml soil sample with 20 ml deionised water.
After 10 minutes, pH was determined using a digital pH meter with a glass electrode. Each
suspension was stirred vigorously using a glass rod immediately prior to pH determination.

2.1.3 Analysis of soil samples for available elements
Available nutrients (P, K and Mg) were determined for all samples using Morgans extracting
solution (10% sodium acetate in 3% acetic acid buffered at pH 4.8). A volume of soil was
extracted with a volume of Morgans extracting solution in a 1:5 ratio (Peech and English, 1944).
The suspension was shaken for 30 minutes on a Gyrotory shaker, and then filtered through No.
2 Whatman filter paper. The filtered extract was analysed on a fully automated analysis system.
Phosphorus was measured colorimetrically using the reaction between P and ammonium
molybdate. Potassium was measured by flame photometry, and Mg by atomic absorption
spectrometry. The automated system has an inbuilt computer-controlled quality-control process
3

and statistical analysis of the output of control samples results. Analysis cannot proceed if all
parameters are not complied with.

Additional available nutrients (Cu, Zn, Mn and Mo) were also determined for the 295 samples
collected as part of the south-eastern study, and are reported in the National Soil Database final
data set. Available Cu, Zn and Mn were determined using the EDTA-Quinol method and flame
atomic absorption. Available Mo was determined using ammonium oxalate as the extracting
solution, and measured by spectrometry. However, these results are not considered in the
context of this national report, as equivalent analyses were not conducted for samples collected
when the study was extended to encompass the remainder of the country.

2.1.4 Analysis of soil samples for soil organic carbon and total N
Soil samples (295) collected during the 1995/1996 south-east study were analysed for organic
C using the Walkley Black method (Walkley and Black, 1934). For the remainder of soil samples
(1015) collected during 2003 and 2004, organic C and total N were determined using a Leco
CN-2000 dry combustion analyser, which only became available in 2002. A correction factor of
1.16 (Brogan, 1966) was applied to the south-east organic C data to account for the different
methodologies employed during the 1995/1996 south-east study and the 2003/2004 study.

The dry combustion method employed followed that described by Wright and Bailey (2001) for
organic C. The analyser operated on a dry combustion principle, with infrared detection for C.
The oxygen used in the combustion process was supplied in two controllable ways: (i) by a
lance flow directly over the sample and (ii) by a background purge. It was therefore possible to
vary the furnace conditions according to the analysis required. A furnace temperature of 1040C
was used for organic C (Wright and Bailey, 2001). The instrument was calibrated using
standard weights (i.e. 0.050.500 g) of a Leco EDTA calibrator which contained 95.7 g N kg
-1

and 410 g C kg
-1
. Ceramic combustion boats with nickel liner inserts were used for blank
corrections, standards and soil samples. Glucose and sucrose standards were used to test the
recovery of organic C. Certified reference material supplied by the Leco Corporation (USA) in
addition to samples supplied by the International Soil-Analytical Exchange (ISE) proficiency test
programme in the Netherlands were used routinely during analysis. Soil samples were finely
ground in a mortar and pestle and passed through a 0.42-mm nylon mesh. The amount of
sample used per determination varied according to the organic C content of the material.
Sample weights of 1 g were the norm for the majority of soils, but for highly organic soils this
weight was reduced to 0.5 g to keep within the calibration range of the instrument. For soil
samples (n = 58) with a pH > 7.0, a weighed sample was treated with dilute HCl (HCl:H
2
O in the
ratio of 1:1) to remove carbonate C. The treated sample was dried on a hot plate at a low
setting until dry and allowed to cool. This step was repeated until no further reaction to the dilute
acid solution occurred. The treated sample was then analysed to determine the non-carbonate
4
C, which is usually referred to as Total Organic C. The method employed followed that
described by the Leco Corporation (2003) for organic C in soils and the furnace temperature
was set to 1350C.

As described above, total N was determined using a Leco CN-2000 dry combustion analyser for
soil samples collected during the 2003 and 2004 sampling campaign, and is reported in the
National Soil Database final data set. However, these results are not considered in the context
of this national report, as equivalent analysis was not conducted for the 295 soil samples
collected as part of the south-eastern study.

2.1.5 Analysis of soil samples for all measured elements
All soil samples were finely ground in an agate mortar and pestle and passed through a 0.42
mm nylon mesh. The method used involved the digestion of a representative sub-sample (200
mg) to dryness on a hot-plate with 10 ml hydrofluoric acid (HF), 5 ml perchloric acid (HClO
4
), 2.5
ml hydrochloric acid (HCl) and 2.5 ml nitric acid (HNO
3
). Salts were dissolved in 20% aqua regia
and made up to 10 ml. Indium was added to this solution prior to analysis to serve as an internal
standard. For analysis of Al, Ba, Ca, Cr, Fe, K, Li, Mg, Mn, Na, P, S, Sr, Ti, and V this solution
was introduced to ICP-OES, while for analysis of As, Cd, Ce, Co, Cu, Ga, Ge, La, Mo, Nb, Ni,
Pb, Rb, Sb, Sc, Sn, Ta, Th, Tl, U, W, Y and Zn, this solution was introduced to ICP-MS. The
latter was used because of its greater sensitivity in the determination of elemental
concentrations at the lower end of the spectrum. In terms of quality control, the standard
procedure in ICP analysis included analysis of every 10th sample in duplicate and the inclusion
of a reference material and a blank for every 50 samples. Reference materials included two in-
house standards and certified reference materials (CRM). An external check on accuracy was
made by participation in Quality Proficiency Testing programmes.

Both Se and Hg were measured using atomic fluorescence spectrometry. In the case of Se the
air-dried and ground sample was digested with 3:1 HCl:HNO
3
by standing for at least 16 hours
and refluxing for 2 hours. The digest was made up to a known volume and an aliquot was
diluted into 33% v/v HCl. This dilution was analysed for Se using hydride generation/atomic
fluorescence spectrometry. For analysis of Hg, the sample was digested with aqua regia, and
treated with free bromine. The digested sample was converted using acidic stannous chloride
from Hg (II) into elemental Hg vapour. The Hg vapour was removed from the solution by a
stream of Argon and was detected using atomic fluorescence spectrometry. Quality-control
standards were included in every batch of analysis. An external check on accuracy was also
made by participation in Quality Proficiency Testing programmes.

5

2.2 Microbiological Analysis
2.2.1 Sampling and sample preservation
A sampling protocol, based on the use of a nucleic acids stabilisation buffer, was developed by
the project team and used by field samplers to collect a representative set of soil samples
(Appendix 1). The stabilisation buffer-based approach was used because the normal guidelines
for preservation of nucleic acids in environmental samples, e.g. immediate liquid nitrogen
freezing of samples in the field, could not be followed in a large-scale study of this kind. The
protocol was evaluated fully in the laboratory, with respect to stabilisation of nucleic acids and
microbial community structure, before application in the study and the results of this evaluation
are presented in the final report. Upon receipt in the laboratory, the soils were centrifuged (5000
x g for 15 minutes) and pellets used to obtain samples for the DNA extraction procedures are
described in Section 2.2.3. Unused soils were re-suspended in stabilisation buffer and
maintained in storage at 4C.

2.2.2 Cell counts and determination of lysis efficiency
Total cell counts and cell lysis efficiencies for soil samples were determined using epifluorescent
microscopy. For total microbial count determinations, 1 g of soil was added to 9 ml of sterile saline
and samples were sonicated for three 20-second bursts, at 16 microns wave amplitude using a
Soniprep 150 (MSE Scientific Instruments, Crawley, Sussex, England). The soil suspensions were
then serially diluted to an appropriate range (i.e. < 100 cells/field), then 2 ml of each suspension
and 200 l of Sybr-Gold
TM
(10 x stock solution in TE buffer, pH 8) were added to the filtration
column and incubated for 5 minutes. Samples were drawn onto 0.2 m black Isopore membrane
filters (Millipore, Ireland), before viewing under a Nikon Optiphot-2 UV microscope fitted with a
100 W mercury bulb, using a B-2A excitation filter for blue light and a 100 planar objective lens.
For cell-lysis efficiency determinations, the post-DNA extraction pellets were added to the 9-ml
saline, without sonication, and cell numbers were counted as described above. For each soil type,
three separate extractions were counted in triplicate (n = 9) and at least 50 fields were counted on
each filter. Cell lysis efficiencies (%) for each method were calculated in relation to total counts
observed for each sample.

2.2.3 DNA extraction methods
All soil DNA extraction procedures selected for evaluation during the National Soil Database
study were based on the direct lysis of cells in the soil sample, with subsequent recovery and
purification of nucleic acids. Prior to DNA extraction, all laboratory solutions were rendered
DNase-free by treatment with diethyl pyrocarbonate (DEPC). Ten replicate extractions were
carried out for each soil type with each method.

Methods 1 and 2: were modified from previously published methods (Fuhrman et al., 1988;
6
DeLong et al., 1993).

Method 1 (M1): liquid nitrogen (~10 ml) was mixed with 250 mg of soil (wet weight from pellet) in
a mortar, ground and transferred to a micro-centrifuge tube (Eppendorf, Germany), 1 ml of
CTAB extraction buffer (Griffiths et al., 2000) was then added, followed by vortexing for 30
seconds. After the addition of 500 l of lysis buffer [50 M Tris-HCl [pH 8]; 40 M EDTA pH 8;
750 M filter-sterilised sucrose and 20 l of lysozyme (10 mg/ml; Sigma-Aldrich, Germany)],
mixtures were briefly vortexed (30 seconds) and incubated at 37C for 30 minutes. Sodium
dodecyl sulphate (SDS) was added to a final concentration of 2%; the samples were again
vortexed and then incubated at 70C for 1 hour. Following this, 6 l of proteinase K (Sigma-
Aldrich, Germany) were added. Samples were then vortexed and incubated at 50C for a further
30 minutes followed by centrifugation for 15 minutes (10,000 g). The supernatants were
transferred to fresh micro-centrifuge tubes and the aqueous phase was extracted by mixing an
equal volume of chloroform-isoamyl alcohol (24:1) followed by centrifugation (10,000 g) for 10
minutes. Total nucleic acids were then precipitated from the extracted aqueous layer with 0.6
volumes of isopropanol overnight, at room temperature, followed by centrifugation (10,000 g)
for 15 minutes. The pelleted nucleic acids were washed in 70% (vol/vol) ice-cold ethanol and
air-dried prior to resuspension in 50 l DEPC-treated water.

Method 2 (M2): 250 mg of soil and 1 ml of 1% CTAB were beaten for 2 minutes with 250 mg of
glass beads (0.1 m), in a Mini Beadbeater-8 (Biospec Products, USA) at the minimum speed
setting. 500 l of extraction buffer were then added and the remainder of the extraction
procedure was as described for Method 1.

Method 3 (M3): was carried out on 500 mg soil samples as described by Griffiths et al. (2000).

Method 4 (M4): the MoBio Ultraclean soil DNA kit (Cambio Ltd, Cambridge, UK). DNA was
extracted from 250 mg of soil according to the manufacturers instructions.

To inspect the quality of extracted DNA, 5 l aliquots of crude extract were run on TAE-agarose
gels (1%) containing ethidium bromide for DNA staining and visualisation, with Lambda
DNA/Hind III molecular size marker (Promega, USA). Nucleic acids were visualised on a UV
transillumination table. For the DNA method evaluation phase of the National Soil Database
study, three Irish soils (collected from the top 010 cm) and one marine sediment were used: (i)
Grey Brown Podzolic (Silvermines), (ii) Gley (Corrib) (both sampled using a Dutch auger), (iii)
peat (Inverin) sampled using a Russian Peat corer (Duncan & Associates, UK). All three soils
were stabilised in buffer as described in the National Soil Database protocol (Appendix 1);
however, as this evaluation phase was carried out prior to field sampling, the soils used were
not part of the National Soil Database Archive; (iv) a marine sediment sample (PAP), taken
7

using a multicorer (inside diameter 9 cm; depth penetration >20 cm; Ocean Scientific
International Ltd, Petersfield, Hampshire, UK) from a depth of 4800 m at the Porcupine Abyssal
Plain site (Eardly et al., 2001) in September 1998, 48
0
50N 16
0
30W. The sample was frozen
upon recovery and the 01 cm section was used in this study. The sediment sample was used
in this study as it had been well quantified with respect to DNA yield, and lysis efficiency
previously at NUI, Galway, and thus provided an internal standard for method evaluation.

Following a careful evaluation process, Method 2 was selected as the most appropriate for use
during this study. It was therefore used for the extraction of DNA from all the preserved National
Soil Database soil samples and for generation of the nucleic acids archive.

2.2.4 Quantification of soil DNA yield
The quantity of extracted DNA was estimated using the PicoGreen double stranded DNA assay
(Molecular Probes, USA; Sandaa et al., 1998). Briefly, samples were diluted 400-fold in 1 x TE
and 100 l of these dilutions were added to 100 l of a 200-fold dilution of PicoGreen (Molecular
Probes, USA). These were reacted in the dark, in black microtitre plates (Corning, USA) for 5
minutes at room temperature. Fluorescence was measured using the Genius System 7
(Boehringer Mannheim, Germany) at 485 nm excitation and 530 nm emission. TE buffer was
used as a blank sample and DNA standards were prepared from bacteriophage DNA stocks
(Molecular Probes, USA).

2.2.5 Nucleic acid purification
Crude soil DNA extracts were purified, in order to facilitate successful PCR amplification of DNA
template, using the GELase enzyme kit (Epicentre, USA), according to the manufacturers
instructions.

2.2.6 Denaturing gradient gel electrophoresis (DGGE)
The V3 region of the eubacterial 16S rRNA gene was PCR amplified using primers EBGC 341f
(5-CCT ACG GGA GGC AGC AG; REF) and UN517r (5-ATT ACC GCG GCT GCT GG) or the
primer pair 27f (5-AGA GTT TGA TCM TGG CTC AG) and 1392r (5-ACG GGC GGT GTG
TRC). A 40 base GC clamp was attached to the 5 end of the forward primer to increase the
separation of DGGE bands in DGGE analysis (Muyzer et al., 1993). The ammonium
monoxygenase gene was amplified using the amoA specific primer sets amoA-2F
(AARGCGGCSAAGATGCCGCC)amoA-5R (TTATT TGATCCCCTC) and amoA-3F
(ACCTACCACATGCACTT)amoA-4R (GGGTAGTGYGACCACCAGTA) as described by
Webster et al. (2002). PCR was performed using an Eppendorf Mastercycler Gradient
(Eppendorf, Germany) in a final volume of 50 l, each containing: 25 pmol of each DNA primer
(MWG-Biotech, Germany), 1 ammonium (NH
4
) buffer (Bioline), 3 mM MgCl
2
(Bioline), each
8
dNTP at a concentration of 200 M (Biogene), 0.5 l of BSA (1 mg/ml) (Promega), 1 U Taq
DNA polymerase (Bioline) and 1 l of template DNA. Negative controls were included to ensure
there was no contamination of reagents both before and during preparation of reaction mixtures.
A touchdown PCR protocol was performed, with denaturation at 95C for 45 seconds, an initial
annealing temperature of 65C for 45 seconds and an extension at 72C for 1 minute. The
annealing temperature was dropped by 1C per cycle until it reached 55C and 15 further
amplification cycles were carried out at this annealing temperature. A final extension step was
performed at 72C for 10 minutes. PCR products were checked on TAE-agarose gels (1%)
containing ethidium bromide.

DGGE was performed using the D-Code system (BioRad, USA). Equal amounts of PCR product
were run on polyacrylamide gels with denaturing gradients ranging from 35% to 70% (100%
denaturant contained 7 M urea and 40% formamide) and were run at 60C and 60 V for 15
hours. Following this, electrophoresis gels were stained for 15 minutes using Sybr-Gold
nucleic acid stain (Molecular probes, USA), de-stained for 10 minutes in sterile DEPC-treated
water and photographed on a UV transillumination table. DGGE profiles were created in
duplicate using PCR products derived from 5 of the 10 samples extracted to investigate
reproducibility of each of the extraction methods under investigation.

2.2.7 Sequencing and phylogenetic analysis of DGGE bands
Selected DNA bands were excised from the gel and the DNA eluted and re-amplified as
described elsewhere (Rlleke et al., 1996). DNA sequencing was carried out on an ABI 310-3
capillary sequencer (Perkin-Elmer, USA). Sequences were initially analysed using the BLAST
2.0 programme on the National Center for Biotechnology Information (NCBI) website
(www.ncbi.nlm.nih.gov). The highest scoring sequences were recovered from GenBank and
further representative sequences were downloaded from the Ribosomal Database Project
(RDP) (http://rdp.cme.msu.edu). Multiple sequence alignments were generated using ClustalX
(Thomson et al., 1997) and edited manually using Genedoc (www.psc.edu/biomed/genedoc).
Phylogenetic trees were constructed using PAUP 4.10 b (Swofford, 1996) and a neighbour-
joining method (Saitou and Nei, 1987).
9

10
References
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Appendix 1
Protocol followed by the samplers for the NSDB
Site selection and access
Before accessing agricultural land, direct consultation with the land owner or his/her
representative will normally be necessary.

Soils will be sampled at fixed locations, two from each 10 x 10 km segment of the National Grid:
one location will derive from each intersection and one from each centre point. Each location
has been moved exactly 1 km to the north and 1 km to the east of National Grid intersections.
Each possible sampling location (and thus soil) has been given a unique number which is
recorded on an Ordnance Survey map. The co-ordinates of sampling positions will be made
available to samplers. Corresponding to each sampling number are a letter, a five-figure
number which denotes the easting and a five-figure number denoting the northing. This defines
the target sampling position. Site areas will be reconnoitred using a 1:50000 Ordnance Survey
map and a GPS (compass page). Field maps (1:25000) are available for most sites (on request)
if this system should fail. In the event of it not being possible to take a sample at the target
sampling position, a default procedure will be used. As a general principle, the site sampled will
need to be similar in land use and quality to the target site. Sampling will first be attempted at a
point 50 m N, than E, S and W in turn with the procedure being repeated at a distance of 100 m,
200 m and 400 m from the original point. If this is not successful no sample will be taken. The
actual sampling co-ordinates will be taken from the GPS (position page) and will be written
down at this time. Additional requested information must be written down on the regulation form
before leaving the site.

For safety reasons in particular, a separate procedure will be necessary at forest sites and at
some peatland sites. For forest sites the location (forest roadway or boundary fence) closest to
the target site should first be accessed. The co-ordinate should be noted at this point. From
here one should proceed a short distance into the forest in the general direction of the target
site along a northerly, easterly, southerly or westerly direction. The direction taken and the
distance travelled to the sampling position should be noted. From this information the co-
ordinates of this position can be calculated (GPS is not reliable under tree cover).

For virgin peatland sites access will be from the nearest dry land. A procedure similar to that for
forest soils should be used. Distance travelled across the bog towards the target site should be
the maximum the conditions allow. In most extreme travel conditions it will still be necessary to
move away from the roadway (to avoid pollution): an exception here is where the defined target
position is actually adjacent to the roadway. Final sampling position should be recorded in the
usual way.

Situations where sampling may not be possible
1. No soil within 800m of target location.
2. On an island with no access by land.
3. Site presents a real danger (i.e. mountainous).
4. Access impossible (forest) due to impenetrable growth.
5. Permission to access land is refused.
11

6. Site area is surrounded by large town/city.

When default procedure may be used
1. Site on roadway, quarry, or small body of water.
2. Presence of bull.
3. To avoid damage to cereal crop.
4. In thickly vegetated forests where access is possible only from the edges.

In all instances it is most important to note the exact sampling position

Sampling for chemical analysis
At sampling, single soil cores must be taken to a depth of 10 cm at 5-m intervals on a grid
measuring 20 m x 20 m and with the centre point of the grid at the sample location. Centre and
corner points will need to be made easily visible using the canes supplied. A Dutch 50-mm
diameter auger will be used. A complete core will be taken from each intersection on a 5-m grid
within each location, collecting cores (totalling 25) in the bucket. The composite sample will be
transferred to a polythene bag. It is expected to weigh about 34 kg. A plastic label with the site
number numbered will be included with the sample; the bag will also be clearly labelled using a
felt pen. In exceptional circumstances (generally on bog) the Dutch auger may be ineffective: in
these instances samples (to 10 cm) may be taken using the trowel. The sample will be returned
to Johnstown Castle within one week.

Soil for microbiological analysis
The composite sample will need to be sampled at collection in order to obtain a sub-sample (ca
25g) for microbiological analysis. The composite sample should first be thoroughly mixed within
the large plastic bag. Sub-samples, numbering 25, should then be taken using the spatula,
supplied. They should be taken from different parts of the bag and transferred to a vial
containing 50 ml stabilisation buffer (CTAB buffer + 2.5 M guanidine isothiocyanate). The total
soil content should fit beneath the surface of the liquid in the vial. After addition the stoppered
vial should be shaken gently. Note: Precautions should be observed in handling vials (see
safety sheet) and their contents and they should be kept away from children. Vials should be
numbered by Site No.

Samples should be stored in a cool place and posted to Galway (c/o Dr Vincent O Flaherty,
National University of Ireland, Galway) within 34 days.

Microbiological sampling: safety and sample handling
1. Stabilisation buffer contains guanidine isothiocyanate, which is toxic. Always wear the
gloves provided when handling the containers and avoid spillage. In the event of contact
with skin wash thoroughly with soap water.
2. Place all used gloves, paper, etc. that have come into contact with the buffer into the
autoclave bags provided and return to NUI, Galway.
3. Try to ensure containers are kept right side-up at all times and try to keep the containers
out of direct sunlight and high temperatures for long periods of time.
4. Spray spatulas and everything coming into contact with the soil samples with the
methylated spirits supplied (flammable) and allow to air-dry before re-sampling. Re-
12
spray and wipe down with paper after sampling. This is to ensure there is no
contamination of samples or between samples.
5. Before commencing to add soil to the buffer, shake the containers well.
6. As the buffer is sterile, when adding soil make sure the containers are open for the
minimum time possible.
7. Add 25 level spatulas (spoonfuls) of soil to each container.
8. Buffer may fizz up temporarily when the soil is added, make sure the lid is on tightly
and that containers are right side-up to avoid leakage.
9. Wipe any buffer leaked on the outside of container with paper provided.
10. Please write the relevant reference information on the lids and sides of the containers
with the pens provided. This is very important so we can identify each container with a
particular sample and site when they reach the lab. Also please fill in the data sheets,
provided by Teagasc, for each sample.

Thank you for taking note of all these points during sampling.

Sampling equipment checklist
FIELD
Personal identification
Farmer sheets
OS map as required
Field map as required
GPS Garmin 12XL
Compass
Edelman auger, 5 cm diameter, stopped at 10 cm
Gauge auger, ss, 1 cm diameter, stopped at 10 cm
Trowel
Bamboo canes (6)
Hammer (tack)
Scraper
Bucket 1 gal
Plastic bags 24 cm x 16cm width Plastic disc Felt pen Biro
Containers (4) for micro-biological samples
Notebook
Field sheet
CAR
Identity plate (Windscreen)
Maps
Batteries for GPS
Large box for soils in car boot
Box for micro samples
Field recording sheets Farmer sheets
Plastic discs
13

14
Appendix 2
Field Sheet for the NSDB

Map/Sample No: ___________________________
Area / town: _________________ County: _______________
Target grid reference ________________________________
Sample E______________W______________
Magnitude and direction of change_______________
Reasons for change: ______________________________________________________
Site location in field: ________________________________

Special characteristics: ___________________________________________________
Farmers name: _________________________________________________________
(if offered)

Vegetation:
Grass Crop Fallow Other
(name) (name)

Grass:
Good Fair Poor Rough

Slope:
Flat Undulating Rolling (Steep)
(01) (26) (712) (13 +)

Soil Texture:

Sand Loam Fine Peat

Sampler: ____________________ Date: ___________

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