Protocol for Ameriflux Eddy Covariance Tower Ecosystem Survey Measurements
Desai Lab, University of Wisconsin-Madison
1. Introduction
This protocol details tasks and measurements needed to be done in order to better understand the
carbon flux rates at the US-WCr: Willow Creek, Us-Los: Lost Creek, and US-Syv: Sylvania
Wilderness Area Ameriflux sites and the specific steps needed to be taken in order to complete
them.
2. Tasks to be completed
The following measurements will be taken during visits to the field sites. Not every measurement
will be taken during every visit to the site. When and where each measurements shall be taken
and what tools will be needed to complete each measurement are outlined in section 3.
 Plant Measurements
 Mean Canopy Height
 Diameter at Breast Height (DBH)
 Tree Species
 Leaf Area Index (LAI)
 Leaf Carbon Level
 Leaf Nitrogen Level
 Soil Concentrations
 Soil Temperature
 Soil Bulk Density
 Soil Carbon Level
 Soil Nitrogen Level
3. Data Collection Procedures
The procedures on how to collect the data stated in section two and the tools needed to complete
said procedures will be described in the following.
3.1 Equipment
Tool
Logbook
Data Sheet
Writing Utensil
Flagging Rods
10+ Meter Tape
Global Positioning System
LAI-2200 C Plant Canopy
Analyzer
Purpose
To record events in the field
To record measurements
To write in logbook and data sheets
To mark off measurement plots
To measure out measurement plots and to measure the height
of the canopy
To find and record positions of plots
To measure the leaf area index
DBH Tape
Inclinometer
Aluminum Tags and Nails
Flash Combustion/Elemental
Analyzer
Sieve
Microcentrifuge Tubes
5x9mm aluminum tins
Microtiter Plate
Oven
Brown Paper Bags
Mortar and Pestle*
Shaker*
Microbalance
Soil Corer
2” x 12” Plastic Soil Core
Liners
2” plastic end caps
2” Soil Core Catcher
Plastic Syringe
Extender
To measure diameter of tree trunks
To measure the height of trees
To mark trees that have been measured
To measure the carbon, nitrogen, and soil bulk density
concentration of the soil core samples
To remove organic material and rocks from the soil
To hold soil samples for testing
To roll soil and foliage samples in
To place rolled soil and foliage samples in
To dry soil and foliage samples
To place samples in in the oven to dry
To pulverize soil and foliage samples
To pulverize soil and foliage samples
To weigh soil and foliage samples
To dig into the ground to acquire the soil sample
To capture the soil core sample inside of the soil corer
To cap the soil core extractors/liners
To catch underwater soil core samples
To take subsamples of soil cores
To attach to the top of the corer which then attaches to the
weight
Attached Weight
To assist the soil corer in digging into the ground
Shovel
To dig into the ground to acquire the soil sample (Easier to use
than soil corer and often times works better)
Waders
To wear when retrieving submerged soil core samples
Infrared Thermometer
To measure the temperature of the soil core sample
Cloth/Rag
To clean off soil corer and extractor in between different soil
core extractions
Sealable Plastic Bags
To store soil core and leaf samples
Permanent Writing Utensil
To record on soil and sample bags
Cooler
To store samples in until return to the lab
Shotgun and Shells
To shoot down leaf samples with
(* denotes a tool that is optional to have)
3.2 Logging Trips
Every time a trip to the above mentioned sites takes place, it will be thoroughly logged. The team
visiting the site will have a logbook with them that they will use for the entirety of the summer.
Each time a site is visited, a new page will be started to note the new trip. At the top of the page
the date, listed as month, day, year (00/00/0000), will be noted along with the name of the site.
The next piece of information that will be noted and clearly labeled will be the arrival time of the
team. Below the arrival time, and clearly labeled, the weather for the arrival time will be noted.
The team member recording in the logbook will note the cloud cover to the best of their ability
(ie: clear skies, partly cloudy, full cloud cover) and also the temperature. If the weather changes
throughout the time the team is at the site, they will note it in the logbook. For example, if it
starts to rain, or ceases to rain, the cloud cover drastically changes, the temperature fluxes
significantly, the team will note it in the logbook and at what time the change in weather was
noted. A short block in the sun from the clouds passing by does not need to be recorded.
If any disturbances should occur during the duration of time the team is at the site, it should be
duly noted along with the time such disturbances took place. A disturbance includes things such
as being confronted by people not involved with the team, a tool breaking, an injury of any team
member, any need to leave the site before all measurements are taken, etc.
Once all the measurements have been taken and the team is ready to leave the site, the time of
departure should be taken and noted in the logbook. Once all the necessary information is logged
in the logbook and the visit to the site is over, if there is blank space still on the page, it should
have a slash put through it so as to ensure a new page will be started for a new visit to the site.
The logbook should be stored in such a place to ensure it is remembered every time a visit is
made to the site.
3.3 Creating Plots
The first visit to the site will largely involve picking out and setting up the plots from which to
receive the measurements. Plot setups will vary by site, but in general the plot(s) and subplots
should be representative of the area around the eddy covariance tower or other point of interest.
Below are the plot setups for the three tower sites studied in the summer of 2015 by the Desai lab
undergraduates.
The standard protocol for creating plots according to “Terrestrial Carbon Observations: Protocols
for Vegetation Sampling and Data Submission” provided by Ameriflux was used. Wherever
possible, this protocol should be used. The plot consists of four equally-spaced subplots inside of
a hectare plot.
As shown, the subplot radius and area will vary by which survey measurement is being taken.
The subplot center points will always be the same. Once these points are determined, GPS
coordinates of each point should be recorded. Also, in this case the center subplot (also the very
center of the entire hectare plot) is the point of the eddy covariance tower itself.
Willow Creek: The standard plot setup was used at this site, with the subplots specially arranged
to account for variation across the plot. One subplot was in an area within the forest hectare plot
that had been harvested in the previous year, another was in an area that had been harvested two
years previous, a third was in an area that has not been harvested recently, and the center was the
tower itself. The procedure involving each subplot center lying 35m from the plot center was
adhered to, except for the subplot across the road near the tower which needed to be placed
slightly farther away.
GPS Coordinates:
A: N45 48.310’ W90 04.837’
B: N45 48.323’ W90 04.721’
C: N45 48.393’ W90 04.727’
A: N45 48.356’ W90 04.823’
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Sylvania (Upper Peninsula Michigan): Two hectare plots were created at this site to account
for tree species variability near the tower. The plot around the tower primarily consisted of sugar
maple, while the plot on a nearby peninsula was dominated by hemlock pine. In future references
to these plots in this protocol and in the data, they are dubbed “Maple or Tower Plot” and
“Hemlock Plot”, respectively.
The maple plot’s tower has a fence surrounding it as well as various equipment setups for other
research at different times, so the center of the plot was not used as a subplot. Instead, four
equally-spaced subplots were placed in each of the northwest, northeast, southwest and southeast
directions from the tower.
GPS Coordinates:
Tower: N46 14.534’ W89 20.758’
A: N46 14.524’ W89 20.740’
B: N46 14.537’ W89 20.752’
C: N46 14.522’ W89 20.761’
D: N46 14.515’ W89 20.762’
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The hemlock plot was set up according to the TCO standard.
GPS Coordinates:
E: N46 14.595’ W89 20.668’
F: N46 14.611’ W89 20.690’
G: N46 14.582’ W89 20.660’
H: N46 14.581’ W89 20.678’
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Lost Creek: Because the nature of a wetland site is so different than that of a forested site, the
four subplots at this site will be laid out differently than the other two sites. At this site, we are
trying to look at the difference across moisture gradients so the subplots are set up in a manner
that allows us to do so. One subplot is in a fairly dry area that has a small to no chance of having
standing water on it, one is in a moist area or one that has minimal amounts of standing water on
it (no more than 3-4 inches), and the other two are continually submerged by one foot or more of
water, one with flowing water and one with stagnant water. The dry plot is to the west of the
tower in forested area on the site. Due to the density of the forested section, the subplot here is a
square, 20 meters by 20 meters. The plot that is partially submerged is a 10 meter radius circle
directly around the tower. The final two subplots are 10 meter radius circles in the river, the
stagnant one being northeast of the tower and the flowing one being east of the tower.
GPS Coordinates:
C (Tower): N46°08.274’ W89° 97.855’
A (Forest): N46°04.971’ W89° 59.069’
D (Flowing): N46°08.315’ W89°97.7095’
E (Stagnant): N46°08.317’ W89°97.744’
Each subplot within a plot at each site shall be labeled by a letter. So for example, the first plot
will be plot A, the second plot will be plot B, and so on. A note of the labeled plots and their
GPS coordinates should be marked in the logbook.
3.4 Growth Measurements
3.4.1 Mean Canopy Height
The mean canopy height is the average height of the canopy, or upper layer of trees and other
plant life. In order to get the average height of the canopy, each plant that is a part of the upper
layer in the plots needs to have its height measured. Either an inclinometer or laser rangefinder
can be used to measure tree height. Different instruments have different procedures, so it is
important that the user know how to use the device(s) for certain before taking measurements in
the field.
In wetland sites the canopy will not be very high and can be measured with a tape measurer.
Depending on how tall the plant is, have one person hold one end of the tape measurer at the
ground and the other pull the other end of the tape measure up to the top of the height. If the
plant/tree is leaning in a manner that is adjacent to the ground, do not measure along the stem of
the plant. Instead, measure straight up to the height of the plant. This measurement is not looking
at how long the actual plants are but how high into the air they grow and is why one should not
measure along the stem of the plant if it does not go straight up. Record the height of each plant
that is measured in the logbook. Averaging the numbers to find the median canopy height is not
necessary in the field and can be done back at the field station or the lab. This should be done at
each subplot to determine the median canopy height at each plot. It should, however, not be done
every time a visit to the site is taken. Only once per season is a suitable amount to take this
measurement.
3.4.2 Tree Diameter (DBH)
There are four parts of the tree survey:
 Large trees (DBH >80cm) sampled comprehensively in the 1 ha plot
 The main tree survey (DBH 10-80cm) consists of measurements in each of the four
subplots with radii of about 10 m.
 Sapling survey (DBH 1-10cm) in each of the four subplots with radii of about 5 m.
 Stumps of all sizes sampled in each of the four subplots with radii of 10 m.
DBH is defined as the diameter of the tree at 1.37 m height (DBH=diameter at breast height).
Use the DBH tape to measure the diameter of the tree. If there are unusual features, such as
bulges or forks at breast height, move the measurement as little as possible to avoid these
features. If a fork is below 1.37 m, the two stems are counted as two separate trees. Tag each
surveyed tree with a numbered aluminum tag by driving a nail into the tree facing the plot center.
Record all measured trees. All tagged trees should be recorded for height, DBH and species.
3.4.3 Tree Species
Each tagged tree in the survey should be identified by species. This can be done using a
handbook for Wisconsin tree species.
3.4.4 Leaf Area Index (LAI)
The leaf area index (LAI) measurement should be taken in early to mid-July when the max leaf
area is expected. It would be ideal for LAI to be taken at dawn, dusk, or on a uniformly
cloudy/overcast day to minimize interference from clouds. There is an alternative, however. LAI
will be taken using the LAI 2200C. It is important that each team member read the instrument
manual thoroughly before attempting to take measurements in the field. Testing the device is
also highly recommended to get an understanding of how it works and to ensure that data
collection will be accurate. Working out misunderstandings or malfunctions of the device ahead
of time will ensure that field data collection will go smoothly.
Divide the two wands of the LAI 2200C between two different team members. In non-ideal sky
conditions, the team member with the wand measuring the light above the canopy needs to take
preliminary measurements (known as K-corrections) before any other measurements may be
taken. These corrections account for clouds or other anomalies in the sky that would otherwise
throw off LAI measurements. A diffuser cap should be put on the eye of the wand and a reading
should be taken with it in the direct sunlight. Next, with the diffuser cap still on the eye of the
wand, shade the eye of the wand with either the user’s hand or head and take another reading.
The third preliminary measurement requires taking the diffuser cap off the lens and log a reading
with the lens in the shade with the shadow created by the user’s hand or head again. The fourth
measurement needs to have the cap on the lens that you will be using throughout the
measurements and be pointed in the direction that all the measurements will be taken in. A more
detailed procedure is detailed in the manual. Once the four preliminary measurements are taken,
data logging for both wands can begin.
The person who has the wand that will be measuring the above canopy light will need to stay in a
clearing if the canopy is too high to reach above. For the wetland site, because the canopy will
not be too high, stay in a stationary position while holding the wand above the canopy. Then, the
other person who has the wand that will be measuring the below canopy light needs to make sure
they have the same cap on the lens of their wand as does the wand that is measuring the above
canopy height. With the data logger attached to the below canopy wand, the “below” wand takes
measurements across the entire hectare plot while the “above” wand is disconnected and logs a
data point for each “below” measurement, while stationary. This can be done via auto-logging
(see manual) or by having the two team members physically logging data points by pressing the
wand buttons at the same time (suggested way).
A diagram describing the exact data points in the hectare plot is shown below (diagram provided
by TCO).
After all 35 data points have been logged in a single session, save the data and end the data
logging session on the logger. Plug the “above” wand into the logger and import the wand data
into the file that was just closed. The result should be a file with 70 corresponding data points
(plus the four K-corrections), or more if automatic GPS logging was allowed.
The file can then be uploaded to the LAI 2200C computer program on a field computer.
Complete data pairing and account for K-corrections according to the manual to ascertain a
single LAI data value for the site. If the number seems reasonable, then the team can move onto
the next site, or repeat the process if it does not.
It is important to purge the data from each wand after each session. Failing to do so will result
in erroneous data compilation.
3.5 Retrieving Soil Cores
Soil cores are collected and analyzed for carbon and nitrogen content. Bulk density of the
samples is also calculated. Samples can be collected with a soil corer, but it is highly
recommended that a shovel is used instead (it is a cheaper and more efficient solution).
In general, a one-foot deep hole or core will be dug and three sub-samples will be taken from
depths at 0 cm, 15 cm, and 30 cm. Samples at different depths may be collected if desired. The
sub-samples can be taken using a standard plastic syringe with the top cut off. Sub-samples are
taken at a consistent recorded volume (¼ ounce is the standard used in this procedure), as this
value will be important for calculating bulk density later.
All sub-samples should be stored separately in labeled plastic bags. For example, the surfacelevel (0 in.) sub-sample from the first core of Subplot A at Willow Creek will be marked “WC
A1-1”, and the 6-inch depth sub-sample from the same core will be labeled “WC A1-2” and so
on.
It is recommended that at least six cores (18 sub-samples) are taken from each subplot with radii
of 10 m from the center. In addition, each core’s temperature will be taken with a gauge, and a
simple acid test will be performed on the remaining core (after extracting sub-samples) to
determine the presence of inorganic carbonates. A full procedure for this test can be found at the
URL below:
http://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/ref/?cid=nrcs142p2_053572
The presence of inorganic carbonates may throw off readings from the carbon content analysis.
Soil maps, which can be found on the link below, may also be read for more information on the
soil type at the site in question, including data on inorganic clays and carbonates and existing
values for organic carbons.
http://casoilresource.lawr.ucdavis.edu/gmap/
Core retrieval from different plot types are detailed below.
3.5.1 Retrieving Soil Core from a Dry Plot
Six soil cores will be removed from each dry plot. The location of each soil core will be evenly
placed throughout each subplot according to the discretion of the samplers and the placement of
large roots and rocks, both above and below ground. You want to try to find a representative
sample of each subplot. When the location for the first soil core has been determined, clear away
any foliage or other debris so only the soil is showing in the marked spot. If using the soil corer,
double check to make sure a plastic liner is inside of the corer. Then, screw on the cap of the soil
corer and screw the whole corer to the weight. Gently nudge the corer into the soil to get a
footing. At first, use the weight only gently to make sure the corer goes into the ground in a
straight line. If the weight is used too aggressively in the beginning, the corer will not grab to the
earth and will falter on its side at an angle to the ground. After the corer has grabbed the earth
enough to where the team member feels the corer will no longer fall to its side, they can start
using the weight more aggressively to help the corer into the ground. However, one does not
want to use the weight too forcefully so as to avoid much compression of the soil core sample. If
the corer gets stuck and will not go any deeper, it is likely it has hit a rock or a root. In the event
this happens, bring the corer back up, remove the soil and any other substance the corer may
have brought back up with it, and then start again next to where the original location was. Once
the corer is deep enough in the ground to where the top of it is even with the ground, start to pull
it out. (Often times you will need to put the corer deep enough into the ground to where the top
of the corer is below the surface of the ground to get enough soil in the corer to fill the tube.) Use
a twisting motion when pulling the core out as this will help to cut any tiny roots loose and help
to retain the full sample. If the sample is stuck in the ground to the point where simple pulling
will not bring it up, use the force of the weight to bring the corer back up. Instead of thrusting the
weight downwards, thrust it upwards causing the weight of the weight to pull the corer back up.
The friction of the soil against the corer and the plastic liner will be strong enough to hold the
soil in so there should be no worries about the soil falling out or being left behind. Once the corer
has successfully been removed from the ground, remove the weight and the cap of the corer to be
able to pull the plastic liner from the corer itself. After the extractor has been successfully
removed from the corer, gently push the top of the soil core to push it out of the plastic liner.
Make sure to give the core support on the bottom as it is coming out of the plastic liner so it does
not fraction off. Once the core is out of the extractor, you will have successfully acquired your
first soil core sample. You should then take three subsamples of the soil core. A subsample
should be taken using a plastic syringe at 0 cm (the surface), 15 cm, and 30 cm. Once that is
accomplished, you will have successfully retrieved the different parts of a soil sample.
It is acceptable, and much easier however, to retrieve a soil sample using a shovel instead of a
soil corer. To retrieve a sample going about this method, one need only to dig a hole that is 30
cm deep. Try to find a longer, skinnier shovel to dig the hole so as to avoid more disruption to
the natural environment than what is necessary. Once the 30 cm hole has been dug, you can take
the subsamples using the syringe from the wall of the hole. Take a subsample, from the surface,
15 cm down on the wall in the hole, and then finally 30 cm down on the wall in the hole. Make
sure to avoid mixing the soils from different levels in the subsamples if you go about this method
as it will be easy for the soil to fall in the hole and intermingle with different levels. The
subsamples should then be put into their own plastic bags and labeled accordingly to keep them
separated. After the three subsamples have been taken, you have collected what you need to test
for carbon and nitrogen content.
When you have acquired the soil core subsamples, some initial measurements must be taken
before the next soil core can be retrieved. The base of the hole the soil core was retrieved from
needs to have its temperature taken. Aim the infrared thermometer at the bottom of the hole from
which the core was removed from and allow it to take the reading. Record the reading in the field
notebook as well as on the bag the subsample is stored in. From there, you will take the
remaining portion of the soil core (if the soil corer was used) or portions of the soil that was
removed from the hole (if the shovel was used) to test if there is any inorganic material in the
soil. It does not matter which portion of the remaining soil is used. You need only pour a slight
amount of HCL onto a small portion of the soil to complete this test. When the HCL is poured
onto the soil, if it bubbles, that is indication the soil has inorganic material in it. If the soil does
nothing, there is no inorganic material in the soil you are testing.
After this has been completed, you may leave the remaining soil behind as it will not be needed.
Try to put what remains of the soil back into the hole you created so as to avoid leaving behind
many disturbances to the environment. Once all the baggies have their material in them, they
have been labeled as described above, and everything is cleared and cleaned up from the current
core, the team may move onto the next core in the subplot, disbursing around the subplot in an
even manner, until six soil cores have been removed from the subplot.
3.5.2 Retrieving Soil Core from a Moist/Slightly Submerged Plot
This type of soil retrieval will only occur at Lost Creek and will be right around the tower itself.
If the soil in this plot is only saturated and not submerged, then you should follow the steps
presented for the dry plot in section 3.5.1 to retrieve a soil core sample. However, if the soil is
submerged, even a few inches, you should proceed to the next section, 3.5.3, and follow the steps
for retrieving a soil core sample from a submerged plot.
3.5.3 Retrieving Soil Core from a Submerged Plot
This type of soil core will only be retrieved at Lost Creek and will be taken from the river.
If using the soil corer, make sure to have a plastic liner in the corer before you put it in the
ground. You also need to be sure that you put the plastic soil core catcher at the bottom of the
plastic liner. This piece is not needed in the dry or moist soils but without it in the submerged
plot, you will not be able to retrieve a sample because the soil in the submerged plot is too moist
to have the friction of the plastic liner and the corer bring the sample back up with it. Therefore,
this added piece acts as a catch to not allow the sample to fall out as the corer is being brought
back up. Connect an extension onto the corer once the cap has been screwed on tightly.
Depending on the depth of the river, you may need a long extension or you may not need one at
all,just be sure to avoid submerging the weight when the proper depth is reached. You may then
screw the weight onto the extension pole if one is used. After all the connections are screwed on
tightly, you are ready to head into the water. If the water is deep enough, be sure to have a pair of
waders on to avoid completely soaking yourself while retrieving the sample.
Once you have found the place you are going to put the the corer into the ground, move away
any debris so only the soil remains, then gently nudge the corer until it grabs the earth and then
start using the weight. It is likely you will be able to forcefully use the weight much sooner in the
submerged subplots than you are in the dry subplots. It is unlikely you will hit a rock or other
obstruction in the submerged subplots but in case you do, pull the corer up and remove any
material that may have been brought back up with it before putting it back in in another location.
The water in the corer should be escaping through the holes on the cover of the corer. Once the
corer has reached a suitable depth in the ground to pull out an acceptable core, begin to pull it up.
The black plastic liner on the top of the core should be forced down when you are pulling up by
the pressure of the water against it, creating a vacuum inside the tube, not allowing water to get
in and helping secure the soil sample in the tube. Once the core is out of the water, it is alright to
carry it right side up, as the vacuum and the soil core catcher should be keeping the core in the
liner. When the liner is removed from the corer, make sure to hold it upright as the sediment will
be able to fall out the top of the tube. You can then pour the sediment out of the tube from the
top or from the bottom by removing the soil core catcher from the tube, making sure to keep it in
order so as to be able to grab the subsamples from the surface, 15 cm, and 30cm down on the
sample.
You may also use a shovel to retrieve this sample which is what we suggest as it still appears to
be simpler than using the soil corer. All one must do is dig a hole under the water that is 30 cm
deep. Then pull out sediment from the desired depth and take the needed amount. It is even more
important to watch for cross contamination of different levels of the soil in this situation as it is
very easy for the water to move the sediment in the hole to different levels where it otherwise
may not have been. After the ¼ of an ounce subsamples have been taken at the three different
levels mentioned above, you have retrieved your soil core sample.
Because there would be so many other influential factors when trying to determine the
temperature of the base of the hole when it is underwater, you should take a temperature reading
of the sediment pulled from the base of the hole. When pulling up samples from underwater, you
should also bring up extra to test with the HCL for inorganic compounds in the soil. Because of
the traverse landscape the Lost Creek wetland has, it was not possible to retrieve six soil samples
from each subplot. The forest plot was able to have six samples removed from it. However, the
subplot around the tower at Lost Creek, which was submerged by about 4 inches of water, only
had four cores extracted from it, one from each side of the tower. The two different river
subplots, flowing and stagnant, only had three cores extracted from it, one on each bank and then
one also in the middle of the river.
After all samples have been retrieved and tested, it can be stored and put away in the same
manner as the dry plot samples. After all the plots have had all of their soil core samples
extracted, all the core samples have been correctly stored in a tub/cooler, and everything you
brought with is cleaned up, your team may leave the site for your next destination.
3.6 Measuring Soil Core Concentrations, Bulk Densities
After the team returns to the lab, each soil subsample is to be placed in its own brown paper bag
and then put into an oven at 60 degrees C for a minimum of 48 hours to dry. You may carefully
break the sample into smaller pieces to allow it to dry quicker. After it has dried and cooled,
sieve and weigh the sample. A 2 mm sieve is used to separate the dried soil from other material
such as rocks and roots. With the weight and volume of the dried sample, you can then determine
the dry soil bulk density. To find that, you need to divide the mass of the dry soil (g), by the
original volume of the cylinder of the subsample (cm ). When that is completed, you will have
the soil dry bulk density (g/cm ) of the subsample.
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Once the soil has been measured for dry bulk density, all sub-samples from the same level within
a subplot are combined and homogenized. For example, all sub-samples from the 30 cm layer of
Willow Creek Subplot A are combined. Once homogenized, this combined soil sample it is to be
pulverized to a flour-like consistency. There are a couple different ways this can be done. One
can use a mortar and pestle and physically grind the soil themselves. Or, if you would rather use
a machine to pulverize the soil, you may place the combined subsamples in microcentrifuge tube
containers with a small stainless steel BB or ball bearing and then place them on a modified paint
shaker for a total of 45 minutes in increments of 15 minutes.
After the soil has been pulverized, roll the samples into 5 x 9 mm tins. The weight of these
samples should afterwards be measured on a microbalance (subtracting the weight of the empty
tin) and should be between 8 and 10 mg. The tins then need to be placed on a microtiter plate
with the sample ID, plate location, and weight recorded on a data sheet. There also needs to be
one empty tin per analysis to correct for the content of the material that makes up the tin, as well
as standardization tests. The microtiter plate should then be stored to await testing in the flash
combustion analyzer.
*Throughout the preparation phase, extra care should be taken to avoid any cross contamination
of the soils or any other material involved. Rinse tools with methanol or ethanol solution after
rolling each sample.
At the end of the research period, you will hopefully have carbon and nitrogen concentration data
to show if there is any type of relationship across the different transects of moisture, harvest,
and/or time.
3.7 Retrieving & Analyzing Foliage Samples
Similar to the soil samples, foliage samples will also be collected and analyzed for carbon and
nitrogen content.
The simplest and most accepted method for collecting foliage samples is by shooting branches
down with a shotgun. There are other methods, but this will be the easiest. Be sure to follow all
safety regulations and obtain the proper permits to shoot around the covariance towers.
Samples are collected on a species basis in each subplot. One tree of each species in a subplot is
sufficient. Collect samples from the top, middle and bottom sections from each tree’s crown and
place in labeled bags. Typically only two to three leaves per section of a tree’s crown will be
needed as not much material is analyzed for carbon and nitrogen content, though pine needles
typically need to have a significant amount collected due to their small size. You do not need to
be very specific when retrieving samples from the different areas of the crown; just make sure to
be in the general area of the top, middle, and bottom.
The remaining procedure for analysis is largely the same as for soil samples. After drying the
leaf samples in individual brown paper bags in an oven at 60 degrees C for at least 48 hours,
grind up each sample (which is not an individual leaf but rather all the leaves you collected from
one level of the crown of one tree) and place it in a centrifuge tube. Because bulk density of the
leaf samples is not being taken, you do not need to weigh the dried samples. You also do not mix
the leaf samples together. Each leaf sample, after ground up, is put into its own container. The
leaf samples then need to be pulverized to a powder-like consistency. You can choose to go
about this with a mortar and pestle or a modified paint shaker, or any other way you see fit to
achieve a powder-like consistency. If the modified paint shaker route is chosen, which is the
simplest way, the leaf samples, like the soil samples, need to be in the microcentrifuge tubes on
the shaker for a total of 45 minutes in increments of 15 minutes. Once powder-like consistency
has been achieved, the leaf samples need to be rolled in the 5 x 9 mm tins, just as the soil was.
Although, the leaves need to be measured to 5-6 mg instead of 8-10 mg (with the weight of the
tin not included in the 5-6 mg). There also needs to be one empty tin per analysis to correct for
the content of the material that makes up the tin, as well as standardization tests. After all the
samples have been rolled in the tins and placed in a microtiter plate, the plate can be stored in an
air-tight container until the samples can be run through the flash combustion analyzer. When you
have received the results, testing of the foliage samples will be complete.
4.0 Final Words
Good luck with all of your sampling. Understand that you will run into problems. Whether the
problems are in the field or back in the lab, they will occur. The key thing is to keep going and
find a solution to the problem. All research is is problem solving. The thing that will help you
succeed while completing this research is being able to solve the problems that arise. If you
choose not to give up and to continue to work hard at your research, you will come out of this
with a very successful experience.