Preparing Soil Samples for the Flash CN Analyzer

How to Prepare Your Soil Samples for the Flash CN Analyzer
a Tutorial by Sarah A-J

So, you have just collected your samples, good job! Now, the lab work. First you will want to sieve and grind your samples.  You probably will want a total of around 3-5mL of sieved, dry, ground soil to work with. Depending on how rocky your soil is, you might have to sieve more or less soil. For me, to get around 5mL, I had to start with around 20mL of soil. I used a 2mm sieve.
After sieving I proceeded to dry my samples at 65 degrees Celcius for 2 days.
Post drying, I ground my samples twice using a mortar and pestle. Twice because I was unhappy with how fine the sample was after the first grinding. Because the final sample size that I will use in the Flash CN analyzer is only about 5-15mg, I think the sample has to be very fine and homogenized.

 

From upper right to lower left, you can really see the difference between unsieved, sieved, ground once, and ground twice. 

Once your soil is all ready, you have to prepare them to drop into the Flash analyzer. Make sure you have all the nessicary equipment and stuff before you begin. Also, it's most time efficient if you plan to prep a large batch at a time. Don't prep the samples and then store them for a few days. Try to prep them and then put them in the analyzer soon after. 

You will need tin sheets or pre-made tins, two tweezers, ethanol (for cleaning), a scupula, a high precision balance (preferably to 6 decimal places).

R.I.P. Beloved Muffle Furnace

It's a sad time here in the third floor lab. Dr. Chaudhary's long term lab companion is being moved to lab equipment hospice care: the Thermolyne 62700 Muffle Furnace. Right when I need to use it, too! Grrrr, lab equipment. First I can't get the Flash CN analyzer to work, and now our muffle furnace is on the fritz.

 

 

This is not what you want to see when you are planing to run your samples. So, we have a second, larger furnace in the downstairs lab, but it is also in a bad way. This second furnace is "new to us" and, as such, is missing quite a few parts, including internal shelves and a plug. A freaking plug! The back of the machine looks pretty naked without a plug to hook up to an outlet.

I've contacted ThemoScientific and they said that the thermal control panel needs to be replaced. Unfortunately, this part costs 640$, and that's uninstalled. I'm like, "Okay, where's that money ganna come from?" Dr. Chaudhary said, "No way. That's not ganna happen."

Oh, well. I guess I'll be putting all my eggs in the lets-just-get-a-new-plug-for-the-other-furnace basket. That or I'll have to face the Flash again (ugh *shiver*). I'm really not as upset as I may sound. It's just that I was hoping to run my soil samples back in October, and it is now half way through November, but all you can do is go with the flow.

Presentation to Senn High School Students

This last Monday, Susanna and I presented about green roofs and our experiment to three groups of ecologically minded Senn High School students.

We prepared our talk for two weeks before hand and were rewarded with three great sessions. Of course there were a few students who were falling asleep (tip: if you're going to sleep, don't sit in the front row), but most of the students seemed interested and engaged. I was nervous before hand, and at times the talk got a little bumpy, but over all it was really rewarding when I could catch someone's eye and they were like, "hey, I'm listening to what you're saying, and I like what I hear."
Here are some of my favorite slides:

Thermochron Replacement and Soil Sample Collection

Two weeks ago, Susanna and I made our way back to the green-roof after an unintentional hiatus. The time had come to change out the thermochron ibuttons. They can hold 2048 temperature readings meaning we could leave them out for about 12 weeks before needing to retrieve the data by connecting them to the computer. After downloading the data and starting new missions for each thermochron, we replaced them into their respective trays.

Susanna replanting the thermochrons.

Because we were both out there, we decided it was the best time to collect the soil samples that we both will need for our lab tests. Susanna will be testing the water retention capacity and I will be measuring soil carbon and nitrogen levels. We collected 100 mL of soil from each of 40 trays, giving us more than enough lab work to grind through in the coming weeks.

Me, posing while collecting soil samples.

The plants are looking pretty good:

Green-roof trays of prairie plants.

 Here are some action shots from the afternoon.

Planting thermochrons

Thermochrons and soil collection

Soil collection

Fall Green Roof Research Intro

My focus of our project is on soil carbon content. I have two experimental questions. First: will added live versus sterile inoculum promote an increase in soil carbon? Second: do sedum species versus native prairie plants lead to higher soil carbon levels? To answer these questions I must be able to accurately measure the carbon in the soil. My goal this semester is just that: get reliable soil carbon data for our experimental trays by using the Flash CN Analyzer. 

Before I can run our real samples, I have to practice preparing samples and running standards and samples with the analyzer using excess soil. This coming week, Susanna and I will collect the end-of-season soil samples for our experiment. From there I will need to grind the soil, finalize the Flash analyzer protocol, and begin running samples.  The Flash Analyzer, which I described in detail in a previous post, is intimidating, but I am excited about the potential data sets we could get; that is if I can get good at using the machine.

This semester, I also want to work on scoring the MIP root slides for our mychorrizal data, but this is secondary because the slides are easily preserved where as I need the carbon data to continue my project. I am also looking forward to helping Susanna with her project which deals in part with soil water retention. 

Summer Green Roof Research explanation

Susanna and I assisted with Kelly Ksiazek’s experiment that had two main questions. The first: does adding live versus sterilized prairie inoculum increase mycorrhizal fungal relationships and promote plant growth. The second: do native prairie plants function better as green roof organisms than traditional sedum species in terms of growth and green roof services provided.

We set up a grid of forty trays filled with soil substrate on the Quinlan Life Science terrace. Life prairie inoculum was added twenty trays and sterilized prairie inoculum to the remaining twenty trays. The inoculum was collected from a local prairie, half of which was sterilized to kill all bacteria and fungi.

Of the forty trays, ten were planted with sedum species, ten with prairie species mix A, ten with prairie species mix B, and ten remained unplanted as the control group. The ten trays of sedum were ordered from the green roof distributer and re planted in the experimental trays. Prairie mixes A and B contain different species of native prairie plants, grown in a greenhouse before planted in the trays. The control trays contain live and sterile inoculum but no plants.

Over the course of the summer we took measurements of plant growth, substrate temperature, soil stability, and soil carbon content. For the plant growth data, we measured month to month the individual plant’s max height, tray coverage area, and flower and fruit number. To measure the tray substrate temperature we inserted a thermochron in the center of each tray. These thermochrons were calibrated to take a temperature reading every hour for an extended period of time. Soil stability was measured using the soil slake test procedure. Soil carbon content was measured by ashing the samples in a muffle furnace. Future soil samples will be measured using the Flash soil carbon and nitrogen analyzer.

Possessed beaker!

 

After staining the roots, I had to sterilize the tissue cassettes by boiling them in water for a while. I added a stirring rod to the mix so the cassettes would stir themselves. It took some careful adjusting, but i got that beaker spinning like nobody's business. Also, first gif ever!

Root Staining

Yesterday, we began staining our MIP roots we had collected and washed over the last few weeks. Luckily for us, the new arbuscular ink and vinegar procedure is really simple compared to old fungi staining techniques. At its core, the procedure is only two steps. The first being boiling the roots (in tissue cassettes) in 10% potassium hydroxide (KOH) for 3-5 minutes. This clears the root cells of their contents, leaving the cell walls and fungi unharmed.

Boiling the roots in the 10% KOH solution.

The second major step is boiling the roots in a vinegar and ink solution for 3 minutes. This stains the fungal tissue within the cells, making it possible to see the hyphae (regular fungal body tissue) and vesicles (reproductive capsules).

10% KOH on the left hotplate and vinegar/ink on the right.

Intermediate steps include removing the cassettes from the hot solutions and rinsing them with water several times. Washing after the first step rinses the remaining KOH from the roots. Washing after the second step initially rinses the excess ink which will leach from the roots.

Susanna removing the cassettes from the solution into water.

Because we are boiling the solutions, the beakers and hot plates obviously get really hot. We got the chance to open and use our brand new thermal safety gloves. They are thick, orange, and terry cloth. Embarrassingly, this is the first pair of elbow length gloves I've ever worn.

Me, looking snazzy and being safe.

Our final product is a beaker full of stained roots, all ready to be mounted on slides. We are temporarily storing them in a beaker of water with a splash of vinegar in the fridge.

Purple haze, all in my roots.

Flash C&N Combustion Analyzer

Last week, we got a surprise present for the lab (surprise for me at least). A brand spanking new Flash Carbon and Nitrogen Combustion Analyzer!  The C&N combustion analyzer does just that: it analyzes the carbon and nitrogen in a sample by burning it at 980 degrees celsius and then 850 degrees celsius until the sample is completely vaporized. I know, pretty hot, huh?

The Flash C&N Combustion Analyzer.

Those two shiny cylinders are the furnaces. They get very hot, as hot as an iron at high temperature. I should know, I couldn't resist (everyone was doing it!). The white tube with red caps is the moisture trap, and the large box behind it is the detector.

Our auto-sampler with two carousels.

This is the auto-sampler. It holds the samples and drops them into the first furnace. Our auto-sampler can hold four carousels- 100 total samples! In the main part of the sampler, there is a piston that pushes the sample into the chrome tube underneath that leads to the furnace.

A carousel with 11 samples loaded.

The Flash analyzer can only process samples that are between about 5-100mg. That's very small. Unfortunately, to get acurate results, we have to buy a new balance for the lab that is accurate to the 0.01mg. Our current balance, that we just got at the beginning of the summer, is only accurate to the 1mg which is apparently not good enough for the Flash analyzer (well, la dee da). Once the sample is massed out in a small tin cartage, it must be balled up to fit into the machine.

Susanna, making a perfect sample ball.

In the first 15 seconds after the sample is dropped into the first furnace, an eerie red light flashes and glows from the eye of the auto-sampler. This is the reflection of the sample combusting at almost a thousand degrees celsius.

The burning eye of the auto-sampler is watching.

When the samples have finally dropped into the furnace, vaporized, and gone through the detector, the computer spits out this:

The not-so-impressive-looking C&N data.

It doesn't look like much, but the area under the peaks (the integral) tells us quantifiably how much nitrogen and carbon was in the sample. The first peak is the amount of nitrogen detected and the second peak is the amount of carbon detected. Once you understand the process and what the graph means, it actually is pretty awesome. You know, for nerds like us.

IButton fever

For the last few weeks we have been trying to understand how to use iButtons for our experiment. We got pretty frustrated because someone forgot to include a manual with the equipment (I'm looking at you, Maxum Integrated). IButtons remotely record the temperature, allowing us to gather multiple data points over a period of time. We were  eventually able to set them up to record the temperature once every hour, on the hour.

Susanna showing off her hard work.

We then planted the iButtons in a randomly selected trays of each experimental group. Each iButton was double bagged of course to prevent water damage.

Me, getting ready to plant an iButton.

Digging the hole.

A well planted iButton.

We also placed three iButtons on the bare roof to measure background roof temperature. Through these temperature measurements, we expect to see lower temperatures in the green roof soil compared to the bare roof temperature.

One of three hot roof iButtons.

After 2000 hours (that's all the storage the iButtons have) we will collect them and finally be able to see some data. After a few days, we downloaded the measurements from one of the bare roof iButtons to see if they were working. We were so excited to see beautiful oscillating data between night and day temperatures.

Corn harvest and root washing

Last week, we harvested the MIP corn. We had to do so carefully as to preserve the stalk and root tissues while also separating them for individual analysis.

Dr. Chaudhary demonstrating the harvest method.

We used clippers to cut the stalk at the very base and put it in a paper bag for later. We then put the conetainer, containing the roots, into plastic bag and into the freezer. We put them in the freezer to stop all biological activity, especially decomposition. 

We want to weigh and record the root tissue mass, but we first must wash all of the soil off of the roots (The roots are very dirty). We do this very carefully.

Gaze in wonder at my clean roots

We then collect some of the tissue to be made into slides and save the rest for weighing. 

MIP update and Why Corn?

A lot has happened over the last month, including significant growth of our corn seedlings for our mychorrizal infection potential experiment.

Our MIP corn has grown significantly larger since last post.

As you can see, the corn looks pretty sad though (excuse the anthropomorphism). The stalks and leaves are streaked with a very vibrant purple.

Isn't that purple almost fuchsia?

The purple is because the soils that we are testing [apparently] have very little phosphorus. While not much is known about phosphorus in the life cycle, plants do need small amounts to grow healthily.

Question: why are we using corn in our experiment? 

Through this MIP, we hope to quantify how much arbuscular fungi is present in the different soil types. We can only find out by seeing how many symbiotic relationships the fungi form with the plant roots in the soil. Corn is perfect for this because it is a great host for these mycorrhizal relationships. Corn roots are so happy for the fungi that if there is any fungus in the sample, the corn roots with make relationships with it.
This comes back to why the corn is purple. In the relationship between fungus and plant root, the plant gives up carbohydrates and sugars in exchange for nutrients such as phosphorous.

MIP hypothesis

Two [technically three] possibilities come to mind.

1. There is no phosphorus in the soil samples
2. There is no fungus to help the corn get the phosphorus
3. There is no phosphorus and there is no fungus

We shall conclude the MIP experiment soon and maybe find out what's really going on around here.

Also, say goodbye to the corn, because we are harvesting the MIP tomorrow. A.k.a., cutting the corn stalks at their bases and separating the roots from the soil. 

Green roof sucess

We were able to set up the green roof yesterday. It is not in its ideal location, but man is it great to see it outside :) It took a lot of work but we were able to pull it off (with no cursing!). A good attitude is half of the battle.
                               

Our green roof, finally set up.

Me, watering empty trays again.

Our roof garden with the city view.

Summary of :Erika Orberndorfer, Green Roofs as Urban Ecosystems

This bullet point list will be expanded on in the future.

Green roofs are beneficial in many ways

• increase fire resistance
• increase sound insulation
• increase longevity of the roof itself
• keep buildings cooler
• reduce rain water run-off
• habitat for insects and birds

Stresses on the plants include

• moisture stress
• drought
• high temperatures
• light intensity
• high wind speeds

The plants used have adaptations

• low, mat forming growth
• succulent leaves
• tough twiggy growth

Green roof soil

• mineral based
• light expanded clay granules
• crushed brick

Main benefits

• storm water management
• energy conservation
• urban habitat

Erica Orberndorfer, Lundholm, J. et al. Green Roofs as Urban Ecosystems: Ecological Structures, Functions, and Services. BioScience, Vol. 57, No. 10 (November 2007), pp. 823-833.

Mychorrizal Infection Potential

One aspect of our experiment involves adding inoculum to the roof garden soils to stimulate fungal growth. The inoculum we are using is soil collected from a prairie. This soil contains fungal spores and hyphae that may not be present in the roof garden soils of our main experiment.
At the end of our main experiment, to see if adding the inoculum changed the amount of mychorrizal relationships, we must have a base line measure of the fungal growth of the uninoculated soils.
We are using corn in our MIP experiment.
The first step of the MIP procedure was germinating the corn seeds. We used certified organic corn seeds because we don't want there to be any anti-fungal herbicide on the corn. We used deionized water in the germinating stage to avoid adding any extraneous contaminants to the developing plants.

 Corn kernels half immersed in water.

 After two days , the seed coats began to split and the first root, called the radical, began to emerge.

Root radicals emerging from the seeds.

We chose the seeds with the most developed radicals for our MIP experiment and planted them in our conetainers. 

Planting germinated corn kernels.

At the end of our MIP experiment, we will study the roots under a microscope and score the amount of mychorrizal relationships between the fungus and the corn roots.

Mychorrizal Infection Potential setup after one week.

Bicarbonate extractable soil phosphorus

The procedure for extracting phosphate looks more difficult than that for nitrates :(

OH NO!! Someone took these super helpful videos off of youtube! I can't find any replacements either. Uber sad face!

KCl extractable soil nitrate

Susanna and I are working on writing a protocol to extract ions from soil particles
into a solution that can then be analyzed by the ion chromatograph. This
guy from UCDavisIPO seems to know what he's doing. I am posing this
video for my as well as your benefit.


OH NO!! Someone took these super helpful videos off of youtube! I can't find any replacements either. Uber sad face!

This is the Ion Chromatograph:

The Ion Chromatograph.

Welcome to Frustration City, State of Aggravation. Population: 2, Susanna and myself. We have the exciting task of figuring out how to bend the will of this machine to our desires. Susanna, pouring over manuals and guides, and I, sifting through books and surfing the web, are searching for the answers.

High-precision Analytical Balance

On my first day, one of my tasks was assembling and calibrating this $7,500 analytical balance. I was so excited. Balances like this are by far my favorite laboratory equipment. It was shipped so neatly packed, as to be expected. It took me over an hour to set up and calibrate it but was completely worth it. This thing is accurate to 1mg. That's 0.001g! Astounding!

$7,500 analytical balance.

Before: crazy.

After: clean.

Also on the first day of the internship, Susanna and I unpacked and organized all of the equipment into the new lab space.


On the plus side, I was given my own lab workstation to use.

My humble workspace. 

Pre-internship work

The sedum and native prairie plants that we are going to use for our experiment arrived several weeks ago and needed to be planted quite a while before the start of the internship. And so, we planted them in their respective roof garden trays as the experimental design dictated.

Planting the native species in a predetermined random configuration.

Sedum species planted to cover about 80% of the soil.

I proceeded to water the trays until the start of the internship. I felt rather silly watering the empty, control trays, but it was necessary. The experiment, and really every experiment, need to have a control group as a base line to compare with the treatment groups. The empty trays, containing only soil and inoculum, act as our control groups.

Watering the sedum as well as the empty, control trays.