Leaving The Lab, But Not The Roots

As the semester has wrapped to a close with the rest of my collegiate career, I just want to say thanks to everyone who helped me and expressed their interest in my work over the past 6 months. It has been such a great experience working in the soil lab and I really thank my mentor, Dr. Chaudhary for giving me such a great opportunity. I will be continuing the blog as best I can, even though I will no longer be in Chicago to do much lab work.

This week I have been trying to clean the lab and get some of the root scoring accomplished. So far it has been very interesting-we have seen a lot of mycorrhizal colonization, as shown by the high number of hyphae, arbuscular, and vesicular intersections that I have encountered in the first 10 samples that I have had the time to score. Some of these slides come from the newer, tiled green roofs, which is a good sign!

I will be continuing to work on this data for much of the next semester, so hopefully I will be able to share a bit of that. Until then, I will be taking a little break to celebrate my graduation from Loyola University Chicago and to plan my next move. Thank you for joining me!

Mycorrhizal Root Scoring

After wonderfully busy break, I am back in the lab, trying to finish making slides of the stained roots from the bioassay. I have around 10 more samples left to mount on slides, but I ran out of coverslips for them last week. In the mean time, I am working on root scoring.

The root scoring involves looking at the slides under a compound microscope and making a tally of the different structures found at 100 intersections of the roots. I start in the same location of each slide and then look at the structures present at regular intervals on the slide. In particular, I look for arbuscules, hyphae, and vesicles at each intersection as well as any non-arbuscular mycorrhiza. As the chitin is the part of the roots that actually stains, structures stained blue on the slides indicate that fungi is present. A really interesting website that shows pictures of the structures that I am looking at is the International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi (INVAM). It can be found at: http://invam.wvu.edu/. My first scoring attempt took a while and I am hoping to get faster with more practice and brushing up on the images found on the INVAM site.

 We are hoping to start by scoring all of the samples from the more "extreme" roofs, those that are oldest or youngest. The comparison between these should highlight some major differences between the mycorrhiza of different green roofs such as those that are tiles of sedums versus those that are prairie plant plots. The next couple of weeks will be a bit of a blur, however, as I have final projects and exams. If you are in the Loyola community and would be interested in learning more about mycorrhiza, feel free to stop by the 3rd floor of the Quinlan LSB between 1:00-2:15 on Thursday, December 5 to see a poster presentation for my evolution project on mycorrhizal symbioses!

Root Staining and Slide Preparation

Finally I have a more project-oriented post! As mentioned last week, we have stained the roots harvested from the bioassay. We used a protocol very similar to the following for staining:
http://tmemam.weebly.com/uploads/1/3/0/2/13027468/mycorrhizal_staining_and_root_colonization_protocol_web.pdf .

After following the protocol, the roots remain in the jar of what seem like pickled cassettes until I make each cassette into a slide to view under the microscope.

While the liquid is very purple in color, the roots are actually stained black, enabling us to see the mycorrhizae present. I then use the PVLG to mount the roots on slides, 10 of which are completed and drying for viewing. 

Once I have made slides for all of the bioassay root subsets, I will work on making slides of the sedum/field roots that we gathered from the roots in the summer. The two types of slides can then be compared. We will be following the intersection scoring method to quantify the mycorrhizae in each subset-more about that to come. 

I know you can't exactly see the roots on these drying slides, but once they are actually ready to be seen under the microscope, I'll provide some better pictures. The PVLG is pretty sticky, so the slides can't be viewed until the slides are in a more fixed position.

While the next couple of weeks will be very chaotic with the winding down of the semester and Thanksgiving, I will try my best to keep posting!

Busy Days

I know this post is a week late, but I am just providing an update in the meantime before getting back to the more project-related blog posts that I typically have.

In the lab, we have mainly been working on cleaning and staining the roots from the bioassay in order to mount them on slides for us to do counts of the mycorrhizae present when viewed under a microscope. I will provide the exact protocols in next week's blog post. I am also about to start a side project for my evolution class that will explore the evolution of mycorrhizal symbioses, which should be a very interesting and helpful bit of knowledge to have.

My days have been especially crazy as I am applying to graduate school and am working on polishing my application essays and CV, and I finally have enough letters of recommendation to apply! Last Friday, I visited Penn State University again, which is my top choice for grad school. I am looking to apply to one of the integrated biology programs there, particularly in either bioinformatics & genomics or genetics. After meeting with some really great potential advisors, the plot has thickened, and I am not entirely sure of which program I will be applying to. I am sure it will become clear with a couple more weeks of personal reflection and discussion. If anyone has any advice in the graduate school application process, please let me know.

Soon I will be able to provide some more root-related information! Until then, in the words of Thomas Huxley, "Try to learn something about everything and everything about something".

Root Washing

In the plus column, I have washed 35 samples of roots. I'm over halfway finished washing the roots from the bioassay! It has been quite interesting to see the variety of masses and volumes of the root systems. When looking at a plant, it is very easy to focus on the quality and size of the above ground mass. I have learned that the appearance of the shoots can be very similar, but the root systems of the plants can look completely different. Furthermore, their volumes may look very similar, but they may have a huge variance in masses. By huge, I mean a couple grams. This is quite a bit when the average mass of the roots remaining after the cassette's subsample is made is only 1.5 grams.

On the other hand, we are far from being finished with these roots! There are 25 samples left to wash. The remainder roots from the washed samples are still in the drying oven along with all of the shoots. Once all of the samples have been washed and dried, we will be able to draw conclusions about their composition. It has been pretty hectic for me, both in and out of the lab, so hopefully by the next post, I will have more of an update. In the words of the Dalai Lama, remember, " The roots of all goodness lie in the soil of appreciation for goodness". Washing these roots has given me a much greater appreciation  for the less obvious aspects of growing; all of the unseen parts are still a part of the big picture. 

Harvesting

On Wednesday,  October 2nd, we harvested the Zea mays bioassay that had been growing in the conetainers for 5 weeks. The 59 samples that survived were around a foot and a half tall and healthy, so we knew that the root systems were pretty strong. We were a little bit sad for our experiment to be leaving the Ecodome, but it will be moving on to bigger and better things!

RIP Zea mays bioassay August 29-October 2

The first step was to separate the roots and shoots of the sample. We harvested the plants by putting the shoots in paper bags to dry in the drying oven and then placed the roots in freezer bags in the freezer to wait until it is their turn to be washed. 

The latest task has been to wash all of the roots for staining and mounting on slides for us to examine. Today, while the Environmental Institute was empty and quiet with everyone on fall break, I made the polyvinyl alcohol-lactic acid-glycerol (PVLG) that we will be using to mount and preserve all of the roots so that they can be stored for around 20 years.  We will view these slides under a microscope and quantify the amount of mycorrhizae present in the roots, using a system that Dr. Chaudhary taught me and I practiced over the summer. 

Getting the roots washed and sorted for slide placement has been one of the more time-consuming processes that I have had to do with this project, which is ironic because the slides are the smallest-scale aspect of the project. For starters, we have 60 samples that need to have their roots washed and freed from the soil, detritus, and rocks that they grew into. This involves several transfers.  Below is a root sample after 2 washes, note the brown specks of soil that are still in the water.

Samples are then laid out and cut into 1 to 2 inch sections to ensure that we are getting an accurate representation of the roots included in each slide. Using forceps, I then take samples from each section of the sample to go into the root cassettes for the time being. Once the PVLG is ready and I have finished washing all the samples and they are cleaned up a bit, we will be able to mount them onto the slides.

As one can see, the root cassettes don't end up holding a large amount of roots, but the rest of the roots are not wasted. We have been recording their freshly harvested mass and then putting them in the drying oven with their shoots, so that we will be able to measure their organic carbon mass once they have dried. 

This process of washing and slide preparation will probably take a couple weeks for me to complete, but hopefully I will be able to post some graphs and other aspects of our project. I am about to begin delving deeper into the evolution of mycorrhizal fungi to supplement the current research. If you see me  and I have soil under my nails in the next couple of weeks, I apologize, but according to Anglo-Saxon law, "Whatever is affixed to the soil, belongs to the soil". Keep letting your roots spread deep, readers! 

Bioassay Part 2

Well, the bioassay is still going strong in the Institute of Environmental Sustainability Ecodome. We have only had one casualty in the 4 weeks that the corn has been in there. While they've been in there, we have had everything from 95 degree days to 60 degree days. There have been days where the experiment has needed watering twice a day and days when the soil is still moist after 24 hours. Okay, before I start sounding like James Taylor's 'Fire and Rain', I think you get the picture.

The plants look pretty good-aside from the purple stems and some withered leaves. This could be due to either/both a lack of sunlight or nutrients. We will hopefully have a better answer once we harvest the plants in the next couple weeks. To do so, we will pull the plants out of the cones and separate the roots from the stems/leaves. We will then be boiling the roots to clear out their interiors, then dyeing them for viewing under a microscope. We will be taking the rest of the plant and placing them in drying ovens for a couple days in order to measure the organic carbon that they are composed of. I will post links for these experimental methods once we harvest! 

I am also continuing to make graphs to see the correlation between different variables for the roof plots from which the samples were taken. Starting to see some really interesting graphs that will certainly contribute a lot to our experimental results. 

Planting

Thursday, August 29, we began the bioassay experiment.

The purpose of the bioassay is to study the impact of the different soil samples from Loyola's green roofs on the growth of mycorrhizal fungi in the soil. We germinated the Zea mays before planting it in the conetainers of soil, and had to replant a couple on September 3rd as they had not yet sprouted. I took note of which samples those were, and now most of the samples look healthy and strong.

It's been so fun to watch these guys grow up! I will post more detail about the qualities of plants of different roots soon-as they are all the same species of plant, it is pretty easy to see subtle differences in the leaves and size of the individual samples. With the opening of the new Institute of Environmental Sustainability that houses our research lab, it's been a little chaotic to gather a lot of data!

Beyond watching the bioassay, we have been working to add more data elements to the data sheets we have been creating all summer regarding the different green roofs. As with all science, there is much to do and so many great ideas of things we can do, but time also flies by!

In the words of one of my favorite scientific creatives, Albert Einstein, " If we knew what we were doing, it wouldn't be called research, would it?"

                             

 

Here We Go!

As today was my first school day of my senior year, it is also my first day as an official undergraduate researcher in the soil lab of Dr. Bala Chaudhary. Therefore, I am posting some details and goals of the project that are long overdue, as I joined the research group in June!

The project focuses on the mycorrhizal fungi of green roofs on Loyola University Chicago's campuses. So far we have taken soil samples and stability measurements at a variety of locations and elevations and are now trying to determine the fungal diversity of a variety of green roof types-from pre-made sedum tiles to prairie plants planted 5 stories up. We hope that our findings on the fungal community can be useful in the implementation of green roofs, improving their sustainability, and providing guidance on options for green roofs.

Using a bioassay of corn, we hope to see the diversity of fungi that associate with the corn plants. Today I started germinating the Zea mays in the hopes that we can grow the corn in the next couple weeks in Loyola's new Ecodome in the Institute of Environmental Sustainability. I will have more details about the specifics of the project as our lab, the Ecodome, and Institute itself are finished and ready for some very interesting research!