Natalie's Blog

Blog Post 1

For the past two weeks, I have been fully immersed in the world of fungi over in the Peay Lab in the Herrin Biology building. Prior to my internship, all I knew about fungi was that they were different from plants and had their own Kingdom (the others being animals, plants, bacteria and archaea). Now, after two weeks in the lab working with and reading about fungi, I not only can define what fungi are but also their importance as a subject for scientific and ecological study.

Prior to my internship, the only type of fungi I knew of was mushrooms. I now know that the mushroom one sees in their backyard is just the fruiting body of an organism mainly underground in a complex lattice of thin almost-microscopic roots called hyphae that compose the mycelium or root system of the fungi. The mushroom or the fruiting body of a fungus simply serves to disperse its spores (produced in the gills found beneath the mushroom cap) above ground so that it can reproduce. However, as I quickly learned my first day in the lab, there are other types of fungi besides mushrooms, namely the ectomycorrizal fungi that I have been working with as part of graduate student Laura Bogar's research project.

Ectomycorrizal fungi are just as their name suggests: they live on the outside (ecto) of the roots (riza) of a fungus (myco) in a symbiotic relationship with a plant. In the case of Laura's project, the ectomycorrizal fungi have a symbiotic relationship with pine sapling roots. Laura, however, wanted to know how much fungi actually benefit from their so-called "symbiotic" relationship with pine trees. To test the biochemical effectiveness of this mutualism, she collected and grew many pine saplings (~140), grafted fungi onto the roots and divided the root system of each individual sapling into two different containers where one fungus, the "good fungus" had been exposed to nitrogen while the other "bad" fungus (of the same species) had not. The pine saplings were then placed into airtight chambers in which the nutrient and oxygen flow could be controlled. The isotope Carbon-13 or heavy carbon was then pumped into the chambers so that later in the experiment, Laura could quantify the amount of photosynthesis by checking the carbon levels in her root-and-fungi samples and thereby quantify how much nutrients the fungi gave to the saplings and vice versa.

I joined this project at the end of the "root dissection" phase in which half-root systems of pine saplings were sorted under the dissecting scope in order to identify different types of roots present. These root types-absorptive, coarse, "happy", "unhappy" and other-serve to categorize the roots based off of the presence of fungi (happy vs. unhappy) on the root ends of the roots while absorptive vs. coarse categorize whether or not "uncolonized root ends" (places where mycorriza or fungi could live) are present. For my root dissections, I would divide a given half-root system into three subsections and from each subsection take out five random roots with forceps. I would then scrutinize the roots under the scope and categorize them into labeled coin envelopes and put all the remaining roots into the "other" folder along with any alternative roots. I also took care to take out any of the artificial nutrient pellets such as perlite (white chunks) and vermiculate (golden flakes) from the root systems before putting them into coin folders.

After two days of intensive microscopy for the sake of root dissections, I was introduced to the world of root weighing. I had already done a pipette tutorial in which I had to use a finicky microbalance that never seemed to want to settle on any hundredth of a milligram value and thus knew to expect flickering data values for the root samples. My protocol for root weighing was as follows:

Wipe down the working counter space with ethanol and take care to also sterilize the forceps. Take out a piece of weigh paper and make sure to tare the microbalance to it. Carefully open the coin envelope of roots (taking care to record the root system number and root type on the data sheet before taking measurements) and empty all contents onto the weigh paper, scraping out any stuck root bits with the forceps when necessary. Place the weigh paper with roots and wait until the weight value seems to have stabilized at a particular value to the hundredth decimal place; take out the root sample and if absorptive, "live" (for the happy or alive mycorriza samples) or "dead". Pour some of the contents of the weigh paper into a test-tube with glass beads (so as to expedite the coming grinding process) and pour the rest of the contents of the weigh paper back into its original folder. REPEAT.

Although root weighing may seem extremely repetitive and dull, it provides critical information about the mass of the different root types that will be used in later stages of the project in order to find the ratio of carbon content to the entire mass for the different root systems (essentially determining the answer to Laura's research question). I also got to do data entry from all of the root dissections in Excel, which allowed me to have a much better sense for the sample size and scope of Laura's experiment than if she just tried to explain it to me in words.

The next step of Laura's project is to "tin-ball" the weighed root samples (the samples ground up when necessary). To tin-ball a sample, one must take 2 mg of sample simple and insert the material into a premade tin ball (thus the name) and fold it in such a way so as to not let any of the sample escape. Tin-balling is necessary for data quantification procedures such as gas mass-spectrometry, in which the root samples will be incinerated so that the speed of the particles moving around in a separate chamber can quantify the presence and abundance of certain particles such as Carbon-13 (since the heavier isotope of Carbon moves slower relative to other particles at a molecular level). Although I will participate some in the tin-balling stage of the research project, I will have already made a transition into the world of culturing with my supervisor, the lab technician Nora Dunkirk.

Thanks to Nora I have read multiple interesting chapters on ecology and fungi these last two weeks, since working in the Peay Lab requires good understanding of both ecology and the study of fungi (mycology). These two fields are intrinsically linked to terrestrial ecology since fungi cycle the nutrients and help support plant life necessary for healthy ecosystems (e.g. forests). Nora is in the process of working on a massive research project of her in own in which she studies the different types of fungi that grow on dung.

Where I fit into this equation is that in order to identify the different types of coprophilous fungi (copro = Latin for "dung" while philous is Greek for "loving"), Nora needs to sequence the DNA of her specimens. Rather than just sequencing the genes of the fungi, which would require the typical PCR (polymerase chain reaction) protocol used for general DNA extractions and amplification, Nora wants to sequence the entire genome, which equates to hundreds of base gene pairs of DNA. In order to get enough tissue to sequence the entire genome of any particular fungi, Nora needs to make many new subcultures, requiring the use of cellophane. Just today I had the opportunity to make cellophane plates in the blower hood, where I carefully used sterilized forceps to lift out individual squares of cellophane that I had previously prepared and autoclaved (to sterilize) with the help of Nora. I then carefully placed the square of cellophane onto an agar plate and repeated the process many times. I also cultured a couple of previously made plates with some "fungal plugs" (the name of chunks taken from fungal cultures for reculturing).

If I had to say what the most surprising thing that I learned was in these last two weeks, I'd say the fact that lichen, an bacterial/algae-like organism, has a symbiotic relationship with fungi. This fact was reinforced by a talk by a visiting graduate student that specializes in ecological research on lichen that I went to with Laura in the Herrin Building. Finally, I've already taken to thinking of things as being the "mushroom" of a fungus rather than the classic expression the tip of the iceberg, which reflects that I still have a lot to learn about fungi these next few weeks.

My work station for root weighing in the lab

Blog Post 2

Fungi can be fluffy. That's just one of the many observations I have made while helping to culture, identify and quantify different species of fungi with Nora the last couple of weeks. Although I have worked principally worked with Nora (when not learning about and studying geomorphology with Professor George Hilley at the Marin Headlands or visiting the Hopkins Marine Station in Monterey), I have also continued to help with Laura's project by learning the art of tin-balling.

Although I covered the big-picture scientific function of tin-balling in the previous blog post, I had yet to experience the painstaking precision I now associate with the word "tin-ball":

To prepare small tinballs of tissue between 2-3 mg, I use forceps to carefully select a premade tin-capsule out of its container and tare it on a microbalance with actual micro-scales (see image below). I then carefully pinch powder from its weighed tube into the tiny opening of the capsule, gently squeezing apart the opening with the forceps if necessary. I then measure the filled tin capsule on the microbalance to see if the sample mass is in the desired range. Although this process may be reminiscent of the root weighing protocol described previously, the fine motor skills required for pinching and "Z-fold"-ing the tin capsule in such a way to ensure that no powdered roots escape is not trivial. After all the necessary folds are made and the former capsule is squished so that it appears in the shape as a cube ("tin-ball" is a bit of a misnomer for this capsule). The tin ball is then checked for "tags" or rough corners or edges that could potentially hinder the ball from dropping out of the carousal in the incineration/spectrometry phase of the experiment. Finally, the tin-ball is re-weighed on the microbalance to ensure that significant root powder was not lost during the experiment.

I have helped make tin-balls for both control samples with unenriched (or no C-13) tissue and some enriched samples in order to help Laura get enough data for analysis with her hope of presenting at conferences later this month. While I have may have written in great detail about tin-balling for Laura's symbiosis project, the majority of my time in the Peay Lab since the last blog post has actually been spent in the realm of fungal cultures with Nora.

I have now helped to culture three different genera of fungi (podospora, pilobolus and mucor) via two different methods over the course of my internship. For the podospora and mucor, I used the standard "plug" (or what I call "chunking") method in which I use a sterile scalpel to cut a square of a fungal culture and plate it on a new plate of agar. Alternatively, for pilobolus, I use a sterile needle under the microscope to find pilobolus sporangia in order to plate the little speck of potential fungal material onto a new agar plate. I intentionally write of the above fungi in terms of their genus as their species has yet to be identified via DNA sequencing.

While I have not done PCR or DNA sequencing, I have had the opportunity to do an entire RNA extraction protocol for three fungal cultures (coprinellus pellucidas, coprinopsis sclerotiger and podospora curvicolla) in Nora's dung fungi experiment. RNA or ribonucleic acid provides genetic instructions for the construction of proteins in a cell and thus yields critical information on the diversity and functionality of proteins within a cell. The purpose of RNA extraction then becomes to discover what proteins are more prevalent and active for a specific fungal species. RNA extractions are more difficult than their DNA counterparts due to the extremely unstable single helix structure and tendency to fold inwards on itself. Entire buckets of ice and liquid nitrogen were employed to keep the RNA at a low temperature and therefore stable during the extraction process. By doing the RNA extraction, I got to learn how to use the centrifuge (similar to other shaking devices I have used in the Peay Lab) and also saw the need for incredible sterility in the form of RNaway and Bunsen Burners. Previous DNA quantifications via the Qubit definitely made me appreciate the scientific steps that allow such data to be collected at all.

Since performing the RNA extraction, I have quantified the RNA with the Qubit (only to have it read that "the concentration is too high", indicating that the RNA needs to be diluted with TE (a buffer I helped Nora make)) and quantified the DNA for cultures Nora had already quantified but found inadequate. The contrast between the two data sets for the DNA and RNA extractions reaffirmed that genomics requires a lot of repetition in order for enough fungal tissue to be quantified, particularly as my supervisor aims to sequence entire fungal genomes as mentioned in the last blog post. I have found the need for "redos" also in the world of culturing, as I have now cultured pilobolus twice with Nora: our efforts the seventh and eighth times attempted to culture the feisty fungus successfully.

Amidst all my work with fungi, I have also enjoyed getting to read more about ecology-specifically on the relationship between evolution and ecology-and sharpen my ability to analyze data sets presented in graphs both in the textbook reading and the emerging ones from Laura and Nora's projects in the Peay Lab. Also in addition to culturing new fungal species, I have had the chance to see Nora's seven (DNA) sequenced fungal species and investigate their fungal features both at macro and micro perspectives under the dissecting scope. The most interesting fungus for me tied between the slime mold phaesarum that experiences "plasmodial streaming" [see image below] due to its plasma structure and the black fungi species with the coolest name bombardia bombarda [see below]. Finally, I enjoyed learning how to take pictures of fungi under the microscope with special software in the lab and I hope to use some of the microscopy pictures as well as pictures from my work around the lab in my upcoming presentation.

Figure 1: a tin capsule and forceps on a mirror

(used to keep track of root powder)

Figure 2: the microbalance (notice the micro scales)

Figure 3: Pilobolus the Feisty Fungus

Figure 4: Podospora the Fluffy Fungus

Figure 5: Mucor the Ferocious (out of focus) Fungus

Figure 6: Phaesarum the Plasmodial Slime Mold (it eats oats)

Figure 7: Nora's Drawer

Blog Post 3

Abstracts, Pt. Reyes and Mushroom Families, oh my!

It's hard to believe that just eight weeks ago I only thought of supermarket mushrooms in response to the word "fungi". Thanks to this internship, I have had the opportunity to culture many different genera of fungi, quantify and extract DNA and RNA, dissect root systems to look for fungal symbionts and even make tin-ball capsules with forceps and a microbalance. And thanks to all the papers Nora gave me over the course of the internship, I am knowledgeable not only about fungal ecology but also about ecology in general and how to design ecological experiments, which was ultimately one of the things I hoped to gain out of this experience.

I remember wanting to work with the Peay Lab for this internship back in the spring because of their motto "Ecology from the Ground Up". Even though I knew very little about fungi before this summer, I correctly anticipated back in the spring that I could learn about nutrient exchange and fungi genomics as well as field experimental set-ups, all of which tied in well with my past interests albeit fungi. Regarding field experiments, I had the opportunity within these last couple of weeks to work just with mushrooms through my work with the Mushroom Archive.

The Mushroom Archive

This "the Mushroom Archive" a.k.a. a cardboard box filled with hundreds of mushroom samples from Borneo collected by Kabir previously unorganized are now categorized in Ziploc Bags by their families and individual specimens labeled by genus. To do this, I spent hours searching tag-names of the bagged mushrooms in the Excel spread sheet in order to look for their family and genus within their taxonomy (remember King Phillip Came Over For Good Spagetti?) since the ITS and LSU regions of the fungal genome often give only enough information to verify a genus rather than a species. Through doing this, I got exposed to over twenty different fungal families and countless genera. Most importantly, however, I got to see just how diverse mushrooms can be morphologically and how DNA sequencing can validate family classification far different than what a trained mycologist might guess just visually.

Besides working on the Mushroom Archive, I spent these last few weeks tying up loose ends in the lab - namely working on my abstract and poster for AGU. Writing my abstract helped me realize just how complex the two experiments are that I have gotten so used to in the lab and how much I have learned as well as have yet to learn. The catch to the two experiments I worked on is that both have made major headway this summer, neither of them are truly finished. Both Laura and Nora have presented on their experiments at conferences, so while I may lack data sets and graphs for my AGU poster, I am confident I will be able to make a good presentation out of my work this summer. Although I only helped with parts of both experiments (which is to say I didn't do PCR or gas mass spectrometry), through focusing on specific skills such as pipetting (for DNA/RNA extractions and quantification), culturing and forcep-work, I have much greater confidence in my ability to execute a biology experiment in tandem with ecological perspective (i.e. symbiosis, species diversity, morphology, etc.).

Tulk Elk watching us wearily at Pt. Reyes

On top of everything else, I had the awesome opportunity drive up to Point Reyes for a day of fieldwork where I learned firsthand how Nora collects her Tule Elk dung. It sounds gross but waiting for the contemplative Tule elk to lose interest in us strange humans on the hill so we can collect fresh dung samples is surprisingly fun.

Finally, I'd like to thank Jenny Saltzmann and Megan D'errico for coordinating this internship program as well as everyone in the Peay Lab (mainly Nora and Laura, of course) for always being willing to let me participate in their projects through data collection endeavors, sterilization, data input and ask them questions not just about their own projects but about fungi and life in general. I fittingly referred to my experiences this summer in my AGU abstract as a "foray into fungal ecology": a foray that I will never forget.