Fall 2015 Week 4

Lab has been so productive this week!

On Thursday (9/24/2015) I ran an experiment using the alcohol sterilization method I've been working with all summer on silver leaved nightshade leaves (S. elaeagnifolium). Four days later I knew there was a problem. On Monday I came back to lab and noticed that quadrants A-D all had 1-2 cm sized green, hilly fungal colonies growing on them, which is far too rapid of growth for endophytes.  Quadrant H also had a fungi growing on it that appeared similar to the one that digested my dead grasshopper. All the fungi were growing directly on the leaf suggesting it wasn't contamination introduced because of me but living on the surface of the plant. Based on the physical characteristics of these fungi, I believe they are species I've grown before.

I decided I needed to take a look at my chosen sterilant, 100% ethyl alcohol. When I asked Cori she said that, contrary to assumption, alcohol is a disinfectant not a sterilant, and even then it actually works better with a little water. Thus 70% alcohol is a better disinfectant than 100%. Clearly this isn't going to work the way I thought. I went back to the literature and decided to use bleach again, in the same manner as most current research. Last semester I had a problem with 1.25% bleach killing everything, including potential endophytes, so to find a concentration to work with in the future, I decided to make multiple strength bleach solutions and test which concentration would surface sterilize without penetrating the interior of the leaf. I await conclusive results but nothing has grown on any plates yet, a good sign.

I need to take more photos of my plates so I can remember what these fungi look like, even if they are contaminates. If I remember correctly the microscope photos I took were of spores from green contaminates I grew over the summer.

I made some photos of my plates. This is what I keep growing that looks like contamination:

This is what looks like the same thing that ate the grasshopper: It has the same black spores that bunch in heads and white mycelium. 

Fall 2015 Week 3

This week in lab I took pictures of possible endophytes in the leaves of silver leaved nightshade using a video equipped light microscope. I am attempting to compile some form of photo identification library for endophytes but am wondering if this is too large a scope for this project. Either way I need photos of my fungi in their native habitat so I will continue this for awhile.

One thing I've been struggling with while making my wet mount slides is the density of the leaves I'm looking through. They are so thick not much light is able to shine through making the photos dark and the structures indistinct. Also at very high magnifications small bumps and waves on the thick surface of the leaves translate to huge changes in elevations under the microscope. This means that to see the structure of a three dimensional object, such as a fungus inside a trichome, I have to adjust the fine focus of the microscope to get an idea of the structure. While this may be just a feature of using a microscope, I want to see what kinds of solutions I can come up with to make it easier for me to take 2 dimensional photos of 3 dimensional things. I am soaking a leaf in acetone to clear it and see if it makes it easier to see through the leaves.

Fall 2015 Week 2

This week in lab has been focused on introspection. Despite long hours contemplating, and many more hours at the bench learning techniques for it, I'm not entirely satisfied with my project. I'm finding it hard to stay motivated studying fungi when my mind so readily wanders to insects instead.

I want to change the topic of my project but I've already invested heavily in my current project. However I'm not entirely sure what a good, viable insect based project would look like. Thus I would be spending a large chunk of my time floundering around looking for a topic and learning completely new techniques before being able to really start a new project. The semester is already getting old...

A compromise would be to have a small side project, such as building an insect terrarium, to keep me occupied while I finish out my project. My endophytes are a great project with so much potential for growth, both personal and within the project. I'm just so uncertain.

I also moved my lab cabinet so I can access my supplies even when there's class. Here's a photo of all my lab supplies:

Fall Week 1

How time flies! School has been in session for nearly three weeks, all my classes are now in full swing and I'm finally getting back into the rhythm of things.
Most of this week has been spent catching up with all the lab people and getting to know some new faces. Other than that I've been using the light microscope to try and find endophytes in silver leaved night shade leaves before sterilizing and plating them. It hasn't been as easy as during the summer because the health status of the individuals I'm taking samples from keeps changing. First I thought they were all completely dried out and dead but, now my brother has started to water the front lawn, where these plants grow, there are tons of healthy, unstressed plants without endophytes. I feel like I need to control their growing conditions more stringently.

Here is a photo of Silver leaved nightshade or Solanum elaeagnifolium:

Summer Week 12

This week I have not been in lab. I haven't even been in Phoenix for over a week. Hurray!!!

Instead of sitting in a boring, stuffy lab all day (haha not really), I have been running all around Colorado. My friends Luke and Jason moved here over two years ago and this is the first time I've seen them since then. Its been so great getting caught up with them and adventuring around Colorado.

I'm looking forward to getting back in the lab when school starts again. For now, here is a photo of beautiful Boulder:

Summer Week 11

I'm doing my blog early this week because I'm headed to Denver WOOT!

I anticipate most of my time will be spent working on my final project. Thankfully the structure of my project is similar to the one I did in the Spring, so much of the background information and literature citations do not need to be reexamined.

One thing that I've been more concerned about since looking at my past research is my sterilization method. Most of the literature concerning endophytes says to use a low concentration of sodium hypochlorate, bleach, to sterilize the surface of plant tissues. I, however, found that the bleach was so caustic that it killed all possible endophytes.

In this table from Spring 2015 shows what kinds of fungi I grew based on how long I soaked the tissues in bleach. C means contaminate, + means possible endophyte and - means no growth at all.

As can be seen after even one minute of soaking in bleach nothing is growing on my plates. There are two possible explanations for this: the bleach solution used last semester was incorrectly diluted making it more concentrated than expected, or bleach is caustic to fungi at any concentration.

I have been getting better results using 100% ethanol for 5 minutes but have only found one other paper that uses exclusively ethanol, which makes me wary...

Summer Week 10

This week in lab has been spent in contemplation of my summer. As it nears to an end, and I begin to write my final paper, I am compelled to think about all I have accomplished:

• Staining plant tissues to identify endophytes microscopically
• Perfecting my sterilization technique to include the laminar hood
• Moving my fungi from bengal agar to potato dextrose agar
• Using tape mounts to stain fungal structures 
• Using super cooled ethanol to freeze dry fungal tissues
• Extracting DNA with a kit

I certainly have had a productive summer. But I think the most important thing I did this summer was sit down and discuss with Cori how to organize my data. I have been struggling with this since I got into this program. What to write down and in what format. Cori really helped me understand how it is people in labs organize data and helped me make more sense of my lab notebook. She's a great mentor

This is a video showing my freeze drying setup