Summer Week 9

This week in lab was spent learning to make microscope tape mounts. This is another, superior way of staining fungal cultures for investigation under the microscope.

When making a normal wet mount slide from a culture grown in a dish a researcher will scrape a small sample off the colony. Unfortunately this distorts the morphology of the mycelium, and bunches the sample into a mass, making observations fuzzy at best.

With tape mounts this frustration is eliminated. Instead of scraping the colony, a strip of tape is gently pressed against the top of the petri plate, preserving the shape of the specimen.

Here are some photos of a colony that was sporulating:

Summer Week 8

For the first time in my life I extracted DNA! Naturally, I started with one of the most notoriously difficult organisms to extract DNA from- fungi. Fungi are similar to plants in that their structure and support comes from rigid cell walls. However, in fungi the macro-molecule that the cell wall is made of is usually chitin instead of cellulose.

These macro-molecules must be broken down to extract any DNA, which is difficult because of how tough they are. Broadly, there are two ways to break down the cell walls; chemical or mechanical. Chemical degradation of the cell walls would be things like adding enzymes whereas mechanical is more grinding of samples or vortexing with glass beads to shred the walls. The method I used did a little of both.

To make grinding easier and more productive I flash froze mycelium samples in a bath of super cooled isopropyl alcohol. This makes the cytoplasm freeze and the walls brittle so grinding with a pestle is easier. After that I used an E.Z.N.A. Fungal DNA extraction kit that was full of buffers to further degrade the cell walls. I ran a gel on my resulting DNA, popped it in the UV viewer, and this is what I got:

Summer Week 7

This week in lab I did a variety of things.

I've noticed in the past that the agar in my petri plates will dry out and crack, a process called desiccation. To prolong the life of my endophytes, I have decided to move cultures to new plates. Because I no longer need to select against any bacteria or fast growing fungi I have decided to move my cultures to plates filled with straight potato dextrose agar, no antibiotics or rose bengal dye. So on Monday I made PDA and filled my plates with it.

On Tuesday I moved my cultures. I have also noticed that some of my plates will grow a black fungal contaminate which I assume is coming from the air. To prevent this from happening again I used the laminar hood when moving my cultures. Hopefully by sterilizing the air with UV light there will not be any contamination on my newest plates.

Summer Week 6

This week in lab I spent a lot of time staining the fungi that I've grown to identify morphology and hopefully the species.

I have been using lactophenol cotton blue dye in accordance with a protocol from Leck (1999). It is a relatively simple protocol, you basically just add dye to a sample on a slide, but I was really struggling with it. The samples of mycelium I was taking from my plates were too small at first, then they were too thick and only a small portion of the sample absorbed dye.

Finally, after much trail and error, I was able to get a sample clear enough to see structures. The trick was to use dissecting needles to tease out the mycelial mat and wait five min for the dye to absorb into the cell walls. I also discovered that the commassie blue dye works about as well as the lactophenol cotton blue and is a less toxic compound, so I may begin to use that more.


Preparation of Lactophenol Cotton Blue Slide Mounts:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/

Summer Week 5

Week of June 22nd-25th

I haven't spent much time in lab this week. It was finals week for my math class and I needed to study. I did notice that a lot of my plates are beginning to grow some pretty lovely cultures. I now have at least 4 species of fungi growing in my plates but I am still unable to classify any of them.

I've been giving a lot of thought to where I want to take this project in the future. Depending on what I want to do my project can take me a lot of places. I could work with Cel and learn how to extract DNA and try to use that procedure on my endophytes. I could learn how to stain and identify the morphology of my fungal colonies. Or I could try and infect a different species with an endophyte and see if it changes the growth of the plant. Choices Choices

Summer Week 4

Week of June 15th-18th

On the 3rd of June I found out that the method I was using to stain tissues to look for endophytes was not as effective as it could have been. By adding a drop of stain instead of excess alcohol when mounting my slide I am better able to see endophytic hyphae because the plant cells are not dehydrated from the alcohol. After altering my staining method I began to see more endophytes. I found three this week in silver leafed nightshade, mint and a succulent euphorbia plant.

The plates made last week began to grow endophytes and the ones from the week before continued to grow larger. The growth rate for all the colonies appears to be very slow which is a good sign they are genuine endophytes.

This week I also found a really neat new resource call the Journal of Video Experiments or JOVE. They have detailed videos of how to set up and conduct different experiments including the formulas used for the dilution of chemicals and things of that nature. One I watched was about how to inoculate plants with endophytes that kill insects. Here is the link, I don't know how to embed videos.

 http://www.jove.com/video/50360/establishing-fungal-entomopathogens-as-endophytes-towards-endophytic

Summer Week 3

The week of June 8th-11th

On Monday June 8th I decided to look at any of my Show Low specimens that looked fresh enough to still absorb dye under a microscope. I looked at six specimens and only one had anything that looked like an endophyte. A big problem I was having is seeing inside the leave of plants have have a low surface area such as pine needles and juniper. The shape of the leaves requires a lot of light pass through a dense area making things blurry and dark. It was also difficult to cut the leaves so I think I need to learn how to clear plant tissues using chemicals.

On the 8th I also noticed that one of my previous plates had some white fungal growth on it. The hyphae were clearly coming out of the interior of the leaf. There has been no evidence of contamination on any of the plates so I know my new sanitation method is working well.

I found another endophyte in a sample from a tree near D building, I'm not sure what the species is. As I was walking to the lab on Monday they were trimming the trees and I picked up a sample. I think it maybe some kind of African Acacia tree but I have no idea. The leaves were so covered in debris that it was difficult to see much inside the leaves. However in more than one place I found had what looked like fruiting bodies connected to hyphae most likely on the surface of the leaf instead of inside it. I plated some samples anyway.