Lauren Kalns's PLPA 631 Lab Notes: October 24 Lab

The goals for this lab were to:

Pick Neurospora ascospores
Look at Mushrooms/Chlorophylum sp.

Find Basidia with spores.
Find Buller's Drop.
Find clamp connections on hyphae.

Fluorescence microscopy (Nuclei, actin, Mak-2/50-> Ping Pong)

Fluorescence microscopy

Today in lab we got the chance to work the original strains of Neurospora under the fluorescence microscope. Working with this scope was a really awesome experience. I've never worked with anything GFP tagged, so getting to see this was very interesting. Also, just observing the rate of growth in Neurospora was quite amazing. When looking at these fungi on squash mounts, you don't really get the sense of fungi being such a responsive being, but observing the responses to light and pressure under the scope today was eye-opening.

We worked on the scope in teams with Dr. Shaw. My group was the first to go, so spore picking would have to wait. In the microscopy room, Dr. Shaw taught us how to gently remove a large agar block and place it on a slide. You want to make sure to have the farthest edge of the growing fungi in the center of this agar block so you can better see growth and not cut the tips of the hyphae. Once you have transfered the agar to a slide, gently place a long cover slip over the agar. Do not press down on this cover or else you will be disturbing the fungi more than necessary. Then we transfered the slide to the microscope and Dr. Shaw handled the scope and setting the software up. The first strain that we looked at was supposed to have GFP tagged microtubules, but when we looked at this on the output, the entire hypha glowed and it was difficult to distinguish the microtubules. We were able to capture a video of the growing tip with a time series capture, but this unfortunately file is too big to upload, and also does not work on my computer so I cannot edit it further. We were able to get a video of a strain with GFP tagged nuclei, and you can see in a couple of places where the nuclei squeeze through the septa and shoot through. It is also interesting just how many hundreds of nuclei are present in just this small portion of the hypha.

Picking Neurospora ascospores
After we were finished in the microscopy room, we went back to the lab to attempt to pick ascospores and transfer them to agar slants. First, we needed to obtain the ascospores from a mature plate. To do this, we took a sterile loop and collected sterile water in the loop. Then the wet loop can be touched to ascospores to pick them up. Once they have been picked up in the loop they need to be transfered to a clean water agar block that is on a slide. Use the loop to spread the spores around so that there is plenty of space in between each spore to cut it from the agar. Next, a spore picker was used to cut the spores from the agar. For beginners, you want to cut three sides around the spore and then scoop it up. As you get better, the cutting of the sides is not necessary. Once you have the cut agar on the picker you can then gently transfer this to the agar slide. From here, these spores were going to be left in the refrigerator over night to hydrate, and then be subject to a heat shock to kill any vegetative growth and activate the spore. We will look at these next time to see if we have germinated any spores.

An example of the cut agar block that the spores will temporarily be transferred to.

Through the microscope, you can see the three cuts that have been made around a single ascospore.

Looking at mushrooms, Chlorophylum spp.

Lastly, we looked at some mushrooms that Dr. Shaw had picked from his yard that morning. The goals were to see clamp connections, Boller's drop, and basidia with spores. I first dissected a portion of the mushroom to look at the gills and see if I could visualize Boller's drop. I unfortunately was not ever able to see this under the scope. I was able to see basidiospores, but was not able to get a good image of them attached to the basidium. Finally, Dr. Ebbole was able to locate a clamp connection for the class to look at, and I was able to get a good picture of this to share.