Lab 7

Introduction

Our goals for this lab included counting and picking ascospores from our Neurospora crosses, observing the mushroom, Chlorophylum sp., and observing hyphal growth via fluorescence microscopy. The corn plants inoculated with Ustilago maydis several weeks ago were also available for observation.

Continuation of the Neurospora Drama

If you will remember from last week, our Neurospora crosses had not matured to the point that we could count and pick ascospores. So instead, we crushed perithecia and observed various stages of immature ascospores. This week the Neurospora did seem to have more ascospores that had been shot onto the lids of the plates, however the number was still very small (given that 10+ people made at least 1 Neurospora cross). 

Picking Random Ascospores

In order to pick ascospores from the crosses, it was first necessary to cut a water agar block and place it on a slide. We used old 1.5% water agar because the 4% agar plates we were supposed to use were not suitable for our purposes. A loopful of ascospores from the lid of a Neurospora culture plate where then spread onto the agar block (Fig. 1) and examined under a dissecting scope (Fig. 2). The goal of this part of the lab was to pick at least 10 ascospores. It took a few different Neurospora plates before I could actually even find 10 ascospores. A metal picker was flamed briefly before slicing out individual ascospores from the water agar block (Fig. 3). Each ascospore was transferred to an agar slant (Fig. 4). My first 5 inoculations were of SMRP10 x GFP, and my next 7 were Sheila's NCAL cross. After class, these slants were supposed transferred to a refrigerator to allow spores to hydrate. The next day, the slants were supposed to be heat shocked by placing them in a 65C water bath for 45 minutes (all done by TA, I presume). 

Fig. 1
Water agar block inoculated with ascospores (and other debris) on a glass slide

Fig. 2
Viewing an agar block inoculated with ascospores under a dissecting microscope.

Fig. 3
Metal ascospore picker

Fig. 4
Agar slants inoculated with Neurospora ascospores

Observation of  Chlorophylum sp.
The goal for observing the mushroom Chlorophylum sp. (Figs. 5-7)  was to make a slide mount of the gills and cap and try to find the basidia with spores, Buller's drop, and clamp connections on the hyphae. In order to do this, I tried to section out gills as thinly as possible (Fig. 8). Maybe I just need more practice in observing Basidiomycetes under the compound scope because I didn't see any of the structures I was supposed to see. The first mount I made was a mess (Fig. 9), and I couldn't really tell anything about it. I was able to see hyphae with a new slide mount (Figs. 10-12), but other than this, I was not able to see anything. :/

Fig. 5
Chlorophylum sp.

Fig. 6
Chlorophylum sp.

Fig. 7
Chlorophylum sp.

Fig. 8
Gills of Chlorophylum sp. 

Fig. 9
Horrible mess of a slide (Chlorophylum sp.)

Fig. 10
Hyphae of Chlorophylum sp. at 10x magnification

Fig. 11
Hyphae of Chlorophylum sp. at 40x magnification

Fig. 12
Hyphae of Chlorophylum sp. at 100x magnification (oil immersion).

Fluorescence Microscopy

During the second part of lab, we journeyed into Dr. Shaw's secret lair in which fluorescence microscopy magic is performed. The goal of this activity was to observe hyphal growth of Neurospora as well as the fluorescent nuclei within the cells.  First, Maxwell sliced an agar block from a Neurospora culture (Fig. 13) and placed it on a glass slide (Fig. 14). As the cells on this block were still alive, we were hoping to observe it growing upon placement under the microscope. We chose 30 timepoints at which pictures would be taken (1 picture/second). Unfortunately, it seemed as though everything we tried did not work, as if the cells were starting to die.But we were able to see what the other groups had done. Team B had an excellent video of hyphal tip growth (Video 1) and Team A had a very nice video of fluorescent nuclei traveling through septa in the cell (Video 2).

Fig. 13
Maxwell slicing a block of agar from a Neurospora plate

Fig. 14
The agar block Maxwell made and placed on a glass slide

Video 1. Video from Team B showing hyphal tip growth in Neurospora

 Video 2. Video taken by Team A showing nuclei traveling through septa of hyphal cells.

 Continuation of Corn Observations
The SQ/Um 9/19 inoculation appeared to have a large tumor at the base of the plant, which was obviously dying (Figs. 15, 16). My mutant plant was completely gone (Fig. 17), all of my inoculated plants were dead, and all of my control plants were still alive (Figs. 18-20).

Fig. 15
Large tumor at base of SQ/Um 9/19

Fig. 16
Closer view of tumor at the base of SQ/Um 9/19

Fig. 17
Container previously holding mutant corn 

Fig. 18
Plant inoculated with U. maydis is dead, while control is still alive

Fig. 19
Plant inoculated with U. maydis is dead, while control is still alive

Fig. 20
Plant inoculated with U. maydis is dead, while control is still alive

Conclusion
I would like to say that I didn't think picking ascospores was that bad. The hardest part was finding the ascospores on the lids. That is mostly due to the sparse numbers of spores generated by our cultures. I actually picked the ascospores pretty quickly. As for the fluorescence microscopy, I thought it was really cool! I just wish my group had better luck with cells. I didn't realize how easy it was to visualize hyphal tip growth.

All for now.

C