Lab 5

Introduction
For lab 5, we were to observe conidiating Hyphomycetes, especially Pestalotia sp., Epicoccum sp.,  Curvularia sp., and Nigrospora sp. We were also supposed to look at the Neurospora contamination on the Mucor cups from last week, make observations of all of our corn plants, and check on our Neurospora crosses. After observing the plants for tumors, we were supposed to stain the tumors for observation under the microscope. Also, Drs. Ebbole and Shaw made a point to spot-check our blogs and give us feedback.

Observations of Conidiating Hyphomycetes

For the first half of lab 5, I observed different cultures of the conidiating Hyphomycetes. I tried to be as efficient as possible, but I was only able to observe four of these cultures (I believe there may have been 8-10 total) during the given time. The first genus I observed Pestalotia sp., exhibited a distinct (memorable) morphology when compared to other fungi I have observed. Pestalotia  spores were somewhat elongate, tapered at both ends, tended to have 4 septations, and exhibited multiple appendages at one end (Fig. 1). What I observed looked generally like what is found in the Illustrated Genera of Imperfect Fungi lab manual (Fig. 2).

Fig. 1
Pestalotia spores at 40x magnification.

Fig. 2
Drawings of Pestalotia sp. from the Illustrated Genera of Imperfect Fungi lab manual.

These appendages may aid in dispersal. The hyphae of this genus were long, slender, and exhibited many very clear septations (Fig. 3). Dr. Shaw commented that this may be because the hyphae produce arthrospores.

Fig. 3
Hyphae of Pestalotia sp. at 40x magnification.

The second genus I observed was Curvularia sp. To me, the spores of this fungus looked like chubby boomerangs (Fig. 4, 5). Hyphae were long and slender, but septa were not clearly visible (Fig. 6).

Fig. 4
Spores and hyphae of Curvularia sp. at 40x magnification. 

Drawings of Curvularia sp. from Illustrated Genera of Imperfect Fungi lab manual.

Fig. 6
Hyphae of Curvularia sp. at 40x magnification.

The third genus I observed was Epicoccum sp. This taxon was also quite distinct, as the spores were spherical, darkly pigmented, and contained many cells (dictyosporic) (Figs. 7, 8).  The conidiophores were compact and exhibited short branches (Figs. 9, 10). I really like this fungus; it’s cute.

Fig. 7
Spores of Epicoccum sp. at 40x magnification.

Fig. 8
Drawings of Epicoccum sp. from  Illustrated Genera of Imperfect Fungi lab manual.

Fig. 9
Epicoccum sp. conidiophores at 40x magnification.

Fig. 10
Epicoccum sp. conidiophores at 40x magnification.

The fourth species I observed was Botrytis cinera. Conidia of this species were ovoid and clear (hyaline) (Fig. 11), and the conidiophores were long, extremely slender, and also clear (Fig. 12, 13). Clusters of conidia could be seen randomly along the length of the hyphae, as well as at the apical portions.

Fig. 11
Conidia and hyphae of Botrytis cinera at 40x magnification.

Fig. 12
Conidiophores and conidia of Botrytis cinera at 40x magnification.

Fig. 13
Drawings of Botrytis cinera from the Illustrated Genera of Imperfect Fungi lab manual.

Observation of Mucor rouxii Contamination
As I spent most of the lab looking at the fungi mentioned above, as well as the corn experiment which will be detailed below, I was not able to observe the Neurospora contaminated Mucor rouxii cups. I will be sure to observe this over the weekend.

Observation of Corn Plants
The second portion of the lab was spent observing the previously inoculated corn plants and the plants I inoculated from last week (Fig. 14). To recap, last week we observed corn plants that had been inoculated with Ustilago maydis, a rust fungus. I also inoculated my on corn plants (see Lab 4 blog).
U. maydis  causes galls to form in the plant tissue and can infect the ear of the corn, as well as the reproductive tissues of the kernels. This fungus works by causing unregulated cell growth and division, which then leads to the galls or tumors. Only one of the previously inoculated plants (SQ/Um 9/19) seemed to exhibit outward symptoms of the disease (tumors) (Fig. 15). Only one of my plants exhibited these tumors (Fig. 16), while the other inoculated and control plants had no signs of the disease (Figs. 17, 18).

Fig. 14
Previously inoculated corn plants.

Fig. 15
SQ/Um 9/19 corn plant exhibiting disease symptoms of tumors.

Fig. 16
One of my corn plants inoculated with 0.5 mL Ustilago maydis exhibiting tumors.

Fig. 17
My mutant plant without any signs of disease.

Fig. 18
One of my control corn plants without any signs of disease.

Staining of Tumors
After observing the plants, we needed to stain the tumors for observation under the microscope. In order to stain the tumors, I first needed to de-stain the leaves (remove the chlorophyll). I cut samples of leaf tissue containing tumors from my plants (Figs. 19, 20). I then placed 5.0 mL of 2:1 ETOH:Acetic Acid via plastic pipette into a large glass petri dish containing my leaf (Fig. 21). I sealed each dish with parafilm and let them sit over night (Fig. 22).

Fig. 19
Leaf of SQ/Um 9/19 exhibiting tumors.

Fig. 20
Leaf of my inoculated corn plant exhibiting tumors.

Fig. 21
2:1 Ethanol:Acetic Acid solution used for de-staining the chlorophyll form diseased leaves.

Fig. 22
Each leaf soaking in the ETOH:Acetic Acid solution. 

After allowing my leaves to de-stain for approximately 22 hours, I washed the leaves in water and ETOH. I then placed the larger leaf (from SQ/Um 9/19) back into its glass dish and coated it with 0.1% Trypan Blue in Lactophenol (Figs. 23, 24). Because my second leaf (from last week’s inoculation) was much smaller than the first and I was trying to conserve the dye, I decided to place it inside of a 50.0 mL centrifuge tube with only 2 drops of the 0.1% Trypan Blue in Lactophenol (Fig. 25). I then allowed both leaves to sit for approximately 1.5 hours in this mixture.

Fig. 23
0.1% Trypan Bluw in Lactophenol used to stain the tumors of diseased leaves.

Fig. 24
Leaf of SQ/Um 9/19 soaking in 0.1% Trypan Blue in Lactophenol.

Fig. 25
My inoculated corn leaf soaking in 0.1% Trypan Blue in Lactophenol.

After this time period, I mounted each leaf in a drop of Lactophenol (Fig. 26) and observed the tumors under the microscope. The Trypan Blue stained the cells of the tumor blue (Figs. 27-29), as well as other areas in the leaf (Fig. 30).

Fig. 26
Lactophenol used to mount leaves on clean glass slides.

Fig. 27
Stained tumors and other portions of my inoculated corn leaf at 10x magnification.

Fig. 28
Stained tumors  of SQ/Um 9/19 leaf at 10x magnification.

Fig. 29
Stained tumors  of SQ/Um 9/19 leaf at 10x magnification.

Fig. 30
Stained areas of SQ/Um 9/19 which did not contain tumors (10x magnification).

Neurospora Crosses

It was apparent after observing my Neurospora cross plate from the previous week that I had failed to culture one of the most easily culturable fungi known to man (Fig. 31). Not only had I not crossed SMRP-10 and SMRP-11, they appeared not to have grown at all. :*(

Fig. 31
My failed Neurospora cross attempt. Little to no growth of fungus on the plate.

In order to make-up for this failure, I tried the cross again, and this time with all of the available mating types (Fig. 32). Using the SC Mating Media, I made the following crosses:

·      SMRP110xSMRP-11

·      SMRP110xNCAL021-1 (I made two of these)

·      SMRP110xCSP-1-CH

Fig. 32
The four Neurospora crosses after being parafilmed and placed inside a plastic shoebox for storage.

There are a couple of reasons I can think of for why my crosses (and inoculations) did not work the first time. First, I may not have cooled my probe (by sticking it in the agar after flaming) before collecting spores from the culture. This may have killed the spores on contact, meaning that I could have tried to inoculate my plate with dead spores. Second, if I didn’t kill the spores, then I may not have inoculated with the appropriate amount of spores. I was very cautious about not getting too much material the first time I tried the cross. In doing the second series of crosses, however, I made sure to cool my probe and use much more material for my inoculation. Here’s to hoping that it worked.

Blogs
We had a spot-check on our blogs today; I got a 4/5.  I usually try to update this thing before each lab, but this week I was not able to do this. I will try to be more consistent with updates. Also, I will make a conscious effort to have a more logical flow.

Unknowns

I would like to update Dr. Shaw and Dr. Ebbole on my unknowns project. I have spent the whole semester up until last week writing my thesis and preparing my defense. Because of this, I wanted to wait until after I defended to start my unknowns project. I realize that I have waited a long time to begin this, but I literally had no extra time in the first part of the semester. By no means am I using this as an excuse: I signed up for my courses this semester knowing that I would be defending. I just wanted to let you know the reason you haven’t seen any blogs regarding my unknowns. You will see an update on my unknowns shortly.

Conclusion
All in all, I feel pretty comfortable with the conidiating Hyphomycetes and the procedures that we have used to inoculate, observe, and stain the corn. I am a little discouraged that my Neurospora crosses did not work, though. There are a couple of reasons I can think of to explain this. First, I may not have cooled my probe (by sticking it in the agar after flaming) before collecting spores from the culture. This may have killed the spores on contact, meaning that I could have tried to inoculate my plate with dead spores. Second, if I didn’t kill the spores, then I may not have inoculated with the appropriate amount of spores. I was very cautious about not getting too much material the first time I tried the cross. In doing the second series of crosses, however, I made sure to cool my probe and use much more material for my inoculation. Here’s to hoping that it worked. Or, I could just be really bad at inoculations. This is definitely something I will be practicing until I get it right. I guess I won't be a microbiologist or a mycologist when I grow up.

Charity

Lab 4

Introduction

In lab 4, we observed the different spores produced by Neurospora sp. and then made crosses of Neurospora strains. We were then allowed to observe the interesting dimorphism of Mucor rouxii. Lastly, we observe diseased corn and inoculated healthy corn with Ustilago maydis, otherwise known as corn smut.

Neurospora sp. 
Observations of Neurospora sp. Spores
The first activity of the lab was to observe different spores formed by the Ascomycete fungus, Neurospora sp. This genus makes three different types of spores: microconidia and macroconidia (which can be separated temporally in terms of production), and ascospores. I made squash mounts in order to observe Neurospora SMRP 11 for microconidia. A quick observation of the plate revealed that growth was very sparse (Fig. 1). 

Fig. 1
Sparse growth of Neurospora SMRP-11on SC Mating Media. 

Viewing the slide mounts under the scope revealed that there were plenty of macroconidia, but the microconidia were a bit more difficult to find. Once I did find them, it was obvious that they were structurally different from the macroconidia that were present, as they originated from very small branches and exhibited ruptured phialides from which spores would exit (Fig. 2). 

Fig. 2
Microconidia and conidiophore of Neurospora SMRP-11 at 40x magnification.

I next observed macroconidia from a different culture using both squash and tape mounts. Observing the plate of this culture revealed a vastly different scene than the previous plate, as orange macroconidia wear clearly visible, covering the entire surface of the agar (unfortunately, I did not document this via photography). These conidia were morphologically different from the microconidia, as they looked more like strings of pearls on the conidiophore than short, rectangular branches (Fig. 3, wet mount; Fig. 4, tape mount). 

Fig. 3
Wet-mount of the macroconidia and conidiophore of Neurospora sp at 40x magnification.

Fig. 4
Tape-mount of the macroconidia and conidiophore of Neurospora sp. at 40x magnification.

Next, I obtained the centrifuge tube labeled “X_10021gfpa 9 July à A Female 35 June”, which contained ascospores of a Neurospora sp. I again created a squash mount and observed the spores under the microscope. These spores were large (when compared to the spores seen from the previous cultures), dark in color, and shaped like a football (Fig. 5). It was clear from this activity that the morphological structures of conidia and spores can be variable within a genus, which may depend in part on stresses encountered in the environment.

Fig. 5
Ascospore of Neurospora sp. at 40x magnification.

Neurospora Crosses
After observing the different spores, our goal was to then try to cross different mating types of Neurospora sp. I obtained cultures of SMRP10 (A) and SMRP 11 (a), which were contained within glass vials stopped with a cotton ball, and a sterile SC Mating Media agar plate. I then simply inoculated the left side of the plate with SMRP10 (A) and the right side with SMRP11 (a), then sealed the plate with parafilm (Fig. 6). I will determine if I successfully crossed A and a next week in lab.

Fig. 6
My Neurospora SMRP-10 and SMRP-11 cross.

Observe Mucor rouxii Dimorphism

The second activity of this lab was to observe the dimorphism of Mucor rouxii in a single culture. This species utilizes Oxygen and Carbon Dioxide availability to determine whether yeast cells of hyphae will development. Because of this, there should be more hyphae near the top of the agar, and more yeast cells at the bottom of the agar cup. In order to observed this, I first obtained a small cup of agar inoculated with this species of fungus (Fig. 7).  

Fig. 7
Cup containing agar inoculated with Mucor rouxii. 

I then sliced a thin sheet of agar from top to bottom and placed this on a slide with a coverslip on top. With this technique, I should have been able to see a gradient of hyphal growth transitioning into more yeast cells at the bottom of the cross section. However, this is not what I observed.  I did see sporadic hyphal growth throughout the agar (Fig. 8), but I never saw any yeast cells. I did another slice with this same technique, but saw no observable differences (i.e. density of hyphal growth had not changed, no yeast cells present). I then sliced the entire top surface off of the agar to observe under the scope, and still only saw sporadic hyphae (Fig. 9). The same technique was applied to the bottom surface of the agar, but there were still no yeast cells. Dr. Shaw and Dr. Ebbole were under the impression that these cultures were not old enough yet, and that they may just be exhibiting random fungal remnants which could be an artifact of the lab they were culture in.

Fig. 8
Sporadic hyphal growth of Mucor rouxii throughout agar cup.

Fig. 9
Sporadic and fragmented hyphae near surface of agar cup.

Ustilago maydis
Observe Diseased Corn

The last activity of the lab focused on corn smut (Fig. 10). 

Fig. 10
Un-inoculated corn (left) and previously inoculated corn plants. 

We first observed and documented corn that had been inoculated with Ustilago maydis on September 13, 2012 (Figs. 11, 12), and September 19, 2012 (Figs. 13). I will continue to document the progress of these plants in the coming weeks. 

Fig. 11
Leaf of corn inoculated with Ustilago maydis on September 13, 2012.

Fig. 12
Entire leaf of corn plant inoculated with U. maydis  on September  13, 2012.

Fig. 13
Leaf of corn plant inoculated with U. maydis on September 19, 2012.

Inoculation of Healthy Corn with Ustilago maydis
We then each inoculated our own corn plants with U. maydis. I obtained two plastic containers of corn plants, each of which had two plants. I then injected 0.5 mL of a solution of U. maydis and dH₂O into the shoot of one plant in each container. The second plant was left as a control in each container. I then obtained a container that held a single corn plant mutant which was bred to be particularly vulnerable to U. maydis. I then injected this pant with 0.30 mL of the same U. maydis solution. (Note: I wanted to treat the mutant the same as I had the other plants, e.g. with 0.50 mL of solution. However, this plant was smaller than the others and literally could not hold more than 0.30 mL).  I then labeled all of my plants and placed them at the front of the room (Figs. 14, 15). I will be observing/documenting these plants in the coming weeks as well.

Fig. 14
My three containers holding inoculated and control corn plants. The left and right containers contain two plants each, with only one in each container inoculated with 0.5 mL of U. maydis (the other plant served as a control). The middle plant is a mutant which has been bred to be especially susceptible to U. maydis. This plant was inoculated with 0.3 mL U. maydis.

Fig. 15
Again, here are my three containers holding the inoculated and control corn plants.

Conclusion
It is important to understand the different spores that can be produced by a single species. Neurospora provided the best example of this, with spores that differed in size, pigmentation, and morphology. I feel comfortable with being able to identify these different spore types. As for the Neurospora strain crosses, there should be successful mating at both mating types (A and a) were used. Even though the Mucor rouxii activity did not go as planned, I thought that the ability of a single organism to exhibit such dimorphism at the same time and over a very small distance was amazing. I am sorry that I did not get to actually witness this in class. As for the corn smut, I am excited to see if the inoculations worked!

All for now.