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.

An example of the basidiospores from this mushroom.

A clamp connection in the hyphae.

Hyphae protruding from a small sample that I dissected off of the mushroom.

October 17 Lab

The goals for this lab were to:

• Examen our Neurospora crosses
• Look at various field collected materials

Neurospora crosses

Our Neurospora crosses have been very slow to develop beaked perithecia containing ascospores, but today it seemed like there was development for us to see the ascospores. First, we simply looked at the plates under the dissecting scopes to find perithecium that were mature enough, then we removed a perithecium from the crossing plate. None of the perithecium on my plate were mature enough, so thankfully Lorna had several mature perithecia and let me take one. To release the ascospores, you can take a dissecting needle to scoop it up and on to a slide, and then attempt to pierce the perithecium with the needle from the very top. You can also take two dissecting needles and use one to hold the perithecium down and use the other to poke it open.  I instead used a cover slip to smash the perithecium and release the ascospores. Because many people in the lab did not have entirely mature perithecia, we waited to do the transfer of the ascospores. However, we did get to take our slides and look for fluoresce if we had a GFP strain. You can see very clearly the nuclei in the hyphae, and if you look closely you can also see four nuclei glowing faintly in the ascospores.

You can see here some ascospores on a squash mount of a perithecium. 

Fluorescence microscopy of ascospores and hyphae. 

 We will work with these crosses again next week if they are finally mature enough.

Field collected materials

Also in lab today were various samples of field collected materials for us to observe.

Xylaria species on wood.

A group of unknown fungi.

Nidula species on wood, also called "Bird's Nest Fungi".

Cyathus olla, another "Bird's Nest Fungi".

Coriolus versicolor also known as "Turkey Tail".

Daedaleopsis confragosa 

Phallus species also known as "Stinkhorn".

Ganoderma lucidum  or the "Lingzhi Mushroom".

Laetiporus sulphureus the "Sulfur Shelf" or "Chicken of the woods".

Ganoderma tsugae or "Hemlock Varnish Shelf"

We were also presented with a Morel mushroom, but this can often be mistaken for other, often poisonous mushrooms. First is a picture of the real Morel, followed my some false Morels.

Morchella esculenta,  and example of a real, common Morel mushroom

A Thick-stalked False Morel

Some more images of false Morels.

A Conifer False Morel.

Saddle-shaped False Morels. 

October 10 Lab

     Today we did not have an actual lab; instead we took the lab practical. There were 16 stations throughout the lab with questions regarding fungal identification, structure identification, a life cycle question and some techniques. The test was not exactly what my expectations had been. I planned on there being fungal identifications, perhaps not as many, and I thought we were supposed to know the species name as well. I had also prepared to know more structures, but this did not seem to be the focus of the test. I was completely caught off guard with the life cycle question, but I suppose I should have expected a question like that. Finally, I thought there were going to be more technique questions. Before we can even start to identify a fungus, we must know the techniques of culturing or mounting or making riddell mounts. I thought theses were fairly important things to know and take away from the class. Overall, it was not a terrible test, I just wish I would have been better prepared for this test.

Unknowns Project

This is still under construction!

One of our goals for this semester was to isolate and identify 3 unknown fungi (unknown to us, anyway). There is a list of common fungi that we cannot turn in, but we can collect from pretty much anywhere for this project. I work with endophytic entomopathogenic fungi in cotton targeting thrips and spider mites, and so I was curious if these arthropods harbor any fungi in heathy conditions that I should know about when doing work like this. I was also curious if they share similar fungi as the food they feed on. In this case, my colonies feed on excised bean leaves, so I did a comparison of endophytes in freshly cut bean leaves and leaves that had been fed upon for one week. The specific groups that I plated out to look for fungi were:

1. Surface sterilized adult spider mite females
2. Non surface sterilized adult spider mite females
3. Surface sterilized adult female thrips
4. Non surface sterilized adult female thrips
5. Surface sterilized immature thrips
6. Non surface sterilized immature thrips
7. Freshly cut surface sterilized bean leaves
8. One week old surface sterilized bean leaves

Adult female Western Flower Thrips.

Two-spotted spider mite adult male. 

To surface sterilize the spider mites and thrips I placed 10 individuals in 70% ethanol for 1 minute, removed them and placed them into 2% bleach for 1 minute, and then removed them into a sterile water wash. The sterilized individuals were then placed evenly throughout a water agar plate. Each group was plated separately, i.e. thrips were not plated with spider mites.

Surface sterilization of plant material followed the same order and dilution, however, leaves remained in ethanol for 2 minutes and in bleach for 3 minutes. Leaves were placed directly on a water agar plate, and with a sterile scalpel, cut into sections to expose more internal surface area to the agar.

The non sterilized mites and thrips were simply pulled from the colony with a sterile tool and placed on a clean agar plate. All of this work was done under a sterile laminar flow hood on September 27, 2012. Once plated, the petri dishes were sealed with parafilm and placed at room temperature near a window to simulate a natural photoperiod.

The laminar flow hood where I did my work, along with surface sterilization materials, a thrips colony and a fresh bean plant.  

Adult female thrips in a beaker containing 70% ethanol. 

A series of beakers for surface sterilizations. From back to front: 70% ethanol, 2% bleach, sterile water.

Adult female thrips across a water agar plate.

A "condo" containing a thrips colony that has been feeding for one week.

Previously fed upon leaves undergoing their surface sterilization.

Adult female spider mites on their water agar plate. I promise they are there, you just have to look closely.

Fresh bean leaves on water agar that were cut to expose internal leaf tissue to the agar.

After six days, the only plates that had growth were the non sterilized adult female mites, the non sterilized female thrips, and the one-week-old bean leaves. 

From the plates that showed growth, agar plugs were removed from areas with clean boundaries that did not visibly have contamination. They were transfered under the flame of a bunsen burner to new, clean 1/2 PDA plates.

A spider mite showing fungal growth.

What appears to be a thrips egg showing fungal growth.

At one week on the 1/2 PDA, there appears to be three distinct fungi growing in my plates. The used leaf and spider mite palates each have a unique fungus, but the thrips plate appears to have a fungus that has also shown up on the spider mite plate. When I get time, I will put these on slides under the microscope to identify what fungi I might have. I have a feeling that they may be on the common list, so I better get to finding new sources of fungus!

Fungi growing from non sterilized thrips.

Two types of fungi from non sterilized spider mites.

Previously fed upon bean leaf on 1/2 PDA growing an unknown fungus.

 

October 3 Lab

The goals for this lab were to:

• Familiarize ourselves with some of the Hyphomycetes
• Check in on our Neurospora crosses from last week
• Check in on our Ustilago infected corn from last week and stain for the fungus

The Hyphomycetes

The hyphomycetes are a broad term for conidiating fungi. These are very common and it would be a good idea to become familiar with the group in general. We looked at 12 different species throughout the lab to get a better idea of their spore shapes, hyphal forms, conidiophores and other various structures. I drew pictures of each of these fungi, and took a few photographs of some. The following images were observed from either a tape or squash mount slide. 

Trichoderma viride showing a conidiophore on the right, and after looking at the Illustrated Genera of Imperfect Fungi (IGIF, from here on out), what I can only assume is hyphae layered with conidia on the left and bottom. 

The coolest conidia we looked at today. Pestalotia conidia that bear appendages on both sides.  Hyphae threaded together on the right.

Fusarium graminearum conidia. They remind me of compartmentalized bananas. 

Monilinia fructicola. This looks like a section of the branched conidiophore on top, loose conidia in the center and an incompletely drawn hypha on bottom. IGIF shows a much better image.

Rhizoctonia solani does not have conidia or asesexual fruit bodies. My sample appears to be a bit dried out, but similar in appearance to IGIF. 

Thielaviopsis brasicola showing its aleuriospores. This also produces phialospores, but they are not shown here.

Aleuriospores of Thielaviopsis brasicola. 

Epicoccum spp. The conidia did not remain with the conidiophore in my images.

Microscopic image of Epicoccum conidia.

This Curvularia spp. reminds me of alternaria, except these are a more grey conidia. The conidia can be straight or bent under the scope.

Not the best Aspergillus niger mount that I have done. You can see conidia in chains or part of the conidiophore here. 

Botrytis cinera. The only useful part of this image is my attempt to demonstrate the slender nature of this fungus. A better image can be seen in IGIF.

Alternaria brassicola. We have seen this before, but here again is a simple drawing. The conidia are brown.

We were also going to look at Colletotrichum coccodes and Nigrospora, but they were contaminated with an unknown fungus. 

Neurospora crosses from last week

We each checked in on our Neurospora crosses from last week to see if any perithecia containing ascospores had formed. You can see growth on my plate, and you can see were the two fungi have met in the center of the plate. Interestingly enough, my plate did not bear perithecia in the center of the plate, but instead on the far ends of the plate. They were not completely developed this week, but I will try to get a better image next week.

My Neurospora cross one week after plating. 

Ustilago infected corn 

Last week we infected one-week-old corn plants with Ustilago maydis. This fungus causes corn smut and leads to large, bulbous growth on the plant after affecting plant cell growth and division. After just one week there were already visible signs of the disease.  

Damage present on the corn plant after just one week.

A better view of a small gall forming on one of my infected plants from last week. 

To confirm that a fungus was causing these galls, I removed a portion of the plant and began a staining protocol that binds a blue dye to the chitin in the fungal cell wall. To begin, I placed the excised leaf material in a plastic petri dish and submerged this leaf in a 2:1 Acetic acid: ethanol solution to clear the leaf. The leaf remained in this solution for 24 hours, was then removed from the solution, rinsed with ethanol, and placed in a microcentrifuge tube with lactophenol blue. This solution has three purposes. It kills any living specimens, preserves fungal components, and stains chitin blue. I kept the leaf in this solution for 4 hours, and then removed it into a jar of ethanol to remove any excess blue dye. I kept the leaf in this solution for several hours, then placed the leaf onto a slide, covered with a long cover slip, and then observed the locations of fungus in the plant.

The excised leaf material freshly placed in acetic acid/ethanol mix.

My stained corn leaf showing pockets of blue where the fungus is present.

The color is a bit off on my camera, but you can see blue color has remained in the veins of the plant. 

The location of a gall. You can see a congregation of blue mycelia.

A small blue lesion, likely the start of a gall.

Closed stomata on the underside of the leaf. You can see the cells dotted with blue spots,  spores perhaps?