Lab 3

Introduction. This week in lab, we simultaneously learned baiting techniques and were able to observe  motile fungi. We first observed motile zoospores from a baited culture of Saprolegnia sp., which is a freshwater mould in the class Oomycota. We then attempted to isolate Allomyces, a chytrid water mould, from a baited soil sample. Lastly, we were able to observe the Riddell mounts from the previous week.

Zoospores

Zoospores are asexual spores that posssess a flagella for locomotion. I tried to observe these from a culture of a  Saprolegnia sp.  [S1; trans. 9.ix.2012]. This proved to be a very difficult task. I did see tiny organisms swimming erratically in samples of this culture, but after comparing to some of my classmate’s zoospores (particularly to the size of the organisms), I came to the conclusion that I was probably observing some sort of bacterium. I looked at several different cultures of Saprolegnia sp. of various ages, but had no luck. It wasn’t until I tried the Phytophthora sp. cultures (trans. 17.ix.2012) that I actually saw zoospores. This gave me so much satisfaction that I decided to take an extremely high quality video in order to share this glorious experience with the rest of the world (Video 1). I was actually able to see, but not capture with video, the flagella of these spores. 

Video 1. 

The dark spots zooming around in circles and changing directions are most likely zoospores (40x).

Allomyces Isolation (attempt)
In this part of the lab, we were supposed to isolate Allomyces from seeds (popcorn or hemp) that had been previously submerged in a soil sample/ distilled water solution. Seeds placed in this solution acted as a bait for fungal growth. The first step was to see whether or not we could observe any mitospores  around the seed. After observing this, we were supposed to then try to isolate these mitospores. I definitely was not successful at any of these tasks. I was not able to see any zoospores, so, under the direction of Dr. Shaw, I transferred the seeds to a micro petri dish filled with DS solution. I then labeled the petri dish, sealed it with parafilm, and placed it in a box in the back of the room so as to check on it next week. Perhaps then I will be able to isolate and culture the Allomyces at that time.

Riddell Mounts
The last thing I accomplished in this lab was observing the Riddell mounts I made of Aspergillus flavus and A. paraciticus from the previous week. Hyphae were definitely prolific on the cover slips (Fig. 1), but I was not able to locate many conidiophores. I’m not sure if the photos I took depict conidiphores or just balls of conidia that clustered together when I made my slide mount (Figs. 2, 3). It definitely looks like Fig. 3 is structured like a conidiophore, but I would really need a professional opinion on this to feel comfortable saying that with any degree of certainty. My A. paraciticus culture was very difficult, and actually impossible in most cases, to observe because there was so much condensation inside on the petri dish. I took a couple of pictures (Fig. 4), but mostly I was not able to see anything on those coverslips.

Fig. 1
Aspergillus flavus hyphal growth on cover slip. Riddell Mount, 40x. 

Fig. 2
A. flavus conidia and perhaps conidiophore? Or maybe just a bubble with clusters of conidia on the periphery?
 Riddell mount, 40x.

Fig. 3
Again, A. flavus and a putative conidiophore. Riddell mount, 40x.

Fig. 4
A. paraciticus conidia and perhaps conidiophore. Several drops of condensation can be seen on the slide.
Riddell mount, 40x.

Conclusion
I feel like I can recognize the zoospores from other organisms because of their more or less spheroidal shape and their flagella. However, since I did not have any luck with Allomyces, I am still uncomfortable with what these types of mitospores should look like. As for my Riddell mounts, I understand how to do them and what they should look like, I just need more practice.

All for now.

Lab 2

Introduction

In this laboratory, we learned a new mounting technique called a Riddell mount, which is a good tool for visualizing hyphal growth and conidiophores. We were also allowed to observe several different Aspergillus species in order to ascertain the morphological differences between them. We then discussed different sampling and isolating procedures for our unknowns project, as well as different media type available for use.

Techniques

Riddell Mount

This technique revolves around the idea of allowing the fungal hyphae to grow onto the coverslip. This is particularly helpful in identifying down to species level, as the arrangement of conidiophores may be different for each taxa. The Riddell mount has a few different variations, but basically consists of inoculating a chunk of agar with the fungus and allowing it to grow for a specified period of time. The first method consisted of taking a clean petri dish with a sterile filter paper soaked with dH₂O in the bottom, placing a bent glass rod inside with a coverslip on top, placing a chunk of inoculated agar on top of that, and then adding another coverslip over the agar. Covering the petri dish and placing in the incubator will allow for growth of the hyphae and conidiophores over the coverslip. I found that method to be rather tedious and inefficient, so I chose the second method. This method consisted of taking a plate of water agar and sterilely cutting 4-5 small chunks of agar out and placing them on top of the entire bed of agar. I then inoculated each chunk with the same fungus and placed a coverslip on each (Fig. 1). I liked this method because it gave me a better chance of growing something useful (something I can see/identify) on a coverslip. I made 4-5 Riddel mounts of Aspergillus flavus  and A. paraciticus.

Fig. 1. Variation of Riddell mount in which agar chunks are placed on the surface of the agar bed and are inoculated with fungi. Coverslips are then placed over each inoculated chunk and the conidiophores are allowed to grow onto the coverslip for a specified period of time (1 week for these particular mounts). 

Observations
For the rest of lab, I made squash mounts of the other Aspergillus  species (A. flavus [Fig. 2], A. sojae [Fig. 3], A. paraciticus [Fig. 4], A. nidulans [Fig. 5], A. tamari [Fig. 6], A. niger [Fig. 7]) and attempted to document differences in morphology via photography. It was difficult for me to determine the difference between these species. This could definitely be due to my inexperience with inoculating the slides with fungi. There were a couple of species that I was able to see clear conidiophores containing the metulae, phialides, and conidia. However, I was not able to see this in all of my mounts.

Fig. 2
Aspergillus flavus conidiophore at 40x magnification.

Fig. 3
Aspergillus sojae conidiophore at 100x magnification.

Fig. 4
Aspergillus  paraciticus at 100x magnification.

Fig. 5
Aspergillus nidulans at 40x magnification.

Fig. 6
Aspergillus tamari at 100x magnification.

Fig. 7
Aspergillus niger at 10x magnification. 

            

Unknowns Project Discussion

Different methods for collection, sterilization, and isolation of fungi were discussed for the remainder of the lab period. Drs. Ebbole and Shaw recommended collecting fungi from various environments, including soil, water, food, and plants. After collection of most substrates, surface sterilization is necessary. If we are fairly certain that a fungus is contained within a certain substrate or organism, it is important to surface sterilize that substrate/organism before trying to plate it, as other incidental taxa may be found on the external surfaces. For substrates like soil and even water, it is probably best to use a serial dilution before plating. For the kind of sampling I want to do, which is sampling the external surface of a substrate, sterile swabs would probably be the most appropriate tool to use.

We also discussed the different types of media we could use for our unknowns project. These included Potato Dextrose Agar (PDA), which is very nutrient rich and is conducive for growth of many different taxa; water agar, which is low in nutrients, but may be better for sporulation of some taxa; cornmeal agar, which is good for plants; and Rose-Bengal Agar (RBA), which contains antibiotics and is good for fungi collected from soil as it inhibits growth of soil microbes. Dr. Ebbole made a point to discuss the importance of utilizing different agars and perhaps even trying obscure recipes depending on the type of fungus we collect. 

All for now.

C

Lab 1

Introduction
For the first actual lab, I learned many new techniques used for viewing and identifying morphological structures of fungi. These techniques include slide mounting with both squash mount and tape mounts, cell-counting with a hemacytometer, and utilizing a dichotomous key for identification of fungi. Several cultures of various fungal genera were also available for observation and identification. Below, I will summarize my experience with each technique as well as provide information regarding the different fungal cultures I observed.

Techniques.
Squash-mount.
The first technique I learned was the squash mount. A drop of sterile water is first placed in the middle of a clean glass slide. While keeping the culture plate at the base of the bunsen burner, a probe is then sterilized via open flame, the lid of the plate is opened slightly, and the probe is cooled by sticking the tip in a clean area of agar. Fungal material is then gently scraped from the culture and the lid of the plate is closed again. The material that has adhered to the probe can then be deposited into the water droplet on the slide. After this transfer, the coverslip is then placed on top of the inoculated droplet and air bubbles are pressed out from under the slip with the blunt end of the probe. Now the slide is ready for viewing under the compound microscope. Hooray! I made approximately 20 different slides with this technique.

Tape-mount.
Another slide-making technique that I had never heard of prior to last week is called a tape mount. Again, a drop of sterile water is placed in the center of a clean glass slide (the water is optional, but I found that it helps with the quality of the image produced by the microscope). Instead of inoculating with a sterile tool, a length of scotch tape (approximately 2cm) is taken and is just barely allowed it to make contact with the fungal substrate. This is then placed over the drop of water on the slide. Ideally, the ends of the tape are supposed to stay dry and adhere to the slide. This type of mount is supposed to be good for seeing conidiophores; however, I found out very quickly that I am not very good at making these types of mounts as I kept collecting a ridiculous amount of spores. This made it difficult to see anything under the microscope. I made around 5 slides this way and decided that I was going to have more luck using the first technique. However, I will practice this technique every lab period.

Cell counting.
We watched a video on the methodology behind counting cells using a hemocytometer (http://www.youtube.com/watch?v=pP0xERLUhyc). A dilution is made of the appropriate fungus and squirted into a special (very expensive) glass slide with tiny quadrants. These quadrants (and the use of a hand counter) allow for simple counting of both viable and dead cells. I practiced making one dilution and looked at it under the microscope; however, I did not actually try to count any cells.  I definitely need more practice on this and hope to gain more experience as the labs continue.

Dichotomous key
We were introduced to several keys that will be helpful in identifying fungus this semester. The one I will likely be utilizing the most is the Illustrated Genera of Imperfect Fungi by Barnett and Hunter. I have already purchased a copy and am excited to start using it in the next lab. Although I did look through the pictures in the key to compare with what I was seeing on my slides, I did not actually start going through the couplets. I would like to think that I am wise enough now to know that it is more time-efficient and less stressful to get acquainted with a new type of organism by a few casual observations before trying to delve into details that I am not familiar, or comfortable, with identifying just yet. As Lab 1 was the very first time I had ever observed a fungus under the scope, I was not about to try to take it through a key. I will begin keying out specimens this week in lab and hopefully become proficient at it by the end of the semester.

Just so it doesn't seem like I am a TOTAL noob, I do have a lot of experience working with both compound and dissecting scopes. I have made thousands of slides of nearly microscopic mites and extensively used dichotomous keys to ID numerous types of arthropods. So, even though I have never used these particular techniques before doesn't mean that they are completely foreign to me.


Observations of Fungal Cultures
We were allowed to observe different fungal cultures in this lab in order to start getting familiar with basic morphology of different genera. I observed the followings fungi:
1. Aspergillua niger
2. Alternaria brassicicola
3. Thielaviopsis basicola
4. Cladosporium sp.

Fungus 1: Aspergillus niger.
I was able to see many conidia, but it was very hard to find the conidiophore of this fungus. I spent a lot of time doing different types of mounts, but I never saw one on my own. The conidia were spherical and formed long chains (Fig. 1).

Fig. 1
Hand-drawing of the spherical conidia of Aspergillus niger from my lab notebook.

Fungus 2: Alternaria brassicicola. 
I was still only able to see the conidia on my own slides, but I believe I saw the conidiophore of these on some of my labmate's slides. Conidia were linked in short and long chains, had a more rectangular appearance, and usually terminated in a knob-like structure (Fig. 2).

Fig. 2
Hand-drawing of the conidia of Alternaria brassicocola from my lab notebook.

Fungus 3. Thielaviopsis bassicola. 
I finally saw a conidiophore with this species! I was very excited about this. Conidia occurred in what seemed like two different types of spores. The first type consisted of very long, thin, rectangular cells, and the second type consisted of more oval-like spores. I believe the second type is referred to as the aleuriospore, in which the spore is further subdivided by a variable number of cells (Fig. 3).

Fig. 3
Hand-drawing of conidia and conidiophore of Thielaviopsis bassicola from my lab notebook.

Fungus 4. Cladosporium sp.
I was also able to easily see the conidiophore on this fungus. It was very long, slender, and rectangular, and terminated in many small spherical conidia (Fig. 4).

Fig. 4
Hand-drawing conidiophore and conidia of Cladosporium sp. from my lab notebook. 

Conclusion
Overall, I learned many new techniques for visualizing and identifying fungi in this first laboratory. However, it will definitely take time to get used to looking at these structures and placing a name to them. I look forward to honing my morphological identification skills in this class.

-C