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