Reference no: EM133125001

Practical: Mycology

All culturing sections of this practical session will be completed in pairs, however all other procedures and observations need to be conducted individually.

Background Information
Fungi are a large, varied and diverse group of organisms that are distributed worldwide. They are found in diverse habitats and commonly survive on organic debris of plant, animal or microorganism origin. Fungi function, with bacteria and other small organisms, to break down organic debris into simpler forms consequently recycling essential nutrients. All fungi require preformed organic substrates as an energy source. Fungal activity is particularly strong in acid soils where bacterial activity is minimal.

Fungi are capable pathogens. This action is a particularly significant activity in the plant kingdom where fungi are the most important group of microbial parasites; and in the animal kingdom, the significance of mycotic diseases is increasing. Additionally fungi have created issues of poisoning, with fungal toxins found in items of food for human and animal consumption.

The economic importance of fungi overall is as great as that of bacteria. Fungal use in industrial applications is substantial, ranging from use within the food industry to the production of industrial products, alcohol, organic acids, vitamins and antibiotics.

Objective Part A
To examine three divisions of the kingdom fungi including:
• Zygomycota
• Ascomycota
• Basidiomycota
With the intent of developing skills to identify taxonomic characteristics

Identification information for Part A of this practical can be found at the end of the practical after the information on writing up your practical report. Where the instructions say to confirm with figure X, please look up the identification information.

Part B
To compare two different fungi with respect to growth characteristics in selected environmental conditions. This is the information that will be written up as a full practical report.

Part A
For the recording of your observations please include in your illustrations: the Phyla, Genus, the viewing magnification; and where possible label the fungal structures observed. All observations of demonstration slides will be carried out using digital images on learnJCU prior to entry into the lab. The same demonstration slides will be available in the lab for assistance with any queries only.
Complete all mycology manipulations in a certified Biological Safety Cabinet (BSC) II.

Terminology: Division(phylum) - order - genus - species (if known)

A1. Zygomycota - Entomophthorales - Entomophthora
Entomophthora muscae is a member of the Zygomycota that rarely produces zygospores. It more commonly produces a series of sporangia with a single spores at the tip, pushing out from the cuticle of the infected host. These are often described as conidiophores bearing a single conidium at the top. The demonstration slide of Entomophthora shows these fungal fruiting bodies bursting through the integument of the fly. No sexual stage is present. The fungal infection coming through the exoskeleton of the insect instigates the production of packed masses of sporangiospores.

A1.1 Label from the demonstration slide, the conidiophores bearing a single apical conidium, hyphae inside the host and the host exoskeleton (use figure
for support).

A3 Zygomycota - Mucorales - Rhizopus stolonifer

A3.1 Draw and label from the demonstration slide of Rhizopus stolonifera (NB 2 images to use here), the sporangium, columnella, colarette and sporangiospores (asexual stage), suspensors and zygospores (sexual stage) (use figure 6.4 for support)

A3.2 Within a BSC II, mount some of the mycelium from the prepared culture of Rhizopus in trypan blue, using a sterile toothpick to manipulate the specimen.
• Tease apart the mycelium and add a coverslip.
• Examine your prepared slide, draw relevant images and label any sexual and asexual structures you observe. Note any any differences between the slide that you have prepared and the demonstration slide provided.
Note: Sexual structures may not be present in prepared cultures as sexual stages often require very particular conditions to develop

A4 Ascomycota - Schizosaccharomycetales - Schizosaccharomyces octosporus
A4.1 Draw and label from the demonstration slide of Schizosaccharomyces octosporus. sexual reproductive features: ascus containing 8 ascospores. This needs to be done at maximum magnification to see spores. Select an ascus where you can see most or all spores to draw. (compare with figure 6.5).

A5 Ascomycota - Eurotiales - Aspergillus nidulans/flavis
A5.1 There are two images to view (sexual and asexual) Draw and label from the demonstration slides of Aspergillus nidulans, the cleistothecium containing multiple asci, each with 8 ascospores (sexual), NB. Hulle cells are not obvious. Also draw and label the asexual conidiophore, phialides (these look like fingers that the conidia come off the top of) and conidia. (compare with figure 6.6a and b).

A5.2 Within a BSCII, Use a sterile toothpick to mount some of the mycelium from the prepared culture of Aspergillus nidulans/flavis (or A. flavus) in trypan blue.
• Tease apart the mycelium and add a coverslip.
• Gently put pressure on the coverslip to release the asci.

Examine your prepared slide, and observe the sexual and asexual structures and record all of your observations, including noting any differences between the slide that you have prepared and the demonstration slide provided.
Note: Sexual structures may not be present in prepared cultures as sexual stages often require very particular conditions to develop.

A6 Ascomycota - Pezizales - Tuber aestivum (black summer truffle) A6.1 Draw and label from the demonstration slide of Tuber aestivum, the thin walled asci containing between 1 and 3 spiky ascospores. Also note the surrounding thick hyphae. Compare with the laminate provided on LearnJCU and draw your observations.

A7 Basidiomycota - Ustilaginales - Ustilago cynodontis
A7.1 Draw and label from the demonstration slide of Ustilago cynodontis infecting plant tissue ustilospores alongside plant tissues. (compare with figure 6.8).

A8 Basidiomycota - Agaricales - Chlorophyllum molybdites & Agaricus bisporus
A8.1 Draw and label from the demonstration slide of Chlorophyllum molybdites the hyphae and basidiospores
A8.2 Draw and label from the specimens of Agaricus bisporus provided in the lab the stipe, pileus, gills, and annulus (compare with figure 6.9).

A9 Basidiomycota - Agaricales - Lycoperdon
A9.1 Draw and label from the demonstration slide of Lycoperdon, hyphae with cavities (lacunae) lined with basidia (compare with figure 6.10).

Part B Environment and Microbes
Biological factors and physico-chemical conditions influence the activity of microbes in the environment and their general survival. The interaction among the environment, host and microbe is significant in disease development.

The temperature tolerance range of mesophiles and thermophiles dramatically illustrates the significance of temperature in microbial activity.

1. Within a BSCII, use a sterile toothpick to inoculate Fusarium onto 4 potato dextrose agar (PDA) plates labelled 5°C, 25°C, 37°C and 45°C on both base and lid. Note: Use non-sporing fungi if possible.
2. Within a BSCII, use a sterile toothpick to inoculate Humicola insolens
onto 4 PDA plates labelled 5°C, 25°C, 37°C and 45°C. (Figure 6.11).

3. Seal the agar plates with parafilm. On the lid, draw around the edges of the subculture square/s with a fine marker, to assist with the growth measurements in the following days. Place a line across the lid through the plug of agar to aid in measuring and place a dot to measure your zero point at the edge of the agar (see figure 6.12) and incubate the plates at the relevant temperatures right side up.
4. Measure colony radius on day 2 (Wednesday), day 3 morning and day 3 afternoon(Thursday). Please see Figure 6.12. Make observations regarding contamination and other issues that might affect reliability of your results.

5. Graph these results as per the instructions below.

Process your measurements

1. Plot radius (y axis) vs time (x axis) for the different temperature plates for Fusarium and Humicola (you should have up to 8 separate lines and time should be reported in hours to take into account different return times)
2. Calculate the linear growth rate (change in radius over time in a linear area of the curve - the gradient of the curve) for each temperature and species
3. Plot growth rate (y axis) vs temperature (x axis) for each species
4. Answer the following as part of the discussion
a. What temperature provides the best conditions for growth rate for each species?
b. What relevance does this have to people?

Attachment:- Mycology.rar