| Abstract |
|
|
|
|
|
|
|
|
|
|
Fungi are eukaryotic microorganisms that are responsible
in a large part for the decay of dead organic material. They are also
a major cause of food spoilage, forming coloured, fuzzy mats on spoiled food
products.
The identification of most fungi is quite difficult. Classification
of the different species in the Fungi Imperfecti, of which most common
molds are a member, is based primarily on microscopic features such as
morphological structure of reproductive structures and the types of spores
produced. The Fungi Imperfecti are a group of filamentous fungi which
have important industrial and food uses, the most common being the molds
which are responible for spoilage (Atlas, 1994).
The most common type of fungi found on food
are molds or filmentous fungi. These fungi are multicellular organisms
that reproduce in a variety of ways. The major part of a mold mat
is made up of thin strand-like structures called hyphae. At the end
of the hyphae are the reproductive structures that produce spores.
Spores are produced on different structures depending on species.
Conidiospores are spores that are produced in a chain from conidia.
The conidium rests on the end of a specialized hyphae called a conidiophore
(Atlas, 1994).
Spores can also be produced in a cluster by
budding from a conidium. Spores produced by this method are referred
to as blastospores (Atlas, 1994).
Another method by which spores are produced is to enclose
the spores within a sac-like structure called a sporangium. The spores
contained within are called sporangiospores.
By using microscopic features of fungi such
as reproductive structures and macroscopic features such as colour, a rough
identification of molds can be made (Atlas, 1994).
For this experiment, common mold was allowed to grow
on bread, cheese and lime and was examined using scanning electron microscopy
with an aim towards identification. Also a comparison of two preparation
techiniques, osmium vapour fixation with air drying, and osmium vapour fixation
with alcohol dehydration and critical point drying was performed.
The fungi samples were grown from airborne
spores on bread, cheese and lime in a moist environment at room temperature.
The specimens were fixed using the osmium vapour technique. The samples
were placed in a sealed dish with osmium tetroxide crystals overnight.
After osmium fixation, half the samples were allowed
to air dry, while the other half was placed in Karnovsky’s fixative (2.5% Gluteraldehyde,
4% Formaldehyde in 0.1 M Cacodylate buffer at pH 7.2) for 24 hours. The
specimens were removed and washed in 0.1M, pH 7.2 Cacodylate buffer and subjected
to an alcohol dehydration series (50%, 70%, 85%, 95%, and 100%). After
dehydration, the samples were critical point dried (Bozzola & Russell, 1991).
All specimens were mounted on specimen stubs, sputter coated and viewed under
the SEM. Stereo pairs of the cheese fungi were produced using standard
methods.
Macroscopically, the molds produced on
the specimens formed in fuzzy mats, coloured either black or green.
The bread produced both types of mold in even amounts. The cheese
samples produced primarily green molds with small amounts of black while
the lime produced only black mold.
Microscopically, the two types of mold on the bread
were confirmed by examining the reproductive structures. The molds either
produced long single chains of spores on tree-like structures (Figure
1) which budded from a terminal hyphae one at a time (Figure
2) or produced clustered spores (Figure 3).
Closer examination of the spores showed a knobby appearance, but where exactly
on the hypha the spores were produced could not be determined (Figure
4).
The formation of spores on the cheese specimens primarily
occurred in chains on the ends of specialized hyphae (Figure
6 & Figure 7). In addition to single
chain spores, a variety of mold found on cheese produced double-chain spores
(Figure 8). As before, both types of fungi are
produced by budding from the ends of branched hyphae (Figure
9).
The black mold on lime had a very high concentration
of mycelia but produced only a small amount of spores (Figure
10). These spores were produced only in a clustered fashion (Figure
11).
The individual spores were examined on a separate stub.
A samle of bread chain-like spores shows that they are joined end to end (Figure
12). Successive close ups of the spores show that they have a wrinkled
appearance with many protrusions (Figure 13).
An extreme close up (Figure 14) shows the site of
attachment similar to the budding scar on yeast. The specimens which are
osmium fixed followed by air drying seemed to survive more intact than specimens
that were alcohol dehydrated and critical point drying.
The hyphae on the air-dried cheese appear to be normal
(Figure 15), whereas the hyphae on the dehydrated
bread sample are withered and ribbon-like (Figure 3).
As well, the dehydration series was so harsh that the lime specimen did not
survive intact. The spores that were processed by air-drying (Figure
16) and those that were dehydrated and critical point dried (Figure
17) shows no difference in size. Both techniques left the spores in
Figure 18 withered in appearance. The knobby
appearance of the spores might have been increased by the air-drying process.
The green mold, with the chain producing structures
appears to be a deuteromycete of the genus Penicillium. Penicillium
is a common fungus of foods and is characterized by brush shaped conidiospores.
The spores of these fungi are produced in chains from specialized hyphae called
sterigmata (Atlas, 1994).
The black mold is almost certainly a species of the
genusAspergillus. The members of Aspergillus produce black
mods and their spores are radically arranged blastospores. These members
are also some of the most abundant food molds found.
Stereo pairs of the cheese fungus Penicillium
sp. were made at 400x, 600x 900x and 1200x magnifications to show the topography
of the mold.
I would like to thank Doug Bray for his help
in preparing and fixing the specimens and for allowing me the opportunity
to learn about SEM. I would also like to thank Roy Pescador for helping
me with the preparation of my stereo pairs and for his endless computer
knowledge. Finally, I would like to thank my wife, Natalie, for coming
to pick me up after late nights at the lab.
Atlas, R.M. (1994), Principles of Microbiology, Mosby-YearBook, Inc., St. Louis,
Missouri.
Bozzola, J.J., and Russell, L.D. (1991) Electron Microscopy, Jones
& Bartlett Publishers, Inc., London.
