Lab Exercise: Diversity of Eukaryotic Microbes
OBJECTIVES
1. To observe representatives of major types of microbes.
2. To cultivate select representatives of major types of microbes.
3. Understand key characteristics of the different eukaryotic microbes and groups found
within these Kingdoms.
INTRODUCTION
Eukaryotic organisms have a nucleus in a membrane. They are typically more complex than
prokaryotic organisms. They make up the Domain Eukarya and include the major kingdoms of
Protista, Fungi, Plantae and
Animalia. Protista is a diverse group
that includes many different types
of organisms, divided into the
animal-like protists, or protozoa,
and the plant-like protists, or algae.
Protozoa are examples of Protistans
that we will survey in this lab. Fungi
are a kingdom of organisms that
may be unicellular or multicellar,
yeasts or molds. All fungi absorb
their nutrients from their
environment. Animals are
multicelluar organisms that ingest
their nutrients. This lab will be
presented in three parts, one
focusing on Protozoa, one on Fungi
and one on Helminths (parasitic worms of the animal kingdom).
Protozoa
These are unicelluar eukaryotes. They do not have cell walls, but do have a membrane called a
pellicle surrounding the cell. They have a nucleus and membrane bound organelles. They typically
form cysts, a hardy dormant life-form that allows survival of harsh environments. Protozoa are
classified into four phyla or groups based on their means of locomotion:
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Flagellates (or phylum Mastigophora)
Amoebae (or phylum Sarcodina)
Sporozoans (or phylum Apicomplexa) and
Ciliates (or phylum Ciliophora).
Flagellates have flagella for locomotion. Amoebae move by use of a pseudopod. Ciliates move with
the aid of multiple cilia. The sporozoans have no means of locomotion and therefore have a lifestyle
that makes them all internal parasites.
Pond water is a great source of assorted protozoan microbes. See the virtual pond
(http://microscopy-uk.org.uk/index.html?http://microscopy-uk.org.uk/ponddip/).
Fungi
Fungi can exist as unicellular yeast or multi-cellular molds. The mold species consist of numerous
filaments called hyphae. A mass of filaments is called mycelium and is what gives the mold its
“fluffy” appearance. They live either as parasites or as
saprophytes, absorbing organic material from their environment.
Their cell walls generally have chitin, a polysaccharide unique to
fungal cell walls.
Taxonomy has changed in recent years, currently based more on
genetic similarity than morphological characteristics, and the
Kingdom Fungi has been rearranged to include and exclude many
groups. For organisms we will work with in this lab, the proper terminology is that of the Kingdom
Eumycota. Within this Kingdom there are three phyla: Chytridiomycota (e.g. aquatic fungi),
Zygomycota (e.g. Rhizopus), and Dikaryomycota, including the subphyla Ascomycotina (e.g. the
antibiotic producing Penicillium and the yeast Saccharomyces cerevisiae) and Basidiomycotina (e.g.
the mushroom Agaricus). In addition, there are lichens, which are actually composite organisms in a
symbiotic relationship---fungi and photosynthetic algae or bacteria.
Most fungi reproduce via spores that are made either sexually or asexually.
Phyla Zygomycota and Dikaryomycota (including the Ascomycotina and
Basidiomycotina) are based mainly on the type of sexual spore that is
produced: zygospore, ascospore, or basidiospore. The sexual spores are
produced by meiosis, and are often contained within a spore forming
structure. Even yeasts produce sexual spores, although they more commonly asexually reproduce by
budding. On the other hand, asexual spores are the more commonly produced spore, their function
being dispersal so that the fungus can disseminate itself throughout the environment. Asexual
spores that are enclosed in a structure are called sporangiospores and ones not enclosed are
condiospores.
For this lab, we will be looking at fungi as molds and as unicellular yeast.
Web resources:
The Amazing Kingdom of Fungi, http://waynesword.palomar.edu/ww0504.htm
UC Berkeley's Introduction to Fungi http://www.ucmp.berkeley.edu/fungi/fungisy.html
Helminths
Helminths are parasitic worms within the Kingdom Animalia. There are many phyla of worms in this
kingdom, but we are looking at just a couple---Platyhelminthes and Nematoda. Platyhelminthes are
flatworms, divided into the 2 classes of Cestodes (tapeworms) and Trematodes (flukes). They are
hermaphroditic, meaning both egg and sperm are contained in the same organism. Mainly, you will
see genital organs inside of them under the microscope, probably with lots of eggs. The Nematodes
are roundworms. They are sexually dimorphic (two sexes), although some also contain
hermaphroditic individuals, and are a bit more evolved than the platyhelminthes.
LAB EXERCISES
I. Protozoa: the animal-like protists
Team supplies
Prepared slides of Trypanosoma
Prepared slides of Plasmodium
Living specimen of Amoeba
Living specimen of Paramecium
Other assorted specimens as
available
Individual supplies
Microscope slides
Cover slips
Protocol:
1. Make wet mounts of live Paramecium, Ameoba, other available protozoa and the pond water.
NOTE: To get a good sample, use the pipette and draw up from the bottom of the container or in
“the gunk” to get your specimen. Do NOT stir the specimen container first.
2. Start with the 10x lens and go to 40x lens. Oil-immersion will magnify too much for most pond
water protozoa.
3. Once you have found your specimen, adjust the iris diaphragm for even better viewing.
4. Look at the prepared blood slides of Trypanosoma and Plasmodium using the 100x oil immersion
lens. Trypanosoma will be easy to see: it is far larger than the red blood cells. However,
Plasmodium will be difficult since the parasite will be inside of the RBCs.
II. Fungi: yeasts and molds
Team supplies
Culture of Saccharomyces cerevisiae
(ale yeast)
1 lactose broth with Durham tube
1 sucrose broth with Durham tube
1 glucose broth with Durham tube
Prepared slides of Rhizopus
Prepared slides of Penicillium
Fresh Agaricus mushrooms
Moldy bread
Example plates of various fungi
Lactophenol cotton blue dye
Cover slips
Razor blades
Individual supplies
Microscope slides
Protocols:
Saccharomyces cerevesiae
1. Make a wet mount of the culture by mixing a small drop of S. cerevisiae into a drop of lactophenol
cotton blue.
2. Observe under the microscope.
3. As a team, inoculate S. cerevisiae into 3 sugar broth tubes: lactose, sucrose, and glucose. Next
class, you will be observing these broths for evidence of fermentation with production of CO2
Prepared slides of Rhizopus and Penicillium
1. For Rhizopus, identify hyphae, sporangia, and sporangiospores. Also differentiate between the
sexual zygospores and the asexual sporangiospores on the slides.
2. For Penicillium, identify hyphae, conidia and conidiospores.
Agaricus mushrooms
1. Using your razor blade, cut a thin gill section from the mushroom cap. Make a wet mount of 1
thin gill section in a drop of lactophenol cotton blue.
2. Observe under the microscope, 10X and 40X, looking for basiospores and basidia.
Plates of various fungi
1. Make macroscopic observations of any molds growing on plates, noting color and reproductive
hyphae.
Bread mold
1. Make a wet mount of your mold mold by mixing a small sample with a drop of lactophenol cotton
blue.
2. Observe under the microscope.
3. Try to identify fungal class (and perhaps even the genus) based on spore characteristics.
Compare to previous examples of molds and see Alexander Atlas pages 19-27.
III. Helminths
Team supplies
Preserved Taenia (tapeworm)
Preserved Ascaris (roundworm)
Protocol:
1. Observe the specimens of both tapeworms and roundworms. Note differences in
shape.
2. Note components of the tapeworm: the scolex and proglotids. See the Alexander
Atlas pages 160-167
3. Compare male and female Ascaris worms. See adjacent photograph, the female is
on the left, male on the right.
DATA AND OBSERVATIONS
I. Protozoa
1. Draw representatives of the following protozoa:
Amoeba
Total magnification _______
Trypanosoma
Total magnification _______
Paramecium
Total magnification _______
Plasmodium
Total magnification _______
II. Fungi
1. Draw S. cerevisiae:
S. cerevisiae
Total magnification _______
2. Observe S. cerevisiae growth in sugar tubes. Note presence of growth, color of medium, and
presence/absence of a gas bubble in the Durham tube:
Glucose broth:
Lactose broth:
Sucrose broth:
3. Draw and label representatives of spores/ spore structures in Rhizopus, Penicillium , and Agaricus:
Rhizopus
Total magnification _______
Penicillium
Total magnification _______
Agaricus
Total magnification _______
4. Make macroscopic observations of molds provided:
Name _________________
Name _________________
Name _________________
5. Draw observations of the wet mount of bread mold:
Total magnification _______
DISCUSSION
I. Protozoa
1. Compare and contrast the three mechanisms of motility displayed by protozoa.
2. Trypanosoma and Plasmodium are both found in blood. What diseases do these microbes cause?
II. Fungi
1. Define the term yeast. Why are yeast cells larger than bacterial cells?
2. Was S. cerevisiae capable of fermenting all sugars? What were the indications of fermentation,
and which tubes showed positives? Relate your results to what you know about the normal
biotechnological use of this yeast.
3. Complete the following table
Organism
Sexual spores
Rhizopus
Phylum
Aspergillus
Penicillium
Agaricus
4. Which phylum and genus did your bread mold belong to?
5. How do fungal spores differ from bacterial endospores?
III. Helminths
1. How does a male roundworm differ from a female roundworm?
2. Name a disease caused by a roundworm.