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ALVEOLATA
Phylum Ciliophora
Introduction
Cilia have the same basic structure as flagella, but are smaller, with more than two per cell, and
they beat in a different way than flagella. How is the ciliary beat different from the flagellar
beat in protists? Ciliate specimens included in this lab are: Paramecium, Blepharisma,
Spirostomum, Euplotes, Didinium, Stentor, and Vorticella. For each ciliate we have live
specimens and prepared slides. You’ll see different structures in each and you might consider
doing the two preparations at the same time. Obviously you’re going to see movement in the
live specimen but the nucleus and the larger organelles will be easier to see on the prepared
slides that have been stained to highlight these structures. Depending on our supplier we may
also have vitally stained specimens. Non-toxic dyes have been added to the culture medium in
an attempt to highlight cell organelles and structures. I’ve said “in attempt” because this
material is often variable in quality. When it works it’s great, when it doesn’t there is no real
difference between the two different types of specimens.
Prepare a wet mount for your observations of the live specimens. Many of the ciliates are visible
to the naked eye and you can fish them out of sample containers placed against a black
background. For smaller ones you might want to use a dissecting scope to help snag a ciliate for
your observations. You may also want to add a drop of “protoslow”, quieting solution, to slow
down movement. How does “protoslow” work? As you search the slide for a ciliate try and
remember how big they are. Will you find them under 10X magnification compared to 100X?
It’s an important distinction because it takes a lot longer to scan your slide at 100X compared
to 10X. For your convenience average sizes have been provided.
Things to consider as you make your observations:
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Do the ciliates swim in the same way flagellate specimens do?
Are cilia on the surface all similar or are there different types distributed in special
locations.
Describe the swimming motion of each specimen and make a note of the differences
between the specimens.
Are there special ciliary movements for feeding and swimming?
What shape does each ciliate have, and how easily does it changes it shape, or can it? Why
do ciliates have consitent shapes?
With the preserved slide be sure you have the illumination of your microscope properly aligned.
Too much light flooding through the optics will hide most of the detail. Remember also the way
these slides are made will distort the shapes of the specimens. Ciliates differ from other protists
by having two different types of nuceli; a macronucleus and a micronucleus and these will
usually be easier to see in prepared slides. What are the different roles of the micronucleus and
macronucleus? As their names imply the macronucleus is large and will be easy to see.
Macronuclei come in a variety of shapes; big blobs, beaded chains for example. The
micronucleus is often much harder to see.
In the simplest ciliates the cilia on the surface of the body are all similar in their shape and
appearance. In the most advanced groups the cilia have been modified into membranelles,
undulating membranes, or combined to form cirri. Membranelles are composed of a few rows
of cilia that are structurally fused together so that they end up functioning like paddles.
Undulating membranes are rows of cilia that function together as single unit and cirri are fused
cilia that form large hair-like structures. Most of these modifications can be found around the
cytostome of the protist.
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Paramecium
Fig. 1. Major anatomical structures of Paramecium. ©
BIODIDAC
Paramecium is a good starting point for ciliate observations. Its used in almost every zoology
labs and it introduces some of the main features of of this protist phylum. Paramecium, like all
ciliates has a unique shape, 180 - 300 µm in length, that is maintained by the underlying pellicle
visible as lines the run the length of the organism. As the specimen rolls, try and keep it in focus
and you’ll see the prominent food groove along the side which leads to the cytostome. Cilia
lining the groove propel food, small organic particles, bacteria and other small microorganisms
to the base of the food groove and the cytostome. When sufficient food has accumulated it is
ingested by phagocytosis. If you look closely you may actually see your Paramecium feed by
phagocytosis. If not, be sure to watch the Digital Zoology video of Paramecium feeding. Once
the food vacuole forms it then circulates through the body of the organism riding the
cytoplasmic flows inside the cell. Is there any direction to that flow? If available make a wet
mount of Paramecia that have been feeding in milk stained with a few crystal of congo red. The
food vacuoles have different colors, Why?
As you watch Paramecium swim is there an anterior and posterior end to the protist? Are
they different from each other? How do they differ? Observe how your specimen moves
through the medium - how would you describe it - smooth, jerky, directed, random? Compare
how it moves when trapped in debris. When Paramecium is trying to swim through debris
how does it do it?
Careful observations should reveal the two water expulsion vesicles. One is located at the
anterior end of the ciliate the other at the posterior end. A central vacuole is connected to a series
of radiating canals that branch into even smaller channels that extend throughout the cytoplasm
of Paramecium. Water is removed from the cytoplasm of the cell, collected in the channels,
passing to the radial canals and central vacuole that empties at regular intervals. Why does
Paramecium have to remove water from its cytoplasm? Again careful, and patient observations
should reveal the water expulsion vesicle at work, if not take a look at the video in Digital
Zoology.
Prepare a wet mount of Paramecium leaving one side open. Place the wet mount on the
microscope and locate the paramecia. Place a drop of 2% acetic next to the open edge and, as
the acetic acid diffuses into the preparation watch for the release of the trichocysts. If you don’t
see it happen you’ll certainly see the result, thin needle like-structures surround the ciliate.
What is the role of the trichocysts?
The nuclear material is best seen in the prepared slides. Ciliates have a unique form of sexual
reproduction referred to a conjugation. During conjugation, and after a reorganization of the
nuclear material, two Paramecia join and exchange a haploid set of genetic material. Once they
separate a series of mitotic events follows and the macronucleus is reformed. You won’t be
able to see any of this nuclear exchange in the conjugation slides, only the unique position of
the two partners. How does it differ from binary fission? Paramecium also undergoes simple
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binary fission. There are prepared slides of both conjugation and fission. The large bean shaped
macronucleus is easy to see and if the micronucleus is visible you’ll find it in the groove of the
macronucleus.
Blepharisma
Fig. 2. Major anatomical features of Blepharisma. ©
BIODIDAC
Because of its pink coloration and large size this ciliate is easy to see with the naked eye, from
50 µm to other over a millimeter in length. How many µm’s are there in a mm? It feeds on
other smaller protists and it’s no uncommon for cultures to contain Euglena as a food source.
Don’t confuse the two as you make your observations. Blepharisma is an excellent specimen
for observing the large complex ciliature that surrounds the cytostome. Why do we call it a
cytostome and not a mouth? Prepare a wet mount of this specimen using “protoslow” solution.
The cilia in this organism have been modified into undulating membranes. If you place the
microscope on high power you will see these specialized cilia in the buccal region. Watch
closely for the metachronal wave that passes across the surface. While on the higher power
take a close look at the surface of Blepharisma. Is it smooth? At the posterior end of the
organism you will also see the large water expulsion vesicle and if you watch your specimen
for a few minutes, you may see it slowly swell and then empty. One of the characteristics of
ciliates is the large macronucleus. In this species the macronucleus is a long bead-like chain
that extends down the length of the organism. The micronucleus is usually too small to be
easily seen in the live specimens.
Be sure to compare structures in your live wet mounts to the prepared slides that are also
available,
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Spirostomum
Fig. 3. Major features of Spirostomum. ©
BIODIDAC
Spirostomum is a very large, 150 µm to 4 mm in length, and you’ll easily see them swimming
at the bottom the cultures that have been provided. The pellicle and underlying myonemes of
the long, sausage shaped body is easily seen as a series of lines that wind gently around the body
from the anterior to the posterior end of the ciliate. Compare how this organism moves through
water and when it is trapped in debris. How easily can Spirostomum change its shape? This
is also a good specimen for observing the beaded macronucleus; the micronuclei are next to
impossible to see
As Spirostomum rolls through you field of view watch for the complex ciliary membranelles
at the anterior end that capture bacteria and small flagellates ingested by phagocytosis. Be
careful as you make your observations, small protists in the culture that feed this ciliate are
many magnitudes smaller the Spirostomum. The water expulsion vesicle identifies the
posterior end of this ciliate
Euplotes
Fig. 4. Major anatomical features of Euplotes.
© BIODIDAC
At first glance Euplotes may appear to be circular but if you have a specimen that rolls the right
way you’ll see that the dorsal surface is convex and the ventral concave and the body is dorsal
ventrally flattened. This gives this little protist, between 50 and 100 µm in diameter, a saucershaped appearance. Euplotes is cilia modified into very prominent cirri with distinct
distribution patterns on the dorsal and ventral surfaces, even between the anterior and posterior
ends of the organism. Watch this specimen move and you will see that the cirri are used almost
like walking legs, locomotion and not food aquisition. Keep watching for a while and you may
get to see the water expulsion vesicle fill and empty.
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The cirri are much easier to see than the membranelles that propel food to the cytostome.
Euplotes feeds on small protists. The membranelles may be easier to see in the prepared slides.
But, be warned though in preparing the slides the cap-shaped appearance will be distorted.
Internally you should also be able to see the elongate sausage shaped macronucleus and the
water expulsion vesicle. In stained specimens you may also be able to locate the micronucleus
but because of its size this may be difficult. If you do see it it will be located near the membranes
and the entrance into the cytopharynx.
Didinium
The freshwater, barrel shaped Didinium are usually between 50 and 200 µm in size and feed on
ciliates often their own size! The anterior end of the ciliate forms a proboscis. Cilia are arranged
in two rows around the body with one near the proboscis and the other near the middle. At the
opposite end from the proboscis the water expulsion vesicle identifies the posterior end.
In this protist cilia are involved only in locomotion, not feeding. If as Didinium spins like a
cartoon Tasmanian devil through the medium its proboscis contacts a potential meal it
immediately attaches using the trichocysts that are concentrated in this region. Potential meals
are engulphed by simple phagocytosis if they are small, or by the swelling proboscis that
surrounds larger meals and engulfs it. You may remember the protist behavior video we looked
at in BIO2125 where one tenacious Didinium swallowed a Paramecium whole, and sideways!
A large horse shoe shaped macronucleus may be visible in the live specimen. If you can’t see
it, use the stained preparations to locate this and the other organelles. Once again the
micronulceus will be difficult to see.
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Stentor
Fig. 5. Major anatomical features of
Stentor. © BIODIDAC
Stentor is another large freshwater ciliate you should be able to see swimming around the
preparations that have been provided. Although they tend to have are more condensed shape
when swimming, they attach to the substrate and extend from 1-2 mm when feeding. Stentor
has a unique blue color from the pigment stentorin that is found in many species. The rows of
the pellicle coincide with the location of the pigment, the cilia that cover the body, and
myonemes that run underneath. Contractions of the myonemes allow for changes in shape and
as you watch a sessile Stentor feed you’ll see it twist and bend as it positiona itself in water. The
only place were cilia are absent is at the base of the organism where the holdfast is used to cling
to the substrate when it settles.
In addition to the cilia that cover the body complex cilia form membranelles surrounding the
peristomal area. As these cilia beat potential food is spun in towards the buccal cavity. Food
selection is not passive and by changing the shape of the outer lip of the peristomal area or the
diameter of the opening to the cytostome Stentor can select between living, non-living and
appropriately sized food particles. Watch your specimen closely and you may see this, if not
there’s a video in Digital Zoology that shows ingested food spinning around the outer edge and
down to the cytostome. In addition to bacteria, small and large protists, small multicellar
animals such as rotifers and larval crustaceans can all be sucked into the feeding vortex of
Stentor.
The water expulsion vesicle is essily seen near the cytostome and has a single long collecting
canal running the length of the ciliate’s body. The macronucleus is beaded and also runs the
length of the animal. It can be seen in the live specimens, and there is a micronucleus usually
associated with each of the beads. The macronucleus is easy to see in stained preparations but
it’s doubtful that you see the micronuclei.
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Vorticella
Fig. 6. Major anitomical features of Vorticella.
© BIODIDAC
Vorticella is a solitary organism but often settles in large gregarious clusters on suitable
substrate. They range in size from 50 - 200 µm in length. Vorticella differs in two ways from
the other ciliates we’ve seen. First, it is sessile and lives most of its life attached to the substrate
by a narrow stalk. About the only time they’re free swimming as after binary fission. As the
two daughter cells develop often only one will gain custody of the parent stalk. The other will
swim away and grow its own stalk as it settles down. In some species both swim off. It’s not
likely you’ll see this in your specimens so be sure to see the video of Vorticella binary fission
available in Digital Zoology.
The second difference; unlike other ciliates with a body covered in cilia most of Vorticella’s
body isn’t. The body consists of two parts, a bell shaped body that is attached to a long, often
coiled, narrow stalk. The stalk lacks cilia and even though the striations of a pellicle are visible
on it and the surface of the bell, cilia are either absent or reduced, and only the contractile
myonemes remain. Rows of ciliary membranelles line the outer rim of the bell and a row on
each side of the oral groove spiral towards the mouth located to one side of the oral disk.
Vorticella feeds on bacteria and smaller protists. Membranelles around the bell margin create
water currents pulling food in towards the the ciliate. Appropriately sized food is moved by the
complex ciliature lining the oral groove towards the cytostome. Anything too large is rejected
and passes by the protist. Although you may not be able to see your specimen feed you should
see the water currents that Vorticella creates, especially if large pieces of organic material are
rejected and speed past the specimen.
If the Vorticella you’ve been observing hasn’t exhibited the avoidance response of retracting,
gently tap the microscope slide. Myonemes run the length of the stalk and their contraction pulls
the bell-shaped body out of harms way. The elasticity of the pellicle returns the stalk to its
original length when the myonemes relax. The water expulsion vesicle is also located near the
surface of the oral disk and empties into the vestibule in front of the cytostome and at the base
of the oral groove.
In prepared slides you’ll see many of the same structures along with the long sausage shaped
macronucleus that takes on a horseshoe shape as it bends through the body. Once again the
smaller micronucleus may be harder to see.
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Phylum Apicomplexa (Sporozoa)
Introduction
By their very nature these are small organisms that have developed complex life cycles
associated with their parasitic existence. Being a parasite requires that the organism pass
successfully between the hosts. To accomplish this requires some very special adaptations of
the body itself and the life cycle of the species.
Plasmodium - Malarial life cycle
Fig. 7.
Life cycle of the malaria, Plasmodium (© BIODIDAC)
The causative organism of malaria is Plasmodium vivax, and its life cycle is a good example
of some of the strategies that parasites use to survive. For parasites there is a danger that the
organism will not succeed in moving between the different hosts in its life cycle. To increase
the chances the transfer will be successful, a series of asexual reproductive stages occur
allowing parasite numbers to increase dramatically in one host before making the jump to the
next. In the malaria life cycle sporogony and schizogony increase the parasite numbers in the
mosquito and human host respectively. Gametogony produces the gametes in the vertebrate
host and once consumed by the mosquito they combine to form the zygote.
Sporozoites in the salivary glands of the Anopheles mosquito are injected into the human host
as the mosquito tales its blood meal. Once in a human the Sporozoites enters liver cells and
increase in number by larval amplification, shizogony, to produce cells filled with
merozoites, the products of shizogony. The infected liver cell containing them is the shizont.
Shizonts rupture releasing merozoites into the blood that then penetrate red blood cells. The
feeding stage in the red blood cell is the trophozoite and is visible as the ring stage. The
numbers inside the cell increases and trophozoite become merozoites, a shizont that ruptures
again releasing merozoites into the blood. This cycle repeats itself and is synchronized between
red blood cells with the resulting chills and fever that malarial patients suffer.
Some of the merozoites don’t invade new red blood cells and instead differentiate into one of
two gametocytes, gametogony. Male and female gametes can’t be distinguished from each
other until they are consumed by the Anopheline mosquito that is attracted to a malaria victim
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by the increased body temperature and perspiration associated with the fever. Once the
gametocytes are in the mosquito gut, their development into the male and female gametes
occurs and they fuse to form the zygote that attaches the gut wall of the mosquito. The zygote
forms an oocyst that protects it from the digestive environment of the mosquito gut. Sporogony
results in large numbers of sporozoites being released into the body cavity of the mosquito
(hemocoel). They eventually penetrate the mosquito’s salivary glands and the cycle repeats
itself.
Prepared slides Prepared slides of the malarial life cycle are becoming increasingly
difficult to obtain and more often than not blood smears are all available for observation. Digital
Zoology contains pictures of the oocyst stage embedded in the gut of the mosquitoes, and
sporozoites in salivary gland squashes from infected mosquitoes.
The small size of these organisms will require the use of the oil immersion lens when you
examine the blood smears. Red blood cells lack nuclei. Infected red blood cells don’t have the
donut shape, appear a bit larger, and contain stained nuclear material. White blood cells have
nuclear material, how do you tell the difference between a white blood cell and an infected red
blood cell? In the early stages of an infection the plasmodial parasite in the red blood cell forms
a ring-like structure from the trophozoite’s nucleus and the surrounding vacuole. This is the ring
stage of the life cycle. As the trophozoite continues to grow its shape changes becoming, ovoid,
elongate, or irregular in appearance. Once it’s reached its final cells it divides to produce
merozoites filling the shell of the red blood cell, the shizont. Locate the ring stage and merozoite
filled shizonts.
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