EXERCISE 1 PROTISTS: OBSERVATION

AND CLASSIFICATION OF
SPECIMENS
Structure
1.1 Introduction
Objectives
1.2 Materials Required
1.3 General Characters and Classification
Super Group – Excavata
Super Group – ‘SAR’ Clade
Super Group – Unikonta
1.4 Observation of Slides of Protists
Amoeba
Euglena
Paramecium
Plasmodium
1.5 Terminal Questions

1.1 INTRODUCTION
In Unit 2 of Block 1 of Animal Diversity course you have already learnt that
animal protists or the protozoans constitute a large assemblage of
microscopic, unicellular organisms. These organisms exhibit all sorts of
symmetry; show varied modes of nutrition and have diverse life histories.
Some are autotrophic (chlorophyll bearing flagellates); others are
heterotrophic including the saprozoic, phagotrophic or holozoic protozoa. They
may be free living or mutualistic or commensals or parasites. They do not
have tissues or organs, rather they have specialised organelles, and possess
one or many nuclei.

In this exercise you will observe prepared slides of some representative

protozoans. You will learn to classify these protozoans giving justification for
placing them within their various groups and classes. You will also draw
diagrams of the specimens displayed in the prepared slides as you observe
them and compare them with figures given in this manual.

Objectives
After performing this exercise you should be able to:

• identify the specimens belonging to the genera – Amoeba, Paramecium,

Euglena, and Plasmodium and give their scientific and common names,

• draw labelled diagrams of the identified genera,

• classify the identified protozoans up to the level of class,

• list characters justifying their classification and mention special features,

if any,
Animal Diversity: • mention the habitat and geographical distribution of the identified
Laboratory
genera, and

• mention the economic importance, if any, of the identified specimens.

1.2 MATERIALS REQUIRED

1. Compound microscope.

2. Prepared slides of Amoeba, Paramecium, Euglena, and Plasmodium.

3. Drawing sheets/note book, an HB pencil and eraser.

1.3 GENERAL CHARACTERERS AND

CLASSIFICATION
Unicellular eukaryotes make up a group of all other eukaryotes that are not
green plants, fungi, or animals. Unlike prokaryotes, eukaryotic cells have a
membrane bound nucleus to enclose their genetic material and other
membrane bound organelles such as mitochondria and Golgi apparatus or
dictyosomes. These provide specific locations in the cell for its diverse
functions, making the structural organization more complicated than the
prokaryotic cell.

Unicellular eukaryotes are a very diverse group. The single celled eukaryotes
or protists are considered to be the simplest eukaryotes but at the cellular
level they are very complex as they perform all the functions in a single cell
that multicellular organisms perform in various organs. Of these the animal like
protists which were traditionally called protozoans have organelles and
cytoskeleton that vary in structure, and they also use different modes of
nutrition. Some are parasitic and others are predatory or mixotrophs
(combining photosynthesis and heterotrophic nutrition). Some may spend their
lifetime in one position and others may be constantly motile. Reproduction and
life cycles are also highly varied. Genetic and morphological studies have
shown that some protists are more closely related to plants, or fungi or
animals than they are to each other; as a result Kingdom Protista in which all
protozoans and plant like protists were placed has been abandoned. Though
various lineages of protists are now recognized, the terms protists and their
animal like representatives, that is, protozoans is still in use for convenience.

Traditionally, the protozoans have been classified as flagellates, amoebae,

sporozoans and ciliates. However, over the past thirty years molecular
phylogenetic studies have led to extensive modifications of the traditional
classification schemes and the most dramatic changes have occurred in the
protist groups. The recent system of classification places all unicellular
eukaryotes in four major supergroups namely Excavata, SAR clade,
Archaeplastida and Unikonta. We advise you to reread the Section on
classification in Unit 1of the Animal Diversity course and refer to Figure 1.1
before you take up this exercise. We will only refer to the classifications of
those protists (that were put under the former Kingdom Protozoa) which you
will observe during this exercise.
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1.3.1 Super Group – Excavata Protists: Observation and
Classification of
All members of Excavata have similar cytoskeletal features. Some of the Specimens
unicellular species that form this group have a distinguishing morphological
feature that is an ‘excavated’ feeding groove found on one side of the cell.
They include photosynthetic, parasitic, heterotrophic and symbiotic taxa.
Mitochondria may be reduced or modified in some groups and have flagella in
some that differ from other organisms. The main protozoans of this super-
group are placed in three phyla as given below one of which is Phylum
Euglenozoa that is of interest to us:

Super Group Excavata

Phylum

Diplomonada Parabasala Euglenozoa

Subphylum

Kinetoplastida Euglenida

Class

Euglenoidea
Phylum Euglenozoa: This phylum includes predatory heterotrophs,
photosynthetic autotrophs, mixotrophs and parasites. Members of this phylum
have a series of longitudinal microtubules that lie below the cell membrane
and help it to stiffen into a pellicle.The main distinguishing feature is the
presence of a rod with either a spiral or crystalline structure inside each of
their flagella. The two best studied groups of euglenozoans are. Subphylum
Kinetoplasta and Subphylum Euglenida.

Subphylum Kinetoplasta: Members of this subphylum have a single large

mitochondrion with a mass of DNA associated with the kinetosome or basal
body of the flagellum forming a unique organelle kinetoplast..

Class Trypanosomatidea: This class includes member which have one or

two flagella arising out of a pocket typically with paraxial rod. All are parasitic;
examples, Trypanosoma gambiense that infects humans causing sleeping
sickness, Trypanosoma cruzithat causes an important disease i.e. Chagas’s
disease. Leishmania that causes kala azar or visceral leishmaniasis which is
widely distributed in eastern India.

Subphylum Euglenida: Members have pellicular microtubules that stiffen

pellicle.

Class Euglenoidea: Members of this class have a pocket at one end from
which arise two flagella, one is small and the other is large. Mostly
photosynthetic but some euglinids are mixotrophs. They have chloroplasts
and these chloroplasts are surrounded by a double membrane and perform
photosynthesis when sunlight is available and become heterotrophic in the
absence of sunlight, absorbing organic nutrients from the environment. Many
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Animal Diversity: other species ingest prey by phagocytosis. The most frequently studied
Laboratory
euglenid in the laboratory is Euglena.A red eyespot or stigma is present that
functions to orient the organism towards light.

1.3.2 Super Group – ‘SAR’ Clade

The SAR clade has been proposed recently (Adl et al. (2012) ) based on the
whole genome DNA sequence analysis which has revealed that the three
major groups of protists Stramenopiles, Alveolata and Rhizaria can be placed
together in a super group. This is a highly diverse collection of protists. Of the
three major groups, Stramenopiles include the photosynthetic diatoms and
golden and brown algae (these will be dealt in the course on Plant Diversity)
and the protozoan members are placed in Alveolata and Rhizaria.

Super Group SAR-Clade

Group

Stramenopiles Alveolata Rhizaria

Phylum Phylum

Ciliophora Dinoflagellata Apicomplexa Cercozoa Foraminifera Radiolaria

Class

Gregarinea Coccidia

Group Alveolata (unranked): They have membrane enclosed sacs or alveoli

under the plasma membrane. They include both photosynthetic and
heterotrophic protists.

Phylum Ciliophora: Ciliates are named because of the cilia that cover them.
A structural system of fibers along with the kinetosomes (basal bodies of the
cilia) forms the infraciliature just beneath the pellicle. They are present in fresh
water and marine habitats as well in moist soils. They are free living, parasitic
and symbiotic. The distinguishing feature is the presence of two types of
nuclei, one large macronucleus and tiny micronuclei. Macronucleus is
responsible for the functioning of the organism and the micronucleus is diploid
and involved only in reproduction. They have a special type of reproduction –
conjugation during which two cells line up and micronuclei are exchanged via
a cytoplasmic bridge between the two cells. Example is Paramecium.

Phylum Apicomplexa: All the species are parasitic and specialised for living
and reproducing in animal tissue; the infectious stage is called sporozoite.
The sporozoites have a system of organelles at one end known as apical
complex that allows the apicomplexan parasite to penetrate the host cell
membrane. Although apicomplexans are not photosynthetic, they retain a N
sa
modified plastid which suggests their algal origin. Most apicomplexans have
fir
elaborate lifecycles involving one or two hosts.
th
Class Gregarinea: Mature gamonts (individuals that produce gametes) are an
8 of
flo
sp
gl
large, extracellular parasites of digestive tract or body cavity of invertebrates; Protists: Observation and
Life cycle with one host. Example, Monocystis and Gregarina. Classification of
Specimens
Class Coccidea: Mature gamonts are small, and typically intracellular. Life
cycle with two hosts. The best known example, Plasmodium that causes
malaria, lives both in the mosquitoes and humans. Another important disease
causing apicomplexan Toxoplasma gondii is a parasite of cats and can infect
humans.

1.3.3 Super Group – Unikonta

Unikonta includes protists that are closely related to fungi and animals. These
protists form two large groups as given in the flow chart below:
Super Group Unikonta
Group

Ameobozoa Opisthokonta
Class

Myxogastrida Dictyostelida Tubulina Archamoeba

1. Group Amoebozoa (Unranked): It includes many Amoeba species that
have lobe shaped or tube shaped pseudopodia. Amoebozoans include
the slime moulds, tubulinids and entamoebas.

a) Class Myxogastrida: This class includes the plasmodial slime

moulds that grow to form a mass called the plasmodium which is
not multicellular but multinucleated. It feeds by pseudopodia and
when it lacks nutrients it stops growing but gives out fruiting bodies
which functions in sexual reproduction.
b) Class Dictyostelida: This group includes the cellular slime
moulds. The feeding stages are individually functioning cells but
when nutrients are depleted the cells come together and form the
slug like aggregate which functions as a unit, but the cells are still
separated by their individual cell membranes. Ultimately the
sluglike aggregate forms asexual fruiting bodies.
c) Class Tubulinea: This class make a large and varied group of
amoebas that have lobe or tube shaped pseudopodia. These
protists are widely present in freshwater, marine environments and
also in soil. Most are heterotrophs that prey on bacteria and other
protists. The most well known example is Amoeba proteus.
d) Class Archamoebea: Most amoebozoans are free living,
however, individuals of genus Entamoeba are parasitic on
vertebrates and some invertebrates. They are unusual in having
flagellated stages in their life cycle and cysts with several nuclei.

2. Group Opisthokonta: It is a diverse group of eukaryotes that includes

the animals and fungi as well as protists that are more closely related to
fungi and animals than to other protists. 9
Animal Diversity:
Laboratory 1.4 OBSERVATION OF SLIDES OF PROTISTS
We had said in the earlier section that a protozoan is not as simple an
organism as one would think it to be. It is so designed that even though it is a
single cell, it still functions as a complete organism.

The slide that you will get for observation is a permanently, stained whole
mount. While observing it you should sketch it as accurately as you can. You
will note that as you sketch the specimen under observation you are more
likely to look for details you would not see otherwise, and if you put those
details in the right places in the sketch and in the right dimensions, you will
recognise the specimen when you see it again.

Some times you may not be able to see all the details in a single specimen but
by observing several specimens and drawing a composite picture from your
observation you will be able to recognise the specimen in any given slide.
Colour coding also helps. You can devise a colour coding system for your self,
for example blue for the nucleus, green for the chloroplast, etc.

1.4.1 Amoeba
Place a slide of Amoeba (common species is A. proteus) under the
microscope and focus it under low power (5X ×5X), and observe it carefully.
Amoeba looks like an irregular colourless drop of gelatinous protoplasm.

Now focus it under high power (10X ×10X) and note the following features:

i) The body is covered by a thin elastic, semi-permeable membrane called

plasmalemma.

ii) Under the plasmalemma, the protoplasm is clearly distinguished into an

outer clear ectoplasm and an inner granular fluid-like endoplasm.

iii) Try to identify the anterior and the posterior ends of Amoeba. The
anterior end is characterised by a thick layer of ectoplasm (the hyaline
cap), while the posterior end is modified in the form of a tail like region
the uroid (The anterior end of Amoeba is the one in which direction a
pseudopodium is given out for progression).

iv) You will see some blunt, finger-like projections. These are the
pseudopodia and are the extensions of the protoplasm. Pseudopodia
help the Amoeba to move in the medium and also in capturing food.

(a) (b)

10 Fig. 1.1: Amoeba proteus. a) light micrograph (160X), b) diagrammatic view.

v) You can also see the single, clearly stained nucleus, a large single Protists: Observation and
contractile vacuole and large number of food vacuoles in the endoplasm. Classification of
Compare your slide with Figure 1.1. Specimens

Habit and Habitat

Amoeba is commonly found in the mud, in fresh water ponds and ditches, and
slow running streams. It is abundantly found in the water with lots of decaying
vegetation and bacteria.

Geographical Distribution: All over the world.

Classification and its Justification

Domain Eukaryota Membrane bound nucleus and organelles

multiple chromosomes complexed with
histones.

Super-Group Unikonta Unicellular eukaryotes closely related to

animals and fungi.

Group Amoebozoa Have lobe shaped or tube shaped locomotory

organelles called pseudopodia.

Class Tubulinea Tubular/finger shaped pseudopodia.

Freshwater heterotrophs.

Genus Amoeba

1.4.2 Euglena
Take the prepared slide of Euglena and focus it first under low power and next
under the high power of the microscope. Observe the following characteristics:

i) Euglena is an oval spindle shaped organism with a blunt anterior end

and a pointed posterior end.

ii) Body is externally covered with a pellicle.

iii) Underneath the pellicle the cytoplasm is clearly differentiated into an

outer ectoplasm and an inner endoplasm.

(a) (b)
Fig. 1.2: a) Photomicrograph showing Eugena (200 x), b) Diagrammatic view. 11
Animal Diversity: iv) Anterior end of the body bears an opening cytostome, which continues
Laboratory
internally as a tubular cytopharynx. The cytopharynx leads into a large
spherical reservoir.
v) From the base of the reservoir a single whip-like flagellum arises.
vi) A single large spherical nucleus is located towards the posterior region
of the body.
vii) Cytoplasm also contains chlorophyll which is contained in bodies known
as chloroplasts.
viii) You can also see a brightly coloured spot – stigma, closely adhered to
the cytopharynx (hence the name Euglena, which means Eu = true +
glena = eye ball).
ix) Compare your slide with Figure 1.2.

Habit and Habitat

Euglena is a solitary protozoan found in fresh water ponds, ditches, lakes and
slow running streams with a lot of vegetation. It is abundantly found in those
ponds, which contain decaying nitrogenous organic matter such as animal
faeces, leaves, etc.

Geographical Distribution: Abundant in warm waters throughout the world.

Classification and its Justification

Domain Eukaryota Membrane bound nucleus and organelles

multiple chromosomes complexed with
histones. .

Super Group Excavata Unicellular eukaryotes having similar

cytosketetal features. Some with
excavated feeding groove on one side of
cell.

Phylum Euglenozoa Have a series of longitudinal

microtubules under the cell membrane;
rod like structure inside their flagella.
Predatory, heterotrophs, photosynthetic
autotrophs, mixotrophs and parasites.

Sub-phylum Euglenida Have peculiar microtubules with stiffen

pellicle.

Class Euglenoidea Have pocket at one end from which

arise two flagella;one small and the other
large. Mostly photosynthetic but some
are mixotrophs.

Genus Euglena

1.4.3 Paramecium
Place a prepared slide of Paramecium under the microscope and focus it
12 under low power (5X × 5X) and observe the following:
i) It has a cigar-shaped or slipper-shaped body, hence commonly called Protists: Observation and
slipper animalcule (=little animal). Classification of
Specimens
ii) Now focus it under high power (10X × 40X): The entire body is covered
with pellicle, which shows rows of tiny depressions.

iii) Look carefully at these depressions. From each such depression arises
a cilium.

iv) These cilia cover the entire body and are uniform in size except at the
posterior end of the body where they are larger and form a caudal tuft.
These cilia help the Paramecium in locomotion.

v) The anterior end is bluntly rounded and the posterior end is slightly
pointed.

vi) One side of the organism has a depression, which leads into an oral
groove. This groove ends in the mouth or cytostome.

(a) (b)

Fig. 1.3: Paramecium bursaria, a) photomicrograph (430 x), b) diagrammatic

representation.

vii) Now observe the protoplasm, which is clearly divisible into an outer
ectoplasm and an inner fluid-like endoplasm. You can see series of rod
like trichocysts under the pellicle embedded in the endoplasm.

viii) The endoplasm contains two nuclei – one large kidney-shaped

macronucleus and the other small dot-like micronucleus.

ix) You can also see two contractile vacuoles – one towards the anterior
end and the other towards the posterior end.

x) Apart from these structures, many spherical food vacuoles can be

seen in the endoplasm.

xi) Compare your stide with Fig. 1.3.

Habit and Habitat

Paramecium is found in fresh water ponds and ditches rich in dead and
decaying vegetation.

Geographical Distribution: World wide. 13

Animal Diversity: Classification and its Justification
Laboratory
Domain Eukaryota Membrane bound nucleus and
organelles, multiple chromosomes
complexed with histones.

Super Group ‘SAR’Clade Unicellular eukaryotes Includes three

groups i.e. Stramenopila, Alveolata and
Rizaria.

Group Alveolata Share a system of sacs called alveoli

under their plasma membrane.

Phylum Ciliophora Cilia cover the body, presence of two types

of nuclei: one large macro-nucleus and
another tiny micro-nucleus.

Genus Paramecium

Species caudatum/
bursaria

1.4.4 Plasmodium
Place a slide of Plasmodium (a malarial parasite of human) under microscope
and focus it gently and nicely, so that you can see this apicomplexan. The
parasites spread through their host as tiny infectious cells called sporozoites.
Apicomplexans are so named because one end (the apex) of the sporozoite
cell contains a complex of organelles specialised for penetrating host cells and
tissues.

Most apicomplexans have intricate life cycles with both sexual and asexual
stages.

i) Plasmodium is an intracellular parasite of human and other vertebrates

and causes malaria.

ii) The life history of Plasmodium (Fig. 1.4) is completed in two hosts, viz
partly in a definite host, the man and partly in an intermediate host, the
female Anopheles mosquito.

iii) When an infected female Anopheles bites a human, sporozites are

introduced in the blood from where they reach in linear cells through
blood stream and multiply to form merozoites.

iv) After a few cycles in the liver the merozoites enter the red blood
corpuscles (RBCs) and feed on its contents.

v) After having undergone 2-8 schizogonous changes in the main blood

stream, the merozoites assume different shapes called gametocytes.

vi) Development of gametocytes cannot proceed further in blood of human,

therefore, they wait for female Anopheles to bite and feed on the blood.

vii) When gametocytes reach through blood of human to Anopheles, they

14
undergo sporogony for further development.
viii) There are 4 species of Plasmodium causing different types of fever. Protists: Observation and
Classification of
i. P. vivax causes benign/tertian
tertian fever. Specimens
ii. P. falciparum causes malignant tertian fever
fever.
iii. P. malariae causes quartan fever.
fever
iv. P. ovale causes mild tertian fever
fever.

(a) (b)

Fig. 1.4: a) Sporozoite of Plasmodium, (b) Life cycle of Plasmodium.

Habit and Habitat

Plasmodium is found as an intracellular parasite in blood of humans

human and other
vertebrates.

Geographical Distribution: It is widely distributed in tropical

t and temperate
countries of the world like India, Srilanka,
Bangladesh,
angladesh, Nepal and Pakistan.

Classification and its Justification

Domain Eukaryota embrane bound nucleus and organelles

Membrane
multiple chromosomes complexed with
histones.

Super Group SAR Clade Includes 3 groups ofunicellular

eukaryotes, Stramenopila, Alveolata and
Rizaria.
Group Alveolata Share a system of sacs called alveoli
under their plasma membrane
membrane.
Phylum Apicomplexa Parasitic, sporozoites have a set of
organ
organelles (apical complex) with the
anterior end specialised for penetrating the
host cell and tissue. Most with one or two
hosts in their life cycle.
cycle

Class Coccidea Mature gamonts small; spores

s or
oocyst present which contains infective
sporozoite. Two hosts in life cycle.
Genus Plasmodium 15
Animal Diversity:
Laboratory 1.5 TERMINAL QUESTIONS
1. How do you distinguish ectoplasm from endoplasm?

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...................................................................................................................

2. What are the functions of contractile vacuole, cytoplasm, pseudopodia

and food vacuoles respectively?

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...................................................................................................................

3. At what level of organisation are the protists placed?

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