Last Update: 4 December 2017

PI
CP- 33
PREPARATION AND STAINING OF PROTOZOAN PARASITES
Preparation of blood film for protozoan parasite:
A small drop of blood is placed on one side of a clean grease free slide. Holding another slide at an
angle of 30 - 40 and in contact with first slide, the drop of blood is touched and leads to spread along the
line of contact between the slides. Second slide is then pushed along the first with a smooth rapid
movement; thus drawing the blood out to form a thin film depends on the size of the drop. The film is then
dried rapidly waving in air.

Fixation and staining of blood film for protozoan parasite:

For best result, parasites require to stain longer time than ordinarily used in differential staining of
blood film. Giemsas stain is probably superior of them.

Staining with Giemsas stain.

I. Preparation of stain.
Certified Giemsas stain is available both as stock solution and as dry powder. The stock solution is
diluted with distilled water and buffered at pH 7.0. the stock powder solution is prepared as following
Giemsa powder : 0.6 gm
Acetone free methyl alcohol : 50.0 ml
Glycene : 50.0 ml
II Staining procedure
The dry blood film is fixed in absolute acetone free methyl alcohol for 10 min. and left to dry. The
stock solution of stain is diluted to 1:30 of pH 7.0. the slide is placed on a staining pack with film side up
and covered with diluted stain. It is left to stand for 30 min adding more stain if necessary to keep the film
well covered.
Washed by pouring natural distilled water over the preparation until colour did not run from it to a
noticeable extent. Drain and stand on end to dry. Although it is not necessary to cover the film, synthetic
resins are used as mounting medium. For covered preparation, a little xylol is placed on the dried film before
the medium and cover glass is added.

CONTENT
1. Leishmania donovoni
2. Leidyana sp.
3. Ascaridia sp.
4. Haemoproteus sp.
5. Paramphistomum sp.
6. Trypanosoma evansi.
7. Raillietina sp.
8. Ascaris lumbricoides.

1. Monomorphic
2. Locomotion by flagella.
Hence phylum : Sarcomastigophora
1. Presence of flagella at tropozoit stage.
2. Chromatophores absent.
3. Body covered by pellicle.
Hence, Class : Zoomastigophora.
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1. Presence of Kinetoplast.
2. Flagellum develops from basal granule.
3. Presence of undulating membrane.
4. Single flagella arise from the basal body and leaves as free flagella.
Hence, Order : Kinetoplastida
1. Intracellular amastigote form present with in the lymphocyte.
2. Round body having large nucleus, kinetoplast and basal body.
Hence, it is amastigote stage of Leishmania donovani

Kinetoplast
Nucleus

Fig: Amastigote stage of Leismania donovani

1. Presence of apical complex at one end of the body.

2. Single type of nucleus (vasicular), lack of cilia.
3. One or more tiny opening for feeding (micropores)
Hence Phylum : Apicomplexa.
1. Conoid (a part of epical complex) is complete.
2. Oocyte formed at the end of the sexual cycle.
3. Sorozoite stage is the infective stage.
Hence Class : Sporozoa
1. One vasicular nucleus, body divided by septa into segments.
2. Anterior most part is mucron
3. Extracellular gomon.
Hence, Subclass : Gregarinia
1. Trophozoite posses mucron
2. Two gamons form Syzygy
3. Asexual reproduction merogony absent in host.
Hence, Order: Eugregarinida
1. Gramont divided into an anterior protometric and a posterior deutometric.
2. Hold fast organallae the epimerite is formed from protomerite.
Hence, Sub Order: Lephalina.
1. Solitary, simple or knob like epimerite.
2. Sporocyst are extruded by sporoduct.
3. Oocyte are board-shaped.
Hence, it is the Leidyana sp.

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 Epimerit Protomerite
e

Trophozoit Gamont Syzygy

stage stage
Fig: Leidyana sp.

1. Body is unsegmented, bilaterally symmetrical and round in section.

2. Body covered by thick cuticle.
3. Blood vascular system is present.
4. Body cavity pseudocoelomate.
Hence, Phyllum: Aschelminthes.
1. Pseudocoelomate animal.
2. Unsegmented or superficially segmented body.
3. Amphids pore like.
Hence, Class: Nematoda.
1. Phasmid present.
2. Amphids pore like and labial in position.
Hence, Subclass: Phasmida.
1. Three large lips
2. Caudal alae are laterally placed.
Hence, Order: Ascaridia.
1. No posterior bulb to the oesophagons.
2. Male possesses a pre anal sucker.
3. Male possesses small aloe and 10 pairs of papillae.
4. Valva is situated at a short distance, anterior to the middle of the body.
Hence, it is Ascaridia sp.

Excretory pore

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 Mouth

Gonopore

Anus

Male Female
Fig: Ascaridia sp.

1. Presence of apical complex at one end of the body.

2. Single type of nucleus (vascular), lack of cilia.
3. One or more tiny opening for feeding.
Hence Phylum : Apicomplexa.
1. Conoid (a part of epical complex) is complete.
2. Oocyte formed at the end of the sexual cycle.
3. Sporozoite is in the infective form.
Hence Class : Sporozoa
1. Intra cellular gamonts.
2. Merogony present.
Hence, Subclass: Coccidia.
1. Merogony, gametogony and sporogony present.
2. Sporozoite formed in the oocyte.
3. Merogamet and microgamet developed individually.
Hence, Order: Eucoccidia.
1. Syzygy and conoid absent.
2. Macro and microgametocyte developed independently
3. Mortile zygote
Hence Suborder: Haemosporidia.
1. Merogony in vertebrate erythrocyte.
2. Haemozoin pigment present.
3. Only macro and micro gametocytes are formed in the RBC.
4. Merogony occur in the internal organ.
Hence, it is Haemoproteus sp.

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 Gametocyte
RBC
Gametocyte

Nucleus

Nucleus
&
parasite

Microgametocyte Macrogametocyte
Fig: Haemoproteus sp. ( &)
1. Dorsoventrally flattened, acoelomate and bilaterally symmetrical.
2. Segmentation absent.
3. No definite anus.
4. Circulatory and respiratory system absent.
Hence, phylum: Platyhelminthes.
1. Two suckers present.
2. Gut consist of mouth, muscular pharynx and bifurcated intestine.
3. Branchial excretory system.
Hence, Class: Trematoda.
1. Anterior oral sucker and posterior ventral sucker.
2. Sucker without hook.
3. Hermaphrodite.
4. Single posterior excretory pore.
Hence, Order: Digenea.
1. Large sub terminal posterior sucker.
2. Genital pore situated at the anterior part of the body.
3. The vitellaria present between the pharynx and posterior sucker.
4. Testes are slightly lobed in tandon anterior to ovary.
Hence, it is Paramphistomum sp.

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 Anterior sucker

Intestine

Testis

Ovary

Vitellaria

Posterior sucker

Fig: Paramphistomum sp.

1. Single type of nucleus.

2. Presence of flagella.
3. Single cell animal.
Hence, Phylum: Sarcomastigophora.

1. Presence of flagella in tropozoite stage.

2. Body covered by pellicle.
3. Chromophore absent.
Hence, Class: Zoomastigophora.

1. Presence of Kinetoplast.
2. Flagellum developed from basal granule.
3. Single flagellum arises from the basal body and lives as free flagella.
4. Oral vesicular nucleus
Hence, Order: Kinetoplastida.

1. Trypomastigote from in the circulatory blood.

2. Multiplication by longitudinal binary fission.
3. Nucleus above the kinetoplast.
Hence, it is Trypanosoma evansi

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 Lymphocyte

Monocyte
Neutrophil

Eosinophil

RBC.

Undulating membrane
Free flagellum

Kinetoplast
Nucleus

Fig: Trypanosoma evansi.

1. Dorsoventrally flattened, acoelomate and bilaterally symmetrical.

2. Segmentation absent.
3. No definite anus.
4. Circulatory and respiratory system absent.
Hence, phylum: Platyhelminthes.

1. No alimentary canal and body cavity.

2. Body divided into numerous proglottids.
Hence, Class: Cestoda.

1. Head is scolex provided with sucker and hook.

2. Body is a strobila provided with number of segments or proglottids.
3. Neck between scolex and proglottids.
4. General pores are unilateral.
5. Rostellum with two hundreds (200) hooks in two rows.
Hence, the specimen is Raillictina sp

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 Head

Proglottid

Raillictina sp.

Fig: Scolex of Raillictina sp.

1. Body is unsegmented, bilaterally symmetrical and round in section.

2. Body covered by thick cuticle.
3. Blood vascular system is present.
4. Body cavity pseudocoelomic.
Hence, Phylum: Aschelminthes.
1. Pseudocoelomate animal.
2. Unsegmented or superficially segmented body.
3. Amphids pore like.
Hence, Class: Nematoda.
1. Phasmid present.
2. Amphids pore like and labial in position.
Hence, Subclass: Phasmida.
1. Mouth with three lips
2. Pharynx with a muscular bulb.
Hence, Order: Ascarida.
1. Body elongate, cylindrical, yellowish brown in colour.
2. Male possesses two equal or almost equal copulatory spineless in ventrally curved tail end.
3. Two needle like chitinoid setae project from the anus.
4. Gonopore on the ventral surface of the vulvar waist, at about one third of the body length from
the anterior end.
Hence it is the Ascaris lumbricoides.

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 Mouth
Excretory pore

Anus

Valver waist
Tail
Male Female
Fig: Ascaris sp.

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