Blood Examination The most commonly used technique for blood
examination is stained blood films. Geimsa stain is usually used to
stain the films. Delafilds haematoxylin stain is used for
microfilariae. Either thick or thin films may be used depending on
the circumstances. The thick film is more sensitive in detecting
parasite and also saves time in examination. The thin film
technique cause very little distortion of the parasite, and permits
species identification when it may not be possible in thick films,
but many fields must be examined to detect parasite when they are
few in number. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 3
Continue Therefore, both thick and thin films must always be
prepared when searching for plasmodia and trypanosomes. If a
precise identification can not be made from thick film, the thin
film will be available. Thick films should be used when searching
for microfilariae. The most economical use of slides is achieved by
making a combination thick and thin slide. However, combination
films must dry thoroughly 8-10 hrs. to overnight before they can be
satisfactorily stained. Slides for malaria should be stained in the
same day. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 4
The thin films will dry quickly and can be stained as soon as
they are dry, and examine for parasites. If parasites are not seen
in the thin film, stain the thick film using Fields stain, and
examine for parasites. Direct wet mounts of fresh whole blood (or
centrifuged blood) are usually used for detection of microfilariae
and trypanosomes, this only gives evidence of infection and stained
films are necessary for confirmation of species present. In areas
where malaria, trypanosomes, and/or microfilariae may all present,
both wet and stained films should be prepared and examined. If
neither trypanosomes nor microfilariae occur in region, only
stained smears need to be made for detection of plasmodia. Raed Z.
Ahmed, Medical Parasitology Lab.,2012 Continue
Slide 5
Examination of Thick & Thin blood smear For optimum
staining, the thick and thin films should be made on separate
slides and different concentrations used for staining. When its
done good quality staining of thick film is of primary importance,
best results are obtained if the blood smear have dried overnight.
Fixation of thin blood film done by adding 3 drops of methanol, or
dipping it in a container of methanol for few seconds, with
prolonged fixation it may be difficult to demonstrate Schuffners
dots and Maurers dots. To permit dehemoglobinization, thick film
should not be fixed; therefore avoid exposure to methanol or
methanol vapor Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 6
Focus on film with 10x objective and search for microfilariae.
They are easily detected with 10x objective. If microfilariae are
present, switch to oil- immersion objective and identify the
species. Also, look for malaria parasites with oil- immersion
objective, at least 100 fields should be examined. Microscopy of
thick film should reveal the following features: 1.The background
should be clean, free from debris, with a pale mottled- gray color
derived from the lysed erythrocytes. 2.Leukocyte nuclei are stained
a deep, rich purple. 3.Malaria parasite are well defined with deep-
red chromatin and pale purplish blue cytoplasm. Raed Z. Ahmed,
Medical Parasitology Lab.,2012 Reading of Thick Film
Slide 7
Focus with the 10x objective on the thin terminal end of the
film where the RBCs are in one layer, put oil drop on the slide and
switch to the oil- immersion objective. When examining fro malaria
parasites and trypanosomes, at least 200 fields should be examined.
Microscopy of thin film should reveal the following features: 1.The
background should be clean and free from debris; erythrocytes are
stained a pale greyish pink. 2.Neutrophil leukocytes have deep
purple nuclei and well defined granules. 3.Malaria parasite are
well defined with deep- red chromatin and pale purplish blue
cytoplasm. 4.Like plasmodia, the cytoplasm of trypanosomes stain
blue, the nucleus and kinetoplast stain red or purple. Raed Z.
Ahmed, Medical Parasitology Lab.,2012 Reading of Thin Film
Slide 8
In thin films, look at the appearance of the parasite and the
appearance of the RBCs containing the parasites. 1.The appearance
of the parasites 2.The appearance of the RBC containing the
parasites: Size: Is the parasitized cell the same size as the blood
cell without parasite or Is it enlarged? Stippling: Is the RBC
filled with pink or red staining dots? Schuffners stippling
Schuffners stippling in the ghost of the erythrocyte can some times
be seen at the edges of the film and indicate infection with
Plasmodium vivax or P. ovale,. Maurers dots Maurers dots show as
stippling in erythrocytes containing the larger ring forms of
Plasmodium falciparum. Raed Z. Ahmed, Medical Parasitology
Lab.,2012 Identification of malarial parasites
Slide 9
Comparison Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 10
Slide 11
Blood Protozoa Raed Z. Ahmed, Medical Parasitology Lab.,2012
Blood Parasite
MicrofilariaeTrypanosomaLeishmaniaPlasmodiumPlasmodium
falciparumPlasmodium vivaxPlasmodium ovalePlasmodium malariae
Slide 12
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 13
Life cycle Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 14
Trypanosoma spp. Trypanosoma cruci (Americans) cause Chagas
disease. Trypanosoma bruci (Africans) cause sleeping sickness
disease. Trypanosoma have many stages: Amastigote, Promastigote,
Epimastigote and Trypomastigote. Reservoir host: mammalian animal.
Intermediate host: Tse tse fly (Glossina spp.) Definitive host:
Human. Infective stage: Metacyclic trypomastigote. Diagnostic
stage: Trypomastigote. Raed Z. Ahmed, Medical Parasitology
Lab.,2012
Slide 15
Continue Raed Z. Ahmed, Medical Parasitology Lab.,2012
Diagnosis: o Detection of Trypanosoma chancer after bite o Blood
smear within 21 days from the bite, it will show the parasites. o
Lymph node aspiration (most reliable). o Lumber puncture if brain
affected. Undulating membrane Flagellum Nucleus
Slide 16
Trypanosoma Trypomastigotes Raed Z. Ahmed, Medical Parasitology
Lab.,2012
Slide 17
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Life cycle Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 19
Leishmania spp. There is many species affect man: Leishmania
tropica : cause skin lesion ( cutaneous ) Leishmania braziliense :
cause muco-cutaneous lesion. Leishmania donovani : cause visceral
lesion. Leishmania have two stages: Amastigote (Leishmania stage),
in man (reticuloendothelial cell). Promastigote (Leptomonas stage),
the infective stage and present in the lumen gut of the sand fly.
Reservoir host: dogs and rodents. Intermediate host: Sand fly
(Phlebotomus). Definitive host: Human. Raed Z. Ahmed, Medical
Parasitology Lab.,2012
Slide 20
Continue Diagnosis: Thick and thin blood film Skin scraping
Blood culture on N.N.N media* Serological tests Raed Z. Ahmed,
Medical Parasitology Lab.,2012 Nucleus Flagellum
Slide 21
Leishmania promastigotes Raed Z. Ahmed, Medical Parasitology
Lab.,2012
Slide 22
Slide 23
Life cycle Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 24
Plasmodium spp. Four species of Plasmodium are the causative
agent of malaria, these are: P. vivax, P. malariae, P. falciparum,
and P. ovale. Intermediate host: Human. Definitive host: Anopheles
mosquitoes. Plasmodium spp. have 4 stages: Ring form (young
trophozoite.) Late ( old ) trophozoite Schizonts Gametocyte.
Infective stage: Sporozoites. Diagnosis: Thick and stained thin
blood film to detect parasites. Raed Z. Ahmed, Medical Parasitology
Lab.,2012
Slide 25
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Slide 29
Ring form P. vivax P. ovale P. malariae P. falciparum Raed Z.
Ahmed, Medical Parasitology Lab.,2012
Slide 30
Trophozoite form P. vivax P. ovale P. malariae P. falciparum
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Slide 31
Schizonts form P. vivax P. ovale P. malariae P. falciparum Raed
Z. Ahmed, Medical Parasitology Lab.,2012
Slide 32
Gametocyte form P. vivax P. ovale P. malariae P. falciparum
Raed Z. Ahmed, Medical Parasitology Lab.,2012