1. Experiment 5
Medical Protozoology

3. Objectives and Requirements
To study morphological structures of these protozoa.
2. To study laboratory diagnostic methods of these protozoa.

4. Entamoeba histolytica
Person-to-person transfer
Trophozoite --active feeding stage,reproductive
stage, pathogenic stage
Cyst resting stage, infective stage

5. Morphology 1.Trophozoite ectoplasm pseudopodium endoplasm
binary fission
ectoplasm
pseudopodium
endoplasm
vesicular nucleus
central karyosome
chromatin granules
Ingested RBC

6. Entamoeba histolytica
trophozoite
Parasites stained with iron--hematoxylin

8. Micrograph of a trophozoite ingesting a red blood cell derived from its host.

9. using oil immersion lens
2. cyst (non-motile, resting stage)
using oil immersion lens
spherical or round，1016μm
cyst wall, 125150 nm
chromatoid body
(rounded end)
1 nucleus cyst
glycogen vacuole
nuclei
4 nuclei cyst

10. iodine stained

12. Diagnosis 1.Stool examination
trophozoite
cyst
specimen
feces
method
direct smear with normal saline
direct smear with iodine stain
diseases
amoebic dysentery
chronic intestinal amoebiasis or carriers
remarks
1.container must clean 2.examined soon after they have been passed. 3.select bloody and mucous portion.
4.keep specimen warm.
5.drug using history.　

13. Diagnosis
2. Serologic studies: indirect hemagglutination(IHA), skin tests, ELISA 3. Tissue examination: sigmoidoscopic biopsy, aspiration
4. DNA probe

14. Promastigote – in the digestive tract of sandfly
Leishmania Donovani
Promastigote – in the digestive tract of sandfly
Amastigote – in the reticuloendothelial system of
man and other vertebrate animals –
visceral leishmaniasis.

15. Morphology 1.Amastigote (Leishman-Donovan body, L.D. body）
Minute round or elliptical body, 2-5µm
No free flagellum
Nucleus – deep red, located at one side
Cytoplasm-blue, contain: kinetoplast; basal body, rhizoplast

16. Leishmania donovani
无鞭毛体
无鞭毛体
stained smear of amastigotes
无鞭毛体

17. amastigote Bone marrow smear -- Giemsa stain
Blue cytoplasm, red nucleus

18. Large numbers of amastigote in a M

19. Promastigote Spindle shaped 1420×1.51.8μm in size Basal body
kinetoplast
1420×1.51.8μm in size
flagellum
nucleus

20. . Promastigote Giemsa stained specimen
in the digestive tract of sandfly
Giemsa stained specimen

21. Spindle shaped with 1 free flagellum
Usually gather together in the medium as a mum
Spindle shaped with 1 free flagellum

22. promastigote rosette

23. 4. Diagnosis (1)Parasitic diagnosis 1) Punctures:
①Direct smear of materials from
a) bone marrow aspiration with 80~90% of detection rate , the safest procedure, first choice
b) lymph nodes aspiration: positive in 60% of cases
c) Splenic aspiration: It is the most sensitive (90% ~ 99.3%) method, but with the risk of life threatening haemorrhage, rarely use

24. ② Animal inoculation
Golden hamster used, for 1-2 months or more,
then impression smears of the spleen .
2) Biopsy: skin
(2) Serologic tests：
ELISA, IHA, IFA, Skin test
(3) Other molecular methods:
PCR，DNA probe

25. Giardia lamblia Giardia lambilia lives in small intestine Giardiasis
Diarrhea
“traveler’s diarrhea”

26. 1. Morphology Trophozoite Inverse Pear-shaped 921×525μm Nucleus 核
Flagellum 鞭毛
(two anterior, posterior, ventral and tail flagella)
Median body 中体
Sucking disk 吸盘
Axostyle 轴柱
Trophozoite

27. Front side flagella
Behind side flagella
ventral flagella
Tail flagella
Ventral surface
Lateral surface

29. Cyst： 10  14µm ×7.5  9µm Nucleus ball-shaped or oval , thick wall ,
2-4 nuclei,
10  14µm ×7.5  9µm
Nucleus
Axostyles
Flagella

31. 4. Diagnosis
(1)Parasitic Diagnosis
1）Stool examination：as same as in E.h
2）Duodenal fluid or bile examination:
①Duodenal aspiration(十二指肠引流)
② Duodenal capsule technique (肠检胶囊法)
(2)Immunologic test：mainly Ab detection
(3)Molecular method:
DNA probe、 PCR

32. Trichomonas Vaginalis
(阴道毛滴虫)
Inhabit --- Female: vagina, urethra
--- Trichomonas vaginitis,
Trichomonas urethritis
--- Male: urethra, prostate gland
--- Trichomonas urethritis

33. 乒乓(ping-pong)感染

34. undulating 1. Morphology Trophozoite colorless pyriform or oval
4 anterior flagella
Trophozoite
1 nucleus
colorless
pyriform or oval
1030μm×1015μm
undulating
membrane
(1 posterior
flagellum)
axostyle

36. Trophozoites – Gimsa stain

37. (1)Parasitic diagnosis： 1） Direct smear ① Saline smear(生理盐水涂片)
② Staining
specimen：
Discharge from posterior vaginal fornix, Urine, Secretion of prostate gland
2）Cultivation：specimen above，37℃，48h，
microscopic exam
(2)Immunologic test：Ag detection
(3)DNA probe

38. Plasmodium Plasmodium vivax （P.v） P. falciparum （P.f）
.Exoerythrocytic stage
.Erythrocytic stage
Malaria

39. Erythrocytic stage ♀ gametocyte ♂ gametocyte Ring form
Trophozoite
Immature schizont
Mature schizont 1 2 3 4
♀ gametocyte
(macrogametocyte） 5
♂ gametocyte
（microgametocyte） 6

40. The ring form (环状体）
The entire organism looks like a ruby-ring, which is about 1/3 that diameter of the infected RBC. Ring like plasma with one nucleus at one side.
P. vivax Thin blood film (Giemsa stained)

41. mature trophozoites (amoeboid form)
The plasmodium grow with pseudopods, more cytoplasm and malarial  pigment presented in the plasma
Red blood cell enlarged and became pale with Schüffner‘s  dots(薛氏小点)

42. Immature schizonts 2-12 nuclei Schüffner‘s dots
Infected RBC enlarge, getting pale

43. Mature schizonts 12～24 merozoits，average 16
The parasite occupied the whole enlarged RBC
Malarial pigments gather together

44. Mature schizonts Merozoites lying free.
Malarial pigment is seen as a clump on one side.

45. Schizonts
increasingly mature schizonts

46. Female gametocyte of P.v
compact nucleus, usually at edge of the parasite
scattered pigment granules
blue staining cytoplasm
The gametocyte is completely filling its host cell and the membrane of the erythrocyte is hardly visible

47. cytoplasm
nucleus
P.v
♀gametocyte
(macrogametocyte)

48. Schuffner’s dots hemozoin ♂gametocyte Smaller parasites
large nucleus at the center of the cell
the pink staining cytoplasm (compared to the blue cytoplasm of female gametocytes)

49. The P. falciparum ring forms
P.f: large trophozoite and schizont can’t find at the peripheral blood
They are in the internal organ blood capillary

50. The ring form The ring form P. v The ring form P. f
The ring form of P. vivax is about 1/3 that diameter of RBC, which looks like a ruby-ring
The ring form of P. f is about 1/5 that diameter of RBC
some appear to have two nuclei
appear to have two ring(multiple infection) in one RBC

51. Male gametocyte of P.f Sausage shape
large compact nucleus at the center of the cell
blue staining cytoplasm
malaria pigment diffused distribution around the nucleus
It is bigger than the red cell ,sometimes the red cell may rupture

52. Female gametocyte of P.f
crescent shape
smaller compact nucleus at the center of the cell
blue staining cytoplasm
malaria pigment compacted distribution around the nucleus
It is bigger than the red cell ,sometimes the red cell may rupture

53. (1) Parasitic diagnosis
1）Smear of peripheral blood
with Giemsa or Wright Staining
The procedure of microscope diagnosis
Collecting a finger-prick (or ear-prick) blood sample
Preparing a thick blood smear (in some settings a thin smear is also prepared)
Staining the smear (most frequently with Giemsa)
Examining the smear through a microscope (preferably with a 100X oil-immersion objective) for the presence of malaria parasites

54. Thin smear--used for determining the species, but easy missing
Thick smear
Thin smear
Label
Thick smear--used for concentrating the parasites, increasing the sensitivity, but hard to read, difficult to distinguish the species of P.
Thin smear--used for determining the species, but easy missing

55. When does the blood be collected?
In vivaxinfections all asexual stages, as well as the gametocytes, occur in peripheral blood, but in falciparum infections only the rings and gametocytes may be present.
When does the blood be collected?
Blood examination should be made at the proper time to increase the positive rate, because the malarial parasites appear more abundantly in the peripheral blood at certain time.
Usually, blood examination is made at the beginning of the paroxysm for P. falciparum
Several to about 10 hours after paroxysm for P.vivax or P.malariae

56. Toxoplasma gondii
It will probably infect almost any mammal.
It parasitizes in all kinds of cells except RBC.
Man is its intermediate host --- toxoplasmosis.

57. 1. Morphology
(1)Trophozoite
crescent shaped 4~ 7 × 2 ~4 μm

58. Trophozoites from peritoneal fluid of an infected mouse;
Scanning electron microgragh of toxoplasma trophozoites

59. Pseudocyst -- intracellular tachyzoites of Toxoplasma gondii

60. Cyst (bradyzoite, 缓殖子), tissue cyst
The cyst have a elastic wall. They contain several to several hundreds trophozoites which called bradyzoite. Under certain conditions, the cyst may be break, and the bradyzoites invade a new cell and form another cyst
Cyst wall
Bradyzoite

61. 4. Diagnosis (1) Parasitic diagnosis 1）Smears： Specimens
Blood, Sputum, bone marrow, cerebrospinal fluid
-Tissue Biopsy
2）Animal Inoculation：inoculation to laboratory
rats or mice ,for 1w, collecting liquid from the abdomen
3） Cultivation

62. (2) Serological tests
　　 1）Dye test (DT) ---is sensitive and specific
2）IHA, IFA, ELISA
(3) PCR
DNA probe

63. Exercise
l. Draw ring form, trophozoite, schizont and gametocytes of P. vivax.
2. Draw ring form and gametocytes of P. falciparum.
3. Draw trophozoite of morphological structures of Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia, Toxoplasma