A LABORATORY GUIDE TO
PARASITOLOGY
(THE FIFTH EDITION)
Department ofParasitology
CONTENTS
Care of themicroscope…………………………………….…………………1
Clonorchissinensis…………………………………….……………….…….2
Paragonimusflukes…………………………………….……………….……5
Fasciolopsisbuski…………………………………….……………….….…..7
Schistosomajaponicum…………………………………….………………...8
Tapeworm…………………………………….……………….………….…14
Ascarislumbricoides & Trichuris trichiura ………………………………..17
Ancylostomaduodenal & Necatoramericanus……………………………..20
Enterobiusvermicularis……………………………………………….…….22
Wuchereriabancrofti & Brugia malayi……………………………………..23
Trichinellaspiralis…………………………………….……………….……25
Examination of alimentaryhelminths…………………………………….…26
Entamoebahistolytica, E.coli& other Amoebae…………………………...27
Giardialamblia…………………………………….……………….……….30
Trichomonasvaginalis…………………………………….………………...31
Leishmaniadonovani…………………………………….……………….…32
Plasmodia…………………………………….…………………….…….…35
Opportunistic pathogenicprotozoan………………………………….……..40
Mosquitoes…………………………………….……………….…….……...43
Fly…………………………………….…………………….…….…….…...45
Sandfly, fleas, lice & other bloodsucking insects…………………………..46
Care of the microscope
Do's
1. Do take special care toprotect the microscope from dust in hot dry periods.
2. Do take special care to protectthe microscope lenses and prisms from fungal growth in hot humid periods.
3. Do clean the immersion oilfrom the immersion objective each time; use lens tissue dampened with ethanol.
4. Do clean the oculars with lenstissue.
5. Do use the microscope retainingscrew fitted at the base of the microscope box to prevent damage to theinstrument while in transit.
Don't
1. Don't use the tissue used forthe oil immersion objective to clean the oculars.
2. Don't dismantle or try toclean parts of the microscope that are difficult to reach.
3. Don't leave the lens partsempty; use the appropriate cover to cover the empty port.
4. Don't exchange lenses frommicroscopes of different manufacture.
CLONORCHISSINENSIS
Objectivesand Requirements
1. Tostudy the morphology and the life history ofC. sinensis as an example of general feature of trematodes.
2. Tostudy the infection route, infection mode, parasitized site and the pathogenesisof C. sinensis infection.
3. Tostudy the characteristics of ovum of C. sinensisand the pathogenic diagnosis of C. sinensisinfection.
4.To learn the basic skill of drawing eggs.
Observationand Experiment
1. Adult worm
(1) Observe the preserved specimen(Demonstration).
Note the size, color, shape of theorganism. Spindle-shaped, transparent.
(2) Observe the general feature ofC. sinensis in stained specimen(Manipulation).
First with naked eye, then study undermicroscope, note and identify following structures.
A.Adherent organs: oral and ventralsuckers, compare their size.
B. Digestive system: mouth, pharynx,esophagus and ceca.
C. Excretory system: excretory bladder andpore.
D.Reproductive system: hermaphroditism.
Male: observe the number, size, shape andposition of testes.
Female: observe ovary, seminal receptacle,vitellarium, Mehlis’ gland and uterus.
What is meant by hermaphroditism?
2. Larva and intermediate hosts (Demonstration)
(1) See live sporocyst, rediaand cercaria (or stained sporocyst to replace live one if necessary).
(2)See the stained cercaria.
Note the eye-spots and characteristicsof tail.
(3)Live metacercaria.
Note its ellipsoidal shape, 138μm×115μm, with two suckers (oral sucker andventral sucker) and excretory bladder containing black granules.
(4) See the first intermediatehosts (Parafossarulus, Alocinma, Bithynia snails), and secondintermediate hosts, fresh water fish and crayfish. What are their names?
3. Ovum (Manipulation)
(1) Study the ova. Note the shape, color,shell, operculum resting on a rim which takes the shape of distinct shoulders,a small protuberance at abopercular end, an asymmetrical miracidium inside theegg. One of the smallest helminth ova, yellow-brown, thicker eggshell.
Material examined: feces and drawnliquid from duodenum.
Examining method: see examination ofalimentary helminths.
(2)See the scanning electron-microscopic (SEM) photograph of ovum (Demonstration).
4. Pathology
(1)See specimen, note the parasites in billiary passages (Demonstration).
(2)See adult worms inhabiting in billiary passages of a reservoir host (Demonstration).
Exercise
1. Drawegg of C. sinensis in detail.
2. Labelin full an adult worm of C. sinensis.
3. Draw a life cycle draft of C.sinensis, including final host, intermediate host; adult parasitized site,main injured organs; excretory route of ovum, pathogenic diagnostic methods;the first and second intermediate hosts and in which how the larva development;the infective stage, infection mode and infection route.
Thinking
1.Why one can obtain parasitic disease after eaten uncooked fish?
2.Record the results of pressing fish flesh and the dissection of the experimentalanimal.
Reference
1. Thefertilization process and egg formation
In male, sperm produced in a pair oftestes are collected in the seminal vesicle through the vas efferens and vasdeferens and are ejected through the ejaculatory duct, around which there is aprostate gland covered by a cirrus sac. In female, a short oviduct comes out ofthe ovary and joins with the seminal receptacle and Laurer’s canal opening onthe dorsal side, and further proceeds to the ootype with surrounding Mehlis’gland. Then, it becomes the uterus, which extends with many contortions to thecommon genital pore. Small ducts from the vitelline glands, gradually gather tobecome the right and left vitelline ducts and then one common vitelline duct,which opens at front of the ootype.
An egg cell from ovary and a sperm fromtestis are united, additional yolk cells provided by the vitelline gland join,and eggshell formation is started in the ootype by the effect of Mehlis’ glandand completed during passage through the uterus. In the egg one miracidium has alreadybeen formed.
2. Examinefor encysted metacercaria in fish flesh
Press a small piece of freshwater fishflesh between two slides, examine for encysted metacercaria of
3. Isolationof encysted metacercaria
Take the 2nd intermediate host-freshwaterfish and smash, digest with 250 ml digestive solution per 10 g flesh for 4 h～12 h (Digestive solution:pepsin 9.8 g, 1 NHCl 164 ml, NaCl 17 g, addwater to 2 000 ml). Discard rough matter by sieve filtration. After severaltimes sedimentation of the filtrated solution, take the sediment and examineunder microscope.
4. Examination adult from infected animal
Dissect the guinea pig (or white rat)which were infected with the aid of stomach tube approximately 30 C
PARAGONIMUSFLUKES
Objectivesand Requirements
1.Tostudy the life cycles of P. weste中國(guó)衛(wèi)生人才網(wǎng)rmani,to learn more about the life history of trematodes.
2.Tostudy the main parasitized sites and major pathogenisis.
3.To study the laboratory diagnosis of these flukes.
Observationand Experiment
1. P.westermani
(1)Adult worm
A.See the preserved specimen (Demonstration).
B. Study the internal structures ofstained specimen (Manipulation).
Note the position of thesuckers. Are they equal in size? The branched testes are situated in posteriorof the body, side by side, and posterior to uterus and ovary. The lobulatedovary is in the opposite side of uterus (fully filled with ova). Are these twofemale reproductive organs situated anterior or posterior to the worms?
C.See the SEM photographs of Paragonimusspines.
(2)Larva and intermediate hosts (Demonstration)
A.See the first and second intermediate hosts-Melania snail and crayfish.
B.See the stained cercaria.
C.See the live cysts.
D.Isolation of the encysted metacercaria from infected crabs (Students work ingroups).
Crushed a crab in a mortar. Add some0.45% NaCl solution or tap water into motar and filter. Allow the filtratestand and settle, examine sediments for encysted metacercaria under microscope.
(3)Egg
A. Morphology of egg(Manipulation).
Measures 80～118μm×48～60μm. Note its ellipsoidal shape, uneventhickness of eggshell, slightly oblique operculum and yellowish brown in color.A germinal cell with more than ten yolk cells inside the egg.
Materials examined: sputum, feces.
Examining methods: direct sputum smear,sputum concentration and direct fecal smear.
B.See the SEM photograph of egg.
(4)Pathology
A.See the preserved infected lungs (Demonstration).
Note the capsules and their surroundingdamaged tissues.
B.See the infected liver (Demonstration).
2.Pagumogonimus skrjabini
(1)Adult worm (Demonstration).
A.See the preserved specimen.
B.See the stained specimen.
(2)Intermediate host (Demonstration)
See the first intermediatehost-snail (Tricula humida).
Exercise
1. Draw P. westermani egg in detail and label.
2. Label the adult worm of
3. Write the result report ofcyst isolation, to have an analysis of epidemiology of P. westermani.
4.Draw a life cycle draft of P. westermani.
Thinking
1. Howto explain paragonimiasis is a natural epidemic source disease in mountainousregion and forest? And what are the epidemic characteristics of paragonimiasis?
Reference
1. Detectionof sputum for egg of P. westermani
(1) Sputum smear
Take a clean slide, place a drop ofsaline in the center of it, pick up a small amount of the sputum and mix itwith the drop of saline, examine under microscope.
(2) Egg concentration
Collect the sputum of 24 h, pour into ameasuring glass and add equal quantity of 10% NaOH, mix, sediment naturally for6 h～8h, remove supernatant, take the sediment and examine under microscope.
2. Surveybiological hosts of P. westerman inepidemiology
Choose one or more brooks, capture
(1) Break up Melania snail and place the internal organs on a slide, open themand put a drop of 0.45% saline, find sporocyst, redia and cercaria undermicroscope.
(2) Dissect stream crab or crayfishand find encysted metacercaria.
3. Infection of P. Westermani for dog
Isolation of cysts from crayfish and mixingwith meat, feed dogs, each one with 100～200 cysts according to thesizes of dogs. Kill the infected dogs 2～3 months later, open chest andexpose the lungs, on which the nodular cysts can be observed, dissect them tofind adult worms.
FASCIOLOPSISBUSKI
Objectivesand Requirements
1.To study the life history and feature in morphology of F. buski.
2.To study the laboratory diagnosis of this fluke.
3.To gain some knowledge of plant vectors and the importance in prophylaxis.
Observationand Experiment
1. Adultworm
(1)See the preserved specimens (Demonstration).
Note the shape, size, color and thepositions of oral and ventral suckers and their sizes, large and thick.
(1) Studythe stained specimen (Manipulation).
First with naked eye, then under lowpower microscope, note the adhesive organ, ceca and reproductive system of bothsexes. The intestine is composed of two blind tracts without side branching,and the ventral sucker is very large.
2. Larva, intermediate host and vector (Demonstration)
(1) See the snail intermediate host(Planorbis).
(2)See the cercaria.
(3)Seethe stained specimen of cyst.
(4)Seethe vector: aquatic plants (caltrop, water chestnut etc.).
3.Ovum
(1) See the preserved specimens(Manipulation).
Note its large size, 130～140μm×80～85μm, oval shape, pale yellow in color, thinshell, small operculum, an germinal cell surrounded by yolk granules.
Materials examined: feces.
(2)See the SEM photograph of ovum.
Exercise
1.Drawand label F. buski egg in detail.
Thinking
1.Drawa life cycle draft of F. buski.
SCHISTOSOMAJAPONICUM
Objectivesand Requirements
1. Tounderstand characteristics of life cycle and morphology of its developmentalstages.
2. Toobserve the pathological changes of schistosomiasis in infected animal.
3. Tostudy morphology of ova, laboratory diagnostic methods and their principle.
4. Togain fundamental knowledge of infective stage, infection mode, methods and significanceof immunologic tests commonly used.
Observationand Experiment
l. Adult worm
(1) See the live specimen(Demonstration).
(1) See the preserved specimen (Demonstration).
Adults are dioecious, a male and a femalelive together.
(3)Study stained specimen (Manipulation).
Note the shape, size and color of bothsexes, oral and ventral suckers and gynecophoral canal of male, the number,shape, size and arrangement of testes, ovary and uterus of female. Females arelong and slender, the posterior half of the body is a little thick and darkbrown in color owing to the vitelline gland and intestine. Near the anteriorend of the body, there are two suckers. The part of the male body behind theventral sucker is flattened, and rolled ventrally along the length to form agroove, canalis gynecophorus, into which the female worm fits. What is themeaning of dioecious?
(2) See the SEM photograph ofadult worm (Demonstration).
2. Larvaand intermediate host (Demonstration)
(1)Seethe live miracidia.
Note the feature of movement inwater.
(2)Studythe structure of stained specimen of miracidia.
(3)Seethe SEM photograph of miracidia.
(4)Identifythe intermediate host Oncomelania snail.
(5)Seethe live cercariae.
Note the posture of the bodyresting beneath water surface and its movement. Study stained specimen, notethe internal structures and the forked tail.
(6)See the SEM photograph of cercariae.
3. Ovum
(1)Place a small drop of suspension containingS. japonicum ova on a slide.
Is there a miracidium inside theegg? Do you find operculum or process of eggshell?
Materials examined: feces.
(2)Study the preserved specimen of S. japonicumova (Manipulation).
Ovoidal, 54～63μm×40～58μm, thin eggshell and lackingthe operculum, on the side near one end there is depression from which thereextends a small spinose process.
(3) See the SEM photograph of egg(Demonstration).
(4) See the ova of
Compare the ova of these threeblood flukes.
4. Pathology
(1) See the cirrhotic liver andenlarged spleen (Demonstration).
Note the size and nodular appearanceof the liver and spleen.
(2)See the sections showing pathological foci in liver (Demonstration).
A. Acutestage, note the infiltration of eosinophils, leucocytes and radiatingacidophilic streaks around the eggs.
B.Chronicstage, note the dead or calcified eggs surrounded by epithelioid cells, giantcells and fibroblasts.
(3)Dissection of infected experimental animal (Students work in groups).
Kill the rabbits infected with
A.Is there ascites or not?
B.Where adult worms live in?
C. Observethe lesions of intestine.
D. Observethe lesions of liver.
E. Biopsy:Remove a small piece of rectal mucosa from infected rabbit. Press it betweentwo slides and examine under microscope. The ova of various developmental stagesof embryogenesis are deposited singly or in clumps sometimes in chainsfollowing the vessel.
5. Laboratory diagnosis
(1)Pathogenic examination: fecal examination.
(2) Hatching of miracidium.
A. Principal
Under suitable condition, within24 h～48h, the miracidium in egg can be hatched. According to the tendency to light andto upside of miracidium, which move on the water surface by straight linemovement.
B. Method
Use a sieve to filter 30 g of fresh feces. Add water to the filtratein conical cylinder to 1 000 ml. Let it stand for 15～20 minutes, decant supernatantfluid and add clear water. After decantation, transfer sediment to a flask with500 ml water. Incubation 6 h～12 h at 37℃ and examine the free swimming miracidia.If negative, repeat the examination after 24 h.
6. Immunodiagnosis
(1)Circumoval precipitin (COP) test, cercarien Hüllenreaction (CHR).
When live eggs, miracidia, or cercariaeare mixed into patients’ sera, precipitate formation on their surfaces andimmobillization of miracidia or cercariae will take place. These reactions arecalled the circumoval precipitin (COP) test, miracidial immobilization test,and cercarien Hüllen reaction (CHR).
(2)Fast-ELISA (fast enzyme-linked immunosorbent assay).
A. Introduction.
Fast-ELISA is an in-vitroimmunodiagnostic test for detection of pathogen infection, which combinesrapid, reliable and simple characteristics.
B. Names of reagents.
No①:Enzyme-coupled reagent; No②: washing reagent;
No③:bottom reagent; No④: developing dye reagent;
No⑤:diluted serum; No⑥: stopped reagent
C.Methods.
In this application, SEA antigen solutionof S. japonicum was used to coatmicroplates overnight at 4℃. Plates were incubated for 3～5 minutes at room temperaturewith one drop of diluted serum or S.japonicum-infected individual serum, or negative serum. The plates werewashed three times with washing reagent and incubated for 3～5 minutes with one drop ofenzyme (alkaline phosphatase)-coupled reagent at room temperature. Incubate for3～5minutes at room temperature and washed five times by tap water. Add one drop ofbottom reagent, one drop of developing reagent respectively. After 30 secondsto 3 minutes at room temperature, add one drop of stopped reagent and observethe result.
D.Criteria of diagnosis.
Grade the result according to the developingdye degree on a white color background.
+++ —++++: the color is much deeper than the positive control.
++: the color is similar to thepositive control.
+: the color is darker than thenegative control and lighter than the positive control.
-: the color is similar to the negativecontrol.
All sample graded higher than “+” areconsidered positive.
7. Epidemiology (Demonstration)
(1) See photographs of patients.
(2) Environment of endemic area.
(3) Eradication of Oncomelnia snails.
Exercise
1.Label the general morphology of adult Schistosoma.
2.Draw an egg of S. japonicum in detail and label.
3.Record the result of dissection of infected experimental rabbit.
4.Record the result of Fast-ELISA detection.
Thinking
1. By which way and route a personcan be infected by S. japonicum? Whatare the differences from other flukes?
2. Can we find the eggs of
3. To compare the prevention andcure measures of schistosomasis japonicawith other flukes.
Reference
1. Infectionof experimental rabbits (Studentswork in groups)
Cover a shaved area of 10 sq. cm with acover glass up-side-down, which contains 10 live cercariae, add a drop of waterto the side of cover glass. After about 10 minutes remove the cover glass andcount the remaining cercariae. Observe the affected skin, is there any papuleor petechiae? 5 weeks later sacrifice the rabbit for observation on adult wormsinhabiting the mesenteric veins, and the gross pathological changes in liverand intestines.
2. Methods for identification of dead andliving eggs
Acridine orange method. Place intestinalmucosa scraps (or other tissue) into Kahn-test tube 0.5 ml～l ml of 1:10 000 acridineorange solution. Keep shaking for a while and then incubate at 37℃ for 2 h forstaining. Wash twice with PBS. Take out the tissue, lay flat on glass slide andexamine under fluorescent microscope. Live ova orange-red or reddish-green;dead ova yellow.
3.Indirect fluorescent antibody method (IFA) for diagnosis of schistosomiasis
(1)Principle
IFA is a method for diagnosis ofschistosomiasis japonica with highsensitivity and specificity. It is also of advantages of easy manipulation andsaving reaction materials (such as antibody, antigen etc.). The simplifiedprinciple is as follows. The given antigen in tissue section is fixed on theslide by conventional method and allowed to react to the examined serum (orsuspension with first antibody). When the antibody is present, the immunocomplexwill form at the specific site where there is corresponding antigen. And thesecond antibody against first antibody is labeled with fluorescein (usuallyisothiocynate, FITC) and consequently allowed to react to the specificimmunocomplex to from secondary immunocomplex which can be easily observedunder fluorescent microscope with violet light source.
(2)Materials
A. Antigen.
The antigen will be presented in tissue section. S. japonicum adults are removed from the liver and portal system ofinfected rabbit, after washing, fixed with Rossman's solution overnight. Then,pick out the parasites and wash them with 75% alcohol. The parasites can now bemade into tissue section by conventional paraffin embedding method or kept in75% alcohol solution for further use. Tissue sections are 5μm in thickness.
B.First antibody.
a. 34B7 monoclonal antibody against
b. Serum from S. japonicum infected rabbits.
c. Normal rabbit serum.
C.Second antibody.
a. Sheep IgG against rat, labeled withisothiocynate.
b. Sheep IgG against rabbit, labeledwith isothiocynate.
c. Contrast staining solution.
10 mg Evan's blue is dissolved in 10 ml 0.5 M PBS pH7.8, and stored in refrigerator.
D.PBS.
0.05 M PBS pH7.8; 0.1 M PBS pH7.8.
E.Buffered glycerin.
Add 1 ml 0.1 M PBS pH7.8 to 9 ml glycerin, mix well.
(3)Manipulation
A. Circle the tissue section onthe slide with a marker pen and drop 10μl1st antibody or examined serum onto the tissue within the circle. The slide isthen transferred to a damp box at 37℃ and allowed to react for 30 min.
B. Gentlywash slide with PBS to remove the 1st antibody and let to air-dry.
C. Dilutethe second antibody with PBS containing Evan's blue to work solution, withEvan's blue concentration at 0.01%. Then add 10μldiluted second antibody to immunocomplex on the slide and transfer again to thedamp box at 37℃ for 30 minutes.
D. Wash the slide with PBS,air-dry.
E. Addone drop of buffered glycerin onto the slide and cover it with a coverslip.Take care to avoid air bubbles between the slide and coverslip.
(4) Observation
Observe the resultant slideunder a fluorescent microscope. The secondary immunocomplex with second antibodylabeled with isothiocynate is present in bright yellow-green color. Theoccurrence of bright yellow-green color implies the 2nd antibody present incombination with 1st antibody or 1st antibody present in the examined serum.Dark red color means negative result.
Try to analyse the specificityof antigen and antibody according to the difference in intensity and specificfluorescence that you observed.
4.Circulating antigen (CAg)detection
(1)Introduction
Diagnosis of schistosomiasis in endemicareas depends mainly on microscopic detection of eggs in stool. The highday-to-day fluctuation in egg excretion necessitates examining stool samples onconsecutive days. Adult schistosomes cannot be directly counted in man becausethey live in inaccessible positions in the mesenteric vasculature. Detection oftwo schistosome adult worm circulating antigens, circulating anodic antigen(CAA), and circulating cathodic antigen (CCA), that show better performanceregarding quantitation of infection intensity and follow up after chemotherapy.CAA levels have been found to correlate with S. japonicum worm burden in experimental animals. The circulatingsoluble egg antigen (CSEA) is 15～20 times more concentrated in eggs thanin adult worms.
(2)Method
Polyvinylchloride, flat-bottomed,microtitration plates were coated by incubation overnight at 4℃ with 100μl/wellof solution containing MAb in 0.035 MPBS, pH 7.8. The plates were washed with 2 mM PBS and nonspecific binding sites were blocked by incubationfor one h at 37℃ with 120μl/well of 0.1% (w/v) bovine serum albumin(BSA) in PBS. Plates were washed and incubated for one hr at room temperaturewith 80μl/well of two-fold serialdilutions of S. japonicum infectedmouse serum, or untreated urine from infected individuals, and an antigenstandard. Control wells were incubated with assay buffer. The plates werewashed and incubated for 1 h at room temperature with 80μl/well of a solution containing 1.2μg/ml of MAb-FITC. The plates were washedagain and incubated for 1 h at room temperature with 80μl/well of anti-FITC/AP. Wash platesthoroughly and incubated overnight at 4℃ with 80μl/well of PNPP in DEA buffer and theabsorption at 405 nm was measured.
５.Cercarial dermatitis
Take one mice, fix its four legs on awooden, shave of a piece of area on abdominal skin, place a slide containing 10live cecariea on the skin, keep wet. Remove the slide 10 minutes later, observethe located rash, edema and pruritus caused by penetration of schistosomecercariae through the skin.
6 Schistosomal Hatching Test
If feces containing viable schistosome eggs are diluted with approximatelyvolumes of water, the eggs hatch within a few hours, releasing miracidia. Themiracidia are positively phototrophic. The following procedure takes advantageof this characteristic.
1). A stool specimen is homogenizedby shaking in normal saline and then strained through two layers of gauze.
2). The material is allowed tosediment, the supernatant is decanted and the sediment resuspended in saline.This process is repeated least twice. I
3). The saline is decanted andreplaced with distilled water, and is suspension is placed in a flask. Theflask is covered to 1 cm belowthe level of fluid in the neck of the flask. Additional water is added ifnecessary.
4) The flask is allowed to stand at room temperature for several hours insubdued light.
5) detect the presence of free-swimming miracidia.
The typical miracidiumis a tiny, ciliated organism. It is long round form. Miracidium swimsceaselessly during its short life. Free-swimming miracidia are very active,although the miracidia do not have eyespots, they apparently havephotoreceptors, and they are positively phototrophic.
TAPEWORM:Taeniasolium, Taeniasaginata,
Objectivesand Requirements
1.To study the general morphology and characteristics of life cycle of tapeworm.
2. To study the diagnosticmethods and morphological comparison between these tapeworms.
3. To understand the dangerousof taeniasis, cysticercosis, hydatid disease and sparganosis to human being.
4.To study the morphology of hydatid cyst of E.granulosus.
Observationand Experiment
1.Taenia Solium and Taenia Saginata.
(1)Adult worm
A.See the preserved specimen of T. solium(Demonstration).
B.See the preserved specimen of T. Saginata(Demonstration).
Note the size, ribbon shape, scolex and segmentation.
D. Observethe stained specimen of scolex of T. solium(Manipulation).
Spherical, four suckers, with rostellumarmed with two rows of hooklets, numbering 25～30.
E.Observethe stained specimen of scolex of T. saginata(Manipulation).
Quadrate, four suckers, no rostellum orhooklets.
What are the differences between the scoleces of these two tapeworms?
E.See the stained specimen of segment of T.solium (Demonstration).
F. Seethe stained specimen of segment T. saginata(Demonstration).
Note each segment has 2 full sets ofreproductive organs, one of male and another female. The testes are follicular,about 100 innumber. Ovary with two lobes (T. saginata)or three lobes (T. solium). Uterusclub shaped. No digestive tract.
G. Observe the ink-stainedspecimen of gravid segment of T. solium (Demonstration).
Count the number of lateral branches ofuterus on one side. 7～13 lateral branches on each side ofuterus unequal in length.
H. Observe the ink-stainedspecimen of gravid segment of T. saginata(Demonstration). Count and compare the number of lateral branches with thatof T. solium. 15～30 lateral branches, equal inlength.
I. Observethe preserved specimen of tapeworm (Demonstration)
Press a gravid segment between two slides,examine and count the number of lateral branches under lower-lens microscope.
J.See the SEM of T. solium scolex (Demonstration).
(2)Larva
A. Study the stained specimen ofcysticercus cellulose.
Note the rostellum, hooklets,sucker and bladder.
B. Seethe stained specimen of cysticercus bovis and compare with the scolex ofcysticercus cellulose (Demonstration).
C. Seethe preserved specimen of cysticerci (Demonstration).
D. See cysticercus bovis incardiac muscle of ox (Demonstration).
E. Seecysticercus cellulose in cardiac muscle of pig (Demonstration).
(3)Ovum (Manipulation)
Observe the live specimen of taenia ova.Note the size, color, characteristics of embryophore and contents of the ova.Nearly spherical, eggshell is easily ruptured, only embryophore is seen.
Materials examined: feces or swab ofanus.
2. Spirometramansoni
(1)Adult worm (Demonstration)
A.See the preserved specimen of adult worm.
B. Study the stained specimen ofadult worm.
Note globular testes (over 300～500 innumber) in the lateral margins of dorsal side, and two lobulated ovary and overlappinguterus in the mid-portion of the segment.
C. See the stained specimen ofscolex.
D. See the SEM photograph of
(2)Larva and intermediate hosts (Demonstration)
A. See the procercoid.
B. Observethe live spargana in frog flesh. Note the spargana lodging in muscles, they arelong whitish worms. The scolex is similar to that of adult, body unsegmented,strong motility.
C. Seethe first intermediate host cyclop and second intermediate host frog.
(3)Ovum
Study ova of S. mansoni. Note its spindle shape, operculum, thin shell, and agerminal cell with numerous yolk granules.
(4)Dissection of frog for sparganum (Studentswork in groups).
Sacrifice the frog by destroying thespinal cord with a small drill. Fix the frog on a board ventral side up. Make amidline incision on the abdominal wall. Skin it and search the flesh forspargana.
3. Echinococusgranulosus
(1) See the stained specimen of adultworm (Demonstration).
Note the four segments (usually) in anadult worm and the difference in maturity, and the characteristics of scolex.
(2) See the preserved hydatid cystin liver (Demonstration).
(3) Study the scoleces of hydatidcyst (Demonstration).
Note the ovoidal scolex armed withsuckers and hooklets.
(4)Study the section of hydatid cyst (Demonstration).
Note the outermost layer, falsecystic wall, resulting from reaction of host, the external layer of true cysticwall is noncellular hyaline cuticular and inner layer is the germinal layer,from which the brood capsules, and daughter cysts and detached germinalfragments are formed.
Exercise
1.Draw taenia ova and gravid segment.
2. Label the scolices of
3. Record the result of dissecting frog forsparganum.
4.
Thinking
1.To compare T. solium and T. saginatabased on their morphology, life cycle, pathogenesis and epidemiology(epidemiologic factors and epidemiologic distribution).
2. Why the curative effectexamination should be taken for treating tapeworm diseases? What is thecurative basis?
ASCARISLUMBRICOIDES &
TRICHURISTRICHIURA
Objectivesand Requirements
1. To gain some knowledge of thelife cycle and common morphology of nematodes.
2. To identify ova of
3. To study the pathogenesis of
4. To further understand the lifecycle and pathogenesis of A. lumbricoidesthrough animal experimental observation.
Observationand Experiment
l. Ascarislumbricoides
(1)Adult (Demonstration)
A. See the preserved male andfemale adult worm of A. lumbricoides.
Note their size, shape and color. Thefemale worm is rather large, being 20～35 cm long. The male worm is rathersmall and 15～30 cm long, with the tail endturning to the ventral side. The front end of the body in both male and femalesis equipped with three lips.
B. Seethe dissected male and female adult worm ofA. lumbricoides.
(2)Study the transverse section of A. lumbricoides.
Note intestinal canal and two sexes ofreproductive system. The female has two sets of genital organs. There is amoderate construction at about 1/3 of the body from the front end forming thegenial girdle, at the ventral side of which the vulva is open. The anus islocated far from the vulva, close to the tail end on the ventral side. The malehas a set of genital organs. The cloaca joining the peripheries of thedigestive and the genital tract is located near the end.
(3)Study the cross section of A. lumbricoides.
Note the internal and external structuresof body wall and body cavity (pseudocele).
A. The cuticle: A body wallconsisting of a protoplasmic syncytial layer called the hypodermis surmountedby an apparently non-living, mainly collagenous cuticle in which no separatecells can be distinguished and underlain by groups of longitudinal muscles.Nuclei are present only in four thickened chords or "line," onedorsal, one ventral, and two lateral. In these chords run nerve fibers. Betweenthe cords there is a single layer of longitudinally spindle-shaped muscle cellsof very peculiar structure (striated contractile portion of muscle cell,protoplasmic portion of muscle cell).
B. Intestine:a flat or cylindrical tube, straight, and is lined by a single layer of cells.
C. Cavity:Between the muscles and the gut wall is a relatively spacious body cavity inwhich the reproductive organs lie, unattached except at their externalopenings. This cavity is not lined by an epithelium as is a true celome, and itis a pseudocoelomate body cavity which plays an important hydrostatic skeletalrole in locomotion. It contains a fluid which serve as distributing medium fordigested food and for collection of waste products. It is provided with a smallamount of "mesenterial" tissue and a few large phagocytic cellscalled celomocytes.
(4)Ova
A. Studythe morphology of fertilized ova of A. lumbricoides(Manipulation).
Note the characteristic shape, size, color,and the shell with special reference to albuminoid membrane. 45～75μm×35～50μm, yellow-brown color.
B.Study the morphology of unfertilized ova of A.lumbricoides (Manipulation).
Compare the appearance and structurewith those of fertilized ova. Measuring 88～98μm×39～44μm.
C.See the infective stages of ova, including a larva (Demonstration).
D. See the SEM photograph offertilized and unfertilized ova (Demonstration).
(5)See the SEM photograph of lips and copulative spicula (Demonstration).
(6)Pathologic specimens (Demonstration)
A. See the gross specimen ofintestinal obstruction due to ascariasis
B. See the gross specimen ofbiliary ascariasis
2.Trichuris trichiura
(1) See the preserved adult worms(Demonstration).
Note whip-like, the thin anterior part(three-fifths) is occupied by the esophagus and the thick posterior part (twofifths) contains intestine and genital organs.
(2) Study the preserved ova(Manipulation).
Note the shape, size, color and shell.Observe particularly their bipolar plugs and germinal cell. 50～54μm×22～23μm in size, barrel-shaped, eggshell isbrown in color, with pores at both ends that are stoppered with colorlessmucoid substance, an egg cell is uncleaved.
(2) Pathologic specimens.
Adults parasitic in human cecal mucosa,posterior part of the body being free.
Exercise
1.Draw the fertilized and unfertilized ova of A.lumbricoides.
2.Draw the ovum of T. trichiura.
Reference
1. Experimental A. lumbricoides infection of animal (Students work in groups)
(1)Placeone female worm Ascaris lying on itsventral surface on a wax pan. Fix with pins at both ends, make an incision atthe lateral side of vulva and cut caudally so as to open the body lengthwisefrom anterior to posterior end. Using a needle, lay the cut edges flat by pinningthem to the wax, add a few saline and separate internal organs carefully.Remove about 1.5 cm of oneuterus near the vagina with needle, put a sprinkling of ova in a drop of salineand prove that are fertilized under microscope.
(2)Putall fertilized ova on a penicillin bottle, and add 2 ml of 2% formalin.Incubate at room temperature or 22℃～33℃for 3 weeks. Observe and record the embryogenetic development every week, andadd 2% formalin to maintain optimal moisture, if necessary.
(3) Three weeks later when theova are mature, remove the formalin by a pipette and mix the ova with a fewsaline to make up suspension, pick up 0.5 ml ovum suspension with a pipette andinoculate it orally into the stomach of mouse.
(3) One week later, dissect theinfected mouse, remove the liver and lung, transfer them onto a petri dish,wash blood away with saline, observe the surface carefully for bloodyextravasation. Lacerate the organs and isolate the larvae under dissectingmicroscope.
ANCYLOSTOMADUODENALE&
NECATORAMERICANUS
Objectivesand Requirements
1.To learn the differential characteristicsof two species of hookworm.
2.To learn the morphologicalcharacteristics of ovum.
3.To gain some knowledge regarding theenvironmental factors that might affect the development of hookworm larvae. Tostudy the methods commonly used in laboratory diagnosis and epidemiologicsurvey.
4.Based on the structural characteristics, tounderstand the pathogenesis of hookworm diseases.
5.To understand the relationship ofhookworm epidemiology with natural factors and crops cultivating.
Observation and Experiment
1. Adult
(1) See the preserved adult worm(Demonstration).
Note the shape, size and gross appearanceof bursa of male worm. The body is thin and long.
(2)See the male and female worms in copulation (Demonstration).
(3)Study the characteristic structure of buccal capsule of Ancylostoma duodenale.
Note two pairs of ventral teeth and apair of accessory teeth. On the dorsal side of the mouth, a pair of smalldorsal teeth is seen in the center.
(4) Study the characteristicstructure of buccal capsule of Necatoramericanus.
Note one pair of ventral cutting platesand a pair of dorsal cutting plates.
(5) See the bursa of
The female is with a small mucro at thetail end, while the male is with a characteristic copulatory bursa at the tailend, which looks like an opened umbrella, the ribs of which are called rays.
(6) See the bursa of
In the male, the copulatory bursa isdeep, with the two branches of the lateral ray tightly adhered to each other,and there is a hook on the tip of each spicule. In the female, the vulva opensa little anterior to the center of the body, and no spine exists at the tailend.
(7)See the SEM photographs of buccal cavity and bursa of hookworm.
2. Ovum and larva
(1)Studythe ova (Manipulation).
Note the shape, size, color, shell andinternal structure. 36～40μm×56～76μm insize, thin eggshell, nearly colorless and transparent. It contains egg cellsunder cleavage.
(2)Seethe ova. Note 2-, 4-, 8-cells, morula and embryonated ova (Demonstration).
(3)Studythe embryogenesis of rhabditiform larvae (Demonstration).
(4)Seethe filariform larvae (Demonstration).
(5)Seethe activities of filariform larvae on water film of soil surface, waiting forsuitable hosts (Demonstration).
(6)Seethe SEM photographs of ova and filariform larvae.
3. Pathological specimens and tissue section
(1)Seethe gross specimen of intestine showing hookworm grasping mucous membrane (Demonstration).
Note the hemorrhagic petechiae andulcers.
(2) See the section of the headend of hookworm grasping mucosa membrane of small intestine (Demonstration).
The worms support themselves bybiting at the root with cutting plate or ventral teeth, and suck blood.
Exercise
1.Draw the hookworm ovum.
2.Draw and label the buccal capsules of A. duodenaleand N. americanus.
Thinking
1. Howdoes a hookworm infect host? And which dangerous can be caused to the host?
2. Whatrelationship between hookworm infection and natural environment is?
Reference
1. Methods used in laboratory diagnosisof hookworm larva.
(1) Direct fecal smear and NaClsaturated floatation (the methods are described in details at Examination ofAlimentary Helminths on page 268 of the Chinese textbook).
(2) See demonstration showing cultivationof larvae.
2. Methods used in epidemiologic surveyof hookworm.
(1) Baermann'stechnique. Put triple-layered gauze on a sieve. To the gauze add the soilsample. Put the sieve on a funnel with clamped rubber tube connecting its lowerend. Fill the funnel with 40℃water until its level is raised to touch the lower portion of the soil. After20 minutes, open the clamp and let out the lowest portion of water to a beakeror centrifuged tube. Let the collected water stand for 10～20 minutes or centrifuge for 1～2 minutes, then examine thesediment for larvae.
(2) Pad method. Soak a pad ofsix to seven layers of gauze in 40℃ water. Cover the soil with the warmpad to attract the larvae. Cover the pad with a petri dish to keep the formerwarm. After 20 minutes, rinse the pad in water repeatedly. Examine sediment forlarvae.
ENTEROBIUSVERMICULARIS
Objectives andRequirements
(1) Studythe structure of E. vermicularis ovaand methods used in the diagnosis of E. vermicularisinfection.
Observationand Experiment
(1) See the preserved adultworm of E. vermicularis (Demonstration).
Note more or lessspindle-shaped, whitish in color. The female worm is 8 mm～13 mm long and the male is only 2 mm～5 mm. The male has its posteriorend strongly curved ventral, and the female tail portion is sharply pointed.
(2) See the whole mount ofadult worms (Demonstration).
(3) Study the preserved specimenof ova (Manipulation).
Note their shape, size, color,shell and content. 50～60μm×20～30μm in size, elongate-ovoidal, flattened onthe ventral side, thick and transparent eggshell, fully embryonated.
Why the shape looks different inlateral and dorsal views?
(4)See the SEM photograph of the anterior end of adult.
(5) See the swab method fordiagnosis of E. vermicularis (Demonstration).
Wet a cotton swab with normalsaline and rub corrugations in perianal area. Then stir the swab in NaClsaturated solution in a test tube (or penicillin bottle), and add NaClsaturated solution up to rim of test tube, cover the test tube with a cleanslide in contact with the solution. Let it stand for 5～10 minutes. Examine the slidefor ova.
Exercise
1. Draw and label the ovum of
Thinking
1.Why there are more infections of E. vermicularisin rural area than in city?
Reference
The rat lungworm, A. cantonensis, is widespread, and theinfection seems to be increasing in rats and bandicoots in the tropics andsubtropics, very probably through the agency of infected rats traveling asstowaways on ships.It has been found in rats in New Orleansand in Egypt,where human cases are not known to occur. Human infections that causeeosinophilic meningitis have been reported from Hawaii,Tahiti, Japan,mainland China, Taiwan,Thailand,Vietnam, Malaysia, Indonesia,Vanuatu, American Samoa, and the Ivory Coast. Unconfirmed cases have been notedfrom other Pacific islands, Hong Kong, the Philippines,Papua New Guinea, Australia, New Caledonia,Réunion, Mauritius,Cuba, and Puerto Rico.
A.cantonensis is a slender worm, up to 25 mm long. Larval stages
develop in slugs and land snails. When eaten by rats, thelarvae migrate to
the meninges and develop in the brain for about a month.Young adults then
migrate to the pulmonary artery, where they attainmaturity. The incidence
of this infection in rats and snails may be quite high inendemic areas.
In human hosts, Angiostrongylus does not complete its developmental
cycle. When third-stage larvae are ingested, theypenetrate into blood vessels
in the intestinal tract and are carried to the meningesbut are unable to
migrate to the lungs, as they do in rats. Rarely, wormsdevelop to the young
adult stage in the meninges, but they soon die, and it isthe death of the
larvae or young adults and the inflammatory reactionprovoked by the dead
worms that causes the characteristic signs and symptomsof human infection.
A presumptive diagnosis may be made in patients from areas where the
disease is endemic on the basis of meningitis with bloodand spinal fluid
eosinophilia. Lesions may be seen in the meninges bycomputed tomography
(CT), with serologic confirmation of the infection byELISA.
Sources of human infection are slugs, land snails, or fresh-water prawns
and other paratenic (transport) hosts (Fig. 9-28), whichare often consumed
raw in islands of the Pacific, in Thailand, and in Vietnam. In Thailand and
Malaysia snails of thegenus Pila are eaten raw, either mixed with vegetables
or as a form of medication. The giant African land snail,Achatina fulica, has
spread throughout the Pacific islands and is apparently acommon vector.
Raw prawns are a frequent article of diet in Tahiti, and larvae found in them
are infective to laboratory rats. The contamination offresh vegetables by
carnivorous land planarians that have fed on infectedsnails appears to
be another important means of infection in New Caledonia, andperhaps
elsewhere.
Symptoms. The incubation period ofthe disease varies and apparently
can be as long as 47 days. Infectionin man is usually benign and se]f-limited,
although fatalities have occurred.Symptoms of meningitis or meningoen-
cephalitis--headache and stiff neck,often with sensorial changes--are of
abrupt onset. Aradiculomyeloencephalitis, with pains and paresthesias of the
lower trunk and legs, bowel andbladder dysfunction, and one death, were
seen in 16 of 21 Korean fishermen whoate giant African land snails in Samoa
and in none of five who ate cookedsnails. A CSF pleocytosis of more than
500 per ul occurred in 80 per cent ofinfected persons, and on autopsy worms
were found in the subarachnoid spaceof the lumbar cord, with invasion of
the white and gray matter. The spinalfluid usually contains 100 to 2000
white blood ceils per ul generallyaccompanied by a marked eosinophilia.
Other common symptoms include nauseaand vomiting, fever, and, early in
the infection, abdominal pain,malaise, and constipation. A blood eosinophilia
is also common; total leukocytecounts are moderately elevated.
Eye invasion is marked byvisual impairment, ocular pain, b]epharo-
spasm, circumcorneal injection,keratitis, cells and flares in the anterior
chamber and vitreous, iritis, andretinal edema. Living worms have been
noted, and on occasion removedsurgically.
Pathogenesis. Little isknown about the effects of this parasite on the
central nervous system, since mostpatients recover uneventfully. On autopsy,
sections of immature Angiostrongylushave been seen in the cerebrum and
cerebellum, as well as the spinalcord, associated with infiltrates of eosino-
phi]s,-monocytes, and foreign-bodygiant cells. Marked tissue necrosis has
been seen in some areas in connectionwith dead worms. Immature worms
have been found in spinal fluidobtained by lumbar puncture; adult worms
have been found in the eye and thepulmonary artery.
Treatment. Trials ofspecific therapy are still inconclusive. Thiabenda-
zole and mebendazole have some effectin animal infections, but thiabenda-
zole was found to be ineffective inreported human cases. Symptomatic treatment
is all that is needed in the majorityof these infections, but where specific anthelminthic treatment
seens necessary, mebendazole, 100mgtwice daily for t days, is a recommended investigational regimen
for adults.
WUCHERERIA BANCROFTI &
BRUGIAMALAYI
Objectivesand Requirements
l.To study the life cycle characteristics and epidemiologic features of filariae.
2.Tostudy the morphology of microfilariae and laboratory diagnostic methods.
Observationand Experiment
1. Adult
(1)Seethe preserved adult worm of Wuchereriabancrofti (Demonstration).
Adults look like thin and long threads.
(2)See the preserved adult worm of Brugiamalayi (Demonstration).
The adults resemble that of
(3)See the preserved adult worm of Loa loa(Demonstration).
The adults take a thread-likeform, and there are small lumps on the body surface.
(4)See the preserved adult worm of Dirofilariaimmitis (Demonstration).
The adults look like thin white noodles,and the tail of the male is coiled.
2. Larvae
(1)Studythe structures of W. bancroftimicrofilariae stained specimen (Manipulation).
Note the shape, appearance, sheath, cellularcolumn and caudal nuclei. The larva is sheathed by egg membrane, the excretorypore is adjacent to the excretory cell, one of the genital cells (G-cells) isfar apart from the others, and there is no nucleus at the tail end.
(2)Study the structures of B. malayimicrofilariae stained specimen (Manipulation).
Compare the structures with those of
(3)Observe the microfilariae in unstained blood film.
(4)See the infective larvae in mosquito thoracic muscles (Demonstration).
(5)See the infective larvae in mosquito proboscis (Demonstration).
3. The intermediate host (Demonstration)
Culexquanquefasciatus and Anophelessinensis.
4. Pathogenesis (Demonstration)
(1) See the photograph showingthe patient with elephantiasis of lower extremity and scrotum.
(2)See the photograph showing the patient with hydrocele.
Exercise
1.Label the microfilariae.
Thinking
1.What are the epidemiologic distribution characteristics of filariasis of
2.To describe the pathogenesis of W. Bancrofti and B.Malayi.
Reference
1. Laboratorydiagnostic methods for filaria
(1)Thick blood film
Sterilize the ear lob with 75%alcohol and puncture with needle. Place on a clean slide two to three dropsblood approximate 60 mm3and spread it with the corner of another slide to equal the diameter of two-fencoin. Let it dry. Put it in water, the thick film becomes whitish in color andexamine it under microscope.
(2)Examination of live microfilariae (Demonstration)
Place two drops of patient's blood(or dog infected with Dirofilaria)and cover with coverslip. Note their wiggle among the RBC.
(3)Examination of venous blood for microfilariae (Demonstration)
Mix 1ml of venous blood (taken atnight) with o.1 ml 3.8% sodium citrate in a centrifugal tube, then add 9 ml ofdistilled water to break the RBC. Centrifugalize for 2 minutes at 3 000 rpm.Decant the supernatant and examine the sediment for microfilariae.
TRICHINELLASPIRALIS
Objectives andRequirements
1. To learn the diagnostic methodof T. spiralis.
2. To understand the life cycleand pathogenesis of T. spiralisthrough animal experiment.
Observationand Experiment
1. Seethe preserved adult worms (Demonstration)
The adults look like pieces of thread,being 2.2 mm～3.0 mmlong in the female and 1.2 mm～1.5 mm long in the male.
2. Seethe stained specimen of cyst (larva) in muscular tissue (Demonstration)
Larvae that reach striated muscles invademuscular fibers to be encysted. With the development of the larva, the cystwall is thickened to form a cyst of characteristic spindle form, its major axisbeing in parallel with the muscle fibers. Usually, each cyst has one coiledlarva.
3. See the live cyst (Demonstration)
4. Experimental infection of mice andexamination (Studentswork in groups)
Take buccal muscle or diaphragm ofinfected mice, cut into pieces and feed the normal mice. Four weeks later, killthe infected mice. Examine (l) buccal muscle, (2) diaphragm and (3) heartmuscles by placing a small piece of the referred tissue between two slides andexamining under microscope for cysts. (4) dissect and wash the small intestineto find the adults.
Exercise
1.Draw the cyst of T. spiralis.
2.Record the result of the infection experiment and give a brief analysis.
Thinking
1. Which kinds of domestic animals can bethe reservoir host of human parasites?
2. What are the differences existed in thedistribution of biohelminth and geohelminth?
EXAMINATION OFALIMENTARY
HELMINTHS
Objectivesand Requirements
1 Tolearn methods commonly used in stool examination.
2.Self-stool examination for helminth eggs and therapy.
Methods
1．Direct fecalsmear.
Transfer a small amount of fecal sampleto 2 drops of saline on a slide. Mix to obtain a fairly dense uniform smearfree of large lumps. Some practice is necessary to judge the density, that is:one can see the strokes of words in the newspaper through the transparentsmear. Identify parasite ova and distinguish them from food residues such asvarious kinds of plant cells, yeast, pollen, plant fiber etc. The fecal smearmust be kept wet during examination.
2． NaClsaturated solution floatation.
Mix small lumps of fecal material (aboutpeanut-sized) with small amount of NaCl saturated solution in a penicillinbottle. Then add up to the rim of bottle, cover with a coverslip and allow tocontact with it (avoid any air bubble). After 20 minutes, examine the coverslipon a slide for ova.
3． Sedimentation.
Mix 5 g～10 g of fecal material with smallamount of water in a beaker (or test tube). Dilute with water and filter awaythe bulky undigested residues. Let the filtrate stand for 15～20 minutes. Discard thesupernatant and add water again, thus repeat several times until thesupernatant is clear. Decant the supernatant fluid and examine sediment forova.
Exercise
1. Recordthe result of self-stool examination.
2. Tryto compare the advantages and disadvantages of the three methods used in the practice.
Reference
1. Qualitycontrol for fecal examination.
Reliable and accurate parasite identificationdepends on:
(1)Collectingsatisfactory specimens.
(2)Preparingand maintaining reagent correctly.
(3)Carefulperformance of appropriate techniques and thorough examination of finishedpreparation.
ENTAMOEBAHISTOLYTICA,
OTHER AMOEBAE
Objectivesand Requirements
1.To study morphological structures of trophozoite and cyst of
2.To study laboratory diagnostic methods ofE. histolytica.
3.To understand the pathogenesis of E. histolytica.
4.To learn differentiating E. hisiolyticafrom E. coli.
Observationand Experiment
1.E. histolytica
(1)Trophozoite
A. Studythe iron-haematoxylin stained specimen ofE. histolytica (Manipulation).
Find the parasite under high power, thenobserve with oil immersion lens. Note ectoplasm and pseudopodia, endoplasm withfine granules. Some of the trophozoites may contain RBCs which appearancevaries with process of digestion. Food vacuoles can also be found in theendoplasma. The spherical nucleus has definite nuclear membrane, the innersurface of which is lined with uniform and closely packed chromatin (peripheralchromatin). The central karyosome is deeply stained.
B. Studythe living trophozoites in fecal material (Manipulation).
With a toothpick, remove some material frommucus or flecks of blood in patient's stool (or from culture medium), mix with salineon a slide, cover and examine. It is advisable to use rather weak illumination.Observe with care the appearance, motility and the cytoplasmic inclusion, e. g.RBC (if specimen obtained from patient) or starch (if specimen obtained fromculture). The fecal samples must be examined immediately after collection or bekept at optimal temperature so as to maintain the motility of the trophozoite; itis particularly so during winter.
(2)Cyst
A. Study the iron-haematoxylinstained specimen of E. histolytica cysts(Manipulation).
Using oil immersion lens, study thespherical cyst with a hyaline cystic wall, the number of nucleus may be single,two or quadrate. The structure of nucleus is the same as trophozoite. Theglycogen is readily dissolved in stained specimen and only vacuole is left.Chromatoid bodies are rod-like masses with round ends. In fully mature cysts,the chromatoid bodies are often lacking.
B. Studythe iodine stained specimen of cysts (Manipulation).
Stain fresh fecal material by adding adrop of iodine solution to the edge of coverslip. The cysts stain yellowish,the glycogen is brownish yellow, the nuclei are hyaline unstained sphere-like.
(3)Cultivation of trophozoite (Students work in groups).
With sterilized technique, add 0.5 ml ofinactivated serum and a small sprinkling of rice starch and a few drops of penicillinsolution to the medium, inoculate the fecal sample containing trophozoites (about0.1 ml). Incubate at 30℃for one week and examine.
(4)Pathologic specimens (Demonstration).
A. See the gross lesions in the intestine.
Note the ulcers vary from pin point tothe size of rice grain, with central erosion surrounded by elevated edematoustissues forming a niche of a volcano. Superficial ulcers are formed due todetachment of necrotic mucosa, or large ulcerated patches due to coalescence ofsmall ulcers. Mucosa between ulcers is usually normal.
B. Seethe section of intestinal lesions.
Note flask-shaped ulcer, numerous trophozoitesand infiltration of leukocytes can be seen.
C. Seethe amebic hepatic abscess.
Abscess of various size can usually be seenin the right lobe, singly and discrete. The abscesses are surrounded bynecrotic tissue, thus the edge of the abscess appears irregular and spongy,while the abscess fluid is brown in color.
(5)See the photographs of trophozoite and cyst.
2. E.coli
(1) See the iron haematoxylinstained E. coli trophozoites (Demonstration).
Compare the size of trophozoite, the nuclearchromatin, position of karyosome and endoplasmic inclusions with those of
(2) Study the iron haematoxylinstained specimen of E. coli cysts(Manipulation).
Note and compare the size, numberof nuclei, nuclear structure and chromatoid bodies with those of
3. Other non-pathogenic amoebae in humanalimentary tract
(1) See the E. gingivalis trophozoites (Demonstration).
Note the distinct ectoplasm and centralor slightly eccentric large karyosome.
(2) See the live
Note the small size, sluggishmotility, blunt pseudopodia large central or eccentric karyosome, peripheral chromatinseldom seen; See demonstration of E. nanacyst. Note small size, oval shape, l～4 nuclei, karyosome large andirregular without peripheral chromatin, usually no chromatoid bodies.
(3) See the live
Note the small size, spherical shape,sluggish motility, blunt pseudopodia, large central karyosome surrounded byrefractive granules, usually no peripheral chromatin. Food vacuole in endoplasmcontains bacteria.
(4) See the I. butschlii cysts (Demonstration).
Note the irregular or sphericalshape, one nucleus, large eccentric karyosome with peripheral chromatin, nochromatoid bodies. The most conspicuous feature is the large glycogen vacuole,stains golden brown with iodine.
(5) See the E. polecki cysts
This is an incidental parasiteof man. Note spherical shape, one nucleus, central or eccentric karyosome,dense uniform peripheral chromatin, rod-like chromatoid bodies.
Exercise
1. Draw the trophozoite and cystof E. histolytica and cyst of
Thinking
1. How does amebic dysentery betransmitted?
2. How to diagnose the intestinallesion and the exo-intestinal lesion of E.histolytica?
GIARDIALAMBLIA
Objectivesand Requirements
1.To study the general structure of G. lambliatrophozoite and cyst.
2.To learn the diagnostic method.
Observationand Experiment
1. Study the iron haematoxylin stainedtrophozoites (Manipulation).
Note inverted pear-shape, convex dorsalsurface and concave ventral surface (sucking disc). Two nuclei with large karyosomeat the anterior, two axostyles and four pairs of flagella.
2. Study the iron haematoxylinstained cysts (Manipulation).
Note shape, well defined wall and two tofour eccentrically located nuclei.
3.See the SEM photographs of G. Lamblia (Demonstration).
Exercise
l.Draw the trophozoite and cyst of G. lamblia.
Thinking
1. Why people infected
TRICHOMONASVAGINALIS
Objectivesand Requirements
1.To study the general structure of T.vaginalis trophozoite and cyst.
2.To learn the diagnostic method.
Observationand Experiment
1. Studythe stained trophozoites of T. vaginalis (Manipulation)
Broad end of the body, 4 flagella, aposterior flagellum, a short flagellum, a nucleus near the origin of theanterior position, axoneme.
2. Seethe live trophozoites of T. vaginalis(Demonstration)
3. Examination of trophozoites ofT. vaginalis
Direct wet film, using vagina excretory.
Exercise
l.Draw the trophozoite of T. vaginalis.
Thinking
1.What are the transmission routes of
Reference
1. Directexamination of vaginal and urethral smears
(1)With a sterile cotton swab, collect the vaginal or urethral discharge.
(1) Put the swab immediately intoa sterile tube containing about 3 ml of sterile saline.
(3) Smears on a slide, andsmear for staining can be made if desired. Finding of T. vaginalis in vaginal secretions, prostatic secretions and urine.
2. Centrifugedor sediment material
(1)Ifa swab in saline is received, remove the excess fluid from the swab bysqueezing it against the side of the tube.
(2)Centrifugethe tube for 2 minutes. If a centrifuge is not available, let the tube standfor 10 minutes to allow any sediment to settle on the bottom.
(3)Witha pipette remove the supernatant fluid.
(4)Takea drop of the sediment and put on a microscope slide.
(5)Coverwith a coverslip and examine with 10×and 40×objectives for motileflagellates.
LEISHMANIADONOVANI
Objectivesand Requirements
1.To study morphological structures of amastigotes and promastigotes of
2.To understand methods used in the diagnosis.
3.To understand the pathogenesis of L. donovanifrom parasitized site.
Observationand Experiment
1. Studythe stained smear of amastigotes (or non-flagellar forms) (Manipulation)
Look for reticulo-endothellial cellsharboring the amastigotes. Note the number of amastigotes in one host cell.They are very small and have the appearance of granules in the cytoplasm of macrophages.When the host cells are ruptured, amastigotes are discrete. Study carefullyunder microscope with oil immersion lens, note the discrete amastigotes, itssize in respect to the size of host cell. Identify the blue cytoplasm, rednucleus, basal body and kinetoplast.
2. Seethe stained specimen of promastigote (or flagellar from) (Demonstration)
Note the spindle shape, flagellum,nucleus, basal body, rhizoplast and kinetoplast, the last three can hardly beseparated from each other and often seen as one purple dot.
3. Seethe live promastigote (Demonstration)
Place a drop of culture medium on aslide. Cover and note the rapid, lashing movement of the flagellum thatactively propels the parasite forward.
4.See the SEM photographs of promastigoteand amastigote (Demonstration)
5. See adult sandfly, the vector of
Exercise
1. Draw the macrophage containsamastigotes, and a free promastigote of L.donovani.
Thinking
1. A person suffered fromvisceral leishmaniasis, how to diagnose it pathogenically?
Reference
1. Cultureof L. donovani
Inoculation of NNN culture medium withspleen juice, blood, or bits of excised dermis is a reliable procedure. Here weparticularly recommends seeding 3 or 4 NNN culture tubes with the sedimentobtained by centrifuging 2 ml～5 ml of blood added to four times itsvolume of citrated saline. The tubes are incubated at 22℃～24℃, and flagellates appear in 7 days orlater in 90 per cent of untreated cases.
(1) Materials.
Difco blood agar base 8 g
Distilled water 200 ml
Defibrinated rabbit blood 0.6 ml in each 5 ml of medium
(2) Preparation of defibrinatedrabbit blood.
Collect 20 ml of rabbit blood into asterile flask containing about 100 glass beads of 4 mm diameter. Defibrinate the blood by rotating the flask for 5minutes. Add 200 u of penicillin, 200 mg of gentamicin, and 2 mg ofstreptomycin per ml of defibrinated blood.
(3) Preparation.
A. Pour 200 ml of water into aflask, add the agar to the water, mix, and warm the flask in boiling wateruntil the agar is completely dissolved.
B. Dispense the medium in 5 mlamounts into screw-cap bottle (20 ml capacity). Sterilize by autoclaving (withcaps loosened) at 121℃ for 15 minutes and allow the agar tocool to 45℃～50℃.
C. Add 0.6 ml of steriledefibrinated rabbit blood to each bottle and mix gently. Allow the medium tosolidify with the bottles in a sloped position.
D. Leave the bottles in anupright position at room temperature for 24 h to allow fluid of condensation toform. The bottles should be stored at 4℃～6℃ untilrequired.
Note: The medium should be prepared inaseptic working conditions.
(4) Use.
A. Inoculate about 0.1 ml ofspecimen aseptically into the fluid of condensation of each of 2 bottles atroom temperature.
B. Incubatethe cultures at 24℃(±2℃) in the dark.
C. Examineevery 4 days. Transfer a drop of the culture using a sterile wire loop to aslide for examination for promastigotes.
Note: Negative cultures must besubcultured after 8 days into fresh medium and examined every 4 days for afurther 20 days.
2. Detectionof Leishmania in lymph node aspirates
Parasites may be demonstrated inaspirates of spleen (98% positivity), bone marrow (54%～86%), or enlarged lymph nodes(64%). When the spleen is small and soft or impalpable, bone-marrow orlymph-node aspiration is recommended. For diagnosis of Leishmania it is especially using bone-marrow or lymph-nodeaspiration.
(1) Prepare the syringe; pull thepiston as far back as possible.
(2) Ask the person to sit down.Disinfect the chosen site on the neck with 70% ethanol.
(3) With left hand, take the glandbetween the thumb and index finger and make it stand out.
(4) Holding the needle betweenthumb and finger, introduce it at right angles into the center of the gland.First pierce the skin, then penetrate the center of the gland.
(5) With left hand, gently kneadthe gland. With right hand, revolve the needle in both directions.
(6) The glandular fluid will oozeinto the needle. The operation should last about one minute.
(7) Withdraw the needle in onerapid movement, holding index finger over the hub. Apply a swab dipped indisinfectant to the point of entry.
(8) Attach the needle to thesyringe, with the piston pulled back. Push the piston gently half way down thebarrel to discharge the glandular fluid in the needle on to the slide.
(9) Cover the preparation with acoverslip. Examine at once under the microscope at a magnification ofapproximately 400×, using the 40× objective.
(10) Wait until the convectioncurrents stop. Then examine the preparation.
PLASMODIA
Objectivesand Requirements
1.Tostudy the life cycle of Plasmodia andunderstand their pathogenic mechanism.
2.To study laboratory diagnostic methods of malarial parasites.
3. To study morphologicalstructures of Plasmodia, to identifymorphological structures of developing stages of erythrocytic schizogony and gametocytesof P. vivax, and to differentiate thering-form and gametocytes of P. vivaxfrom P. falciparum.
4.To identify of insect vector of malaria—Anopheles.
5.To learn simple method of experiment with Plasmodium.
Observationand Experiment
1.P. vivax (Erythrocytic stage) (Manipulation)
(1) Ring form
Compare its size in respect to theinfected RBC. Note the delicate blue-stained ring of cytoplasm and a red chromatindot.
(2) Trophozoite
Compare the small and large trophozoites,do they have definite form? Note the vacuole, changes of chromatin, pigment,alteration of infected RBC and Schuffner's dots. The cytoplasm with the nucleusbegins to enlarge while the ring form becomes irregular in shape and sometimesprojects pseudopodia.
(3) Schizont
Note the number and arrangement ofchromatin, distribution of cytoplasm and aggregation of pigment granules. Itbecomes oval or round body, and its nucleus begins to segment. The number ofthe nucleus in the mature schizont is from 12～24, usually 16.
(4) Gametocytes
Note the size, shape, position ofchromatin, distribution of pigment granules and alteration of infected RBC.What are the morphological differences between male and female gametocytes?
2. P.falciparum (Erythrocytic stage) (Manipulation)
(1)Ringform
Note its small size and delicate ring,about 1/5 the diameter of erythrocyte. Sometimes there are two chromatin dotsin one ring form or multiple infection in one RBC.
(3) Gametocytes
Note their particular sausage or crescentshape, position of chromatin, distribution of pigment granules and alterationof infected RBC.
3. P.malariae (Erythrocytic stage) (Demonstration)
(l)See the ring form.
Note the large chromatin dot, densecytoplasm and its size in respect to infected RBC.
(2)See the trophozoite.
Note the band form, its cytoplasmcompact, pigment coarse and early appearance.
(4) See the schizont.
Note mature schizont, the merozoites 6～12 in number, arranged as "rosette"central mass of dark brown pigment granules.
(5) See the gametocytes.
Resemble those of
4. Seethe SEM photographs of P. vivax and
5. Exo-erythrocytic stage (Demonstration)
6. Development of Plasmodia in mosquito host (Demonstration)
(1)See the oocyst in the intestinal wall of mosquito.
(2)See the sporozoites in the salivary gland smear of infected mosquito.
7. Inoculation of P. berghei (Studentswork in groups)
Obtain blood from infected mouse bycardiac puncture (or orbital puncture), dilute in 1 ml saline inject 0.1 mldiluted blood to a mouse intraperitoneally. Rear the mouse and examine nextweek.
8. Preparation of blood film of malarialparasites and staining (Manipulation)
(l) Preparation of both thinand thick blood films at one slide. Collect two drops of blood from the cuttail of infected mouse and place on one end of a clean slide. Spread the dropnear end to the size of two-fen coin with the corner of another slide to make upthick film. Holding another slide at an angle of 30°～45°,and in contact with the other drop of blood, the first touch the drop of bloodand let it spread along the line of contact between the slides. Then push theslide along, with a smooth, rapid movement, thus drawing the blood out to forma thin film. Then let it dry. Its thickness will depend on the size of thedrop, the angle between the slides and rapidity with which the smear is made.
(2) Giemsa's staining. Fix thedried thin film with methyl alcohol (avoid fixing the thick film) and let itdry. Cover with 2% Giemsa's solution on the smears. Let stay 30~60 minutes.Wash by pouring neutral distilled water over the slide until color does not runfrom it to a noticeable extent. Drain and stand on end to dry.
9. Vector of malarial parasites—
(1)An. sinensis
(2)An. Minimus
(3)An. dirus
10. See the SEM photographs of merozoite (Demonstration)
Exercise
l.Draw ring form, trophozoite, schizont and gametocytes of P. vivax.
2.Draw ring form and gametocytes of P. falciparum.
3.Label SEM photographs of merozoite.
4.Record the result of artificial infection of P. berghei in mice.
Thinking
1. According to the life cyclesof Plasmodium, explain the paroxysm,recrudescence and relapse.
2. What are the epidemiccharacteristics of malaria?
Reference
1. Acridineorange fluorescent stain
Fix the thin smear with methyl alcohol,then immerse it in 0.01% acridine orange solution for 4 minutes, rinse with water,then immerse in distilled water for one minute. Add one drop of water and coverit, examine under fluorescent microscope. DNA of chromatin shows yellowishgreen fluorescence, while RNA of cytoplasm orange-yellow fluorescence.
2. Indirectfluorescent antibody test
(l) Antibody:rabbit-anti-human IgG fluorescent antibody to be diluted to 1:8 (or 1:16) withPBS, pH8.0.
(2) Antigen sample: dry bloodfilms made with blood collected right after clinical attacks. Hemolyse theslide in 0.1N HCl for 5 minutes, wash in running water, immerse in PBS for 5minutes, then air dry.
(3)Serum for examination: serum sample to be diluted with PBS to 1:20.
(4)Staining.
A. Spread one drop of dilutedserum sample on the antigen blood film, put in a moistened box and transfer to anincubator at 37℃ for 30minutes to allow antigen-antibody reaction.
B. Washthe blood film with PBS for 1 minute, immerse in PBS for 5 minutes, repeat once,then air dry.
C. Addone drop of antibody containing Evans blue (in dilution 1:10 000) on the blood filmwhere Ag-Ab reaction took place, put the slide in a moistened box intoincubator at 37℃ for 30minutes.
D. Wash away the excessivefluorescent antibody as step B.
(6) Mount the dried blood filmwith carbonate or phosphate buffered glycerin, or just add a small drop of PBS(pH8.0) onto it and cover. Examine under fluorescent microscope.
(7) Criteria of diagnosis.
Grade the result according to thefluorescence density on the schizont or trophozoite.
+++ —++++: fluorescence on plasmodial cytoplasm is bright to brilliant, structuresclear.
++: fluorescence bright,structures clear.
+: cytoplasm clearly visible butstructures not very clear.
±: only faint fluorescence visible on theplasmodial cytoplasm and RBC shadow, parasite structures not clear.
-: no fluorescence visible on plasmodialcytoplasm.
All sample graded higher than “+” areconsidered positive.
3. ParaSight-Frapid manual diagnostic test of P.falciparum
(1)Introduction
Current diagnostic methods are based onmicroscopic examination which takes 5～10 minutes for staining and upto 20 minutes for reading, and requires a well-maintained microscope and anexperienced microscopist. New techniques, such as hybridization with DNAprobes, are too sophisticated for routine use in the field.
(2)Test principal
ParaSight-F test is a qualitativediagnostic test of P. falciparum,which is based on the detection by monoclonal antibody of a species-specificsoluble antigen histidine-rich protein (HRP-Ⅱ),a special glycoprotein of P. falciparumsecreted during the parasite's erythrocytic cycle, with a peak during schizontrupture in whole blood and which can be performed without special equipment. Avisual reading is given by a polyclonal antibody coupled with dye-loadedliposomes; when positive, a pink line appears.
4. Arapid dipstick antigen capture assay for the diagnosis of P. falciparum
(1)Principal
Recent advances in the diagnosis of
(2)Steps
A. Addlysing fluid to tube.
B. Takeblood sample from fingerprick.
C. Mixblood with anticoagulant in capillary tube.
D. Transferblood sample to lysing fluid.
E. Placedispensing tip on sample tube.
F. Placelysed blood sample in well.
G. Standdipstick in blood sample.
H. Adddetection agent to well.
I. Addwashing fluid to well.
J.Inpositive cases, a pink line develops almost simultaneously at the monoclonalantibody deposit site with a pink broken line above it as the reagent control.In negative cases, only the pink broken line occurs.
5. ICTMalaria P.f / P.v test
(1)Introduction
Malaria diagnosis has been a difficultprocess requiring the skills of highly trained microscopists to detect
(2)Testprinciple
The ICT Malaria P.f / P.v test is anin-vitro immunodignostic test for the detection and speciation of
6. Qualitycontrol for blood examination
(1)Equipmentmust be clean.
(2)Filmsmust be of correct density.
(3)Filmsmust be allowed to dry in a horizontal position and for the correct time toensure good results.
(4)Thestain dilutions and the buffered water used for staining must be accuratelyprepared and the stock stain must be of good quality.
(5)Thestaining procedure should be followed very carefully.
OPPORTUNISTIC PATHOGENICPROTOZOAN
Pneumocytiscarinii,Toxoplasma gondii
&
Objectivesand Requirements
1.Toobtain a primary understanding of the relation between the immunity of infectedhost and opportunistic pathogenic protozoan.
2.Toobserve the structural features of several species of opportunistic pathogenicprotozoan.
Observationand Experiment
1. Pneumocytiscarinii
(1)See the stained specimen (Demonstration)
A. Trophozoite, note its size,various shape of nucleus, and the color of both cytoplasm and nucleus.
B. Cyst, note its shape, size andthe number of sporozoites within it.
(2)Animal experiment (Students work in groups)
A. Introduction
The establishment of parasitic infectionimplies that the parasites come together with their hosts and the interactionbegins. As soon as the parasites enter the host, first thing is that they haveto struggle against the host immunity before they multiply enough to injure thehost and usher in clinical symptoms. In some cases, for example in theopportunistic pathogenic protozoan, the outcome of the interaction is usuallyestablishment of the balance between the parasites and their host. That is thecertain numbers of the parasites survive within the host but none of therelated clinical symptoms are observed, which is called suppressive infection.However, the balance requires existence of certain conditions of both host andthe parasites. Any change of the related conditions would result in theappearance of imbalance. For instance, the decrease of the host immunity mightlead to dramatic multiplication of the protozoan in the state of suppressiveinfection, and consequently the occurrence of acute attack. The protozoan ofthese characteristics, including P. carinii,T. gondii and species of
In this experiment, the immunity ofobserved animals are artificially suppressed by administration ofimmuno-suppressed drugs before they are infected with P. carinii, and the observations are carried out on the changes inthe body weight, physiological state, pulmonary pathological change of thehosts, as well as examination for pathogens in the hosts.
B.Materials
① Experimental animal: whiterat.
② Homogenate of infected lungtissue with P. carinii.
③ Immuno-suppressed drugs: dexamethasone,cortisone acetate.
④ Nourishment: low proteindiet; drinking water with tetracycline 1 mg/ml.
C.Methods
① Experimental Group. Six white rats areused in the experimental group. The animals are orally administrated with lmg/L dexamethasone in drinking water or intradermally injected with cortisone12.5 mg/100g bodyweight two times a week for six weeks. Two weeks after drug administration,three of them are transtracheally inoculated with 0.15 ml homogenate ofinfected lung tissue with P. carinii.At sixth week, the animals are stopped feeding of immuno-suppressed drug andone or two out of both transtracheally inoculated infected and the othernon-infected subgroups are dissected respectively for observations on thepulmonary pathological changes and examination for P. carinii by stained smear of lung tissue. The remainders of the twosubgroups are left till they are dissected at the eighth week for the sameobservation as conducted at the sixth week. Each of the animals treated withimmuno-suppressed drug is muscularly inoculated once a week with PG 40 000 uand streptomycin 20 000 u.
② ControlGroup. Three white rats are used in the control group. They are free from theimmuno-suppressed drug and two of them are transtracheally inoculated with 0.15ml homogenate of infected lung tissue with P.carinii. All three animals are then dissected at the sixth week for thesame observations as experimental group.
③ Stopimmuno-suppressed drug at the 6th week, dissect one or two rats out of both experimentand control groups, observe the pathological changes in the lungs and smear onslide for examination of parasites under microscope.
④ The remainders are left for two weeks morebefore they are dissected and observed as described in step C.
⑤ Bothexperimental and control groups of animals are weighed and observed forphysiological state once a week. The results of weighing and observation arerecorded.
⑥ Theroute of the experiment is outlined as the following graph.
Experimental group Control group
.gif)
2 weeks normaldiet 2 weeks normaldiet
+cortisone
transtracheal transtracheal
inoculation inoculation normal
of P.carinii of P.carinii diet
4 weeks 4 weeks4 weeks 4 weeks
dissect stop stop dissect dissect dissect
drug drug
2 weeks2 weeks
dissect dissect
2. Cryptosporidium
(l)See the stained oocysts (Demonstration).
Noteits size, shape and number of sporozoites within it.
3. Toxoplasmagondii
(l) See the stainedtachyzoites and pseudocysts (Demonstration).
Note its size, shape, and the color ofboth cytoplasma and nucleus. Pseudocyst is factually a macrophage containingtachyzoites with the number from several to tens.
(2)See the stained cysts (Demonstration).
Note its size, shape, cystic walland the number of bradyzoites within it.
Exercise
1. Based on the recorded results,to write the experimental report on the relationship between opportunisticpathogenic protozoan infection and the immunity of the host, with analysis,discussion and conclusion.
Reference
1. Thepathogenic examination of T. gondiiand animal inoculation
(1)Liquidmaterial: Blood, using blood film. Cerebrospinal fluid (CSF) or ascites,examining the sediment after centrifugation. The live trophozoites arecrescentic in shape, move actively.
(2)Solidmaterial: Examined by cutting section pathologically. If negative, using animalinoculation. Method: Grinding tissue to become paste-like, add saline to diluteit, inject into mouse abdomen, tap the abdomen 7 days later and examine ascitesfor trophozoites.
MOSQUITOES
Objectivesand Requirements
1.To study morphological characteristics of developing stages of mosquitoes.
2.To understand mosquitoes as vectors of mosquito-borne diseases.
3.To learn differential characteristics of Anopheles,Culex and Aedes.
Observationand Experiment
1. See three genera of live adultmosquitoes (Demonstration)
(1)Anopheles
(2)Culex
(3)Aedes
Note the differences in color, wingspots, position of head at rest, abdominal segments covered with scales ofvarious colors.
2. Studythe desiccated specimens of adults of three genera of mosquitoes (Demonstration)
Identify organs and appendages of head,thorax and abdomen. A pair of compound eyes, antennae, maxillary palps and aproboscis (mouth parts) of head; the thorax bearing 3 pairs of legs and onepair of wings; 10 segmented abdomen (the last two segments are modified to malegenitalia).
3. Structureof mosquito heads
(1)Study whole mount of head of Anopheles (Manipulation).
Identify compound eyes, 15 jointedantennae with intersegmental hairs. The mouthpart consists of a tubular labiumterminating with two tiny labella, one labium and one hypopharynx, one pair of mandiblesand one pair of maxillae with all these to form a cannula during blood sucking.
Differentiate the male and femaleAnopheles. Note the head for the plumose antennal hairs of males and pilosehairs of females. Compare the length of antenna in respect to that of proboscisof both sexes.
(2)See Culex head, both male and female,and compare (Demonstration).
4. Seethe egg of Anopheles,
Which of them is floating on water inform of raft or scattered, with or without floats? Note the shape and size.
5. Seethe live larvae of three genera (Demonstration)
Note the siphon or spiracles and restingposition of larvae.
6. Seethe live pupae of three genera (Demonstration)
Note the large anteriorportion-cephalothorax with two respiratory trumpets which extend above thesurface film and enable the pupae to obtain its air supply and the curveabdomen consists of visible segments assuring a "comma" shape.
7.See the photographs of breeding and restingplaces of mosquitoes (Demonstration)
8. Study the relation of mosquito to thediseases
(1)Transmitting malaria (Demonstration).
A. See the plasmodial oocysts onthe intestinal wall of mosquito.
B. Seethe plasmodial sporozoites in the salivary gland smear of infected mosquito.
(2)Transmitting filariasis (Demonstration).
See the infective filarial larvaein labium of infected mosquito.
(3)TransmittingDengue virus (Demonstration).
See the fluorescent stained specimen of Denguevirus in the salivary gland smear of the infected mosquito.
Exercise
l.Label head of Anopheles.
Thinking
1. How to identify the arthropodof Arachnida and Insecta?
2. Which kinds of arbo-diseases(entomophilous diseases) can be prevented by eliminating mosquitoes in city?
FLY
Objectives andRequirements
1. To understand the basicmorphology of different stages of common flies and their role as vector.
Observationsand Experiment
1. Studythe desiccated specimen of housefly (Manipulation)
Identify head, thorax and abdomen. Note apair of compound eyes and proboscis in the head; thorax bearing three pairs of legsand a pair of wings, 4 longitudinal black strips on the dorsum of thorax,abdomen segmented.
2. See the dry specimen of
3. See the dry specimen of
4. Study the external structures of head (Demonstration)
Note the sucking mouthpart consisting ofthree parts. The proximal part, the rostrum, bears a pair of spin maxillary palpsand is considered as a part of head proper; the middle region, the haustellum,is supposed to be homologous to labium; the expanded distal part is the oral platesmade up of fleshy labella with tracheal structures.
5. Studythe structure of leg (Demonstration)
Note the hair appearance of legterminating in pad and claws. Observe carefully minute hairs on the pad.
6. Seethe fly larvae (Demonstration)
Note the shape, size, segmentation,spiracular plate and movement of living larvae.
7. See the preserved specimen of pupae (Demonstration)
8. See the fly eggs (Demonstration)
9. See the helminthic ova carried byhousefly footpads (Demonstration)
Exercise
1.Label the head and leg of fly.
SANDFLY, FLEAS, LICE& OTHER
BLOOD-SUCKING INSECTS
Objectives and Requirements
1. To understand the basicmorphology of different stages of sandfly, the vector of pathogen of kala azar.
2.To learn the morphological characteristics of flea, louse, bedbug, tick andmite.
3.To understand the relation of above arthropods to the diseases.
Observationand Experiment
1. Sandfly (Demonstration)
(1)See the adult.
(2)See the eggs.
(3)See the larva.
(4)See the pupa.
2. Flea (Demonstration)
(1) See the adult.
Note the shape, size and color. The wholebody is laterally compressed with bristles and spines in posterior direction,genal or/and pronotal comb in some species, suctorial mouth parts, wingless andthree pairs of legs (Genal comb-dark brown teeth on the ventral margin of gena.Pronotal comb–dark brown on the posterior margin of the dorsum of the firstthoracic segment.).
(2) See the powerful legterminating in two curved claws.
The last pair is being greatlyelongated for leaping.
(3)See the eggs.
3. Louse (Demonstration)
(1)See the whole mount of body louse.
(2)See the whole mount of head louse.
(3)See the whole mount of crab louse.
Note the general features of these threespecies. The head bears a pair of eyes, a pair of 5-jointed antennae and an extensilepiercing mouthpart. The thorax is composed of three fused segments. Each ofwhich bears a pair of legs terminating in a single hook-like claw and tibialprocess for grasping hairs or fibers of clothes. The body and head lice differonly in size, the crab louse is small in size, indistinct segmented abdomen andlarge heavy claws.
(4)Seethe eggs.
Note the ellipsoidal, operculatedwhite eggs firmly attached to the hairs or fibers.
4. Cockroach (Demonstration)
(1) See Periplaneta americana.
(2) See Blattella germanica.
5. Tick (Demonstration)
(1) See the whole mount of adult hardtick.
Note its large size, fused cephalothorax,piercing mouthpart and the chitinous (shield-shaped) covering the entire dorsalsurface in the male and anterior part in the female; 4 pairs of legs.
(2) See the whole mount of adult softtick.
Note its largbhskgw.cn/yishi/e size, and fused cephalothoracicportion. The mouthpart is situated ventral to anterior end and can not bevisible in dorsal view. No scutum.
6. Mite (Demonstration)
(1) Sarcoptesscabiei
A. See the whole mount of adult.
Note its small size, oval shape, dorsal surfaceof the body with transverse ridges, spines and bristles. The mouthpart consistsof toothed chelicerae. Pedipalps and labial palp fused to the hypostome, 4pairs of short but stout legs, the first two pairs terminating in long tubularprocesses each with a bell shaped sucker and claws.
(2) Leptotrombidiumdeliense
A.See the eggs (Demonstration).
B. Seethe whole mount of larvae (Demonstration).
Note its tiny size, oval shape,capitulum, scutum and body hairs.
C. Collectand observe live larvae.
Anesthetize one mouse and inspect theears for larval mites. Remove them, if any, with dissecting pen to a petri dishcontaining water, observe under low power lens.
D.See the live nymphs (Demonstration).
E.See the whole mount of adult (Demonstration).
F.See the live larvae grouping at the tip of a conical object (Demonstration).
(3) Dermatophagoides(Demonstration)
See the whole mount of adult. Note itssmall size, compact wrinkles of integument, two anterior pairs and twoposterior pairs of legs terminating in long tubular processes each with a bellshaped sucker and claws.
(4) Demobex
A. See the whole mount of adult (Demonstration).
B.Self-survey for Demodex.
7. Bedbug (Demonstration)
(1) See the whole mount of adult.
Note dorsoventrally flattened shape, color,body covered with short serrated hairs; a pair of prominent compound eyes, a slenderand flexible mouthpart of head; three pairs of legs of thorax; ten segmented abdomen.Do you find the stink-glands between the basal parts of the second and thirdlegs?
(2)See the eggs.
(3)See the larvae.
Exercise
1.Report the result of examining demodex.
