(THE FIRST EDITION) - 海南医学院精品课程gzy.hainmc.edu.cn/jingpin/jsc/content/laboratory guide to... · A A LABORATORY LABORATORY GUIDE GUIDE TO TO PARASITOLOGY PARASITOLOGY

AAA

A

LABORATORYLABORATORYLABORATORY

LABORATORY

GUIDEGUIDEGUIDE

GUIDE

TOTOTO

TO

PARASITOLOGYPARASITOLOGYPARASITOLOGY

PARASITOLOGY

(THE(THE(THE

(THE

FIRSTFIRSTFIRST

FIRST

EDITION)EDITION)EDITION)

EDITION)

LULULU

LU

GANGGANGGANG

GANG

DepartmentDepartmentDepartment

Department

ofofof

of

ParasitologyParasitologyParasitology

Parasitology

HainanHainanHainan

Hainan

MedicalMedicalMedical

Medical

CCC

C

ollageollageollage

ollage

,,,

,

SeptemberSeptemberSeptember

September

,,,

,

200200200

200

777

7

1

CONTENTSCONTENTSCONTENTS

CONTENTS

Care of the microscope…………………………………….…………………1Ascaris lumbricoides & Trichuris trichiura………………………………..17Ancylostoma duodenal & Necator americanus……………………………..20Enterobius vermicularis……………………………………………….…….22Wuchereria bancrofti& Brugia malayi……………………………………..23Trichinella spiralis…………………………………….……………….……25Clonorchis sinensis…………………………………….……………….…….2Paragonimus flukes…………………………………….……………….……5Fasciolopsis buski…………………………………….……………….….…..7Schistosoma sp…………………………………………….………………...8Tapeworm…………………………………….……………….………….…14Examination of alimentary helminths…………………………………….…26Entamoeba histolytica, E.coli& other Amoebae…………………………...27Giardia lamblia…………………………………….……………….……….30Trichomonas vaginalis…………………………………….………………...31Leishmania donovani…………………………………….……………….…32Plasmodia…………………………………….…………………….…….…35Opportunist ic pathogenic protozoan………………………………….……..40Mosquitoes…………………………………….……………….…….……...43Fly…………………………………….…………………….…….…….…...45Sandfly, fleas, lice & other blood sucking insects…………………………..46

2

CareCareCare

Care

ofofof

of

thethethe

the

microscopemicroscopemicroscope

microscope

Do'sDo'sDo's

Do's

1. Do take special care to protect the microscope from dust in hot dry periods.2. Do take special care to protect the microscope lenses and prisms from fungal growthin hot humid periods.

3. Do clean the immersion oil from the immersion objective each time; use lens tissuedampened with ethanol.

4. Do clean the oculars with lens tissue.5. Do use the microscope retaining screw fitted at the base of the microscope box toprevent damage to the instrument while in transit.

Don'tDon'tDon't

Don't

1. Don't use the tissue used for the oil immersion objective to clean the oculars.2. Don't dismantle or try to clean parts of the microscope that are difficult to reach.3. Don't leave the lens parts empty; use the appropriate cover to cover the empty port.4. Don't exchange lenses from microscopes of different manufacture.

3

ASCARISASCARISASCARIS

ASCARIS

LUMBRICOIDESLUMBRICOIDESLUMBRICOIDES

LUMBRICOIDES

&&&

&

TRICHURISTRICHURISTRICHURIS

TRICHURIS

TRICHIURATRICHIURATRICHIURA

TRICHIURA

ObjectivesObjectivesObjectives

Objectives

andandand

and

RequirementsRequirementsRequirements

Requirements

1. To gain some knowledge of the life cycle and common morphology of nematodes.2. To identify ova of A. lumbricoides and T. trichiura.3. To study the pathogenesis ofA. lumbricoides infection.4. To further understand the life cycle and pathogenesis of A. lumbricoides throughanimal experimental observation.

ObservationObservationObservation

Observation

andandand

and

EEE

E

xperimentxperimentxperiment

xperiment

l.l.l.

l.

AscarisAscarisAscaris

Ascaris

lumbricoideslumbricoideslumbricoides

lumbricoides

(1) Adult (Demonstration)A. See the preserved male and female adult worm ofA. lumbricoides .

Note their size, shape and color. The female worm is rather large, being 20～35 cmlong. The male worm is rather small and 15～30 cm long, with the tail end turning tothe ventral side. The front end of the body in both male and females is equipped withthree lips.

B. See the dissected male and female adult worm of A. lumbricoides.(2) Study the transverse section ofA. lumbricoides.

Note intestinal canal and two sexes of reproductive system. The female has two setsof genital organs. There is a moderate construction at about 1/3 of the body from thefront end forming the genial girdle, at the ventral side of which the vulva is open. Theanus is located 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 the digestive and thegenital tract is located near the end.

(3) Study the cross section ofA. lumbricoides.Note the internal and external structures of body wall and body cavity (pseudocele).

A. The cuticle: A body wall consisting of a protoplasmic syncytial layer called thehypodermis surmounted by an apparently non-living, mainly collagenous cuticle inwhich no separate cells can be distinguished and underlain by groups of longitudinalmuscles. Nuclei are present only in four thickened chords or "line," one dorsal, oneventral, and two lateral. In these chords run nerve fibers. Between the cords there is asingle layer of longitudinally spindle-shaped muscle cells of 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.

4

C. Cavity: Between the muscles and the gut wall is a relatively spacious body cavity inwhich the reproductive organs lie, unattached except at their external openings. Thiscavity is not lined by an epithelium as is a true celome, and it is a pseudocoelomatebody cavity which plays an important hydrostatic skeletal role in locomotion. Itcontains a fluid which serve as distributing medium for digested food and forcollection of waste products. It is provided with a small amount of "mesenterial"tissue and a few large phagocytic cells called celomocytes.

(4) OvaA. Study the morphology of fertilized ova of A. lumbricoides (Manipulation).

Note the characteristic shape, size , color, and the shell with special reference toalbuminoid membrane. 45～75μm×35～50μm, yellow-brown color.

B. Study the morphology of unfertilized ova ofA. lumbricoides (Manipulation).Compare the appearance and structure with 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 of fertilized and unfertilized ova (Demonstration).(5) See the SEM photograph of lips and copulative spicula (Demonstration).(6) Pathologic specimens (Demonstration)A. See the gross specimen of intestinal obstruction due to ascariasisB. See the gross specimen of biliary ascariasis2.2.2.

2.

TrichurisTrichurisTrichuris

Trichuris

trichiuratrichiuratrichiura

trichiura

(1) See the preserved adult worms (Demonstration).Note whip-like, the thin anterior part (three-fifths) is occupied by the esophagus andthe thick posterior part (two fifths) contains intestine and genital organs.

(2) Study the preservedova (Manipulation).Note the shape, size, color and shell. Observe particularly their bipolar plugs andgerminal cell. 50～54μm×22～23μm in size, barrel-shaped, eggshell is brown incolor, with pores at both ends that are stoppered with colorless mucoid substance, anegg cell is uncleaved.

(1) Pathologic specimens.Adults parasitic in human cecal mucosa, posterior part of the body being free.

ExerciseExerciseExercise

Exercise

1. Draw the fertilized and unfertilized ova ofA. lumbricoides.2. Draw the ovum of T. trichiura.

ReferenceReferenceReference

Reference

1.1.1.

1.

ExperimentalExperimentalExperimental

Experimental

A.A.A.

A.

lumbricoideslumbricoideslumbricoides

lumbricoides

infectioninfectioninfection

infection

ofofof

of

animalanimalanimal

animal

(Students work in groups)

5

(1) Place one female worm Ascaris lying on its ventral surface on a wax pan. Fix withpins at both ends, make an incision at the lateral side of vulva and cut caudally so asto open the body lengthwise from anterior to posterior end. Using a needle, lay thecut edges flat by pinning them to the wax, add a few saline and separate internalorgans carefully. Remove about 1.5 cm of one uterus near the vagina with needle,put a sprinkling of ova in a drop of saline and prove that are fertilized undermicroscope.

(2) Put all fertilized ova on a penicillin bottle, and add 2 ml of 2% formalin. Incubate atroom temperature or 22 ℃ ～ 33 ℃ for 3 weeks. Observe and record theembryogenetic development every week, and add 2% formalin to maintain optimalmoisture, if necessary.

(3) Three weeks later when the ova are mature, remove the formalin by a pipette and mixthe ova with a few saline to make up suspension, pick up 0.5 ml ovum suspensionwith a pipette and inoculate it orally into the stomach of mouse.

(2) One week later, dissect the infected mouse, remove the liver and lung, transfer themonto a petri dish, wash blood away with saline, observe the surface carefully forbloody extravasation. Lacerate the organs and isolate the larvae under dissectingmicroscope.

6

AAA

A

NCYLOSTOMANCYLOSTOMANCYLOSTOMA

NCYLOSTOMA

DUODENALEDUODENALEDUODENALE

DUODENALE

&&&

&

NECATORNECATORNECATOR

NECATOR

AMERICANUSAMERICANUSAMERICANUS

AMERICANUS


Objectives

andandand

and


Requirements

1.To learn the differential characteristics of two species of hookworm.2.To learn the morphological characteristics of ovum.3.To gain some knowledge regarding the environmental factors that might affect thedevelopment of hookworm larvae. To study the methods commonly used in laboratorydiagnosis and epidemiolog ic survey.

4.Based on the structural characteristics, to understand the pathogenesis of hookwormdiseases.

5.To understand the relationship of hookworm epidemiology with natural factors andcrops cultivating.


Observation

andandand

and

ExperimentExperimentExperiment

Experiment

1.1.1.

1.

AdultAdultAdult

Adult

(1) See the preserved adult worm (Demonstration).Note the shape, size and gross appearance of bursa of male worm. The body is thinand long.

(2) See the male and female worms in copulation (Demonstration).(3) Study the characteristic structure of buccal capsule ofAncylostoma duodenale.

Note twopairs of ventral teeth and a pair of accessory teeth. On the dorsal side of themouth, a pair of small dorsal teeth is seen in the center.

(1) Study the characteristic structure of buccal capsule of Necator americanus.Note one pair of ventral cutting plates and a pair of dorsal cutting plates.

(2) See the bursa of A. duodenale (Demonstration).The female is with a small mucro at the tail end, while the male is with acharacteristic copulatory bursa at the tail end, which looks like an opened umbrella,the ribs of which are called rays.

(3) See the bursa of N. americanus (Demonstration).In the male, the copulatory bursa is deep, with the two branches of the lateral raytightly adhered to each other, and there is a hook on the tip of each spicule. In thefemale, the vulva opens a little anterior to the center of the body, and no spine existsat the tail end.

(7) See the SEM photographs of buccal cavity and bursa of hookworm.2.2.2.

2.

OvumOvumOvum

Ovum

andandand

and

larvalarvalarva

larva

7

(1) Study the ova (Manipulation).Note the shape, size, color, shell and internal structure. 36～40μm×56～76μm insize, thin eggshell, nearly colorless and transparent. It contains egg cells undercleavage.

(2) See the ova. Note 2-, 4-, 8-cells, morula and embryonated ova (Demonstration).(3) Study the embryogenesis of rhabditiform larvae (Demonstration).(4) See the filariform larvae (Demonstration).(5) See the activities of filariform larvae on water film of soil surface, waiting for

suitable hosts (Demonstration).(6) See the SEM photographs of ova and filariform larvae.3.3.3.

3.

PathologicalPathologicalPathological

Pathological

specimensspecimensspecimens

specimens

andandand

and

ttt

t

iii

i

ssuessuessue

ssue

secsecsec

sec

ttt

t

ionionion

ion

(1) See the gross specimen of intestine showing hookworm grasping mucous membrane(Demonstration).Note the hemorrhagic petechiae and ulcers.

(2) See the section of the head end of hookworm grasping mucosa membrane of smallintestine (Demonstration).The worms support themselves by biting 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. duodenale and N. americanus.

ThinkingThinkingThinking

Thinking

1. How does a hookworm infect host? And which dangerous can be caused to the host?2. What relationship between hookworm infection and natural environment is?


Reference

1.1.1.

1.

MethodsMethodsMethods

Methods

usedusedused

used

ininin

in

laboratorylaboratorylaboratory

laboratory

diagnosisdiagnosisdiagnosis

diagnosis

ofofof

of

hookwormhookwormhookworm

hookworm

larvalarvalarva

larva

...

.

(1) Direct fecal smear and NaCl saturated floatation (the methods are described in detailsat Examination ofAlimentary Helminths on page 268 of the Chinese textbook).

(2) See demonstration showing cultivation of larvae.2.2.2.

2.


Methods

usedusedused

used

ininin

in

epidemiologicepidemiologicepidemiologic

epidemiologic

surveysurveysurvey

survey

ofofof

of

hookwormhookwormhookworm

hookworm

...

.

(1) Baermann's technique. 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 lower end.Fill the funnel with 40℃ water until its level is raised to touch the lower portion ofthe soil. After 20 minutes, open the clamp and let out the lowest portion of water to abeaker or centrifuged tube. Let the collected water stand for 10～ 20 minutes or

8

centrifuge for 1～2 minutes, then examine the sediment for larvae.(2) Pad method. Soak a pad of six to seven layers of gauze in 40℃ water. Cover the soil

with the warm pad to attract the larvae. Cover the pad with a petri dish to keep theformer warm. After 20 minutes, rinse the pad in water repeatedly. Examine sedimentfor larvae.

ENTEROBIUSENTEROBIUSENTEROBIUS

ENTEROBIUS

VERMICULARISVERMICULARISVERMICULARIS

VERMICULARIS


Objectives

andandand

and


Requirements

(1) Study the structure of E. vermicularis ova and methods used in the diagnosis of E.vermicularis infection.


Observation

andandand

and


Experiment

(1) See the preserved adult worm of E. vermicularis (Demonstration).Note more or less spindle-shaped, whitish in color. The female worm is 8 mm～13mm long and the male is only 2 mm～5 mm. The male has its posterior end stronglycurvedventral, and the female tail portion is sharply pointed.

(2) See the whole mount of adult worms (Demonstration).(3) Study the preserved specimen of ova (Manipulation).

Note their shape, size, color, shell and content. 50～60μm×20～30μm in size,elongate-ovoidal, flattened on the ventral side, thick and transparent eggshell, fullyembryonated.Why the shape looks different in lateral and dorsal views?

(4) See the SEM photograph of the anterior end of adult.(5) See the swab method for diagnosis of E. vermicularis (Demonstration).

Wet a cotton swab with normal saline and rub corrugations in perianal area. Then stirthe swab in NaCl saturated solution in a test tube (or penicillin bottle), and add NaClsaturated solution up to rim of test tube, cover the test tube with a clean slide incontact with the solution. Let it stand for 5～10 minutes. Examine the slide for ova.


Exercise

1. Draw and label the ovum of E. vermicularis.


Thinking

1. Why there are more infections of E. vermicularis in rural area than in city?

9

WUCHERERIAWUCHERERIAWUCHERERIA

WUCHERERIA

BANCROFTIBANCROFTIBANCROFTI

BANCROFTI

&&&

&

BRUGIABRUGIABRUGIA

BRUGIA

MALAMALAMALA

MALA

YYY

Y

III

I


Objectives

andandand

and


Requirements

l. To study the life cycle characteristics and epidemiolog ic features of filariae.2.To study the morphology of microfilar iae and laboratory diagnostic methods.


Observation

andandand

and


Experiment

1.1.1.

1.

AdultAdultAdult

Adult

(1) See the preserved adult worm of Wuchereria bancrofti (Demonstration).Adults look like thin and long threads.

(2) See the preserved adult worm of Brugia malayi (Demonstration).The adults resemble that of W. bancrofti in morphology, but is thinner and shorter.

(3) See the preserved adult worm of Loa loa (Demonstration).The adults take a thread-like form, and there are small lumps on the body surface.

(4) See the preserved adult worm of Dirofilaria immitis (Demonstration).The adults look like thin white noodles, and the tail of the male is coiled.

2.2.2.

2.

LarvaeLarvaeLarvae

Larvae

(1) Study the structures of W. bancroftimicrofilar iae stained specimen (Manipulation).Note the shape, appearance, sheath, cellular column and caudal nuclei. The larva issheathed by egg membrane, the excretory pore is adjacent to the excretory cell, one ofthe genital cells (G-cells) is far apart from the others, and there is no nucleus at the tailend.

(2) Study the structures of B. malayimicrofilar iae stained specimen (Manipulation).Compare the structures with those of W. bancroftimicrofilar iae. The larva is sheathedand shorter, the excretory cell is located separately from the excretory pore, all of thefore genital cells are situated far ahead of the anus, the first cell (G-1) being especiallylarge, and the tail end has two terminal nuclei.

(3) Observe the microfilar iae in unstained blood film.(4) See the infective larvae in mosquito thoracic muscles (Demonstration).(5) See the infective larvae in mosquito proboscis (Demonstration).3.3.3.

3.

TheTheThe

The

intermediateintermediateintermediate

intermediate

hosthosthost

host

(Demonstration)Culex quanquefasciatus and Anopheles sinensis.

4.4.4.

4.

PathogenesisPathogenesisPathogenesis

Pathogenesis

(Demonstration)(1) See the photograph showing the patient with elephantiasis of lower extremity andscrotum.

10

(2) See the photograph showing the patient with hydrocele.


Exercise

1. Label the microfilar iae.


Thinking

1. What are the epidemiolog ic distribution characteristics of filariasis of W. bancrofti?2. To describe the pathogenesis of W. Bancrofti and B. Malayi.


Reference

1.1.1.

1.

LaboratoryLaboratoryLaboratory

Laboratory

diagnosticdiagnosticdiagnostic

diagnostic

methodsmethodsmethods

methods

forforfor

for

filariafilariafilaria

filaria

(1) Thickblood filmSterilize the ear lob with 75% alcohol and puncture with needle. Place on a cleanslide two to three drops blood approximate 60 mm3 and spread it with the corner ofanother slide to equal the diameter of two-fen coin. Let it dry. Put it in water, the thickfilm becomes whitish in color and examine it under microscope.

(2) Examination of live microfilar iae (Demonstration)Place two drops of patient's blood (or dog infected with Dirofilaria) and cover withcoverslip. Note their wiggle among the RBC.

(3) Examination of venous blood formicrofilar iae (Demonstration)Mix 1ml of venous blood (taken at night) with o.1 ml 3.8% sodium citrate in acentrifugal tube, then add 9 ml of distilled water to break the RBC. Centrifugalize for2 minutes at 3 000 rpm. Decant the supernatant and examine the sediment formicrofilar iae.

11

TRICHINELLATRICHINELLATRICHINELLA

TRICHINELLA

SPIRALISSPIRALISSPIRALIS

SPIRALIS


Objectives

andandand

and


Requirements

1. To learn the diagnostic method of T. spiralis.2. To understand the life cycle and pathogenesis of T. spiralis through animalexperiment.


Observation

andandand

and


Experiment

1.1.1.

1.

SeeSeeSee

See

thethethe

the

preservedpreservedpreserved

preserved

adultadultadult

adult

wormswormsworms

worms

(Demonstration)The adults look like pieces of thread, being 2.2 mm～3.0 mm long in the female and1.2 mm～1.5 mm long in the male.

2.2.2.

2.

SeeSeeSee

See

thethethe

the

stainedstainedstained

stained

specimenspecimenspecimen

specimen

ofofof

of

cystcystcyst

cyst

(larva)(larva)(larva)

(larva)

ininin

in

muscularmuscularmuscular

muscular

tissuetissuetissue

tissue

(Demonstration)Larvae that reach striated muscles invade muscular fibers to be encysted. With thedevelopment of the larva, the cyst wall is thickened to form a cyst of characteristicspindle form, its major axis being in parallel with the muscle fibers. Usually, each cysthas one coiled larva.

3.3.3.

3.

SeeSeeSee

See

thethethe

the

livelivelive

live

cystcystcyst

cyst

(Demonstration)4.4.4.

4.

ExperimentalExperimentalExperimental

Experimental

infectioninfectioninfection

infection

ofofof

of

micemicemice

mice

andandand

and

examinationexaminationexamination

examination

(Students work in groups)Take buccal muscle or diaphragm of infected mice, cut into pieces and feed the normalmice. Four weeks later, kill the infected mice. Examine (l) buccal muscle, (2)diaphragm and (3) heart muscles by placing a small piece of the referred tissuebetween two slides and examining undermicroscope for cysts. (4) dissect and wash thesmall intes tine to 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 be the reservoir host of human parasites?2. What are the differences existed in the distribution of biohelminth and geohelminth?

12

CLONORCHISCLONORCHISCLONORCHIS

CLONORCHIS

SINENSISSINENSISSINENSIS

SINENSIS


Objectives

andandand

and

RRR

R

eqeqeq

eq

uuu

u

irementsirementsirements

irements

1. To study the morphology and the life history of C. sinensis as an example of generalfeature of trematodes.

2. To study the infection route, infection mode, parasitized site and the pathogenesis of C.sinensis infection.

3. To study the characteristics of ovum of C. sinensis and the pathogenic diagnosis of C.sinensis infection.

4. To learn the basic skill of drawing eggs.


Observation

andandand

and


Experiment

1.1.1.

1.

AdultAdultAdult

Adult

wormwormworm

worm

(1) Observe the preserved specimen (Demonstration).Note the size, color, shape of the organism. Spindle-shaped, transparent.

(2) Observe the general feature of C. sinensis in stained specimen (Manipulation).First with naked eye, then study under microscope, note and identify followingstructures.

A. Adherent organs: oral and ventral suckers, compare their size.B. Digestive system: mouth, pharynx, esophagus and ceca.C. Excretory system: excretory bladder and pore.D. Reproductive system: hermaphroditism.

Male: observe the number, size , shape and position of testes.Female: observe ovary, seminal receptacle, vitellarium, Mehlis ’ gland and uterus.What is meant by hermaphroditism?

2.2.2.

2.

LarvaLarvaLarva

Larva

andandand

and


intermediate

hostshostshosts

hosts

(Demonstration)(1) See live sporocyst, redia and cercaria (or stained sporocyst to replace live one if

necessary).(2) See the stained cercaria.

Note the eye-spots and characteristics of 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 intermediate hosts (Parafossarulus, Alocinma, Bithynia snails), andsecond intermediate hosts, fresh water fish and crayfish. What are their names?

3.3.3.

3.

OvumOvumOvum

Ovum

(Manipulation)(1) Study the ova. Note the shape, color, shell, operculum resting on a rim which takes

13

the shape of distinct shoulders, a small protuberance at abopercular end, anasymmetrical miracidium inside the egg. One of the smallest helminth ova, yellow-brown, thicker eggshell.Material examined: feces and drawn liquid from duodenum.Examining method: see examination of alimentary helminths.

(2) See the scanning electron-microscopic (SEM) photograph of ovum (Demonstration).444

4

...

.

PathologyPathologyPathology

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. Draw egg of C. sinensis in detail.2. Label in full an adult worm of C. sinensis.3.3.3.

3.

Draw a life cycle draft of C. sinensis , including final host, intermediate host; adultparasitized site, main injured organs; excretory route of ovum, pathogenic diagnosticmethods; the first and second intermediate hosts and in which how the larvadevelopment; 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 experimental animal.


Reference

1.1.1.

1.

TheTheThe

The

fertilizationfertilizationfertilization

fertilization

processprocessprocess

process

andandand

and

eggeggegg

egg

formationformationformation

formation

In male, sperm produced in a pair of testes are collected in the seminal vesicle throughthe vas efferens and vas deferens and are ejected through the ejaculatory duct, aroundwhich there is a prostate gland covered by a cirrus sac. In female, a short oviductcomes out of the ovary and joins with the seminal receptacle and Laurer’s canalopening on the dorsal side, and further proceeds to the ootype with surroundingMehlis’ gland. Then, it becomes the uterus, which extends with many contortions tothe common genital pore. Small ducts from the vitelline glands, gradually gather tobecome the right and left vitelline ducts and then one common vitelline duct, whichopens at front of the ootype.An egg cell from ovary and a sperm from testis are united, additional yolk cellsprovided by the vitelline gland join, and eggshell formation is started in the ootype bythe effect of Mehlis ’ gland and completed during passage through the uterus. In the eggone miracidium has already been formed.

2.2.2.

2.

ExamineExamineExamine

Examine

forforfor

for

enenen

en

cystedcystedcysted

cysted

metacercariametacercariametacercaria

metacercaria

ininin

in

fishfishfish

fish

fleshfleshflesh

flesh

14

Press a small piece of freshwater fish flesh between two slides, examine for encystedmetacercaria of C. sinensis under lower-lens microscope.

3.3.3.

3.

IsolationIsolationIsolation

Isolation

ofofof

of

encystedencystedencysted

encysted

metacercariametacercariametacercaria

metacercaria

Take the 2nd intermediate host-freshwater fish and smash, digest with 250 ml digestivesolution per 10 g flesh for 4 h～12 h (Digestive solution: pepsin 9.8 g, 1 N HCl 164 ml,NaCl 17 g, add water to 2 000 ml). Discard rough matter by sieve filtration. Afterseveral times sedimentation of the filtrated solution, take the sediment and examineundermicroscope.

444

4

...

.

ExaminationExaminationExamination

Examination

adultadultadult

adult

fromfromfrom

from

infectinfectinfect

infect

ededed

ed

animalanimalanimal

animal

Dissect the guinea pig (or white rat) which were infected with the aid of stomach tubeapproximately 30 C. sinensis cysts 40 days ago. Expose the common bile duct, gallbladder and search for the adult worms in the billiary tree.

15

PARAGONIMUSPARAGONIMUSPARAGONIMUS

PARAGONIMUS

FLUKESFLUKESFLUKES

FLUKES


Objectives

andandand

and


Requirements

1. To study the life cycles of P. westermani , to learn more about the life history oftrematodes.

2. To study the main parasitized sites and major pathogenisis.3. To study the laboratory diagnosis of these flukes.


Observation

andandand

and


Experiment

1.1.1.

1.

P .P .P .

P .

westermaniwestermaniwestermani

westermani

(1) Adult wormA. See the preserved specimen (Demonstration).B. Study the internal structures of stained specimen (Manipulation) .Note the position of the suckers. Are they equal in size? The branched testes aresituated in posterior of the body, side by side, and posterior to uterus and ovary. Thelobulated ovary 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 Paragonimus spines.(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 in groups).Crushed a crab in a mortar.Add some 0.45% NaCl solution or tap water into motar andfilter. Allow the filtrate stand and settle, examine sediments for encysted metacercariaunder microscope.

(3) EggA. Morphology of egg (Manipulation).Measures 80～118μm×48～60μm. Note its ellipsoidal shape, uneven thickness ofeggshell, slightly oblique operculum and yellowish brown in color. A germinal cellwith 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) PathologyA. See the preserved infected lungs (Demonstration).Note the capsules and their surrounding damaged tissues.

16

B. See the infected liver (Demonstration).2.2.2.

2.

PagumogonimusPagumogonimusPagumogonimus

Pagumogonimus

skrjabiniskrjabiniskrjabini

skrjabini

(1) Adult worm (Demonstration).A. See the preserved specimen.B. See the stained specimen.(2) Intermediate host (Demonstration)

See the first intermediate host-snail (Tricula humida).


Exercise

1. Draw P. westermani egg in detail and label.2. Label the adult worm of P.westermani.3. Write the result report of cyst isolation, to have an analysis of epidemiology of P.westermani.

4. Draw a life cycle draft of P. westermani .


Thinking

1. How to explain paragonimiasis is a natural epidemic source disease in mountainousregion and forest? And what are the epidemic characteristics of paragonimiasis?


Reference

1.1.1.

1.

DetectionDetectionDetection

Detection

ofofof

of

sputumsputumsputum

sputum

forforfor

for

eggeggegg

egg

ofofof

of

P .P .P .

P .

www

w

estermanestermanesterman

esterman

iii

i

(1) Sputum smearTake a clean slide, place a drop of saline in the center of it, pick up a small amount ofthe sputum and mix it with the drop of saline, examine undermicroscope.

(2) Egg concentrationCollect the sputum of 24 h, pour into a measuring glass and add equal quantity of 10%NaOH, mix, sediment naturally for 6 h～8 h, remove supernatant, take the sedimentand examine undermicroscope.

2.2.2.

2.

SurveySurveySurvey

Survey

biologicalbiologicalbiological

biological

hostshostshosts

hosts

ofofof

of

P .P .P .

P .

www

w

estermanestermanesterman

esterman

ininin

in

epidemiologyepidemiologyepidemiology

epidemiology

Choose one or more brooks, capture Melania snail and stream crab or crayfish .(1) Break up Melania snail and place the internal organs on a slide, open them and put a

drop of 0.45% saline, find sporocyst, redia and cercaria under microscope.(2) Dissect stream crab or crayfish and find encysted metacercaria.3.3.3.

3.

InfectionInfectionInfection

Infection

ofofof

of

P .P .P .

P .

WWW

W

estermaniestermaniestermani

estermani

forforfor

for

dogdogdog

dog

Isolation of cysts from crayfish and mixing with meat, feed dogs, each one with 100～200 cysts according to the sizes of dogs. Kill the infected dogs 2～3 months later, openchest and expose the lungs, on which the nodular cysts can be observed, dissect themto find adult worms.

17

FFF

F

ASCIOLOPSISASCIOLOPSISASCIOLOPSIS

ASCIOLOPSIS

BUSKIBUSKIBUSKI

BUSKI


Objectives

andandand

and


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.


Observation

andandand

and


Experiment

1.1.1.

1.

AdultAdultAdult

Adult

wormwormworm

worm

(1) See the preserved specimens (Demonstration).Note the shape, size, color and the positions of oral and ventral suckers and their size s,large and thick.

(1) Study the stained specimen (Manipulation).First with naked eye, then under low power microscope, note the adhesive organ, cecaand reproductive system of both sexes. The intestine is composed of two blind tractswithout side branching, and the ventral sucker is very large.

2.2.2.

2.

Larva,Larva,Larva,

Larva,

iii

i

ntermediatentermediatentermediate

ntermediate

hosthosthost

host

andandand

and

vectorvectorvector

vector

(Demonstration)(3) See the snail intermediate host (Planorbis).(2) See the cercaria.(3) See the stained specimen of cyst.(4) See the vector: aquatic plants (caltrop, water chestnut etc.).3.3.3.

3.

OvumOvumOvum

Ovum

(1) See the preserved specimens (Manipulation).Note its large size, 130～140μm×80～85μm, oval shape, pale yellow in color,thin shell, small operculum, an germinal cell surrounded by yolk granules.Materials examined: feces.

(2) See the SEM photograph of ovum.


Exercise

1.Draw and label F.buski egg in detail.


Thinking

1.Drawa life cycle draft of F.buski.

18

SCHISTOSOMASCHISTOSOMASCHISTOSOMA

SCHISTOSOMA

JAPONICUMJAPONICUMJAPONICUM

JAPONICUM


Objectives

andandand

and


Requirements

1. To understand characteristics of life cycle and morphology of its developmental stages.2. To observe the pathological changes of schistosomiasis in infected animal.3. To study morphology of ova, laboratory diagnostic methods and their principle.4. To gain fundamental knowledge of infective stage, infection mode, methods and

signif icance of immunologic tests commonly used.


Observation

andandand

and


Experiment

l.l.l.

l.

AdultAdultAdult

Adult

wormwormworm

worm

(1) See the live specimen (Demonstration).(1) See the preserved specimen (Demonstration).

Adults are dioecious, a male and a female live together.(3) Study stained specimen (Manipulation).

Note the shape, size and color of both sexes, oral and ventral suckers andgynecophoral canal of male, the number, shape, size and arrangement of testes, ovaryand uterus of female. Females are long and slender, the posterior half of the body is alittle thick and dark brown in color owing to the vitelline gland and intestine. Near theanterior end 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 a groove,canalis gynecophorus, into which the female worm fits. What is the meaning ofdioecious?

(2) See the SEM photograph of adult worm (Demonstration).2.2.2.

2.

LarvaLarvaLarva

Larva

andandand

and


intermediate

hosthosthost

host

(Demonstration)(1) See the live miracidia.

Note the feature of movement in water.(2) Study the structure of stained specimen of miracidia.(3) See the SEM photograph of miracidia.(4) Identify the intermediate host Oncomelania snail.(5) See the live cercariae.

Note the posture of the body resting beneath water surface and its movement. Studystained specimen, note the internal structures and the forked tail.

(6) See the SEM photograph of cercariae.3.3.3.

3.

OvumOvumOvum

Ovum

(1) Place a small drop of suspension containing S. japonicum ova on a slide.Is there a miracidium inside the egg? Do you find operculum or process of eggshell?

19

Materials examined: feces.(2) Study the preserved specimen of S. japonicum ova (Manipulation).

Ovoidal, 54～63μm×40～58μm, thin eggshell and lacking the operculum, on theside near one end there is depression from which there extends a small spinoseprocess.

(3) See the SEM photograph of egg (Demonstration).(4) See the ova of S. mansoni and S. Hematobium (Demonstration).

Compare the ova of these three blood flukes.4.4.4.

4.

PathologyPathologyPathology

Pathology

(1) See the cirrhotic liver and enlarged spleen (Demonstration).Note the size and nodular appearance of the liver and spleen.

(2) See the sections showing pathological foci in liver (Demonstration).A. Acute stage, note the infiltration of eosinophils, leucocytes and radiating acidophilic

streaks around the eggs.B. Chronic stage, note the dead or calcif ied eggs surrounded by epithelioid cells, giant

cells and fibroblasts.(3) Dissection of infected experimental animal (Students work in groups).

Kill the rabbits infected with S. japonicum formore than 40 days. Note the followingfeatures after dissection:

A. Is there ascites or not?B. Where adult worms live in?C. Observe the lesions of intestine.D. Observe the lesions of liver.E. Biopsy : Remove a small piece of rectal mucosa from infected rabbit. Press it between

two slides and examine under microscope. The ova of various developmental stagesof embryogenesis are deposited singly or in clumps sometimes in chains followingthe vessel.

5.5.5.

5.

LaboratoryLaboratoryLaboratory

Laboratory


diagnosis

(1) Pathogenic examination: fecal examination.(2) Hatching of miracidium.A. Principal

Under suitable condition, within 24 h～48 h, the miracidium in egg can be hatched.According to the tendency to light and to upside of miracidium, which move on thewater surface by straight line movement.

B. MethodUse a sieve to filter 30 g of fresh feces.Add water to the filtrate in conical cylinder to1 000 ml. Let it stand for 15～20 minutes, decant supernatant fluid and add clear water.After decantation, transfer sediment to a flask with 500 ml water. Incubation 6 h～12

20

h at 37 ℃ and examine the free swimming miracidia. If negative , repeat theexamination after 24 h.

6.6.6.

6.

ImmunodiagnosisImmunodiagnosisImmunodiagnosis

Immunodiagnosis

(1) Circumoval precipitin (COP) test, cercarien Hüllen reaction (CHR).When live eggs, miracidia, or cercariae are mixed into patients ’ sera, precipitateformation on their surfaces and immobillization of miracidia or cercariae will takeplace. These reactions are called the circumoval precipitin (COP) test, miracidialimmobilization test, and cercarien Hüllen reaction (CHR).

(2) Fast-ELISA (fast enzyme-linked immunosorbent assay).A. Introduction.

Fast-ELISA is an in-vitro immunodiagnostic test for detection of pathogen infection,which combines rapid, 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 solution of S. japonicum was used to coatmicroplates overnight at 4 ℃ . Plates were incubated for 3 ～ 5 minutes at roomtemperature with one drop of diluted serum or S. japonicum-infected individualserum, or negative serum. The plates were washed three times with washing reagentand incubated for 3～5 minutes with one drop of enzyme (alkaline phosphatase)-coupled reagent at room temperature. Incubate for 3～5 minutes at room temperatureand washed five times by tap water. Add one drop of bottom reagent, one drop ofdeveloping reagent respectively. After 30 seconds to 3 minutes at room temperature,add one drop of stopped reagent and observe the result.

D. Criteria of diagnosis.Grade the result according to the developing dye degree on a white color background.+++ — ++++: the color is much deeper than the positive control.++: the color is similar to the positive control.+: the color is darker than the negative control and lighter than the positive control.-: the color is similar to the negative control.All sample graded higher than “+” are considered positive.

7.7.7.

7.

EpidemiologyEpidemiologyEpidemiology

Epidemiology

(Demonstration)(1) See photographs of patients.(2) Environment of endemic area.(3) Eradication of Oncomelnia snails.

21


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 person can be infected by S. japonicum? What are thedifferences from other flukes?

2. Can we find the eggs of S. japonicum from the feces of a patient while the adult wormsinhabit in the mesenteric vessels? Why?

3. To compare the prevention and cure measures of schistosomasis japonica with otherflukes.


Reference

1. InfectionInfectionInfection

Infection

ofofof

of

experimentalexperimentalexperimental

experimental

rabbitrabbitrabbit

rabbit

sss

s

(Students work in groups)Cover a shaved area of 10 sq. cm with a cover glass up-side-down, which contains 10live cercariae, add a drop of water to the side of cover glass. After about 10 minutesremove the cover glass and count the remaining cercariae. Observe the affected skin, isthere any papule or petechiae? 5 weeks later sacrifice the rabbit for observation onadult worms inhabiting the mesenteric veins, and the gross pathological changes inliver and intestines.

2.2.2.

2.


Methods

forforfor

for

identificidentificidentific

identific

ationationation

ation

ofofof

of

deaddeaddead

dead

andandand

and

livinglivingliving

living

eggseggseggs

eggs

Acridine orange method. Place intestinal mucosa scraps (or other tissue) into Kahn-testtube 0.5 ml～ l ml of 1:10 000 acridine orange solution. Keep shaking for a while andthen incubate at 37℃ for 2 h for staining. Wash twice with PBS. Take out the tissue,lay flat on glass slide and examine under fluorescent microscope. Live ova orange-redor reddish-green; dead ova yellow.

3.3.3.

3.

III

I

ndirectndirectndirect

ndirect

fluorescentfluorescentfluorescent

fluorescent

antibodyantibodyantibody

antibody

methodmethodmethod

method

(IFA)(IFA)(IFA)

(IFA)

forforfor

for


diagnosis

ofofof

of

schistosomiasisschistosomiasisschistosomiasis

schistosomiasis

japonicajaponicajaponica

japonica

(1) PrincipleIFA is a method for diagnosis of schistosomiasis japonica with high sensitivity andspecificity. It is also of advantages of easy manipulation and saving reaction materials(such as antibody, antigen etc.). The simplified principle is as follows. The givenantigen in tissue section is fixed on the slide by conventional method and allowed toreact to the examined serum (or suspension with first antibody). When the antibody ispresent, the immunocomplex will form at the specific site where there is correspondingantigen. And the second antibody against first antibody is labeled with fluorescein

22

(usually isothiocynate, 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) MaterialsA. Antigen.

The antigen will be presented in tissue section. S. japonicum adults are removed fromthe liver and portal system of infected rabbit, after washing, fixed with Rossman'ssolution overnight. Then, pick out the parasites and wash them with 75% alcohol. Theparasites can now be made into tissue section by conventional paraffin embeddingmethod or kept in 75% alcohol solution for further use. Tissue sections are 5μm inthickness.

B. First antibody.a. 34B7 monoclonal antibody against S. japonicum eggs.b. Serum from S. japonicum infected rabbits.c. Normal rabbit serum.

C. Second antibody.a. Sheep IgG against rat, labeled with isothiocynate.b. Sheep IgG against rabbit, labeled with isothiocynate.c. Contrast staining solution.10 mgEvan'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) ManipulationA. Circle the tissue section on the slide with a marker pen and drop 10μl 1st antibody or

examined serum onto the tissue within the circle. The slide is then transferred to adamp box at 37℃ and allowed to react for 30 min.

B. Gently wash slide with PBS to remove the 1st antibody and let to air-dry.C. Dilute the second antibody with PBS containing Evan's blue to work solution, with

Evan's blue concentration at 0.01%. Then add 10 μ l diluted second antibody toimmunocomplex on the slide and transfer again to the damp box at 37℃ for 30minutes.

D. Wash the slide with PBS, air-dry.E. Add one 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 slide under a fluorescent microscope. The secondary

23

immunocomplex with second antibody labeled with isothiocynate is present in brightyellow-green color. The occurrence of bright yellow-green color implies the 2ndantibody present in combination with 1st antibody or 1st antibody present in theexamined serum. Dark red color means negative result.Try to analyse the specificity of antigen and antibody according to the difference inintensity and specific fluorescence that you observed.

4. CirculatingCirculatingCirculating

Circulating

antigenantigenantigen

antigen

(CAg)(CAg)(CAg)

(CAg)

detectiondetectiondetection

detection

(1) IntroductionDiagnosis of schistosomiasis in endemic areas depends mainly on microscopicdetection of eggs in stool. The high day-to-day fluctuation in egg excretionnecessitates examining stool samples on consecutive days. Adult schistosomescannot be directly counted in man because they live in inaccessible positions in themesenteric vasculature. Detection of two schistosome adult worm circulatingantigens, circulating anodic antigen (CAA), and circulating cathodic antigen (CCA),that show better performance regarding quantitation of infection intensity and followup after chemotherapy. CAA levels have been found to correlate with S. japonicumworm burden in experimental animals. The circulating soluble egg antigen (CSEA) is15～20 times more concentrated in eggs than in adult worms.

(2) MethodPolyvinylchloride, flat-bottomed, microtitration plates were coated by incubationovernight at 4℃ with 100μl/well of solution containing MAb in 0.035 M PBS, pH7.8. The plates were washed with 2 mM PBS and nonspecific binding sites wereblocked by incubation for one h at 37℃ with 120μl/well of 0.1% (w/v) bovineserum albumin (BSA) in PBS. Plates were washed and incubated for one hr at roomtemperature with 80μ l/well of two-fold serial dilutions of S. japonicum infectedmouse serum, or untreated urine from infected individuals, and an antigen standard.Control wells were incubated with assay buffer. The plates were washed andincubated for 1 h at room temperature with 80μl/well of a solution containing 1.2μg/ml of MAb-FITC. The plates were washed again and incubated for 1 h at roomtemperature with 80μl/well of anti-FITC/AP.Wash plates thoroughly and incubatedovernight at 4℃ with 80μl/well of PNPP in DEA buffer and the absorption at 405nm wasmeasured.

５...

.

CercarialCercarialCercarial

Cercarial

dermatitisdermatitisdermatitis

dermatitis

Take one mice, fix its four legs on a wooden, shave of a piece of area on abdominalskin, place a slide containing 10 live cecariea on the skin, keep wet. Remove the slide10 minutes later, observe the located rash, edema and pruritus caused by penetrationof schistosome cercariae through the skin.

666

6

SchistosomalSchistosomalSchistosomal

Schistosomal

HatchingHatchingHatching

Hatching

TestTestTest

Test

24

If feces containing viable schistosome eggs are diluted with approximately volumesof water, the eggs hatch within a few hours, releasing miracidia. The miracidia arepositively phototrophic. The following procedure takes advantage of this characteristic.1). A stool specimen is homogenized by shaking in normal saline and then strainedthrough two layers of gauze.2). The material is allowed to sediment, the supernatant is decanted and the sedimentresuspended in saline. This process is repeated least twice. I3). The saline is decanted and replaced with distilled water, and is suspension is placed ina flask. The flask is covered to 1 cm below the level of fluid in the neck of the flask.Additional water is added if necessary.4) The flask is allowed to stand at room temperature for seve ral hours insubdued lig ht.5) detect the presence of free-sw imming miracid ia.

The typica l miracid ium is a tiny, ciliated organism. It is long round form.Miracid ium swims cease less ly during its short lif e. Free-sw imming miracid iaare very active, although the miracid ia do not have eyespots, they apparentlyhave photoreceptors, and they are positively phototrophic.

TAPEWORMTAPEWORMTAPEWORM

TAPEWORM

:::

:

Taenia solium, Taenia saginata,

25

Spirometra mansoni & Echinococus granulosus


Objectives

andandand

and


Requirements

1. To study the general morphology and characteristics of life cycle of tapeworm.2. To study the diagnostic methods and morphological comparison between thesetapeworms.

3. To understand the dangerous of taeniasis , cysticercosis, hydatid disease andsparganosis to human being.

4. To study the morphology of hydatid cyst of E. granulosus.


Observation

andandand

and

EEE

E

xperimentxperimentxperiment

xperiment

1.1.1.

1.

TTT

T

aeniaaeniaaenia

aenia

SoliumSoliumSolium

Solium

andandand

and

TaeniaTaeniaTaenia

Taenia

SaginataSaginataSaginata

Saginata

...

.

(1) Adult wormA. 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. Observe the stained specimen of scolex of T. solium (Manipulation).

Spherical, four suckers, with rostellum armed with two rows of hooklets, numbering25～30.

E. Observe the stained specimen of scolex of T. saginata (Manipulation).Quadrate, four suckers, no rostellum or hooklets.What are the differences between the scoleces of these two tapeworms?

E. See the stained specimen of segment of T. solium (Demonstration).F. See the stained specimen of segment T. saginata (Demonstration).

Note each segment has 2 full sets of reproductive organs, one of male and anotherfemale. The testes are follicular, about 100 in number. Ovary with two lobes (T.saginata) or three lobes (T. solium). Uterus club shaped. No digestive tract.

G. Observe the ink-stained specimen of gravid segment of T. solium (Demonstration).Count the number of lateral branches of uterus on one side. 7～13 lateral branches oneach side of uterus unequal in length.

H. Observe the ink-stained specimen of gravid segment of T. saginata (Demonstration).Count and compare the number of lateral branches with that of T. solium. 15～30lateral branches, equal in length.

I. Observe the preserved specimen of tapeworm (Demonstration)Press a gravid segment between two slides, examine and count the number of lateralbranches under lower-lens microscope.

J. See the SEM of T. solium scolex (Demonstration).

26

(2) LarvaA. Study the stained specimen of cysticercus cellulose.

Note the rostellum, hooklets, sucker and bladder.B. See the stained specimen of cysticercus bovis and compare with the scolex of

cysticercus cellulose (Demonstration).C. See the preserved specimen of cysticerci (Demonstration).D. See cysticercus bovis in cardiac muscle of ox (Demonstration).E. See cysticercus cellulose in cardiac muscle of pig (Demonstration).(3) Ovum (Manipulation)

Observe the live specimen of taenia ova. Note the size , color, characteristics ofembryophore and contents of the ova. Nearly spherical, eggshell is easily ruptured,only embryophore is seen.Materials examined: feces or swab of anus.

2.2.2.

2.

SpirometraSpirometraSpirometra

Spirometra

mansonimansonimansoni

mansoni

(1) Adult worm (Demonstration)A. See the preserved specimen of adult worm.B. Study the stained specimen of adult worm.

Note globular testes (over 300～500 in number) in the lateral margins of dorsal side,and two lobulated ovary and overlapping uterus in the mid-portion of the segment.

C. See the stained specimen of scolex.D. See the SEM photograph of S. mansoni scolex.(2) Larva and intermediate hosts (Demonstration)A. See the procercoid.B. Observe the live spargana in frog flesh. Note the spargana lodging in muscles, they are

long whitish worms. The scolex is similar to that of adult, body unsegmented, strongmotility.

C. See the first intermediate host cyclop and second intermediate host frog.(3) Ovum

Studyova of S. mansoni. Note its spindle shape, operculum, thin shell, and a germinalcell with numerous yolk granules.

(4) Dissection of frog for sparganum (Students work in groups).Sacrifice the frog by destroying the spinal cord with a small drill. Fix the frog on aboard ventral side up. Make a midline incision on the abdominal wall. Skin it andsearch the flesh for spargana.

3.3.3.

3.

EchinococusEchinococusEchinococus

Echinococus

granulosusgranulosusgranulosus

granulosus

(4) See the stained specimen of adult worm (Demonstration).Note the four segments (usually) in an adult worm and the difference in maturity, andthe characteristics of scolex.

27

(5) See the preservedhydatid cyst in liver (Demonstration).(6) Study the scoleces of hydatid cyst (Demonstration).

Note the ovoidal scolex armed with suckers and hooklets.(4) Study the section of hydatid cyst (Demonstration).

Note the outermost layer, false cystic wall, resulting from reaction of host, theexternal layer of true cystic wall is noncellular hyaline cuticular and inner layer is thegerminal layer, from which the brood capsules, and daughter cysts and detachedgerminal fragments are formed.


Exercise

1. Draw taenia ova and gravid segment.2. Label the scolices of T. solium and T. saginata.3. Record the result of dissecting frog for sparganum .4.


Thinking

1.To compare T. solium and T. saginata based on their morphology, life cycle,pathogenesis and epidemiology (epidemiolog ic factors and epidemiolog ic distribution).

2. Why the curative effect examination should be taken for treating tapeworm diseases?What is the curative basis?

EXAMINATIONEXAMINATIONEXAMINATION

EXAMINATION

OFOFOF

OF

ALIMENTARYALIMENTARYALIMENTARY

ALIMENTARY

28

HELMINTHSHELMINTHSHELMINTHS

HELMINTHS


Objectives

andandand

and


Requirements

1 To learn methods commonly used in stool examination.2. Self-stool examination for helminth eggs and therapy.

MeMeMe

Me

ttt

t

hodshodshods

hods

111

1

．DirectDirectDirect

Direct

fecalfecalfecal

fecal

smear.smear.smear.

smear.

Transfer a small amount of fecal sample to 2 drops of saline on a slide. Mix to obtain afairly dense uniform smear free of large lumps. Some practice is necessary to judge thedensity, that is: one can see the strokes of words in the newspaper through thetransparent smear. Identify parasite ova and distinguish them from food residues suchas various kinds of plant cells, yeast, pollen, plant fiber etc. The fecal smear must bekept wet during examination.

222

2

．NaClNaClNaCl

NaCl

saturatedsaturatedsaturated

saturated

solutionsolutionsolution

solution

floatation.floatation.floatation.

floatation.

Mix small lumps of fecal material (about peanut-sized) with small amount of NaClsaturated solution in a penicillin bottle. Then add up to the rim of bottle, cover with acoverslip and allow to contact with it (avoid any air bubble). After 20 minutes, examinethe coverslip on a slide for ova.

333

3

． Sedimentation.Sedimentation.Sedimentation.

Sedimentation.

Mix 5 g～10 g of fecal material with small amount of water in a beaker (or test tube).Dilute with water and filter away the bulky undigested residues. Let the filtrate standfor 15～20 minutes. Discard the supernatant and add water again, thus repeat severaltimes until the supernatant is clear. Decant the supernatant fluid and examine sedimentfor ova.


Exercise

1. Record the result of self-stool examination.2. Try to compare the advantages and disadvantages of the three methods used in the

practice.


Reference

1.1.1.

1.

QualityQualityQuality

Quality

controlcontrolcontrol

control

forforfor

for

fecalfecalfecal

fecal

examination.examination.examination.

examination.

Reliable and accurate parasite identif ication depends on:(1) Collecting satisfactory specimens.(2) Preparing and maintaining reagent correctly.

29

(3) Careful performance of appropriate techniques and thorough examination of finishedpreparation.

ENTAMOEBAENTAMOEBAENTAMOEBA

ENTAMOEBA

HISTOLYTICAHISTOLYTICAHISTOLYTICA

HISTOLYTICA

,,,

,

E.E.E.

E.

COLCOLCOL

COL

III

I

&&&

&

OTHEROTHEROTHER

OTHER

AMOEBAEAMOEBAEAMOEBAE

AMOEBAE


Objectives

andandand

and


Requirements

1. To study morphological structures of trophozoite and cyst of E. histolytica.2. To study laboratory diagnostic methods of E. histolytica.3. To understand the pathogenesis of E. histolytica.4. To learn differentiating E. hisiolytica from E. coli.


Observation

andandand

and


Experiment

1.1.1.

1.

E.E.E.

E.

histolyticahistolyticahistolytica

histolytica

(1) TrophozoiteA. Study the iron-haematoxylin stained specimen of E. histolytica (Manipulation).

Find the parasite under high power, then observe with oil immersion lens. Noteectoplasm and pseudopodia, endoplasm with fine granules. Some of the trophozoitesmay contain RBCs which appearance varies with process of digestion. Food vacuolescan also be found in the endoplasma. The spherical nucleus has definite nuclearmembrane, the inner surface of which is lined with uniform and closely packedchromatin (peripheral chromatin). The central karyosome is deeply stained.

B. Study the living trophozoites in fecal material (Manipulation).With a toothpick, remove some material from mucus or flecks of blood in patient'sstool (or from culture medium), mix with saline on a slide, cover and examine. It isadvisable to use rather weak illumination. Observe with care the appearance, motilityand the cytoplasmic inclusion, e. g. RBC (if specimen obtained from patient) or starch(if specimen obtained from culture). The fecal samples must be examined immediatelyafter collection or be kept at optimal temperature so as to maintain the motility of thetrophozoite; it is particularly so during winter.

(2) CystA. Study the iron-haematoxylin stained specimen of E. histolytica cysts (Manipulation) .Using oil immersion lens, study the spherical cyst with a hyaline cystic wall, thenumber of nucleus may be single, two or quadrate. The structure of nucleus is the

30

same as trophozoite. The glycogen is readily dissolved in stained specimen and onlyvacuole is lef t. Chromatoid bodies are rod-like masses with round ends. In fullymature cysts, the chromatoid bodies are often lacking.

B. Study the iodine stained specimen of cysts (Manipulation).Stain fresh fecal material by adding a drop of iodine solution to the edge of coverslip.The cysts stain yellowish, the glycogen is brownish yellow, the nuclei are hyalineunstained sphere-like.

(3) Cultivation of trophozoite (Students work in groups).With sterilized technique, add 0.5 ml of inactivated serum and a small sprinkling ofrice starch and a few drops of penicillin solution to the medium, inoculate the fecalsample containing trophozoites (about 0.1 ml). Incubate at 30℃ for one week andexamine.

(4) Pathologic specimens (Demonstration).A. See the gross lesions in the intestine.

Note the ulcers vary from pin point to the size of rice grain, with central erosionsurrounded by elevated edematous tissues forming a niche of a volcano. Superficialulcers are formed due to detachment of necrotic mucosa, or large ulcerated patchesdue to coalescence of small ulcers. Mucosa between ulcers is usually normal.

B. See the section of intestinal lesions.Note flask-shaped ulcer, numerous trophozoites and infiltration of leukocytes can beseen.

C. See the amebic hepatic abscess.Abscess of various size can usually be seen in the right lobe, singly and discrete. Theabscesses are surrounded by necrotic tissue, thus the edge of the abscess appearsirregular and spongy, while the abscess fluid is brown in color.

(5) See the photographs of trophozoite and cyst.2.2.2.

2.

E.E.E.

E.

colicolicoli

coli

(1) See the iron haematoxylin stained E. coli trophozoites (Demonstration).Compare the size of trophozoite, the nuclear chromatin, position of karyosome andendoplasmic inclusions with those of E. histolytica.

(2) Study the iron haematoxylin stained specimen of E. coli cysts (Manipulation).Note and compare the size, number of nuclei, nuclear structure and chromatoid bodieswith those of E. histolyitca cyst.

3.3.3.

3.

OtherOtherOther

Other

non-pathogenicnon-pathogenicnon-pathogenic

non-pathogenic

amoebaeamoebaeamoebae

amoebae

ininin

in

humanhumanhuman

human

alimentaryalimentaryalimentary

alimentary

tracttracttract

tract

(1) See the E. gingivalis trophozoites (Demonstration).Note the distinct ectoplasm and central or slightly eccentric large karyosome.

(2) See the live Endolimax nana trophozoites (Demonstration).Note the small size, sluggish motility, blunt pseudopodia large central or eccentric

31

karyosome, peripheral chromatin seldom seen; See demonstration of E. nana cyst.Note small size, oval shape, l～ 4 nuclei, karyosome large and irregular withoutperipheral chromatin, usually no chromatoid bodies.

(3) See the live Iodamoeba butschlii trophozoites (Demonstration).Note the small size, spherical shape, sluggish motility, blunt pseudopodia, largecentral karyosome surrounded by refractive granules, usually no peripheral chromatin.Food vacuole in endoplasm contains bacteria.

(4) See the I. butschlii cysts (Demonstration).Note the irregular or spherical shape, one nucleus, large eccentric karyosome withperipheral chromatin, no chromatoid bodies. The most conspicuous feature is thelarge glycogen vacuole, stains golden brownwith iodine.

(5) See the E. polecki cystsThis is an incidental parasite of man. Note spherical shape, one nucleus, central oreccentric karyosome, dense uniform peripheral chromatin, rod-like chromatoidbodies.


Exercise

1. Draw the trophozoite and cyst of E. histolytica and cyst of E. coli.


Thinking

1. How does amebic dysentery be transmitted?2. How to diagnose the intestinal lesion and the exo-intestinal lesion of E. histolytica?

GIARDIAGIARDIAGIARDIA

GIARDIA

LAMBLIALAMBLIALAMBLIA

LAMBLIA

32


Objectives

andandand

and


Requirements

1. To study the general structure of G. lamblia trophozoite and cyst.2. To learn the diagnostic method.


Observation

andandand

and


Experiment

1. Study the iron haematoxylin stained trophozoites (Manipulation) .Note inverted pear-shape, convex dorsal surface and concave ventral surface (suckingdisc). Two nuclei with large karyosome at the anterior, two axostyles and four pairs offlagella.

2. Study the iron haematoxylin stained cysts (Manipulation) .Note shape, well defined wall and two to four 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 infectedG. lambliamay suffer diarrhea?

TRICHOMONASTRICHOMONASTRICHOMONAS

TRICHOMONAS

VAGINALISVAGINALISVAGINALIS

VAGINALIS

33


Objectives

andandand

and


Requirements

1. To study the general structure of T. vaginalis trophozoite and cyst.2. To learn the diagnostic method.


Observation

andandand

and


Experiment

1.1.1.

1.

StudyStudyStudy

Study

thethethe

the


stained

trophozoitestrophozoitestrophozoites

trophozoites

ofofof

of

T.T.T.

T.

vaginalisvaginalisvaginalis

vaginalis

(Manipulation)Broad end of the body, 4 flagella, a posterior flagellum, a short flagellum, a nucleusnear the origin of the anterior position, axoneme.

2.2.2.

2.

SeeSeeSee

See

thethethe

the

livelivelive

live


trophozoites

ofofof

of

T.T.T.

T.


vaginalis

(Demonstration)3. ExaminationExaminationExamination

Examination

ofofof

of


trophozoites

ofofof

of

T.T.T.

T.


vaginalis

Direct wet film, using vagina excretory.


Exercise

l. Draw the trophozoite of T. vaginalis.


Thinking

1.What are the transmission routes of T. vaginalis?


Reference

1.1.1.

1.

DirectDirectDirect

Direct


examination

ofofof

of

vaginalvaginalvaginal

vaginal

andandand

and

urethralurethralurethral

urethral

smearssmearssmears

smears

(1) With a sterile cotton swab, collect the vaginal or urethral discharge.(1) Put the swab immediately into a sterile tube containing about 3 ml of sterile saline.(3) Smears on a slide, and smear for staining can be made if desired. Finding of T.

vaginalis in vaginal secretions, prostatic secretions and urine.2.2.2.

2.

CentrifugedCentrifugedCentrifuged

Centrifuged

ororor

or

sedimentsedimentsediment

sediment

materialmaterialmaterial

material

(1) If a swab in saline is received, remove the excess fluid from the swab by squeezing itagainst the side of the tube.

(2) Centrifuge the 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) With a pipette remove the supernatant fluid.(4) Take a drop of the sediment and put on a microscope slide.(5) Cover with a coverslip and examine with 10 × and 40× objectives for motile

flagellates.

LEISHMANIALEISHMANIALEISHMANIA

LEISHMANIA

DONOVANDONOVANDONOVAN

DONOVAN

III

I

34


Objectives

andandand

and


Requirements

1. To study morphological structures of amastigotes and promastigotes of L. donovani.2. To understand methods used in the diagnosis.3. To understand the pathogenesis of L. donovani from parasitized site.


Observation

andandand

and


Experiment

1.1.1.

1.

StudyStudyStudy

Study

thethethe

the


stained

smearsmearsmear

smear

ofofof

of

amastigotesamastigotesamastigotes

amastigotes

(or(or(or

(or

non-flagellarnon-flagellarnon-flagellar

non-flagellar

forms)forms)forms)

forms)

(Manipulation)Look for reticulo-endothellial cells harboring the amastigotes. Note the number ofamastigotes in one host cell. They are very small and have the appearance of granulesin the cytoplasm of macrophages. When the host cells are ruptured, amastigotes arediscrete. Study carefully under microscope with oil immersion lens, note the discreteamastigotes, its size in respect to the size of host cell. Identify the blue cytoplasm, rednucleus, basal body and kinetoplast.

2.2.2.

2.

SeeSeeSee

See

thethethe

the


stained


specimen

ofofof

of

propropro

pro

mastigotemastigotemastigote

mastigote

(or(or(or

(or

flagellarflagellarflagellar

flagellar

from)from)from)

from)

(Demonstration)Note the spindle shape, flagellum, nucleus, basal body, rhizoplast and kinetoplast, thelast three can hardly be separated from each other and often seen as one purple dot.

3.3.3.

3.

SeeSeeSee

See

thethethe

the

livelivelive

live

propropro

pro


mastigote

(Demonstration)Place a drop of culture medium on a slide. Cover and note the rapid, lashing movementof the flagellum that actively propels the parasite forward.

4. SeeSeeSee

See

thethethe

the

SEMSEMSEM

SEM

photographsphotographsphotographs

photographs

ofofof

of

propropro

pro


mastigote

andandand

and

amastigoteamastigoteamastigote

amastigote


5.

SeeSeeSee

See

adultadultadult

adult

sandfly,sandfly,sandfly,

sandfly,

thethethe

the

vectorvectorvector

vector

ofofof

of

L.L.L.

L.

DonovaniDonovaniDonovani

Donovani

(Demonstration)


Exercise

1. Draw the macrophage contains amastigotes, and a free promastigote of L. donovani.


Thinking

1. A person suffered from visceral leishmaniasis, how to diagnose it pathogenically?


Reference

1.1.1.

1.

CultureCultureCulture

Culture

ofofof

of

L.L.L.

L.

donovanidonovanidonovani

donovani

Inoculation of NNN culture medium with spleen juice, blood, or bits of excised dermisis a reliable procedure. Here we particularly recommends seeding 3 or 4 NNN culturetubes with the sediment obtained by centrifuging 2 ml～5 ml of blood added to fourtimes its volume of citrated saline. The tubes are incubated at 22℃～ 24℃ , andflagellates appear in 7 days or later in 90 per cent of untreated cases.

(1) Materials.

35

Difco blood agar base 8 gDistilled water 200 mlDefibrinated rabbit blood 0.6 ml in each 5 ml of medium

(2) Preparation of defibrinated rabbit blood.Collect 20 ml of rabbit blood into a sterile flask containing about 100 glass beads of 4mm diameter. Defibrinate the blood by rotating the flask for 5 minutes. Add 200 u ofpenicillin, 200 mg of gentamicin, and 2 mg of streptomycin per ml of defibrinatedblood.

(3) Preparation.A. Pour 200 ml of water into a flask, add the agar to the water, mix, and warm the flask in

boiling water until the agar is completely dissolved.B. Dispense the medium in 5 ml amounts into screw-cap bottle (20 ml capacity). Sterilize

by autoclaving (with caps loosened) at 121℃ for 15 minutes and allow the agar tocool to 45℃～50℃.

C. Add 0.6 ml of sterile defibrinated rabbit blood to each bottle and mix gently. Allow themedium to solidify with the bottles in a sloped position.

D. Leave the bottles in an upright position at room temperature for 24 h to allow fluid ofcondensation to form. The bottles should be stored at 4℃～6℃ until required.Note: The medium should be prepared in aseptic working conditions.

(4) Use.A. Inoculate about 0.1 ml of specimen aseptically into the fluid of condensation of each

of 2 bottles at room temperature.B. Incubate the cultures at 24℃(±2℃) in the dark.C. Examine every 4 days.Transfer a drop of the culture using a sterile wire loop to a slide

for examination for promastigotes.Note: Negative cultures must be subcultured after 8 days into fresh medium andexamined every 4 days for a further 20 days.

2.2.2.

2.

DetectionDetectionDetection

Detection

ofofof

of

LeishmaniaLeishmaniaLeishmania

Leishmania

ininin

in

lymphlymphlymph

lymph

nodenodenode

node

aspiratesaspiratesaspirates

aspirates

Parasites may be demonstrated in aspirates of spleen (98% positivity), bone marrow(54%～86%), or enlarged lymph nodes (64%). When the spleen is small and soft orimpalpable, bone-marrow or lymph-node aspiration is recommended. For diagnosis ofLeishmania it is especially using bone-marrow or lymph-node aspiration.

(1) Prepare the syringe; pull the piston as far back as possible.(2) Ask the person to s it down. Disinfect the chosen site on the neck with 70% ethanol.(3) With left hand, take the gland between the thumb and index finger and make it stand

out.(4) Hold ing the needle between thumb and finger, introduce it at right angles into the

center of the gland. First pierce the skin, then penetrate the center of the gland.

36

(5) With left hand, gently knead the gland. With right hand, revolve the needle in bothdirections.

(6) The glandular fluid will ooze into the needle. The operation should last about oneminute.

(7) Withdraw the needle in one rapid movement, holding index finger over the hub. Applya swab dipped in disinfectant to the point of entry.

(8) Attach the needle to the syringe, with the piston pulled back. Push the piston gentlyhalf waydown the barrel to discharge the glandular fluid in the needle on to the slide.

(9) Cover the preparation with a coverslip. Examine at once under the microscope at amagnification of approximately 400×, using the 40× objective.

(10) Wait until the convection currents stop. Then examine the preparation.

PLASMODIAPLASMODIAPLASMODIA

PLASMODIA

37


Objectives

andandand

and

RequiremenRequiremenRequiremen

Requiremen

ttt

t

sss

s

1.To study the life cycle of Plasmodia and understand their pathogenic mechanism.2. To study laboratory diagnostic methods of malarial parasites.3. To study morphological structures of Plasmodia , to identify morphological structuresof developing stages of erythrocytic schizogony and gametocytes of P. vivax, and todifferentiate the ring-form and gametocytes of P. vivax from P. falciparum.

4. To identify of insect vector of malaria—Anopheles.5. To learn simple methodof experiment with Plasmodium.


Observation

andandand

and


Experiment

1.1.1.

1.

P .P .P .

P .

vivaxvivaxvivax

vivax

(Erythrocytic(Erythrocytic(Erythrocytic

(Erythrocytic

stage)stage)stage)

stage)

(Manipulation)(1) Ring form

Compare its size in respect to the infected RBC. Note the delicate blue-stained ring ofcytoplasm and a red chromatin dot.

(2) TrophozoiteCompare the small and large trophozoites, do they have definite form? Note thevacuole, changes of chromatin, pigment, alteration of infected RBC and Schuffner'sdots. The cytoplasm with the nucleus begins to enlarge while the ring form becomesirregular in shape and sometimes projects pseudopodia.

(3) SchizontNote the number and arrangement of chromatin, distribution of cytoplasm andaggregation of pigment granules. It becomes oval or round body, and its nucleusbegins to segment. The number of the nucleus in the mature schizont is from 12～24,usually 16.

(4) GametocytesNote the size, shape, position of chromatin, distribution of pigment granules andalteration of infected RBC. What are the morphological differences between male andfemale gametocytes?

222

2

...

.

P .P .P .

P .

falciparumfalciparumfalciparum

falciparum


(Erythrocytic

stage)stage)stage)

stage)

(Manipulation)(1) Ring form

Note its small size and delicate ring, about 1/5 the diameter of erythrocyte. Sometimesthere are twochromatin dots in one ring form or multiple infection in one RBC.

(3) GametocytesNote their particular sausage or crescent shape, position of chromatin, distribution ofpigment granules and alteration of infected RBC.

3.3.3.

3.

P .P .P .

P .

malariaemalariaemalariae

malariae


(Erythrocytic

stage)stage)stage)

stage)

(Demonstration)

38

(l) See the ring form.Note the large chromatin dot, dense cytoplasm and its size in respect to infectedRBC.

(2) See the trophozoite.Note the band form, its cytoplasm compact, pigment coarse and early appearance.

(4) See the schizont.Note mature schizont, the merozoites 6～12 in number, arranged as "rosette" centralmass of dark brown pigment granules.

(5) See the gametocytes.Resemble those of P. vivax, except smaller in size, coarse dark pigment granules,alteration of infected RBC rare.

4.4.4.

4.

SeeSeeSee

See

thethethe

the

SEMSEMSEM

SEM


photographs

ofofof

of

P .P .P .

P .

vivaxvivaxvivax

vivax

andandand

and

P .P .P .

P .


falciparum

5.5.5.

5.

Exo-erythrocyticExo-erythrocyticExo-erythrocytic

Exo-erythrocytic

stagestagestage

stage


6.

DevelopmentDevelopmentDevelopment

Development

ofofof

of

PlasmodiaPlasmodiaPlasmodia

Plasmodia

ininin

in

mosquitomosquitomosquito

mosquito

hosthosthost

host

(Demonstration)(1) See the oocyst in the intestinal wall of mosquito.(2) See the sporozoites in the salivary gland smear of infected mosquito.7.7.7.

7.

InoculationInoculationInoculation

Inoculation

ofofof

of

P .P .P .

P .

bergheibergheiberghei

berghei

(Students work in groups)Obtain blood from infected mouse by cardiac puncture (or orbital puncture), dilute in1 ml saline inject 0.1 ml diluted blood to a mouse intraperitoneally. Rear the mouseand examine next week.

8.8.8.

8.

PreparationPreparationPreparation

Preparation

ofofof

of

bloodbloodblood

blood

filmfilmfilm

film

ofofof

of

malarialmalarialmalarial

malarial

parasitesparasitesparasites

parasites

andandand

and

stainingstainingstaining

staining

(Manipulation)(l) Preparation of both thin and thick blood films at one slide. Collect two drops of blood

from the cut tail of infected mouse and place on one end of a clean slide. Spread thedrop near end to the size of two-fen coin with the corner of another slide to make upthick film. Hold ing another slide at an angle of 30°～45° , and in contact with theother drop of blood, the first touch the drop of blood and let it spread along the line ofcontact between the slides. Then push the slide along, with a smooth, rapid movement,thus drawing the blood out to form a thin film. Then let it dry. Its thickness will dependon the size of the drop, the angle between the slides and rapidity with which the smearis made.

(2) Giemsa's staining. Fix the dried thin film with methyl alcohol (avoid fixing the thickfilm) and let it dry. Cover with 2% Giemsa's solution on the smears. Let stay 30~60minutes.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.

9.9.9.

9.

VectorVectorVector

Vector

ofofof

of

malarialmalarialmalarial

malarial

parasitesparasitesparasites

parasites

———

—

AnophelesAnophelesAnopheles

Anopheles

(Demonstration)(1)An. sinensis(2)An.Minimus(3)An. dirus10.10.10.

10.

SeeSeeSee

See

thethethe

the

SEMSEMSEM

SEM


photographs

ofofof

of

merozoitemerozoitemerozoite

merozoite

(Demonstration)

39


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 cycles of Plasmodium, explain the paroxysm, recrudescence andrelapse.

2. What are the epidemic characteristics of malaria?


Reference

1.1.1.

1.

AcridineAcridineAcridine

Acridine

orangeorangeorange

orange


fluorescent

stainstainstain

stain

Fix the thin smear with methyl alcohol, then immerse it in 0.01% acridine orangesolution for 4 minutes, rinse with water, then immerse in distilled water for one minute.Add one drop of water and cover it, examine under fluorescent microscope. DNA ofchromatin shows yellowish green fluorescence, while RNA of cytoplasm orange-yellow fluorescence.

2.2.2.

2.

IndirectIndirectIndirect

Indirect


fluorescent

antibodyantibodyantibody

antibody

testtesttest

test

(l) Antibody: rabbit-anti-human IgG fluorescent antibody to be diluted to 1:8 (or 1:16)with PBS, pH8.0.

(2) Antigen sample: dry blood films made with blood collected right after clinical attacks.Hemolyse the slide in 0.1N HCl for 5 minutes, wash in running water, immerse in PBSfor 5 minutes, then air dry.

(3) Serum for examination: serum sample to be diluted with PBS to 1:20.(4) Staining.A. Spreadone drop of diluted serum sample on the antigen blood film , put in a moistened

box and transfer to an incubator at 37℃ for 30 minutes to allow antigen-antibodyreaction.

B. Wash the blood film with PBS for 1 minute, immerse in PBS for 5 minutes, repeatonce, then air dry.

C. Add one drop of antibody containing Evans blue (in dilution 1:10 000) on the bloodfilm whereAg-Ab reaction took place, put the slide in a moistened box into incubatorat 37℃ for 30 minutes.

D. Wash away the excessive fluorescent antibody as step B.(6) Mount the dried blood film with carbonate or phosphate buffered glycerin, or just add

a small drop of PBS (pH8.0) onto it and cover. Examine under fluorescent microscope.

40

(7) Criter ia of diagnosis.Grade the result according to the fluorescence density on the schizont or trophozoite.+++ — ++++: fluorescence on plasmodial cytoplasm is bright to brilliant, structuresclear.++: fluorescence bright, structures clear.+: cytoplasm clearly visible but structures not very clear.±: only faint fluorescence visible on the plasmodial cytoplasm and RBC shadow,parasite structures not clear.-: no fluorescence visible on plasmodial cytoplasm.All sample graded higher than “+” are considered positive.

3.3.3.

3.

ParaSight-FParaSight-FParaSight-F

ParaSight-F

rapidrapidrapid

rapid

manualmanualmanual

manual

diagnosticdiagnosticdiagnostic

diagnostic

testtesttest

test

ofofof

of

P .P .P .

P .


falciparum

(1) IntroductionCurrent diagnostic methods are based on microscopic examination which takes 5～10minutes for staining and up to 20 minutes for reading, and requires a well-maintainedmicroscope and an experienced microscopist. New techniques, such as hybridizationwith DNA probes, are too sophisticated for routine use in the field.

(2) Test principalParaSight-F test is a qualitative diagnostic test of P. falciparum, which is based on thedetection by monoclonal antibody of a species-specific soluble antigen histidine-richprotein (HRP-Ⅱ), a special glycoprotein of P. falciparum secreted during the parasite'serythrocytic cycle, with a peak during schizont rupture in whole blood and which canbe performed without special equipment. A visual reading is given by a polyclonalantibody coupled with dye-loaded liposomes; when positive, a pink line appears.

4.4.4.

4.

AAA

A

rapidrapidrapid

rapid

dipstickdipstickdipstick

dipstick

antigenantigenantigen

antigen

capturecapturecapture

capture

assayassayassay

assay

forforfor

for

thethethe

the


diagnosis

ofofof

of

P .P .P .

P .


falciparum

(1) PrincipalRecent advances in the diagnosis of P. falciparum infection have made it possible toconsider supplementing light microscopy with a standardized dipstick antigen captureassay based on the detection of HRP-Ⅱ . The stability, reproducibility, and ease of useof the assay clearly indicate that it has potential for application in the management ofmalaria, particularly at the peripheral health care level, provided its accuracy can beassured and that it can be made affordable. A specific assay for P. falciparum is nowcommercially available and a similar assay for P.vivax is under development.

(2) StepsA. Add lysing fluid to tube.B. Take blood sample from fingerprick.C. Mix blood with anticoagulant in capillary tube.D. Transfer blood sample to lysing fluid.E. Place dispensing tip on sample tube.

41

F. Place lysed blood sample in well.G. Stand dipstick in blood sample.H. Add detection agent to well.I. Add washing fluid to well.J. In positive cases, a pink line develops almost simultaneously at the monoclonal

antibody deposit site with a pink broken line above it as the reagent control. Innegative cases, only the pink broken line occurs.

5.5.5.

5.

ICTICTICT

ICT

MalariaMalariaMalaria

Malaria

P.fP.fP.f

P.f

///

/

P.vP.vP.v

P.v

testtesttest

test

(1) IntroductionMalaria diagnosis has been a difficult process requiring the skills of highly trainedmicroscopists to detect Plasmodium in stained blood films. In addition to detection ofparasites, the ability to differentiate Plasmodium falciparum from other malarialspecies is important for the early and effective treatment of infection with thispotentially lethal organism. The introduction of the ICT Malaria P.f / P.v test combinesrapid and reliable diagnosis of malaria with the ability to differentiate P. falciparumand P. vivax infections.

(2) Test principleThe ICT Malaria P.f / P.v test is an in-vitro immunodignostic test for the detectionand speciation of P. falciparum and P. vivax in whole blood. Based onimmunochromatographic technology, this test uses antibodies to identify specificantigens associated with P. falciparum and P. vivax infections. A finger prick bloodsample is all that is required for the step procedure which gives results in eightminutes. Malarial antigens are detected using colloidal gold labeled antibodies. In apositive test, pink lines develop in the test window. The location of these linesidentif ies the Plasmodium species detected, while development of a proceduralcontrol line indicates that the test has been performed correctly.

6.6.6.

6.

QualityQualityQuality

Quality

controlcontrolcontrol

control

forforfor

for

bloodbloodblood

blood


examination

(1) Equipment must be clean.(2) Films must be of correct density.(3) Films must be allowed to dry in a horizontal position and for the correct time to

ensure good results.(4) The stain dilutions and the buffered water used for staining must be accurately

prepared and the stock stain must be of good quality.(5) The staining procedure should be followed very carefully.

42

OOO

O

PPORTUNISTICPPORTUNISTICPPORTUNISTIC

PPORTUNISTIC

PATHOGENPATHOGENPATHOGEN

PATHOGEN

ICICIC

IC

PROTOZOAPROTOZOAPROTOZOA

PROTOZOA

NNN

N

PneumocytisPneumocytisPneumocytis

Pneumocytis

cariniicariniicarinii

carinii

,,,

,

ToxoplasmaToxoplasmaToxoplasma

Toxoplasma

gondiigondiigondii

gondii

&&&

&

CryptosporidiumCryptosporidiumCryptosporidium

Cryptosporidium


Objectives

andandand

and


Requirements

1. To obtain a primary understanding of the relation between the immunity of infectedhost and opportunistic pathogenic protozoan.

2. To observe the structural features of several species of opportunistic pathogenicprotozoan.


Observation

ananan

an

ddd

d


Experiment

1.1.1.

1.

PneumocytisPneumocytisPneumocytis

Pneumocytis

cariniicariniicarinii

carinii

(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 and the number of sporozoites within it.(2) Animal experiment (Students work in groups)A. Introduction

The establishment of parasitic infection implies that the parasites come together withtheir hosts and the interaction begins. As soon as the parasites enter the host, firstthing is that they have to struggle against the host immunity before they multiplyenough to injure the host and usher in clinical symptoms. In some cases, for examplein the opportunistic pathogenic protozoan, the outcome of the interaction is usuallyestablishment of the balance between the parasites and their host. That is the certainnumbers of the parasites survive within the host but none of the related clinicalsymptoms are observed, which is called suppressive infection. However, the balancerequires existence of certain conditions of both host and the parasites. Any change ofthe related conditions would result in the appearance of imbalance. For instance, thedecrease of the host immunity might lead to dramatic multiplication of the protozoanin the state of suppressive infection, and consequently the occurrence of acute attack.The protozoan of these characteristics, including P. carinii, T. gondii and species ofCryptosporidium, are called opportunistic pathogenic protozoan. This is why the P.carinii infection is fatal to the AIDS patients.In this experiment, the immunity of observed animals are artificially suppressed by

43

administration of immuno-suppressed drugs before they are infected with P. carinii ,and the observations are carried out on the changes in the body weight, physiologicalstate, pulmonary pathological change of the hosts, as well as examination forpathogens in the hosts.

B. Materials① Experimental anima l: white rat.② Homogenate of infected lung tissue with P.carinii.③ Immuno-suppresseddrugs: dexamethasone, cortisone acetate.④ Nourishment: low protein diet; drinking water with tetracycline 1 mg/ml.C. Methods① Experimental Group. Six white rats are used in the experimental group. The animals

are orally administrated with l mg/L dexamethasone in drinking water orintradermally injected with cortisone 12.5 mg/100g body weight two times a week forsix weeks. Two weeks after drug administration, three of them are transtracheallyinoculated with 0.15 ml homogenate of infected lung tissue with P. carinii. At sixthweek, the animals are stopped feeding of immuno-suppresseddrug and one or two outof both transtracheally inoculated infected and the other non-infected subgroups aredissected respectively for observations on the pulmonary pathological changes andexamination 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 same observation asconducted at the sixth week. Each of the animals treated with immuno-suppresseddrug is muscularly inoculated once a week with PG 40 000 u and streptomycin 20 000u.

② Control Group. Three white rats are used in the control group. They are free from theimmuno-suppressed drug and twoof them are transtracheally inoculated with 0.15 mlhomogenate of infected lung tissue with P. carinii. All three animals are thendissected at the sixth week for the same observations as experimental group.

③ Stop immuno-suppressed drug at the 6th week, dissect one or two rats out of bothexperiment and control groups, observe the pathological changes in the lungs andsmear on slide for examination of parasites under microscope.

④ The remainders are left for two weeks more before they are dissected and observedas described in step C.

⑤ Both experimental and control groups of animals are weighed and observed forphysiological state once a week. The results of weighing and observation arerecorded.

⑥ The route of the experiment is outlined as the following graph.

Experimental group Control group

44

2 weeks normal diet 2 weeks normal diet+cortisone

transtracheal transtrachealinoculation inoculation normalof P.carinii of P.carinii diet

4 weeks 4 weeks 4 weeks 4 weeks

dissect stop stop dissect dissect dissectdrugdrug

2 weeks 2 weeksdissect dissect

2.2.2.

2.

CryptosporidiumCryptosporidiumCryptosporidium

Cryptosporidium

(l) See the stained oocysts (Demonstration).Note its size, shape and number of sporozoites within it.

3.3.3.

3.

ToxoplasmaToxoplasmaToxoplasma

Toxoplasma

gondiigondiigondii

gondii

(l) See the stained tachyzoites and pseudocysts (Demonstration).Note its size, shape, and the color of both cytoplasma and nucleus. Pseudocyst isfactually a macrophage containing tachyzoites with the number from several to tens.

(2) See the stained cysts (Demonstration) .Note its size, shape, cystic wall and the number of bradyzoites within it.


Exercise

1. Based on the recorded results, to write the experimental report on the relationshipbetween opportunistic pathogenic protozoan infection and the immunity of the host,with analysis, discussion and conclusion.


Reference

1.1.1.

1.

TheTheThe

The

pathogenicpathogenicpathogenic

pathogenic


examination

ofofof

of

T.T.T.

T.

gondiigondiigondii

gondii

andandand

and

animalanimalanimal

animal

inoculationinoculationinoculation

inoculation

(1) Liquid material: Blood, using blood film. Cerebrospinal fluid (CSF) or ascites,examining the sediment after centrifugation. The live trophozoites are crescentic inshape, move actively.

(2) Solid material: Examined by cutting section pathologically. If negative, using animalinoculation. Method: Grinding tissue to become paste-like, add saline to dilute it,inject into mouse abdomen, tap the abdomen 7 days later and examine ascites fortrophozoites.

45

MOSQUITOESMOSQUITOESMOSQUITOES

MOSQUITOES


Objectives

andandand

and


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 andAedes.


Observation

andandand

and


Experiment

1.1.1.

1.

SeeSeeSee

See

threethreethree

three

ggg

g

eee

e

neraneranera

nera

ofofof

of

livelivelive

live

adultadultadult

adult

mosquitoesmosquitoesmosquitoes

mosquitoes

(Demonstration)(1)Anopheles(2) Culex(3)AedesNote the differences in color, wing spots, position of head at rest, abdominal segmentscovered with scales of various colors.

2. StudyStudyStudy

Study

thethethe

the

desiccateddesiccateddesiccated

desiccated

sss

s

pepepe

pe

cicici

ci

mensmensmens

mens

ofofof

of

adultsadultsadults

adults

ofofof

of

threethreethree

three

generageneragenera

genera

ofofof

of


mosquitoes

(Demonstration)Identify organs and appendages of head, thorax and abdomen. A pair of compoundeyes, antennae, maxillary palps and a proboscis (mouth parts) of head; the thoraxbearing 3 pairs of legs and one pair of wings; 10 segmented abdomen (the last twosegments are modified to male genitalia).

3.3.3.

3.

StructureStructureStructure

Structure

ofofof

of


mosquito

headsheadsheads

heads

(1) Studywhole mount of head ofAnopheles (Manipulation) .Identify compound eyes, 15 jointed antennae with intersegmental hairs. Themouthpart consists of a tubular labium terminating with two tiny labe lla, one labiumand one hypopharynx, one pair of mandibles and one pair of maxillae with all these toforma cannula during blood sucking.Differentiate the male and female Anopheles. Note the head for the plumose antennalhairs of males and pilose hairs of females. Compare the length of antenna in respect tothat of proboscis of both sexes.

(2) SeeCulex head, both male and female, and compare (Demonstration).4.4.4.

4.

SeeSeeSee

See

thethethe

the

eee

e

gggggg

gg

ofofof

of

AnophelesAnophelesAnopheles

Anopheles

,,,

,

CulexCulexCulex

Culex

andandand

and

AedesAedesAedes

Aedes

(Demonstration)Which of them is floating on water in form of raft or scattered, with or without floats?Note the shape and size.

5.5.5.

5.

SeeSeeSee

See

thethethe

the

livelivelive

live

larvaelarvaelarvae

larvae

ofofof

of

threethreethree

three

generageneragenera

genera

(Demonstration)Note the siphon or spiracles and resting position of larvae.

46

6.6.6.

6.

SeeSeeSee

See

thethethe

the

livelivelive

live

pupaepupaepupae

pupae

ofofof

of

threethreethree

three

generageneragenera

genera

(Demonstration)Note the large anterior portion-cephalothorax with two respiratory trumpets whichextend above the surface film and enable the pupae to obtain its air supply and thecurve abdomen consists of visible segments assuring a "comma" shape.

7.7.7.

7.

SeeSeeSee

See

thethethe

the


photographs

ofofof

of

breedingbreedingbreeding

breeding

andandand

and

restingrestingresting

resting

placesplacesplaces

places

ooo

o

fff

f


mosquitoes


8.

StudyStudyStudy

Study

thethethe

the

relationrelationrelation

relation

ofofof

of


mosquito

tototo

to

thethethe

the

diseasesdiseasesdiseases

diseases

(1) Transmitting malaria (Demonstration).A. See the plasmodial oocysts on the intestinal wall of mosquito.B. See the plasmodial sporozoites in the salivary gland smear of infectedmosquito.(2) Transmitting filariasis (Demonstration).

See the infective filarial larvae in labium of infected mosquito.(3) Transmitting Dengue virus (Demonstration).

See the fluorescent stained specimen of Dengue virus in the salivary gland smear ofthe infected mosquito.


Exercise

l. Label head of Anopheles.


Thinking

1. How to identify the arthropod of Arachnida and Insecta?2. Which kinds of arbo-diseases (entomophilous diseases) can be prevented byeliminating mosquitoes in city?

FLYFLYFLY

FLY


Objectives

andandand

and

RequiremenRequiremenRequiremen

Requiremen

ttt

t

sss

s

1. To understand the basic morphology of different stages of common flies and their role

47

as vector.

ObservationsObservationsObservations

Observations

andandand

and


Experiment

1.1.1.

1.

StudyStudyStudy

Study

thethethe

the

desiccateddesiccateddesiccated

desiccated


specimen

ofofof

of

houseflyhouseflyhousefly

housefly

(Manipulation)Identify head, thorax and abdomen. Note a pair of compound eyes and proboscis in thehead; thorax bearing three pairs of legs and a pair of wings, 4 longitudinal black stripson the dorsum of thorax, abdomen segmented.

2.2.2.

2.

SeeSeeSee

See

thethethe

the

drydrydry

dry


specimen

ofofof

of

ChrysomyiaChrysomyiaChrysomyia

Chrysomyia

megacephalamegacephalamegacephala

megacephala

(Demonstration)333

3

...

.

SeeSeeSee

See

thethethe

the

drydrydry

dry


specimen

ofofof

of

SarcophagidaeSarcophagidaeSarcophagidae

Sarcophagidae

speciesspeciesspecies

species


4.

StudyStudyStudy

Study

thethethe

the

externalexternalexternal

external

structuresstructuresstructures

structures

ofofof

of

headheadhead

head

(Demonstration)Note the sucking mouthpart consisting of three parts. The proximal part, the rostrum,bears a pair of spin maxillary palps and is considered as a part of head proper; themiddle region, the haustellum, is supposed to be homologous to labium; the expandeddistal part is the oral plates made up of fleshy labella with tracheal structures.

5.5.5.

5.

StudyStudyStudy

Study

thethethe

the

structurestructurestructure

structure

ofofof

of

leglegleg

leg

(Demonstration)Note the hair appearance of leg terminating in pad and claws. Observe carefullyminute hairs on the pad.

6.6.6.

6.

SeeSeeSee

See

thethethe

the

flyflyfly

fly

larvaelarvaelarvae

larvae

(Demonstration)Note the shape, size, segmentation, spiracular plate and movement of living larvae.

7.7.7.

7.

SeeSeeSee

See

thethethe

the

preservedpreservedpreserved

preserved


specimen

ofofof

of

pupaepupaepupae

pupae


8.

SeeSeeSee

See

thethethe

the

flyflyfly

fly

eggseggseggs

eggs


9.

SeeSeeSee

See

thethethe

the

helminthichelminthichelminthic

helminthic

ovaovaova

ova

carriedcarriedcarried

carried

bybyby

by

houseflyhouseflyhousefly

housefly

footpadsfootpadsfootpads

footpads

(Demonstration)


Exercise

1. Label the head and leg of fly.

SANDFLY,SANDFLY,SANDFLY,

SANDFLY,

FLEAS,FLEAS,FLEAS,

FLEAS,

LICELICELICE

LICE

&&&

&

OTHEROTHEROTHER

OTHER

BLOOD-SUCKINGBLOOD-SUCKINGBLOOD-SUCKING

BLOOD-SUCKING

INSECTSINSECTSINSECTS

INSECTS


Objectives

andandand

and


Requirements

48

1. To understand the basic morphology of different stages of sandfly, the vector ofpathogen of kala azar.

2. To learn the morphological characteristics of flea, louse, bedbug, tick and mite.3. To understand the relation of above arthropods to the diseases.


Observation

andandand

and


Experiment

1.1.1.

1.

SandflySandflySandfly

Sandfly

(Demonstration)(1) See the adult.(2) See the eggs.(3) See the larva.(4) See the pupa.222

2

...

.

FFF

F

lll

l

eaeaea

ea

(Demonstration)(1) See the adult.

Note the shape, size and color. The whole body is laterally compressed with bristlesand spines in posterior direction, genal or/and pronotal comb in some species,suctorial mouth parts, wingless and three pairs of legs (Genal comb-dark brown teethon the ventral margin of gena. Pronotal comb–dark brown on the posterior margin ofthe dorsum of the first thoracic segment.).

(2) See the powerfulleg terminating in twocurved claws.The last pair is being greatly elongated for leaping.

(3) See the eggs.3.3.3.

3.

LouseLouseLouse

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 three species. The head bears a pair of eyes, a pair of5-jointed antennae and an extensile piercing mouthpart. The thorax is composed ofthree fused segments. Each of which bears a pair of legs terminating in a single hook-like claw and tibial process for grasping hairs or fibers of clothes. The body and headlice differ only in size, the crab louse is small in size, indistinct segmented abdomenand large heavy claws.

(4) See the eggs.Note the ellipsoidal, operculated white eggs firmly attached to the hairs or fibers.

4.4.4.

4.

CockroachCockroachCockroach

Cockroach

(Demonstration)(1) See Periplaneta americana.(2) See Blattella germanica.5.5.5.

5.

TickTickTick

Tick

(Demonstration)(1) See the whole mount of adult hard tick.

49

Note its large size, fused cephalothorax, piercing mouthpart and the chitinous (shield-shaped) covering the entire dorsal surface in the male and anterior part in the female;4 pairs of legs.

(2) See the whole mount of adult soft tick.Note its large size, and fused cephalothoracic portion. The mouthpart is situatedventral to anterior end and can not be visible in dorsal view.No scutum.

6.6.6.

6.

MiteMiteMite

Mite

(Demonstration)(1)(1)(1)

(1)

SarcoptesSarcoptesSarcoptes

Sarcoptes

scabieiscabieiscabiei

scabiei

A. See the whole mount of adult.Note its small size, oval shape, dorsal surface of the body with transverse ridges,spines and bristles. The mouthpart consists of toothed chelicerae. Pedipalps and labialpalp fused to the hypostome, 4 pairs of short but stout legs, the first two pairsterminating in long tubular processes each with a bell shaped sucker and claws.

(2)(2)(2)

(2)

LeptotrombidiumLeptotrombidiumLeptotrombidium

Leptotrombidium

deliensedeliensedeliense

deliense

A. See the eggs (Demonstration).B. See the whole mount of larvae (Demonstration).

Note its tiny size, oval shape, capitulum, scutumand body hairs.C. Collect and observe live larvae.

Anesthetize one mouse and inspect the ears for larval mites. Remove them, if any,with dissecting pen to a petri dish containing 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)(3)(3)

(3)

DermatophagoidesDermatophagoidesDermatophagoides

Dermatophagoides

(Demonstration)See the whole mount of adult. Note its small size, compact wrinkles of integument,two anterior pairs and two posterior pairs of legs terminating in long tubular processeseach with a bell shaped sucker and claws.

(4) DemobexDemobexDemobex

Demobex

A. See the whole mount of adult (Demonstration).B. Self-survey forDemodex.7.7.7.

7.

BedbugBedbugBedbug

Bedbug

(Demonstration)(1) See the whole mount of adult.

Note dorsoventrally flattened shape, color, body covered with short serrated hairs; apair of prominent compound eyes, a slender and flexible mouthpart of head; threepairs of legs of thorax; ten segmented abdomen. Do you find the stink-glands betweenthe basal parts of the second and third legs?

(2) See the eggs.(3) See the larvae.

50


Exercise

1. Report the result of examining demodex.

Documents

(THE FIRST EDITION) - 海南医学院精品课程gzy.hainmc.edu.cn/jingpin/jsc/content/laboratory guide to... · A A LABORATORY LABORATORY GUIDE GUIDE TO TO PARASITOLOGY PARASITOLOGY