A LABORATORY GUIDE TO PARASITOLOGY CTHE FIFTH EDITIONI ZHENG XIAOYING WU ZHONGDAO Department of parasitology Zhongshan Medical collage Sun Yat-Sen University July, 2002
A LABORATORY GUIDE TO PARASITOLOGY (THE FIFTH EDITION) ZHENG XIAOYING WU ZHONGDAO Department of Parasitology Zhongshan Medical collage , Sun Yat-Sen University July, 2002
CONTENTS Care of the microscope Clonorchis sinensis Paragonimus flukes Fasciolopsis buski Schistosoma japonicum Tar Ascaris lumbricoides Trichuris trichiura Ancylostoma duodenal Necator americanus Enterobius vermicularis Wuchereria bancrofti brugia malayi Trichinella spirali Examination of alimentary helminths Entamoeba histolytica. E coli other Amoebae Giardia lam blia Trichomonas vaginalis Leishmania donovani Plasmodia Opportunistic pathogenic protozoan Mosquitoes y Sandfly, fleas, lice other blood sucking insects
1 CONTENTS Care of the microscope…………………………………….…………………1 Clonorchis sinensis…………………………………….……………….…….2 Paragonimus flukes…………………………………….……………….……5 Fasciolopsis buski…………………………………….……………….….…..7 Schistosoma japonicum…………………………………….………………...8 Tapeworm…………………………………….……………….………….…14 Ascaris lumbricoides & Trichuris trichiura ………………………………..17 Ancylostoma duodenal & Necator americanus……………………………..20 Enterobius vermicularis……………………………………………….…….22 Wuchereria bancrofti & Brugia malayi……………………………………..23 Trichinella spiralis…………………………………….……………….……25 Examination of alimentary helminths…………………………………….…26 Entamoeba histolytica, E.coli & other Amoebae…………………………...27 Giardia lamblia…………………………………….……………….……….30 Trichomonas vaginalis…………………………………….………………...31 Leishmania donovani…………………………………….……………….…32 Plasmodia…………………………………….…………………….…….…35 Opportunistic pathogenic protozoan………………………………….……..40 Mosquitoes…………………………………….……………….…….……...43 Fly…………………………………….…………………….…….…….…...45 Sandfly, fleas, lice & other blood sucking insects…………………………..46
Care of the microscope Dos 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 growth in hot humid periods 3. Do clean the immersion oil from the immersion objective each time, use lens tissue dampened 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 to prevent damage to the instrument while in transit Dont 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
2 Care of the microscope 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 growth in hot humid periods. 3. Do clean the immersion oil from the immersion objective each time; use lens tissue dampened 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 to prevent damage to the instrument while in transit. 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
CLONORCHIS SINENSIA Objectives and requirements 1. To study the morphology and the life history of C sinensis as an example of general feature 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 and experiment 1. Adult 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 following 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 sys tem: 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. Larva and intermediate hosts (Demonstration) (1) See live sporocyst, redia and cercaria(or stained sporocyst to replace live one if (2)See the stained cercaria Note the eye-spots and characteristics of tail lipoidal shape, 138 u mX115 u m, with two suckers(oral sucker an entral sucker) and excretory bladder containing black granules (4)See the first intermediate hosts(Parafossarulus, Alocinma, Bithynia snails), and second intermediate hosts, fresh water fish and crayfish. What are their names? 3. Ovum(Manipulation) (1)Study the ova. Note the shape, color, shell, operculum resting on a rim which takes
3 CLONORCHIS SINENSIS Objectives and Requirements 1. To study the morphology and the life history of C. sinensis as an example of general feature 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 and Experiment 1. Adult 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 following structures. 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. Larva and intermediate 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 and ventral sucker) and excretory bladder containing black granules. (4) See the first intermediate hosts (Parafossarulus, Alocinma, Bithynia snails), and second intermediate hosts, fresh water fish and crayfish. What are their names? 3. Ovum (Manipulation) (1) Study the ova. Note the shape, color, shell, operculum resting on a rim which takes
the shape of distinct shoulders, a small protuberance at abopercular end,an asymmetrical miracidium inside 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) 4. Pathology (1)See specimen, note the parasites in billiary passages(Demonstration) (2)See adult worms inhabiting in billiary passages of a reservoir host(Demonstration) Exercise I. Draw egg of C. sinensis in detail 2. Label in full an adult worm of C. sinensis 3. Draw a life cycle draft of C. sinensis, including final host, intermediate host; adult parasitized site, main injured organs; excretory route of ovum, pathogenic diagnostic methods the first and se intermediate hosts and in which how the larva development; the infective 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 I. The fertilization process and egg formation In male, sperm produced in a pair of testes are collected in the seminal vesicle through the vas efferens and vas deferens and are ejected through the ejaculatory duct, around which there is a prostate gland covered by a cirrus sac. In female, a short oviduct comes out of the ovary and joins with the seminal receptacle and Laurers canal opening on the dorsal side, and further proceeds to the ootype with surrounding Mehlis'gland. Then, it becomes the uterus, which extends with many contortions to the common genital pore. Small ducts from the vitelline glands, gradually gather to become the right and left vitelline ducts and then one common vitelline duct, which opens at front of the ootype An egg cell from ovary and a sperm from testis are united, additional yolk cells provided by the vitelline gland join, and eggshell formation is started in the ootype by the effect of Mehlis' gland and completed during passage through the uterus. In the egg one miracidium has already been formed 2. Examine for encysted metacercaria in fish flesh
4 the shape of distinct shoulders, a small protuberance at abopercular end, an asymmetrical 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). 4. Pathology (1) See specimen, note the parasites in billiary passages (Demonstration). (2) See adult worms inhabiting in billiary passages of a reservoir host (Demonstration). Exercise 1. Draw egg of C. sinensis in detail. 2. Label in full an adult worm of C. sinensis. 3. Draw a life cycle draft of C. sinensis, including final host, intermediate host; adult parasitized site, main injured organs; excretory route of ovum, pathogenic diagnostic methods; the first and second intermediate hosts and in which how the larva development; the infective stage, infection mode and infection route. Thinking 1. Why one can obtain parasitic disease after eaten uncooked fish? 2. Record the results of pressing fish flesh and the dissection of the experimental animal. Reference 1. The fertilization process and egg formation In male, sperm produced in a pair of testes are collected in the seminal vesicle through the vas efferens and vas deferens and are ejected through the ejaculatory duct, around which there is a prostate gland covered by a cirrus sac. In female, a short oviduct comes out of the ovary and joins with the seminal receptacle and Laurer’s canal opening on the dorsal side, and further proceeds to the ootype with surrounding Mehlis’ gland. Then, it becomes the uterus, which extends with many contortions to the common genital pore. Small ducts from the vitelline glands, gradually gather to become the right and left vitelline ducts and then one common vitelline duct, which opens at front of the ootype. An egg cell from ovary and a sperm from testis are united, additional yolk cells provided by the vitelline gland join, and eggshell formation is started in the ootype by the effect of Mehlis’ gland and completed during passage through the uterus. In the egg one miracidium has already been formed. 2. Examine for encysted metacercaria in fish flesh
Press a small piece of freshwater fish flesh between two slides, examine for encysted metacercaria of C. sinensis under lower-lens microscope 3. Isolation of encysted metacercaria Take the 2nd intermediate host-freshwater fish and smash, digest with 250 ml digestive solution per 10 g flesh for 4 h"12 h(Digestive solution: pepsin 9.8g, I N HCI 164 mL NaCl 17 g add water to 2 000 ml). Discard rough matter by sieve filtration. After several times sedimentation of the filtrated solution, take the sediment and examine under microscop 4. Examination adult from infected animal Dissect the guinea pig(or white rat) which were infected with the aid of stomach tube approximately 30 C. sinensis cysts 40 days ago. Expose the common bile duct, gal bladder and search for the adult worms in the billiary tree
5 Press a small piece of freshwater fish flesh between two slides, examine for encysted metacercaria of C. sinensis under lower-lens microscope. 3. Isolation of encysted metacercaria Take the 2nd intermediate host-freshwater fish and smash, digest with 250 ml digestive solution 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. After several times sedimentation of the filtrated solution, take the sediment and examine under microscope. 4. Examination adult from infected animal Dissect the guinea pig (or white rat) which were infected with the aid of stomach tube approximately 30 C. sinensis cysts 40 days ago. Expose the common bile duct, gall bladder and search for the adult worms in the billiary tree
PARAGONIMUS FLUKES Objectives and requirements 1. To study the life cycles of P. westermani, to learn more about the life history of trematodes 2. To study the main parasitized sites and major pathogenisis 3. To study the laboratory diagnosis of these flukes Observation and Experiment 1.P A. 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 situated in posterior of the body, side by side, and posterior to uterus and ovary lobulated ovary is in the opposite side of uterus(fully filled with ova). Are these two female 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 and filter. Allow the filtrate stand and settle, examine sediments for encysted metacercaria under microscop (3)Egg A Morphology of egg(Manipulation) Measures 80118 W mX48-60 u m. Note its ellipsoidal shape, uneven thickness of eggshell, slightly oblique operculum and yellowish brown in color. A germinal cell with more than ten yolk cells inside the egg Materials examined; sputum, feces Examining methods: direct sputum smear, sputum concentration and direct fecal smear B. See the SEM photograph of egg (4)Pathology A. See the preserved infected lungs(Demonstration)
6 PARAGONIMUS FLUKES Objectives and Requirements 1. To study the life cycles of P. westermani, to learn more about the life history of trematodes. 2. To study the main parasitized sites and major pathogenisis. 3. To study the laboratory diagnosis of these flukes. Observation and Experiment 1. P. westermani (1) Adult worm A. 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 are situated in posterior of the body, side by side, and posterior to uterus and ovary. The lobulated ovary is in the opposite side of uterus (fully filled with ova). Are these two female 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 and filter. Allow the filtrate stand and settle, examine sediments for encysted metacercaria under microscope. (3) Egg A. Morphology of egg (Manipulation). Measures 80～118μm×48～60μm. Note its ellipsoidal shape, uneven thickness of eggshell, slightly oblique operculum and yellowish brown in color. A germinal cell with more than ten yolk cells inside the egg. Materials examined: sputum, feces. Examining methods: direct sputum smear, sputum concentration and direct fecal smear. B. See the SEM photograph of egg. (4) Pathology A. See the preserved infected lungs (Demonstration)
Note the capsules and their surrounding damaged tissues B. See the infected liver( Demonstration) 2. Pagumogonimus skrjabini (1)Adult worm(Demonstration) A. See the preserved specimen B. See the stained specimen (2)Intermediate host(Demonstration) ee 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. 4. Draw a life cycle draft of P westermani Thinking 1. How to explain paragonimiasis is a natural epidemic source disease in mountainous egion and forest? and what are the epidemic characteristics of paragonimiasis? R eference 1. Detection of sputum for egg of P westermani Take a clean slide, place a drop of saline in the center of it, pick up a small amount of the sputum and mix it with the drop of saline, examine under microscope (2)Egg concentration Collect the sputum of 24 h, pour into a measuring glass and add equal quantity of 10% NaoH, mix, sediment naturally for 6 h8 h, remove supernatant, take the sediment and examine under microscop 2. Survey biological hosts of P westerman in 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 microscop (2)Dissect stream crab or crayfish and find encysted metacercaria 3. Infection of p westermani for 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, open chest and expose the lungs, on which the nodular cysts can be observed
7 Note the capsules and their surrounding damaged tissues. B. See the infected liver (Demonstration). 2. Pagumogonimus skrjabini (1) Adult worm (Demonstration). A. See the preserved specimen. B. See the stained specimen. (2) Intermediate host (Demonstration) See the first 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 mountainous region and forest? And what are the epidemic characteristics of paragonimiasis? Reference 1. Detection of sputum for egg of P. westermani (1) Sputum smear Take a clean slide, place a drop of saline in the center of it, pick up a small amount of the sputum and mix it with the drop of saline, examine under microscope. (2) Egg concentration Collect 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 sediment and examine under microscope. 2. Survey biological hosts of P. westerman in 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. Infection of P. Westermani for 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, open chest and expose the lungs, on which the nodular cysts can be observed
dissect them to find adult worms FASCIOLOPSIS BUSKI Objectives 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 prophylax Observation and experiment 1. Adult worm (1)See the preserved specimens(Demonstration) Note the shape, size, color and the positions of oral and ventral suckers and their sizes. large and thick (1)Study the stained specimen(Manipulation First with naked eye, then under low power microscope, note the adhesive organ, ceca and reproductive system of both sexes. The intestine is composed of two blind tracts without side branching, and the ventral sucker is very large 2. Larva, intermediate host and vector(demonstration) (1)See the snail intermediate host(Planorbis (2)See the cercaria (3)See the stained specimen of cyst (4)See the vector: aquatic plants(caltrop, water chestnut etc.) 3. Ovum (1)See the preserved specimens(Manipulation) Note its large size, 130-140 u mX80-85 H m, 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 Draw and label F. buski egg in detail. Thinking 1. Draw a life cycle draft of f. buski
8 dissect them to find adult worms. FASCIOLOPSIS BUSKI Objectives 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 and Experiment 1. Adult worm (1) See the preserved specimens (Demonstration). Note the shape, size, color and the positions of oral and ventral suckers and their sizes, large and thick. (1) Study the stained specimen (Manipulation). First with naked eye, then under low power microscope, note the adhesive organ, ceca and reproductive system of both sexes. The intestine is composed of two blind tracts without side branching, and the ventral sucker is very large. 2. Larva, intermediate host and vector (Demonstration) (1) See the snail intermediate host (Planorbis). (2) See the cercaria. (3) See the stained specimen of cyst. (4) See the vector: aquatic plants (caltrop, water chestnut etc.). 3. Ovum (1) See the preserved specimens (Manipulation). Note its large size, 130～140μm×80～85μm, oval shape, pale yellow in color, 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.Draw a life cycle draft of F. buski
SCHIASTOSOMA JAPONICUM Objectives and Requirements 1. To understand characteristics of life cycle and morphology of its developmental 2. To observe the pathological changes of schistosomiasis in infected animal 3. To study morphology of ova, laboratory diagnostic methods and their princi 4. To gain fundamental knowledge of infective stage, infection mode, methods and significance of immunologic tests commonly used Observation and Experiment L Adult 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 and gynecophoral canal of male, the number, shape, size and arrangement of testes, ovary and uterus of female. Females are long and slender, the posterior half of the body is a little thick and dark brown in color owing to the vitelline gland and intestine. Near the anterior end of the body, there are two suckers. The part of the male body behind the ventral sucker is flattened, and rolled ventrally along the length to form a groove, canal is gynecophonus, into which the female worm fits What is the meaning of dioecious? (2)See the seM photograph of adult worm( Demonstration) 2. Larva and intermediate 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 Study stained specimen, note the internal structures and the forked tail (6)See the SEM photograph of cercariae 3. ovu (1) Place a small drop n containing S. japonicum ova on a slide
9 SCHISTOSOMA JAPONICUM Objectives 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 significance of immunologic tests commonly used. Observation and Experiment l. Adult 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 and gynecophoral canal of male, the number, shape, size and arrangement of testes, ovary and uterus of female. Females are long and slender, the posterior half of the body is a little thick and dark brown in color owing to the vitelline gland and intestine. Near the anterior end of the body, there are two suckers. The part of the male body behind the ventral 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 of dioecious? (2) See the SEM photograph of adult worm (Demonstration). 2. Larva and intermediate 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. Study stained specimen, note the internal structures and the forked tail. (6) See the SEM photograph of cercariae. 3. Ovum (1) Place a small drop of suspension containing S. japonicum ova on a slide