Medical parasitology lab. Faculty of health sciences Medical Laboratory Sciences Department Medical parasitology lab. Staining of Parasites 2

Cryptosporidium parvum Infect human and most mammals. The infective stage is oocyst containing sporozoites measuring 4-6µ in diameter. The diagnostics stage is oocyst containing 4 sporozoites. Diagnosis: Detecting oocyst in stool. Acid-fast stain http://www.cdc.gov/dpdx/cryptosporidiosis/gallery.html#oocystswetmount http://emedicine.medscape.com/article/215490-overview

Isospora belli Isosporiasis is a human intestinal disease caused by the parasite Isospora belli. The coccidian parasite Isospora belli infects the epithelial cells of the small intestine, and is the least common of the three intestinal coccidia that infect humans. Infection causes acute, non-bloody diarrhea with crampy abdominal pain, which can last for weeks and result in malabsorption and weight loss. In immunodepressed patients, and in infants and children, the diarrhea can be severe. Eosinophilia may be present. Diagnosis: Acid- fast stain

Stool specimen, wet mount Oocysts are spherical and refractile, about 5 micrometer http://www.medicine.cmu.ac.th/dept/parasite/proto/062.htm Notice how easy it would be to miss this on a fecal ova and parasite examination.

http://www.medicine.cmu.ac.th/dept/parasite/framepro.htm Oocysts are elongated ellipsoidal in shape, one sporoblast or two sporocysts appear inside Oocyst wall is thin and colorless, unexperienced microscopist may miss it easily

Cryptosporidium parvum oocyst Oocysts in clinical specimens may be difficult to identify with out special staining. Modified acid-fast (partial acid-fast) stains are recommended for identifying these organisms. This test detects coccidian parasites (Cryptosporidium, Cyclospora, or Isospora belli) in stool. It is used to evaluate chronic diarrhea. Cryptosporidium parvum oocyst Isospora belli oocyst

Principle of acid fast stain The oocysts absorb the red from the carbol-fuchsin stain and may appear in a range of colors from pink to dark purple with bright red being typically seen. The background material typically stains blue or light red. Specimen Collection Concentrated sediment of fresh or formalin preserved stool may be used. Other types of clinical specimens such as bile , duodenal fluid, pulmonary fluid (induced sputum, bronchial washings, biopsy specimens may also be used to stain for organisms.

procedure 1 2 3 4 5 1-carbol-fuchsin for 3-5 mint 2-rinse by tap water 3- add acid alcohol as decolorizer for 1mints 4- rinse by tapewater 5- add methylene blue as counter stain for 1 mint Rinse by tapewater then examine under oil immersion. 4 5

Iron Hematoxylin Stain The  iron hematoxylin stain reveals excellent morphology of the intestinal protozoa. Iron hematoxylin was the stain used for most of the original morphological descriptions of intestinal protozoa found in humans . Under oil immersion power (1,000),one can examine the diagnostic features used to identify the protozoan parasite. Appearance of select protozoan structures and background material on the iron hematoxylin stain Protozoa cytoplasm Blue to purple Protozoa nuclear material Dark blue to dark purple Debris and background material Light blue sometimes with pink tint http://www.uq.edu.au/_School_Science_Lessons/appendixB.html http://www.med-chem.com/para-site.php?url=procedures&subsection=microscopic&tertiary_section=permanent_stained_smear&quad_section=pss_iron_hematoxylin_stain&quint_section=iron_hematoxylin_procedures

Method 1

Place the stool smear in fixative solution for 30 seconds Place the stool smear in fixative solution for 30 seconds. Remove slide from the coplin jar and let air dry. Place approxiamtely 50 ml of fixative solution, solution1, solution 2 in separate jars . Place approximately 50 ml of deionized water in separate coplin jar.

Remove slide from deionized water. And let air dry. Rinse briefly with deionized water. Let air dry. Place slide in solution 2 for 15 seconds . Remove slide from deionzed water. Rinse briefly with deioized water. Let air dry. Place the stablized smear in solution 1 for 15 seconds.

Method 2

Place slides in 50% ethanol for 2 min. Prepare slide for staining (for SAF smears. Place slide in 70% ethanol for 5 min. Place slides in 50% ethanol for 2 min. http://www.atlas-protozoa.com/microscope-exam-ed.php

Place slides under running tap water (best if tepid) for 10 min. Wash slide in running tap water (constant stream of water into the container) for 10 min. Place slide in iron hematoxylin working solution for 4 to 5 min. Differentiate slides (one by one) in destaining solution for 30 seconds. Place slides under running tap water (best if tepid) for 10 min. Ethyl alcohol 95% 70 ml -     Distilled water 30 ml -     Concentrated hydrochloric acid 1 ml Mix well.

Place slide in 95% ethanol for 5 min.* Place slide in two changes of 100% ethanol for 5 min each.* Place slide in two changes of xylene for 5 min each. Add Per mount to the stained area of the slide and cover with a coverslip. Examine the smear microscopically with the 100 x objective. http://www.med-chem.com/para-site.php?url=procedures&subsection=microscopic&tertiary_section=permanent_stained_smear&quad_section=pss_iron_hematoxylin_stain&quint_section=iron_hematoxylin_procedures

Nematodes = roundworm General characters Non segmented Sex are separate Male is smaller than female and its posterior end is curved ventrally. Female are either Larviparous or viviparous: giving birth to larvae Oviparous: laying egg Oviviviparous: laying eggs which contain larvae and which hatch out immediatly

Tissue Nematodes Intestinal Nematodes Trichinella spiralis Entrobius vermicularis Trichuris trichiura Ancylostoma duodenale Strongyloides stercoralis Ascaris lumbricoides http://www.medicine.cmu.ac.th/dept/parasite/nematode/framene.htm

Trichinella spiralis Adult inhabit the small intestine of the rats, pigs and human. Both males and females lie freely in the lumen of the intestine of pigs, rats and human. Fertilized female only penetrate the mucosa where the larviposit, they do not lay eggs.

Trichinella spiralis larvae encysted in muscle

Trichinella spiralis infective stage diagnostic stage. Disease trichinosis, trichiniasis or trichinelliasis infective stage Larvae is live encysted in the flesh of the host, diagnostic stage. Larvae Route of infection eating undercooked pork containing infective encysted larvae. dead end host. Human Diagnosis: Finding larvae in the blood during migration or in muscle after encystation. Immunological tests. X- ray to detect the calcified larvae in muscle.

Entrobius vermicularis Eggs Adults

Young and mature worms are present in small intestine (at terminal ileum till fertilization).

Egg Morphology : 20-50u, transparent with double walled shell. Oval, It may show one side convex and the other flat. Shell: double layered, thick, colorless. Embryo stage of development varies may be unembryonated, embryonated, mature. Microscopically examined the slide under low power. Reduced light is recomeneded as the eggs will appear colorless, making them difficult to detect under high light intensity . http://www.provlab.ab.ca/webbug/parasite/artifact/enterobius.htm

Starch artifact can confused with egg of Entrobius vermicularis These cells are readily differentiated from parasitic forms because they lack internal structures. Starch artifact can confused with egg of Entrobius vermicularis

The tail of female pointed resembles pinhead The female is 0.88-1.3 cm long, it has a long tapering tail resembling 1/3 its length, its straight. Gravid females are present at lower rectum where they lay ova at perianal region around anus (oviparous). The male is shorter than female (2-5 mm) the tail is curved strongly to ventral side, and has a single spicule.

Entrobius vermicularis Alternative names human pin worm and seat worm Disease oxyurdiasis. Infective stage Embryonated Eggs Definitive host human Diagnosis: Note:When found in feces, the eggs are typically unembryonated as seen in this wet preparation. The typical embryonated (infective) eggs are usually seen in the cellulose tape preparation the most reliable means for detecting the parasite . Egg in stool is rarely detected but adults present after purgative. Egg can be detected at perianal area as follow: N.I.H method (National Institute of Health), its based on swabbing the perianal area with a cellophane paper. The adhesive cellulose tape (scotch tape), better result. The Vaseline cloth wiping, the collected ova examined.

scotch tape The specimen should be obtained first thing in the morning before the patient bathes or defecates. Pinworm infection should not be ruled out at least five daily consuecutive specimens are negative. 1- fold the edges of a piece of clear not frosted cellophane tape around the edge of a tongue depressor so that the sticky side faces out. 2- spread buttocks and apply tape to the anal area using a rocking motion to cover as much as the perianal mucosa as possible . 3- remove the tape and apply it to a microscope slide, sticky side down, press firmly so that no air bublles are trapped. NOTE: Pinworm eggs are very infectious.

http://www.practicalscience.com/contents.html http://old.lf3.cuni.cz/mikrobiologie/parazitologie/

http://www.docstoc.com/docs/39019050/PINWORM-COLLECTION-INSTRUCTIONS-CELLULOSE-TAPE-SLIDE---PDF

Trichuris trichiura

Adult inhabit the large intestine in the caecum of man. The adult male smaller than female, male 3.4-4.5 cm, female 4-5 cm. Its commonly called whip worm because of the shape of this worm (anterior thin and posterior thick).

Trichuris trichiura eggs have distinct shape. (oviparous) Shell:smooth,yellow brown color due to bite contact. Hyaline plug at each pole. Diagnosis: Stool examination to detect eggs.