LAB 5

Flotation The flotation procedure permits the

separation of protozoan cysts and certain eggs from excess debris through the use of a liquid with a high specific gravity.

The parasitic elements are recovered in the surface film, and the debris remains in the bottom of the tube.

This technique yields a cleaner preparation than the sedimentation procedure.

Zinc Sulphate techniqueZinc Sulphate technique

Why can’t all helminth eggs be recovered using the flotation concentration rather than the sedimentation concentration?

Some helminth eggs are quite heavy (unfertilized Ascaris eggs) and will not float, even using zinc sulfate with a specific gravity of 1.20. Other helminth eggs are operculated; when the egg is placed in a high specific gravity solution, the operculum “pops” open and the egg fills with fluid and sinks to the bottom of the tube. Thus, both the surface film and the sediment should be examined before reporting the specimen as negative.

Materials and MethodMaterials and Method Zinc sulphate solution with specific gravity 1.18.

Mix 330gm dry zinc sulphate in 670ml distilled water. Use the hydrometer or densitometer to adjust specific gravity

around 1.18 Notes : Warning! May cause skin irritation. May cause eye

irritation and possible burns.Evaporation Rate:< ether Chemical Stability: Stable under normal

temperatures and pressures.

Zinc Sulphate centrifugal Zinc Sulphate centrifugal flotation techniqueflotation technique1. Transfer about 0.5 teaspoon of stool to a test tube

containing 1-2 ml of water and comminute thoroughly then fill the tube to within 2-3 mm of the top with water.

2. Centrifuge at 1500 rpm for 1 mint. Discard supernatant fluid.

If the sample contains a large amount of material that floats in water, you may want to wash the sample before doing the flotation.

3. Add 1-3 ml of zinc sulphate solution to the sediment and resuspend the sediment.

4. Fill the tube within 2-3 mm of rim with additional zinc sulfate solution.

5. Strain the suspension through strainer into paper cup.

6. Return the suspension to the tube, and add enough zinc sulfate solution to fill within 2-3mm of rim .

7. Centrifuge at 1500 rpm for a 1 minute. Allow the centrifuge to come to a stop without interference or vibration.

8. Without removing the centrifuge tube from the centrifuge and using wire loop ,remove 1-2 drops from the center of the surface film and add them to the drop of iodine or water on the slide then make examination .

The surface film must be examined within a few minutes after flotation is complete.

Otherwise the cysts and eggs may distort or collapse.

If zinc sulfate is the only concentration method used, both the surface film and the

sediment should be examined should be examined to ensure the detection of all

possible organisms.

The surface film must be examined within a few minutes after flotation is complete.

Otherwise the cysts and eggs may distort or collapse.

If zinc sulfate is the only concentration method used, both the surface film and the

sediment should be examined should be examined to ensure the detection of all

possible organisms.

Why is the flotation concentration used less frequently than the sedimentation concentration?

There are several reasons. First, not all parasites will float; therefore, you need to examine both the surface film and the sediment before indicating the concentration examination is negative. Second, the organisms must not be left in contact with the high specific gravity zinc sulfate for too long or protozoa will tend to become distorted, so the timing of the examination is more critical. Also, the specific gravity of the fluid will need to be checked periodically.

Dog Tape worm – Echinococcus granulosusFish Tape worm – Diphylobothrium latum

Beef Tape worm – Taenia saginataPork Tape worm – Taenia soliumDwarf Tape worm – Hymenolepis nana

Tissue Cestodes

Ecchinococcus granulosusEcchinococcus granulosus

Ecchinococcus granulosus also known as hydatid worm.Disease: Ecchinococcus (serious disease).Adult lives in intestine of dogs but never in human intestine.Definitive host: Dogs.Dead end host: Human.Intermediate host: sheep, cattle, horse, pigs.Infective stage: ova by fecal oral route.Diagnosis:

Tests involve antigen antibody reaction.X-RAY examination.

Ecchinococcus granulosus stages Ecchinococcus granulosus stages

Intestinal Cestodes

Diphyllobotherium latum • knows as abroad fish tapeworm which can cause Diphylobotheriasis.• Mode of infection: through consumption of raw or undercooked fish

contain plerocercoid larva.• Immature eggs are passed in feces of the mammal host (the definitive host).• After ingestion by a suitable freshwater crustacean (intermediate host), the

coracidia develop into procercoid larvae.• Following ingestion of the crustacean by a suitable second intermediate

host, the procercoid larvae are released from the crustacean and migrate into the fish's flesh where they develop into a plerocercoid larvae

• The plerocercoid larvae are the infective stage for the definitive host (including humans).

• Diagnosis: – Stool analysis to finding typical eggs ( oval, yellow brown with

operculum at one end) and segments.

Diphyllobotherium latum eggsDiphyllobotherium latum eggs

Of the 32 recognized species of Taenia, only Taenia solium and Taenia saginata are medically important.

Taenia saginata, which is also known as the "beef tapeworm", though it also infects humans.

Taenia solium, which is known as the "pork tapeworm". Like Taenia saginata humans serve as its primary host.

Both have mature, immature and gravid segments.Both have scolex provided with four cup- shaped

suckers, but T. saginata has an unarmed scolex while T. solium has an armed scolex

• In both species the infective stage is the cysticercus.• Cows are the intermediate host of T. saginata while pigs are

the intermediate host of T. solium. • Definitive host: Human.• Diagnosis:

– By finding gravid proglotid or eggs in stool.• Eggs present in stool less often are proglottids.

– Direct fecal smear.– Brine flotation technique.

• Taenia saginata and Taenia solium must be differentiated from each other by the following: Mature segment, gravid proglotid and adult worm.

Taenia Egg Taenia Egg

Taenia scolexTaenia scolex

Taenia saginata Taenia solium

Taenia mature segmentTaenia mature segment

Taenia solium3 ovary lobes

Taenia saginata2 ovary lobes

Taenia Gravid Proglotid Taenia Gravid Proglotid

Taenia soliumTaenia saginata

Taenia Adult wormTaenia Adult worm

Taenia soliumTaenia saginata

Comparison Comparison

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Hymenolepis nanaHymenolepis nana This is one of the most common cestodes of humans, especially

children. And it’s the smallest tape worm in man, also called dwarf tape worm.

Eggs measures 30-37u, and have double membrane and contain six hooked oncosphere.

The scolex of  H. nana has four suckers and a short rostellum with hooks.

Infective stage: Eggs. Diagnosis:

Stool examination to detect the eggs.

H. nanaH. nana egg egg

Hymenolepis nana – The Dwarf Tapeworm

30

Hymenolepis nana

Definitive host Man

Intermediate host No

Infective form

Mode of transmission Ingestion

Site of localization Small intestine

Eggs

(Dwarf Tape Worm)

Internal autoinfection

Hymenolepis nana scolex

Mature proglottid

Gravid proglottid