Medical Parasitology Lab. - PowerPoint Presentation

Slide1

Medical Parasitology Lab.

Concentration techniques

Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013

Prepared By:

Mr. Raed Z. AhmedSlide2

The microscopic examination of feces is required for the recognition and identification of intestinal

parasites:Direct Microscopy:AdvantagesUseful for the observation of motile protozoan trophozoites.DisadvantagesMay not detect ova, cysts and larvae which are present in scant numbers.Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide3

Concentration techniques :

AdvantagesMaximizes the numbers of organisms detected which may be too scanty to be seen by direct microscopy alone. Worm eggs, larvae, and protozoan cysts may be recovered.DisadvantagesDestroys trophozoite stages. Most concentration methods destroy trophozoites stages.The purpose of concentrating feces is to increase possibility to finding ova, cyst, or larvae in samples that not be able to seen by direct microscopy.Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide4

Concentration Methods

Sedimentation methodModified Formal- Ether sedimentation techniqueAcid- Ether sedimentation techniqueFlotation methodSaturated Salt Solution techniqueSheather’s Sugar Centrifugal Flotation techniqueZinc Sulphate Centrifugal Flotation techniqueRaed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide5

Modified Formal- ether sedimentation

Sedimentation MethodsRaed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide6

Modified Formal- Ether Sedimentation

Formalin- Ether or Formalin- Ethyl acetate method is the recommended concentration procedures.Most types of worm eggs (round worms, tapeworms, schistosomes, and other fluke eggs), larvae, and protozoan cysts may be recovered by this method. Advantages:Speed: one sample can be processed in 5 minutes.Broad spectrum: it will recover most ova, cyst and larvae.The morphology of most parasites is retained for easy identification.Disadvantages:Requires several pieces of apparatus which does not make it an easy.The preparation contains some debris.Ether is flammable. Formalin is an irritant.Hymenolepis nana and Fasciola spp. do not concentrate well.

Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide7

Materials and Method

LibraApplicator stickGlass centrifugal tubesBeaker Wire sieveVortex or whirlimixer Centrifuge.Reagent: Reagent I: 10% formalin solution in distilled water.Reagent II: diethyl ether or ethyl acetate

.Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide8

Procedures

Emulsify 1 gm. of feces in 7 ml of 10% formalin in a centrifuge tube.Strain the suspension through a brass wire sieve, and collect in beaker.Pour the filtrate into a 15 ml boiling tube and add 3 ml of ether, then mix well 15 sec on vortex or whirlimixer or 1 min by hand.Transfer the ether- formalin suspension back into the washed centrifuge tube, and centrifuge at 3,000 rpm for 1 min.Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide9

Loosen the fatty layer and debris at the top of the tube with an applicator stick and invert the tube quickly to discard the supernatant. On righting the tube, a few drops only should remain with the sediment, mix the sediment well and transfer one drops onto a glass slide and cover it with coverslip.

Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites. Procedures (cont.) Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide10

Acid- ether sedimentation technique

Sedimentation MethodsRaed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide11

Materials and Method

LibraApplicator stickGlass centrifugal tubesBeaker Wire sieveVortex or whirlimixer Centrifuge.Reagent: Reagent I: 15% Hydrochloric acid.Conc. HCl 40 ml + 60 ml Distilled water.

Reagent II:

diethyl ether or ethyl acetate.

Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide12

Mix thoroughly 1 gm. feces with 3 ml of 15% of hydrochloric acid and then mix well.Add and additional 5-6 ml of 15% HCl and mix.

Strain the suspension through a wire sieve into beaker.Place suspension in a glass centrifuge tube and make up to the 10 ml with distilled water.Add 4 ml of ether, stopper the tube and shake vigorously 20 -30 sec using vortex.Procedures Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide13

Centrifuge 2-3 min at 1500 rpm, the suspension now will be layered.Loosen plug of debris with applicator stick and immediately pour off liquid.

Transfer one drops onto a glass slide and cover it with coverslip. Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites. Procedures (cont.) Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide14

Intestinal Protozoa

Giardia lambliaRaed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide15

Giardia lamblia

It is the most common flagellate of the intestinal tract that cause giardiasis, Traveler's diarrhea.There is two diagnostic stages for Giardia lamblia :Cyst is oval measuring 11 – 14u in length and 7 to 10 µm in width with 4 nuclei and remnant flagella, and it’s the infective stage.Trophozoite is described as having a 'tear-drop' shape and are 10 to 20 µm long and 5 to 10 µm wide. The trophozoites contain two nuclei, four pair of

flagella. (motility

by flagella).

Diagnosis:

Stool examination to see cyst stage, or trophozoite stage if the sample is fresh.

Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide16

Life cycle

Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide17

Giardia lamblia

cystRaed Z. Ahmed, Medical Parasitology Lab.,2012-2013Slide18

Giardia lamblia Trophozoite

Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013