Medical Parasitology Lab.

Concentration techniques

The microscopic examination of feces is required for the recognition and identification of intestinal parasites:1. Direct Microscopy:• Advantages

Useful 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

2. Concentration techniques :• Advantages

Maximizes 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.• Disadvantages

Destroys 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

Concentration Methods

1. Sedimentation method Modified Formal- Ether sedimentation technique Acid- Ether sedimentation technique

2. Flotation method Saturated Salt Solution technique Sheather’s Sugar Centrifugal Flotation technique Zinc Sulphate Centrifugal Flotation technique

Raed Z. Ahmed, Medical Parasitology Lab.,2012

MODIFIED FORMAL- ETHER SEDIMENTATION

Sedimentation Methods

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Materials and Method Libra Applicator stick Glass centrifugal tubes Beaker Wire sieve Vortex 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

Procedures

Raed Z. Ahmed, Medical Parasitology Lab.,2012

1. Emulsify 1 gm. of feces in 7 ml of 10% formalin in a centrifuge tube.

2. Strain the suspension through a brass wire sieve, and collect in beaker.

3. 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.

4. Transfer the ether- formalin suspension back into the washed centrifuge tube, and centrifuge at 3,000 rpm for 1 min.

5. 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.

6. 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.

7. Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites.

Procedures (cont.)

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:1. Speed: one sample can be processed in 5 minutes.2. Broad spectrum: it will recover most ova, cyst and larvae.3. The morphology of most parasites is retained for easy identification.

• Disadvantages:1. Requires several pieces of apparatus which does not make it an easy.2. The preparation contains some debris.3. Ether is flammable. Formalin is an irritant.4. Hymenolepis nana and Fasciola spp. do not concentrate well.

Raed Z. Ahmed, Medical Parasitology Lab.,2012

ACID- ETHER SEDIMENTATION TECHNIQUE

Sedimentation Methods

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Materials and Method

Libra Applicator stick Glass centrifugal tubes Beaker Wire sieve Vortex 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

1. Mix thoroughly 1 gm. feces with 3 ml of 15% of hydrochloric acid and then mix well.

2. Add and additional 5-6 ml of 15% HCl and mix.3. Strain the suspension through a wire sieve into beaker.4. Place suspension in a glass centrifuge tube and make up

to the 10 ml with distilled water.5. Add 4 ml of ether, stopper the tube and shake

vigorously 20 -30 sec using vortex.

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Procedures

6. Centrifuge 2-3 min at 1500 rpm, the suspension now will be layered.

7. Loosen plug of debris with applicator stick and immediately pour off liquid.

8. Transfer one drops onto a glass slide and cover it with coverslip.

9. Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites.

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Procedures (cont.)

Intestinal Protozoa

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Giardia lamblia

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 :1. Cyst (infective stage).2. Trophozoite (motile form, 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

Life cycle

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Giardia lamblia cyst

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Giardia lamblia Trophozoite

Raed Z. Ahmed, Medical Parasitology Lab.,2012