1. Medical parasitology lab.
Faculty of health sciences
Medical Laboratory Sciences Department
Medical parasitology lab.
Concentration techniques

2. The microscopic examination of feces is required for the recognition and identification of intestinal parasites:
Direct Microscopy:
Advantages
Useful for the observation of motile protozoan trophozoites.
Disadvantages
May not detect ova, cysts and larvae which are present in scant numbers.

3. In most cases Samples contains some form of debris
In most cases Samples contains some form of debris. Removal of such material is essential to ensure accurate and thorough screening of the specimen, it is for this reason, coupled with the fact that the parasites are often present in low numbers and need to be condenced into one area of the sample that concentration methods have been developed. pa

4. STOOL CONCENTRATION METHODS:  Where heavy infestation is present this method is not needed. Concentration method may be used to seen whether treatment  of  the  parasites  has been successful. To  find ova of S.mansoné or Taenia is  few of other ova and cysts if they have not been seen  in routine  examination  (being very few) and are  suspected  to  be present. To examine stool specimens from patients who do not come from an area where a particular parasite is found.

5. If number of organisms in stool specimen is low, examination of a direct wet mount may not detect parasites.
Thus, whenever possible, the stool should be concentrated.
worm eggs, larvea, and protozoan cysts may be recovered by concentration but protozoan trophozoites will not be seen as they are usually destroyed during the concentration procedures. This makes direct wet mount examination obligatory as the initial phase of microscopic examination.

6. Principles of concentration techniques
The concentration procedure is indicated when the initial wet mount examination is negative despite the clinical symptoms indicating parasitic infection of a patient.
The principle of concentration technique is based on specific gravity . By addition of ethyle acetate to formaline fixed sample and subsequent centrifugation,the parasites present are heavier than the solution and seetle in the sediment of the tube .

7. Sample debrise is typically lighter , relatively speaking ,and rises to the upper layer s of the test tube. subsequent decaning of the supernatant and preparation and examination of concentrate saline or iodine wet preparation of sediment complete this process.

8. 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.

9. Concentration Methods
Sedimentation method
Modified Formal- Ether sedimentation technique
Acid- Ether sedimentation technique
Flotation method
Saturated Salt Solution technique
Sheather’s Sugar Centrifugal Flotation technique
Zinc Sulphate Centrifugal Flotation technique

10. In  sedimentation  methods,  the parasites are  not  floated  but deposited,  usually  by centrifuging. A small  piece of stool is mixed with saline in a tube or  bottle and sieved through  a  strainer.  The  sieved   contents   are centrifuged and the  supernatant fluid poured off. The deposit is re-suspended  in more  saline, mixed, and  centrifuged.  This  is repeated  until the supernatant fluid is clear. The  deposit is examined directly on a slide. By  this  simple method, parasitic cysts, eggs, and  free  living parasites can be concentrated.

11. Modified Formal- ether sedimentation
Sedimentation Methods
This method gives a good concentration of parasitic contents  and is recommended for routine work. This method, however, cannot  be used  to  concentrate  free-living forms as  formalin  kills  the parasites
Modified Formal- ether sedimentation

12. 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.

13. 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.
Reagents: 10 % formal-saline. Add  50 ml conc. formaldehyde solution to 450 ml saline  and  mix well.

14. Caution
Ether is a highly flammable compound and will ignite and explode quickly if there is a flame or spark nearby.

16. 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.

18. Procedures (cont.)
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.
A centrifuge uses centrifugal force (g-force) to isolate suspended particles from their surrounding medium on either a batch or a continuous-flow basis. Increasing the effect of gravity: the centrifuge.
Many particles or cells in a liquid suspension, given time, will eventually settle at the bottom of a container due to gravity (1 x g). However, the length of time required for such separations is impractical. Other particles, extremely small in size, will not separate at all in solution, unless subjected to high centrifugal force. When a suspension is rotated at a certain speed or revolutions per minute (RPM), centrifugal force causes the particles to move radially away from the axis of rotation. The force on the particles (compared to gravity) is called Relative Centrifugal Force (RCF). For example, an RCF of 500 x g indicates that the centrifugal force applied is 500 times greater than Earth’s gravitational force. Table 1 illustrates common centrifuge classes and their applications.

20. Acid- ether sedimentation technique
Sedimentation Methods
Acid- ether sedimentation technique

21. 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.

22. Procedures
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 sec using vortex.

23. Procedures (cont.)
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.

24. Entamoeba histolytica/ dispar
E. histolytica inhabit large intestine and cause amoebic dysentery.
There is two diagnostic stages for E. histolytica/ dispar:
Cyst is regular round measuring measure 10 – 20u in diameter with 4 nuclei, and it’s the infective stage.
Trophozoite is the motile form, measure 15-20u in diameter with large nucleus. (motility by pseudopodia).
Diagnosis:
Stool examination to see cyst stage, or trophozoite stage if the sample is fresh.

26. Entamoeba histolytica/ dispar

27. Entamoeba histolytica/ dispar Trophozoite