Concentration Techniques : Introduction, Principle, Types, Test Procedure

Concentration Techniques for Stool Parasites: Introduction, Principle, Types, Test Procedure and Uses

Concentration techniques principle works on specific gravity. By addition of ethyle acetate to formalin fixed sample and subsequent centrifugation, the parasites present are heavier than the solution and settle in the sediment of

Introduction of Concentration Techniques for Stool Parasites

Fecal specimen direct microscopy is the most common practice in the Parasitology laboratory for the microscopic examination of feces for the recognition and identification of intestinal parasites. It is very useful for the observation of motile protozoan trophozoites whereas may not detect ova, cysts, and larvae which are present in scant numbers. To get rid of fecal debris and recover parasites in low load need concentration techniques. Therefore, the use of concentration techniques increases the chances of detecting parasitic organisms, thus increasing the sensitivity of recovery of organisms.

//Heavy load of the parasite (Trichomonas species) indirect wet mount of feces as shown below//

//Heavy load of Strongyloides in stool and also larva in Gram stain of sputum as shown below//

Principle of Concentration Techniques

The concentration techniques principle works on specific gravity. By the addition of ethyl acetate to the formalin-fixed sample and subsequent centrifugation, the parasites present are heavier than the solution and settle in the sediment of the tube. Sample debris is typically lighter relatively and rises to the upper layer of the test tube. Subsequent decanting of the supernatant and preparation and examination of concentrate saline or iodine wet preparation of sediment complete this process.

Concentration Methods

It is of mainly two types-

Concentration by flotation and
Concentration by sedimentation

Concentration by flotation

Ova and cyst of parasites having a density lower than that of suspending medium are floated at the top of the medium.

Saturated sodium chloride (Brine solution) and 33% zinc sulfate (ZNSO4) are suspending media.

Flotation method

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

Sedimentation method

Concentration by sedimentation can be achieved by the following methods-

Simple sedimentation
Sedimentation by Formal-ether
Formal detergent sedimentation
Acid-ether sedimentation

Formal-Ether Concentration Technique

Formalin- Ether or Formalin- Ethyl acetate method is the recommended concentration technique.
Most types of worm eggs (roundworms, tapeworms, schistosomes, and other fluke eggs), larvae, and protozoan cysts may be recovered by this method.

Principle of Formal-Ether Concentration Technique

This type of concentration technique clears from its name using formalin and ether. Sedimentation techniques use solutions of lower specific gravity than the parasitic organisms, thus concentrating the latter in the sediment. The formal-ether concentration technique takes advantage of the high specific gravity of protozoan cysts and helminth eggs compared to water. Their natural tendency to settle out in aqueous solutions can be accelerated by light centrifugation. Formalin fixes the eggs, larvae, oocysts, and spores, so that they are no longer infectious, as well as preserves their morphology. Fecal debris is extracted into the ethyl acetate phase of the solution. Parasitic elements are sedimented at the bottom.

Test Requirements

Beaker
Wire sieve
Centrifuge tube ( 15 ml capacity)
Centrifuge
Physiological saline (0.85% w/v NaCl)
10% buffered formalin
Diethyl ether or ethyl acetate
Test tubes with stopper
Vortex
Glass rod
Iodine
Microscope
Positive specimen ( optional for quality control

Procedure for Formal Ether Sedimentation Technique

First, wear gloves when handling stool specimens.
In a suitable container, thoroughly mix a portion of stool specimen approximately 1 ml or the size of a walnut into 10 ml normal saline. Mix thoroughly with the help of a vortex.
Filter the emulsion through fine mesh gauze or alternatively wire sieve into a conical centrifuge tube as shown above picture.
Centrifuge the suspension at 2000 rpm for 10 minutes. Note: The suspension should yield about 0.75 ml of sediment for fresh specimens and 0.5 ml for formalized feces.
Decant the supernatant and wash the sediment with 10 ml of saline solution. Centrifuge again and repeat washing until the supernatant is clear.
After the last wash, decant the supernatant and add 10 ml of 10% formalin to the sediment. Mix and let stand for 5 minutes to effect fixation.
Add 1 to 2 ml of ethyl acetate, Stopper the tube, and shake vigorously.
Centrifuge at 1500 rpm for 10 minutes. Four layers should result as a top layer of ethyl acetate, plug of debris, layer of formalin, and sediment respectively.
Free the plug of debris from the side of the tube by ringing with an applicator stick. Carefully decant the top three layers.
Mix the remaining sediment with a pipette
Transfer one drop each to a drop of saline and iodine on a glass slide and mix.
Cover with a coverslip and observe first for the presence of parasitic forms under low power (10X) objective, and then high power (40X) objective under the microscope.

Quality Control

Check solutions whether free from contamination or not.
Run known positive specimen as positive control through the procedure to verify organism recovery. This should be done at least two times per year.

Uses of Concentration techniques

The purpose of concentrating feces is to increase the possibility of finding ova, cyst, or larvae in samples that are not be able to see by direct microscopy.
The concentration method may be used to see whether the treatment of the parasites has been successful or not.
To find ova of S. mansoné or Taenia is a few of other ova and cysts if they have not been seen in routine examination ( due to 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.
If the number of organisms in stool specimens is low, the examination of a direct wet mount may not detect parasites. Thus, whenever possible, the stool should be concentrated.
The concentration procedure is indicated when the initial wet mount examination is negative despite the clinical symptoms indicating parasitic infection of a patient.

//Small, round to oval, pink red-stained bodies measuring 4-6 micrometer as shown in the video at centre-Some are like a bitten apple in shape that is the evidence of being an organism, Cryptosporidium parvum–

//Egg of tapeworm or Taenia under the Microscope of saline preparation//

They are spherical, brown in color (bile stained), and measure 30-40 µm in diameter. They are surrounded by embryo here which is brown, thick-walled, and radially striated. Inside embryo here, the hexacanth embryo (oncosphere) presents with three pairs of hooklets. They do not float in a saturated solution of common salt (brine solution) and they are viable for 8 weeks.

A common advantage of Concentration techniques

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.

The disadvantage of Concentration techniques

Most concentration methods destroy trophozoites.
Worm eggs, larvae, 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.

Keynotes on Sedimentation techniques

The sedimentation procedure can also be used to process polyvinyl alcohol (PVA) fixed specimens. After the procedure by filing one half of a tube with the stool-PVA mixture and add physiological saline ( 0.85% NaCl) almost to the top of the tube. Then filter the mixture through wet gauze into a 15 ml centrifuge tube and follow the remaining standard procedure.
If ethyl acetate is used, swab the insides of the tube with a cotton-tipped applicator stick after the plug of debris is rimmed and the excess fluid is decanted. Excess ethyl acetate in the sediment at the time smears are prepared will lead to bubbles that may obscure the parasitic forms one is attempting to observe.
Errors in interpretation may occur if too much or too little feces is used in the sedimentation procedure. Adhere to the recommended formula of 0.75 mL of sediment for fresh specimens and 0.5 mL for formalized feces.
Allow the centrifuge to reach maximum speed before the time is monitored. If the centrifugation time is too little, certain smaller parasitic forms, such as the oocysts of Cryptosporidium species, may not reach the sediment.

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