Stool examination Direct Saline and Iodine Wet Mount Diagnosis

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Stool is one of the most common sample used to diagnose intestinal parasites,Though some serological methods have been develop,the most commonly performed procedure in parasitology is the ova and parasite (O &P) examination base on stool microscopical observation

Gastro-intestinal infestations (infections) by parasites (Protozoan/Helminthes) are primarily diagnosed by detecting live motile trophozoites (for protozoans); cyst (inactive dormant stage of Protozoa) or eggs (in case of Helminthes) in stool.

Microscope slides made from faecal specimen of the patients can be examined under low and high power. These etiological agents are identified based on their morphological/staining some may be bile stained or not bile stained; in case of eggs of helminthes) basic characteristics yet they are seen as clearly when mounted properly on the microscope

Two basic methods are involve in stool microscopical examination.

The saline mount and staining method

Saline wet mount

from the name,it makes uses of Normal saline and is made by mixing a small well mixed quantity (about 2 mg) of the sample( stool) in a drop of saline placed on a clean glass slide and covered with a cover slide.

The smear is then examined under the microscope with specific objectives X4,X10,X40. The Saline wet mount is used for the detection of trophozoites and cysts of protozoa, and eggs and larvae of helminthes.
It is particularly useful for detection of live motile trophozoites of protozoans like E. histolytica, Giardia lamblia and Balantidium coli and helminths larva like Strongyloides stercolaris and ova's of hookwook,trichuris trichuira etc.

REAGENTS AND EQUIPMENT REQUIRED FOR WET MOUNT

Normal Saline (0.85% NaCl) OR Lugol’s Iodine (Staining method)
Glass slides
Cover slips
Pipettes
Gloves
Microscopes

PROCEDURE:

Completely mix the stool sample to form a homogeneous or heterogenous mixture depending on it constituent

With the help of an applicator stick,pick up the stool sample and make a smear on a clean microscope slide. Remove any gross fibers and particles.

Immediately before the specimen dries, add 1 or 2 drops of saline . Mix

Cover the specimen with a cover slip. (Note: Avoid air bubbles by drawing one edge of the coverslip slightly into the suspension and lowering it almost to the slide before letting it fall. The mount should be just thick enough that newspaper print can be read through the slide.)

If desired the coverslip (s) can be sealed using petroleum jelly and Paraffin oil or other suitable sealing preparations. Sealing the coverslip keeps organisms from moving when using oil immersion objectives and prevents the preparation from drying out.

Examination and Observation:

Examine the sample with the low power objective (10x) and low light. Begin at one corner of the smear and systematically examine successive adjacent swaths with the low power microscope. Low power examination includes entire area of 22 by 22 mm coverslip preparation (both saline and iodine).

If a parasite ,Egg is seen,use the 40X objectives.This makes the object more visible details and clearly with a good resolution. High dry power examination should include at least one third of the coverslip area (both saline and iodine).

Results:

Results from the direct smear examination should often be considered presumptive; however, some organisms could be definitely identified (Giardia lamblia cysts and Entamoeba coli cysts, helminth eggs, and larvae, Isospora belli oocysts). These reports should be considered “preliminary”, while the final report would be available after the results of concentration and permanent stained smear were available.

It should be noted that the use of iodine or iodine wet mount preparation kills the trophozoites forms of the organism hence not motile seen but also stain ova(bile salt stain)