Stool is one of the most common sample used in the laboratory 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 macroscopic and microscopical observation
Gastro-intestinal infestations (infections) by parasites (Protozoan/Helminthes) are primarily diagnosed by detecting live motile trophozoites (protozoans); cyst (inactive dormant stage of Protozoa) or eggs and larva (in case of Helminths) in stool.
Preparation of Microscopical slides from faecal specimen of the patients can be examined under low and high power microscope objectives. These etiological agents are identified based on their morphological and staining. Some may be bile stained or not bile stained; in the case of eggs of helminths 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.Normal saline is an Isotonic solution that maintains the osmotic pressures in the cells and doesn’t not lyse them.
The smear is then examined under the microscope with specific objectives X4,X10,X40. The Saline wet mount is used for the detection of parasitic trophozoites and cysts of protozoa, and eggs and larvae of helminths.
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 ,Enterobius vermicularis etc.
Lugol Iodine Staining Method
It is the same technique as the Saline method but here,Instead of normal saline,lugol iodine is used. Lugol’s iodine stains the nuclei of some cyst form of protozoans like Entamoeba histolystic making easy to view under the microscope.A common Disadvantage with this method is the ability of lugol iodine to kill the motile ( Trophozoites) form of these parasite or protozoans hence not advisable if intended to view motile trophozoites.
REAGENTS AND EQUIPMENT REQUIRED FOR WET MOUNT
- Normal Saline (0.85% NaCl) OR Lugol’s Iodine (Staining method)
- Glass slides
- Cover slips
- 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 or Lugol iodine depending on the method choosed. It is advisable to make 2 mount (Normal saline and stain slide) for more chances to recover parasites. 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 (As shown above) with the low power microscope. Low power examination includes entire area of 22 by 22 mm coverslip preparation (both saline and iodine).
- If a larva, trophozoite or 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 cover slip area (both saline and iodine).
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 no motile form can be seen but also stain ova(bile salt stain)
Uses of Direct Wet Mount
- To assess worm burden of patient
- To provide quick diagnosis of heavily infected specimen
- To check organism motility (primarily protozoan trophozoties)
- To diagnose organisms that might not been seen from permanent stain methods
Common Artifacts in Stool examination
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