Intestinal parasites are commonly predominant as the leading reason of diarrhea specifically between children and frequently linked with illness and death in developing countries. Parasites of diarrheal background are prevalent, infecting a significant percentage of the human population in 3rdworld nations. Most commonly in the subcontinent region. Faeces are the most repeated samples, are typically studied in the laboratory setup by microscopic examination, that is basically contains of Direct wet-mount preparation, concentration, and permanent staining.Microscopically examination of wet-mounts is the utmost broadly used technique for the identification of ova/eggs and cysts of parasites from stool samples. The precise analysis of the situation is organized by evaluation of protozoan cyst or trophozoite; helminthes ova/eggs or less commonly adult worms or larvae in the stool specimens and smears by microscopically examined.
Direct Wet Mount KOH Preparation:
Direct wet-mount preparation of stool specimen is widely used for the parasitological analysis in laboratories and also for the diagnosis of intestinal parasites by microscopically method. KOH – Potassium Hydroxide, is extensively used in the direct wet-mount preparation for different specimens for examples fungal elements and fungi, hair stuffs, skin scales, and nail scraping or other clinical samples with a small drops of 10% Potassium Hydroxide – KOH on a glass slide. The Potassium Hydroxide digests the pertinacious/resolute cellular debris and bleaches pigments, loosens their the sclerotic constituents but don’t damaging their the clinical constituents. So they give excellent view under microscopic examination.
Wet Mount Saline Method:
As the name suggested,
- It is made by the help of Normal saline by mixing a little amount around 2 milligram of stool specimens.
- Take a small drop of normal saline on a glass slide and cover with a cover slip. Normal saline is basically an Isotonic Solution which help to maintain the osmotic pressures inside the cells and doesn’t not lyse or destroy their structure.
- Under the microscope examine the smear, initially at low power 4X and then focus on at 10X objectives.
- The Saline wet-mount procedure is widely used to help for the detection and identification of parasitic cysts and trophozoite and ova and larvae of helminthes parasites.
- It is predominantly valuable for identification of live and motile trophozoite of parasites such as Entamoeba histolytica, Balantidium coli, G.lamblia and helminthes larva such S.stercolaris andova’s of ancylostoma hook worm, trichuris trichuira, Enterobius vermicularis etc.
Lugol Iodine Staining Method:
Lugol Iodine is the same method as the wet Saline preparation but there is only difference is despite the use of normal saline, we use lugol iodine in this method. Lugol iodine help to stains the some cyst’s nuclei of protozoa such as E.histolystica and easily seen under microscope. A basic disadvantage with this technique isthe capability of lugol iodine to kill the motile parasites trophozoite.
Procedure for Wet Mount:
- Fully mix the stool specimen to make a homogeneous or heterogeneous fusion dependent on its integral material.
- Take an applicator stick, take the stool specimen and make a good smear on a clean dry glass slide. Remove all gross fibers, debris and particles.
- Add 1 to 2 drops of Lugol iodine or saline according to the method of choice, and do it before the sample get dry. It is suitable to make 2 smear slides and it has extra advantage to recover parasites.
- Cover the sample with the help of cover slip. (Note: Avoid any air bubbling by draw one side of the cover slip marginally into the suspension and lowering it nearly to the slide before allowing it to fall).
- The quantity must be sufficient thick, a newspaper can easily be read through the glass slide.
- If preferred, the cover slip might be sealed with the help of Paraffin oil or Petroleum jelly. Sealing the cover slip preserves parasites from moving.
Interpretation, Examination and Results:
- Examine the sample initially at 4X and then focus at 10X by lower the light of microscope. Start to view from one edge of the specimen and observe consecutive bands. Low power at 10X examination comprises full area of 22/22 millimeter cover slip (both in saline and iodine preparation).
- If ova/egg, trophozoite or larva is seen, move the objectives lens at 40X and with the help of aperture increase the power from low to high. This will help, object further visible, clear and more detailed with a good resolution. High dry power examination must comprise at least 1/3rd of the cover slip part (both saline and iodine preparation).