Direct Saline/ Iodine Wet Mount for Diagnosis of Intestinal Parasites

Last updated on June 7th, 2021

Stool is the most common specimen submitted to the diagnostic laboratory for the examination of parasitic infestations. The most commonly performed procedure in parasitology is the ova and parasite (O &P) examination.

Egg of Ascaris in Wet Mount
Egg of Ascaris in wet mount

Gastro-intestinal infestations by parasites (protozoan/helminths) are primarily diagnosed by detecting live motile trophozoites (for protozoans); cyst (inactive dormant stage of protozoa) or eggs (in case of helminths) in the stool.

Eggs of helminths are often easier to find and identify because of their size and their distinctive morphological features.

In some cases, the helminths (eggs/ova) are present in sufficient quantities to be found by direct examination of a small amount of feces, i.e. the direct smear.  Microscope slides made from fecal specimens of the patients can be examined under low and high dry power. These etiological agents are identified based on their morphological/staining (bile stained or not bile stained; in case of eggs of helminths) characteristics.

Trophozoites and Cyst of Giardia lamblia
Trophozoite (left) and Cyst of Giardia lamblia (right)

Saline wet mount is made by mixing a small quantity (about 2 mg) of feces in a drop of saline placed on a clean glass slide. The smear is then examined under a microscope. Saline wet mount is used for the detection of trophozoites and cysts of protozoa, and eggs and larvae of helminths.
It is particularly useful for the detection of live motile trophozoites of E. histolytica, Giardia lamblia, and Balantidium coli.

It is usually more efficient for laboratories to do a simple concentration to avoid overlooking parasites that may be present in very small numbers. A modification of the direct smear procedure, the Kato-Katz technique, is especially useful for field surveys aimed to detect schistosome or soil-transmitted nematode (roundworm, whipworm, and hookworm). It also gives an estimation of the intensity of infection.  

Uses of Direct Wet Mount

  1. To assess worm burden of patient
  2. To provide quick diagnosis of the heavily infected specimen
  3. To check organism motility (primarily protozoan trophozoites)
  4. To diagnose organisms that might not be seen from permanent stain methods

Reagents and equipment:

  1. Normal saline (0.85% NaCl);  Lugol’s Iodine
  2. Glass slides
  3. Cover slips
  4. Pipettes
  5. Gloves
  6. Microscope

Procedure

  1. With a wax pencil or other marker, write the patient’s name or identification number and the date at the left-hand end of a clean microscope slide.

2. Place a drop of saline in the center of the left half of the slide and place a drop of iodine solution in the center of the right half of the slide (Fig .1). (Note. Iodine wet mount preparations are most useful for protozoa, less so for helminths.)  

3. With an applicator stick or match, pick up a small portion of faeces (approximately 2 mg which is about the size of a match head) and add it to the drop of saline: add a similar portion to the drop of iodine. Mix the faeces with the drops to form suspensions (Fig. 2).

4. Cover each drop with a coverslip by holding the coverslip at an angle, touching the edge of the drop, and gently lowering the coverslip onto the slide so that air bubbles are not produced (Fig. 3).

Suitable Wet Mount Preparation (Img Source: DPDx)
Suitable Wet Mount Preparation (Img Source: DPDx)


Ideal preparations containing 2 mg of feces are uniform – not so thick that fecal debris can obscure organisms, nor so thin that blank spaces are present. The mount should be just thick enough that newspaper print can be read through the slide.

5. 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

Scanning the stained smear (Image source: dpdx)
Scanning the stained smear (Image source: dpdx)
  1. Examine the specimen with the low power objective (10x) and low light. Begin at one corner of the smear and systematically examine (either up and down or laterally) successive adjacent swaths with the low power microscope. Low power examination includes an entire area of 22 by 22 mm coverslip preparation (both saline and iodine).
  2. When a parasite-like object comes into view, switch to higher magnification to see the more detailed morphology of the object in question. It should be more closely examined and identified under high power(40x) objective. High dry power examination should include at least one-third of the coverslip area (both saline and iodine).

Results

Wet Mount Smear of Stool showing E. histolytica

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.

Limitations 

  1. Once Iodine is added to the preparation, the organism will be killed and motility will be lost.
  2. Oil immersion examination is not recommended (organism morphology not that clear)
About Acharya Tankeshwar 474 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

5 Comments

  1. Sir please provide pneumonics for bile stained anf non bile stained organisms as well as floating and non floating organisms. Thank you.

  2. Please I need to know how to prepare saturated salt solution using Nacl in 100ml of distil water

  3. How do i report Giardia cysts in a sample using a grading scale (+,++) and what is the reference material from which you got the reporting.

    • I got your message thank, please I need information on how to interpret the result on tabular form

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