Iodine Wet Mount Preparation: Introduction, Principle, Procedure, Result

Iodine Wet Mount Preparation: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Introduction of Iodine Wet Mount Preparation

D’Antoni’s Iodine stain is recommended for use in Iodine wet mount preparation for detection of protozoan cysts and helminth eggs and larvae of stool specimens.

Principle of Iodine Wet Mount Preparation

Iodine Wet Mount Preparation is a brown-colored preparation that stains the presence of pale refractile nuclei, yellowish cytoplasm, and brown glycogen material within the cyst. Helminth eggs and larvae are recognizable in wet mounts, though iodine staining occasionally masks the internal morphologic details.

Composition of D’Antoni’s Iodine Stain

Iodine: 1.5 g
Potassium Iodide: 1.0 g
Distilled water/Demineralized/ deionized Water: 100 ml

Test Requirements for Iodine Wet Mount Preparation

D’Antoni’s Iodine
Clean and grease gree glass slides
Coverslips (22×22 mm)
Dropper/Pipettes/applicator stick
Gloves
Microscope
Sample ( stool)
Waste bin
Tissue paper

Procedure of Iodine Wet Mount Preparation

Label a clean grease-free glass slide with a diamond pencil or other marker.
Put a drop of iodine solution in the center of the slide.
With an applicator stick or match, pick up a small portion of feces (approximately 2 mg which is about the size of a match head) and add it to the drop of iodine and mix it properly.
Cover the preparation with a coverslip by holding the coverslip at an angle, touching the edge of the drop, and gently lowering the coverslip onto the slide that minimizes air bubbles formation. Note: Ideal preparations containing 2 mg of feces are uniform – not so thick that fecal debris can masks 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.
Observe the specimen with the low power objective (10X) with low light. Begin at one corner of the smear and systematically examine successive adjacent swaths with the low power microscope. Low power examination includes an entire area of a coverslip preparation.
When a parasite-like object comes into view, switch to higher magnification to grasp the more detailed morphology of the object in question. It should be more closely examined and identified under the high power(40X) objective. A high dry power examination should include at least 1/3 of the coverslip area.

Result Interpretation of Iodine Wet Mount Preparation

Presence of active trophozoite/s killed: Non-motile retractile bodies

Cyst, oocyst, egg, inactive trophozite/s, larvae: Retractile bodies and finally focus at high dry power field

Protozoan cyst cytoplasm: yellow-gold

Glycogen material: Brown

Nucleus: paler refractile nuclei

Keynotes on Iodine Wet Mount Preparation

If desired the coverslip can be sealed using petroleum jelly or 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.
D’Antoni’s Iodine preparation is ready to use and no further dilution is required.
Store prepared D’Antoni’s Iodine stain at room temperature (20-25°C) and protect from light.
Handle Iodine carefully since it may cause an allergic skin reaction, eye, skin, respiratory tract irritation, reproductive, and also fetal effects.
Lugol’s Iodine stain may also be used for Iodine wet mount preparation but it needs further dilution prior to use.
Lugol’s Iodine stain has a shelf life of 26 weeks from the date of manufacture while the diluted working solution of Lugol’s Iodine has a shelf life of approximately 3 to 4 weeks.
Composition of Lugol’s Iodine stain-

Iodine Crystals:5.0 g

Potassium Iodide:10.0 g

Distilled water: 100 mL

Iodine wet mount preparations are most useful for protozoa, less so for helminths.
If the iodine stain lightens or if atypical results are observed with known controls then a fresh working solution should be prepared.
Preserved specimens do not require the iodine wet mount preparation since motile protozoa will not be visible.

Limitations of Iodine Wet Mount Preparation

Once Iodine is added to the preparation, the organism will be killed and no doubt, motility will be lost.
Oil immersion examination is not recommended and therefore organism morphology is not so clear.
The bile staining property of the helminths eggs can not be appreciated in the iodine preparation as it is already colored.
D’Antoni’s Iodine is a nonspecific, contrast dye, added to fresh, unpreserved stool samples to aid in the detection of helminth eggs and larvae and in differentiating protozoan cysts from host leukocytes.
A minimum of 3 stool samples, collected prior to treatment, is recommended for routine examination for intestinal parasites.
Specimens examination is unsuitable for patients who taking barium sulfate, mineral oil, bismuth, nonabsorbable antidiarrheal preparations, antimalarials, and some antibiotics.
Wet mount examinations alone cannot confirm the Identification of protozoan organisms and therefore for Definitive confirmation, a permanent stained smear is required.

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