Stool: Routine Microscopy

Principle:
The direct microscopic examination of a stool specimen has several purposes:
to assess the worm burden of a patient
to provide a quick diagnosis of a heavily infected specimen
to check organism motility
to diagnose parasites that may be lost in concentration techniques
Specimen:
Any fresh stool specimen that has not been refrigerated is acceptable. Since trophozoites
within preserved specimens would exhibit no motility on a direct smear, it is not necessary
to perform this procedure on specimens submitted in preservatives (10% formalin, sodium
acetate-acetic acid-formalin, and various types of polyvinyl alcohol, single-vial preservative
collection systems).
Reagents:
The reagents indicated below are available commercially.
A. 0.85% NaCl
NaCl .....
water .................................. 100.0 ml
1. Dissolve the sodium chloride in distilled water in a flask or bottle by using a stirrer.
2. Distribute 10 ml of the solution into multiple tubes.
3. Label as 0.85% NaCl with an expiration date of 1 year.
4. Sterilize by autoclaving at 121oC for 15 min.
5. When cool, store at 4oC.
B.
Potassium iodide .............................. 10.0 g
Powdered iodine crystals ................ 5.0 g
Distilled water
1. Dissolve the potassium iodide and iodine crystals in distilled water in a flask or bottle.
2. Some excess crystals of iodine should remain on the bottom of the bottle.
3. Store in a brown, glass-stoppered bottle at room temperature.
4. The solution is ready for immediate use. Label as Lu
date of 1 year (the stock solution remains good as long as an excess of iodine crystals
remains on the bottom of the bottle).
5. Dilute a portion 1:5 with distilled water for routine use (working solution).
6. Place this working solution into a brown dropper bottle. The working solution should
resemble strong tea and should be discarded when it lightens in color (usually within
10 to 14 days).
Quality control:
A. Check the working iodine solution each time it is used or periodically (once a week)
1. The iodine should be free of any signs of bacterial and/or fungal contamination.
2. The color should be that of strong tea (discard if too light).
3. Protozoan cysts should contain yellow-gold cytoplasm, brown glycogen material, and paler refractile
nuclei. Human WBCs mixed with negative stool can be used as a QC specimen. The human cells
will stain with the same color as that seen in the protozoa.
B. The microscope should be calibrated, and the original optics used for the calibration should be in
place on the microscope. Post the calibration factors for all objectives on the microscope for easy access.
Although there is not universal agreement, the microscope should probably be recalibrated once each
year. This recommendation should be considered with heavy use or if the microscope has been bumped
or moved multiple times. If the microscope does not receive heavy use, then recalibration is not required
on a yearly basis.
C. Record all QC results.
Procedure:
A. Wear gloves when performing this procedure.

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 B. Place 1 drop of 0.85% NaCl on the left side of the slide and 1 drop of iodine (working solution) on the
right side of the slide.
C. Take a very small amount of fecal specimen (about the amount picked up on the end of an
applicator stick when introduced into the specimen), and thoroughly emulsify the stool in the
saline and iodine preparations (use separate sticks for each).
D. Place a coverslip (22 by 22 mm) on each suspension.
E. Systematically scan both suspensions with the 10X objective. The entire coverslip area should
be examined.
F. If you see something suspicious, use the 40X objective for more-detailed study. At least one-
third of the coverslip should be examined with the 40X objective, even if nothing suspicious has
been seen.

References:
1. A.L. L, editor. 9.3.3 Microscopic Examination of Fecal Specimens: Direct Smears.
In: Clinical Microbiology Procedures Handbook. 4th ed. Washington(DC): ASM Press;
2016. p. [Link]-[Link].

Stool microscopy exercises:

Findings:

Quadrinucleate cyst of
Entamoeba histolytica
seen

Saline mount Iodine mount

Checked by:

5
57
 2. Stool microscopy Exercise 2 (Date: )
13.04.2026 (Batch B)
14.04.2026 (Batch C)
18.04.2026 (Batch A)

Findings:
Cyst of Giardia lamblia
seen

Saline mount Iodine mount

Checked by:

3. Stool microscopy Exercise 3 (Date: )

13.04.2026 (Batch B)
14.04.2026 (Batch C)
18.04.2026 (Batch A)

Findings:

Saline mount Iodine mount

Checked by:

58
 4. Stool microscopy Exercise 4 (Date: )

13.04.2026 (Batch B)
14.04.2026 (Batch C)
18.04.2026 (Batch A)
)
Findings:

Saline mount Iodine mount

Checked by:

5. Stool microscopy Exercise 5 (Date: )

13.04.2026 (Batch B)
14.04.2026 (Batch C)
18.04.2026 (Batch A)

Findings:

Saline mount Iodine mount

Checked by:

5
59