Trichrome Staining for Fecal Smears

Stained fecal films are the single most productive means of stool examination for the detection of intestinal protozoan parasites and eggs of helminths. Because the refractive index of protozoan cysts and some helminth eggs are near that of water, staining procedures are required for optimal detection and for detailed study of their internal structures. Stained smears facilitate the detection of small protozoa missed by wet mount examination and afford a permanent record of encountered protozoa.

Cyst of Giardia lamblia

Permanent stains enhance the identification of Entamoeba histolytica and the detection of Giardia, two of the more commonly encountered and important protozoa. Two types of permanent stains are commonly used to visualize intestinal protozoa in fecal smears:

  1. The iron hematoxylin stain and
  2. The modified (Wheatley’s) Gomori’s Stains
Cyst of Entamoeba histolytica

Principle

The Wheatley trichrome technique for the fecal specimen is a modification of the original Gomari technique for tissue staining. It is a simple yet rapid technique that produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast, and artifact material.
Wheatly trichrome staining technique contains chromotrope 2R and light green SF as the primary staining agents. Trichrome stain is widely used in clinical parasitology laboratories. It is easy to perform, and good results are obtained with both fresh and polyvinyl alcohol (PVA)-preserved fecal material.

Reagents

ReagentsAmount
Chromotrope 2R0.6 g
Light green SF0.3 g
Phosphotungstic acid0.7 g
Acetic acid (glacial)1.0 mL
Distilled water100 mL
  1. Add 1.0 mL of acetic acid to the dry components.
  2. Allow the mixture to sit for 15-30 minutes at room temperature before adding 100 mL of distilled water.
  3. The stain should appear purple.
  4. When properly stored in glass or plastic bottles at room temperature, the shelf life is 2 years.

Iodine-Alcohol Solution (70% ethanol plus iodine)

  1. Add sufficient iodine crystals to 70% alcohol to make a dark, concentrated stock.
  2. At the time of use, dilute the desired amount to the stock solution with 70% alcohol until a port-wine working solution is obtained.
  3. The exact concentration is not critical.

Quality Control

It is recommended to use a control slide of a known protozoan such as Giardia spp. from a PVA preserved specimen with each staining run. Properly fixed and correctly stained smears produce a blue-green cytoplasm of protozoan trophozoites with a slight tinge of purple. Nuclei, inclusions (chromatoid bodies, red blood cells, bacteria), and Charcot-Leyden crystals appear red, often tinged with purple.

Trophozoite of Giardia lamblia

Cloudy preparations may result if dehydration is incomplete owing to failure to change alcohol solutions that may have become contaminated with water. Staining quality can be improved by draining slides between transfers from one reagent to another.

If staining is of poor quality, the smears may have been too thick or poorly fixed, or residual HgCl2 may not have been removed owing to the use of an alcohol-iodine mixture that was too weak.

Procedure

Wear gloves when handling stool specimens.

Preparation of the Smear

  1. With a small portion of the fresh stool specimen, prepare two smears on a microscope slide by using an applicator stick or brush
  2. Immerse smears immediately in Schaudinn’s fixative and allow to fix for a minimum of 30 minutes. Overnight fixation is preferred.
  3. If the specimen is liquid, mix several drops of fecal material with three or four drops of PVA on a glass slide and let it dry for several hours in a 37°C incubator.
  4. If the specimen is in PVA, pour some of the mixtures onto a paper towel to absorb out the PVA. Prepare slides of the material from the paper towels as described.

Staining Procedure

  1. After smears are properly fixed and dried, place slides in the 70% ethyl alcohol for 3 minutes.
  2. Place slides in alcohol-iodine working solution for 2-5 minutes.
  3. Wash with two changes of 70% alcohol, one for 5 minutes and one for 2-5 minutes.
  4. Place in trichrome-staining solution for 10 minutes.
  5. Place in acidified 90% ethyl alcohol for 5 seconds.
  6. Dip slides several times in 100% ethanol.
  7. Place in two changes of 100% ethyl alcohol.
  8. Remove alcohol with two changes of xylene or toluene for 2-5 minutes each.
  9. Add mounting medium and overlay with a #1 coverslip.
  10. Examine under oil immersion for parasitic forms.

Results

The cytoplasm of thoroughly fixed and well-stained cysts and trophozoites are blue-green, tinged with purple. The nuclear chromatin, chromatoid bodies, and ingested red blood cells appear red to red-purple. The background material appears green providing a nice color contrast with the protozoa.

Trophozoite of Entamoeba histolytica with ingested RBC

Procedure Notes

  1. If specimens are improperly fixed, protozoan forms may take a dirty red color.
  2. Incomplete removal of mercuric chloride (Schaudinn’s fixative) may result in the deposit of highly refractive granules that may interfere with the detection of parasitic forms. Be sure to change the 70% ethanol-iodine solution regularly so this does not happen.
  3. Smears that are too green may indicate inadequate removal of iodine by 70% ethanol. Lengthening the time of washing in 70% alcohol and frequent changes of solution will minimize this problem.

References and further readings

  1. Gracia L. Examination of fecal specimens. In Diagnostic Medical Parasitology. Washington, DC. American Society for Microbiology, 2007
  2. Stool Specimens – Staining Procedures. Centers for Disease Control and Prevention.

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

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