Trichrome Staining Procedure ( CDC, 2011 )

It is generally recognized that stained fecal films are the single most productive means
of stool examination for intestinal protozoa. The permanent stained smear facilitates
detection and identification of cysts and trophozoites and affords a permanent record of
the protozoa encountered. Small protozoa, missed by wet mount examinations (of
either unconcentrated or concentrated samples) are often seen on the stained
smear. The Wheatley Trichrome technique for fecal specimens is a modification of
Gomoris original staining procedure for tissue. It is a rapid, simple procedure, which
produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast,
and artifact material.
Specimen:
The specimens usually consist of fresh stool or stool fixed in polyvinyl alcohol (PVA)
smeared on microscope slides and allowed to air dry or dry on a slide warmer at 60C.
Stool preserved in sodium acetate-acetic acid-formalin (SAF) or some of the one-vial
fixatives can also be used.
Reagents:
There are seven steps to this procedure, requiring the following solutions:
1. 70% Ethanol plus iodine: prepare a stock solution by adding iodine crystals to
70% alcohol until you obtain a dark solution. To use, dilute the stock with 70%
alcohol until a dark reddish brown color or strong tea color is obtained.
2. 70% Ethanol (twice)
3. Trichrome Stain: may be purchased commercially
4. 90% Acid Ethanol
90% ethanol

99.5 ml

Acetic acid (glacial)

0.5 ml

5. 95% ethanol
6. 100% ethanol (twice)
7. Xylene or xylene substitute (twice)
Procedure:
1. For PVA smears, place the slide in 70% ethanol plus iodine for 10 minutes. For
other fixatives, follow the manufacturer's instructions. Omit the iodine step for
preservatives that do not contain mercuric chloride.
2. Place slide in 70% Ethanol for 5 minutes.
3. Place in second 70% Ethanol for 3 minutes
4. Place in Trichrome stain for 10 minutes.
5. Destain in 90% ethanol plus acetic acid for 1 to 3 seconds.
6. Rinse several times in 100% ethanol.
7. Place in two changes of 100% ethanol for 3 minutes each.
8. Place in two changes of xylene or xylene substitute for 10 minutes.
9. Mount with coverslip using mounting medium (e.g., permount).
10. Examine the smear microscopically utilizing the 100 objective. Examine at least
200 to 300 oil immersion fields.

Quality Control:
A control slide of a known protozoan such as Giardia spp. from a PVA preserved
specimen should be included with each staining run. When the smear is thoroughly fixed
and the stain is performed correctly, the cytoplasm of protozoan trophozoites will have a
blue green color sometimes with a tinge of purple. Cysts tend to be slightly more
purple. Nuclei and inclusions (chromatoid bodies, red blood cells, bacteria) and CharcotLeyden crystals have a red color sometimes tinged with purple. Glycogen is dissolved by
the solvents and appears as a clear area in the organism. The background material
usually stains green providing a nice color contrast with the protozoa.