TRICHROME STAIN PROCEDURE

PRINCIPLE

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 Gomori’s 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 PVA or MIF smeared on microscope slides and allowed to air dry or dry on a slide warmer at 60°C. Stool preserved in 10% -formalin 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
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
7. Xylene or xylene substitute

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 Charcot-Leyden 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.

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.

INTERPRETATION

The cytoplasm of organisms stains blue-green tinged with purple, whereas nuclear chromatin, chromatoid bodies, erythrocytes, and bacteria stain red or purplish-red. Background material will stain green to blue-green. Organisms undergoing degeneration take a pale stain, and incompletely fixed organisms may stain red. Yeasts and molds may stain either green or red. Cryptosporidium and Cyclospora oocysts are not satisfactorily stained in Trichrome smears and can easily be overlooked; sometimes they appear as “ghosts” in this stain, which can serve as a clue to their presence.

REFERENCE

Ash, Lawarence and Thomas Orihel. Atlas of Human Parasitology. ASCP. 4th Edition. 1997.

1 Response to "TRICHROME STAIN PROCEDURE"

  1. Rahman February 11, 2011 at 7:05 AM
    assalamualaikum
    mo tanya, kalau staining protozoa terutama yang siliata bagusnya pake apa ya? bisa beri saya penjelasan staining untuk common protozoa ?

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