Wheatley Trichrome Staining – Principle, Procedure, Results and Uses

Wheatley trichrome staining is used for the microscopic examination of intestinal protozoa in fixed fecal smears. In this method, a thin smear of the stool specimen is prepared and fixed before staining with trichrome stain. The stained smear is briefly differentiated with acid alcohol, followed by dehydration and clearing. It is then mounted with a coverslip to obtain a permanent stained preparation. The slide is examined under the microscope using an oil-immersion objective.

Routine stool examination for intestinal parasites may include both wet preparations and permanent stained smears. Wet preparations are used to examine fresh stool specimens and observe motile trophozoites. In permanent stained smears, the internal structures of protozoan cysts and trophozoites can be seen more clearly than in unstained wet mounts. These stained slides can also be preserved for later examination.

The fixation and preservation of stool specimens affect the staining procedure. Stool preserved in polyvinyl alcohol (PVA) and certain other fixatives can be used for preparing permanent fecal smears. The pretreatment of the smear depends upon the type of fixative used. Iodine-alcohol treatment is required for certain mercury-containing fixatives, but it is not necessary for specimens preserved in non-mercury fixatives.

What is Wheatley Trichrome Staining?

Wheatley trichrome staining is a permanent differential staining method used for examining intestinal protozoa in fixed fecal smears. It is also known as Wheatley’s trichrome stain. The method is used for staining protozoan cysts and trophozoites. Their internal structures can be differentiated from the fecal material present around them.

In 1951, Wheatley developed this staining method by modifying Gomori’s trichrome procedure. Gomori’s method was originally used for staining tissue sections. Wheatley modified it for the examination of intestinal amoebae and flagellates in stool specimens.

Masson’s trichrome is a different staining method used for the differential staining of tissue components in histological sections, whereas Wheatley’s trichrome is used in intestinal parasitology.

Purpose of Wheatley Trichrome Staining

The following are the objectives of Wheatley trichrome staining:

1. To prepare permanent stained smears of stool specimens for the microscopic examination of intestinal protozoa.
2. To detect protozoan cysts and trophozoites present in fecal specimens.
3. The nuclei and cytoplasm of intestinal protozoa are stained so that their internal structures can be examined clearly.
4. The number of nuclei, karyosomes and peripheral chromatin can be observed in protozoan cysts.
5. To examine the cytoplasmic inclusions and chromatoid bodies present in intestinal protozoa.
6. Wheatley trichrome staining is used to differentiate protozoan cysts and trophozoites from yeast cells, fecal debris and other materials present in the smear.
7. To help in the morphological identification of intestinal amoebae and flagellates.
8. The stained slides can be preserved for future examination and reference.

Principle of Wheatley Trichrome Staining

Wheatley trichrome staining is based on the different affinities of protozoan cellular components for the dyes present in the staining solution. Chromotrope 2R is an acid dye which has an affinity for nuclear chromatin, karyosomes and chromatoid bodies. Light green or fast green dyes stain the cytoplasm of preserved cysts and trophozoites. These dyes produce different colours in the nuclear and cytoplasmic structures.

Phosphotungstic acid is present in the stain and helps in the differential staining of cellular components by affecting the uptake of the dyes. Its exact action is not completely understood. Acetic acid is also added to maintain an acidic medium for staining.

After staining, the excess dye is removed by briefly treating the smear with acid alcohol. This process is known as differentiation. It helps to remove loosely bound stain from the smear without removing the stain retained by the cellular structures.

The smear is then dehydrated using alcohol to remove water before clearing and mounting. The different staining of nuclear and cytoplasmic components allows the internal morphology of protozoan cysts and trophozoites to be examined under the microscope.

Reagents and Materials Required for Wheatley Trichrome Staining

Reagents Required

1. Wheatley trichrome stain (commercial ready-to-use solution or laboratory-prepared formulation).
2. Wheatley trichrome staining solution (classical formulation):
   • Chromotrope 2R – 6.0 g
   • Light Green SF – 1.5 g
   • Fast Green FCF – 1.5 g
   • Phosphotungstic acid – 7.0 g
   • Glacial acetic acid – 10.0 mL
   • Demineralized water – 990.0 mL
3. Iodine-alcohol solution (70% ethanol with iodine, for mercuric chloride-containing fixatives).
4. 70% ethanol.
5. 90% acid ethanol.
6. 95% ethanol.
7. Absolute ethanol (100%).
8. Xylene or xylene substitute.
9. Polyvinyl alcohol (PVA) fixative.
10. Schaudinn’s fixative.
11. Sodium acetate-acetic acid-formalin (SAF) fixative.
12. Permanent mounting medium.
13. Immersion oil.

Materials and Equipment Required

• Clean glass microscope slides.
• Coverslips.
• Stool collection containers.
• Disposable gloves.
• Applicator sticks.
• Glass or plastic transfer pipettes.
• Coplin jars or staining containers.
• Staining racks.
• Slide warmer.
• Forceps.
• Absorbent paper or paper towels.
• Light microscope with a 100× oil-immersion objective.

Preparation of Wheatley Trichrome Stain

Wheatley trichrome stain can be prepared in the laboratory using chromotrope 2R, light green SF, fast green FCF, phosphotungstic acid and glacial acetic acid.

Composition of Wheatley Trichrome Stain

The following reagents are required for the preparation of approximately 1 litre of Wheatley trichrome stain:

1. Chromotrope 2R – 6.0 g
2. Light Green SF – 1.5 g
3. Fast Green FCF – 1.5 g
4. Phosphotungstic acid – 7.0 g
5. Glacial acetic acid – 10.0 mL
6. Demineralized water – 990.0 mL

Preparation Procedure

The procedure for preparing Wheatley trichrome stain is given below:

1. Weigh the required amounts of chromotrope 2R, light green SF, fast green FCF and phosphotungstic acid.
2. Place all the dry ingredients in a clean glass beaker or flask.
3. Add 10 mL of glacial acetic acid slowly to the dry ingredients and mix thoroughly.
4. Allow the mixture to stand at room temperature for 30 minutes.
5. Add 990 mL of demineralized water and mix the solution thoroughly.
6. The prepared staining solution should be dark purple in colour.
7. Transfer the stain into a clean, labelled glass or plastic bottle. Keep the bottle tightly closed and protected from light at room temperature.
8. The staining solution should be tested using a known positive control smear before use.

Commercially prepared Wheatley trichrome stain is also available in ready-to-use form. These solutions do not require further preparation. The composition may vary depending upon the manufacturer.

Procedure of Wheatley Trichrome Staining

The procedure of Wheatley trichrome staining using the CDC method for PVA-preserved stool specimens is given below:

Preparation of Stool Smear

1. Collect the stool specimen in a clean, dry and leakproof container. Urine and water should not be present in the specimen.
2. Fresh stool specimens should be examined immediately or preserved after collection. For permanent staining, mix one part of fresh stool with three parts of PVA fixative.
3. If the specimen is already preserved in PVA, mix it well before preparing the smear.
4. Place 2–3 drops of the preserved stool specimen on a clean glass slide. Spread it with an applicator stick to prepare a thin smear covering an area of approximately 22 × 22 mm.
5. Allow the smear to air dry completely at room temperature. A slide warmer can also be used at 60°C for 5 minutes.
6. PVA helps the fecal material to adhere to the glass slide during staining.

Pretreatment of Fixed Smears

The pretreatment of smears prepared using mercury-containing PVA fixatives is given below:

1. The dried smear is placed in 70% ethanol containing iodine for 10 minutes.
2. Transfer the slide into 70% ethanol for 5 minutes.
3. Place it in a second change of 70% ethanol for 3 minutes to remove the remaining iodine.

Iodine-alcohol treatment is not required for mercury-free fixatives. The pretreatment of specimens preserved in SAF, zinc-based PVA and other mercury-free fixatives depends upon the type of fixative used. SAF-preserved specimens may require an adhesive such as albumin-glycerin for attachment to the slide.

Staining the Smear

The staining procedure is carried out after the pretreatment of the fixed smear.

1. Transfer the slide from the second 70% ethanol solution into Wheatley trichrome stain for 10 minutes.
2. Dip the slide in 90% acid ethanol for 1–3 seconds. It should not be left in the solution for more than 3 seconds.
3. Immediately rinse the slide several times in 100% ethanol to remove the excess acid-alcohol.
4. Place the slide in fresh 100% ethanol for 3 minutes and then transfer it to another change of 100% ethanol for 3 minutes.
5. The slide is then cleared in two changes of xylene or a compatible xylene substitute for 10 minutes each.

In the Remel Wheatley trichrome staining procedure, 90% ethanol is used for rinsing and dehydration. The slide is cleared in two changes of Xylene-S for 5–10 minutes each. These conditions should not be mixed with the CDC alcohol sequence without validation.

Mounting and Microscopic Examination

1. Remove the slide from the final xylene solution and drain the excess clearing reagent.
2. Add a drop of permanent mounting medium such as Permount to the stained smear.
3. Place a clean coverslip over the smear without trapping air bubbles.
4. Allow the mounting medium to set before examining the slide.
5. The stained smear is examined under the microscope using the 100× oil-immersion objective.
6. Examine at least 200–300 oil-immersion fields systematically.
7. Observe the nuclear and cytoplasmic structures of protozoan cysts and trophozoites for morphological identification.

Results and Interpretation of Wheatley Trichrome Staining

Observation

The observations of Wheatley trichrome staining under the microscope are given below:

The staining colour may vary depending upon the fixation of the specimen and the composition of the staining solution. Glycogen is removed by the solvents during staining and appears as a clear area within the organism.

Interpretation

Wheatley trichrome staining is used for the morphological identification of intestinal protozoa in permanent fecal smears. The cysts and trophozoites are examined according to their size, shape, number of nuclei and internal structures.

Giardia duodenalis trophozoites are pear-shaped and usually show two nuclei. Median bodies, flagella and the ventral sucking disc may also be observed. The cysts are oval in shape and contain two or four nuclei. Internal fibrils can be seen in stained cysts.

In the Entamoeba histolytica/dispar complex, the trophozoites usually contain a single nucleus with a small central karyosome and fine peripheral chromatin. Mature cysts generally have four nuclei. Chromatoid bodies may also be present in the cytoplasm.

The presence of protozoan cysts or trophozoites is determined by their morphological characteristics. However, E. histolytica, E. dispar and E. moshkovskii cannot be reliably differentiated by routine microscopic examination. A species-specific test such as PCR is required to distinguish these organisms.

Quality Control of Wheatley Trichrome Staining

The following are the quality control measures for Wheatley trichrome staining:

• A known positive control slide containing intestinal protozoa, such as Giardia spp., should be included with each staining run. The control smear can be prepared from a PVA-preserved stool specimen.
• The staining solution should be checked when a new batch is prepared or a new lot of stain is used. Positive control staining is also recommended at least once every week.
• If protozoan-positive specimens are not available, PVA-preserved stool containing leukocytes or epithelial cells can be used to check the staining reaction.
• The cytoplasm of trophozoites should appear blue-green, sometimes with a purple colour. Protozoan cysts usually stain more purple.
• Nuclear chromatin, karyosomes and chromatoid bodies should stain red to purple-red. The background fecal material is usually green.
• The smear should be properly fixed and completely dried before staining. Poorly fixed protozoa may remain unstained or appear mainly red. Inadequately dried smears may wash off during staining.
• The iodine-alcohol solution used for mercury-containing fixatives should have a dark reddish-brown colour. Incomplete removal of mercury may produce dark crystalline granules in the smear.
• The 70% ethanol rinses should remove the iodine from the smear before staining. Remaining iodine may cause the smear to appear predominantly green.
• Differentiation with 90% acid ethanol should not exceed 3 seconds. Prolonged treatment may cause poor differentiation of the internal structures.
• The alcohol and xylene solutions should be changed when they become contaminated or cloudy. Water present in the xylene requires replacement of the 100% ethanol and xylene.
• Staining containers should be kept covered to prevent evaporation. Drain the slides between solutions to reduce the transfer of reagents from one container to another.
• The stained control slide should be examined using a properly calibrated microscope. The smear is also checked for its attachment to the slide and the staining of nuclear structures.
• Quality control results and any corrective action taken should be recorded. Patient results should not be reported when the control staining is unsatisfactory.

Applications of Wheatley Trichrome Staining

The following are the applications of Wheatley trichrome staining:

• It is used to detect intestinal protozoan cysts and trophozoites in fixed fecal smears.
• To identify intestinal amoebae by examining their nuclear and cytoplasmic structures.
• The cysts and trophozoites of Giardia duodenalis can be examined in permanently stained stool preparations.
• It is used to observe the number of nuclei, karyosomes, peripheral chromatin and chromatoid bodies present in protozoan cysts and trophozoites.
• Small intestinal protozoa which may be missed during wet-mount examination can be detected in the stained smear.
• To differentiate protozoan forms from yeast cells, human cells and other materials present in the fecal specimen.
• It is used in the laboratory examination of stool specimens for suspected intestinal protozoal infections, along with wet-mount and concentration methods.
• The stained slides can be preserved for future examination and reference.
• Permanent stained smears are also used for teaching the morphology of intestinal protozoa and comparing their internal structures.

Limitations of Wheatley Trichrome Staining

The following are the limitations of Wheatley trichrome staining:

• Wheatley trichrome staining is mainly used for the examination of intestinal protozoa. It is not suitable for the routine detection of helminth eggs and larvae.
• The oocysts of Cryptosporidium, Cyclospora and Cystoisospora are not reliably detected by this staining method. Special staining methods such as modified acid-fast staining are required.
• Microsporidian spores are difficult to detect using routine Wheatley trichrome staining. Modified trichrome staining is used for their examination.
• The method cannot differentiate Entamoeba histolytica, E. dispar and E. moshkovskii based on their morphology. Molecular or species-specific antigen tests are required for identification.
• The motility of protozoan trophozoites cannot be observed in permanently stained smears because the organisms are fixed before staining.
• Improper fixation may damage the internal structures of protozoa. Such organisms may remain unstained or appear predominantly red.
• Thick fecal smears may prevent the clear observation of nuclear and cytoplasmic structures. Inadequately dried smears may also wash off during staining.
• Prolonged differentiation with acid alcohol may remove excessive stain from the smear and affect the observation of cellular structures.
• The staining results depend upon the type of fixative used. Some mercury-free fixatives may give poor staining or preservation of protozoan morphology.
• A small number of protozoa present in the stool specimen may not be detected during microscopic examination. A negative result from a single specimen does not exclude intestinal protozoal infection.
• Microscopic identification requires an experienced laboratory worker because protozoan cysts and trophozoites may resemble yeast cells, human cells and other fecal materials.
• The use of mercury-containing PVA or Schaudinn’s fixatives requires special handling and waste disposal due to the presence of mercuric chloride.

Precautions of Wheatley Trichrome Staining

The following are the precautions of Wheatley trichrome staining:

• Collect the stool specimen in a clean, dry container. The specimen must not be contaminated with urine or water.
• Fresh stools should be examined or preserved as soon as possible after collection. Delayed fixation can damage the internal morphology of the protozoa.
• Prepare a thin and even fecal smear on a clean glass slide. Thick smears are not ideal as they make it very difficult to see the nuclear and cytoplasmic detail.
• Allow the PVA preserved smear to dry completely before staining (otherwise the faeces will just wash off the slide).
• Iodine alcohol is required for mercury fixatives. It should not be used on specimens preserved in mercury free fixatives.
• Change the iodine-alcohol solution regularly. If the solution becomes saturated then mercury compounds may remain in the smear.
• The staining time should be as per the method employed. In the CDC method the smear is stained with Wheatley trichrome for 10 minutes.
• Dip the slide in 90% acid ethanol for just 1–3 seconds. Any longer and you risk removing too much stain from the cells.
• Rinse the slide straight away in 100% ethanol following differentiation to remove the residual acid-alcohol.
• Use fresh ethanol solutions for dehydration as the water content in the alcohol may affect the clearing and mounting of the stained smear.
• Don’t transfer any excess reagent from one staining container to another. Keep the staining containers covered when they’re not in use.
• Handle xylene and alcohol in a well ventilated area away from any open flame. Gloves, a lab coat and safety glasses are all required during the staining.
• Mercury containing fixatives need to be handled with care. The fixatives themselves and any contaminated material must be disposed of in accordance with laboratory chemical waste procedures.
• A known positive control smear must be included with each staining run. Patient results must not be reported if the control smear is poor.

References

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2. Flournoy, D. J., McNabb, S. J. N., Dodd, E. D., & Shaffer, M. H. (1982). Rapid trichrome stain. Journal of Clinical Microbiology, 16(3), 573–574. https://doi.org/10.1128/jcm.16.3.573-574.1982
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5. Ridley, J. W. (2012). Parasitology for medical and clinical laboratory professionals. Delmar Cengage Learning. https://books.google.com/books?id=7i_m5OwUi2YC
6. Shoaib, S., Hafiz, A., & Tauheed, S. (2002). Role of trichrome staining techniques in the diagnosis of intestinal parasitic infections. Journal of the Pakistan Medical Association, 52(4), 152–154. https://pubmed.ncbi.nlm.nih.gov/12174479/
7. Wheatley, W. B. (1951). A rapid staining procedure for intestinal amoebae and flagellates. American Journal of Clinical Pathology, 21(10), 990–991. https://doi.org/10.1093/ajcp/21.10_ts.990