Wheatley Trichrome Staining

Wheatley Trichrome Staining is a special permanent staining technique used in parasitology for the detection and identification of intestinal protozoans from stool samples. Protozoans are single-celled organisms that can cause various diseases in humans and animals, such as amoebiasis, giardiasis, and trichomoniasis. Wheatley Trichrome Staining helps to visualize the different morphological forms of protozoans, such as cysts and trophozoites, by staining them with different colors.

Wheatley Trichrome Staining is a modified version of Gomori`s original trichrome staining method, which was developed for staining tissues. Wheatley simplified and improved the procedure by adding fixative and hydration steps, and reducing the staining time. Wheatley Trichrome Staining produces uniformly stained smears of intestinal protozoans, yeasts, artifacts, and human cells, with a high contrast between the background and the organisms.

Wheatley Trichrome Staining is performed on stool samples that are either fresh or fixed in polyvinyl alcohol (PVA) or sodium acetate-acetic acid-formalin (SAF). The stool samples are smeared on microscopic slides and stained with a solution containing chromotrope 2R, light green SF, phosphotungstic acid, and acetic acid. The staining solution colors the cytoplasm of protozoan trophozoites blue-green or light purple, and the cysts more purple. The nuclei and inclusion bodies stain red with a tinged purple. The background stains green. The stained smears are then examined under a microscope at high magnification to identify the protozoans.

Wheatley Trichrome Staining is a sensitive and rapid technique that can detect small protozoans that may be missed during wet mount examination. It is also useful for identifying different yeast cells and human cells in stool samples. However, Wheatley Trichrome Staining has some limitations, such as not being able to stain helminth eggs and larvae, Cryptosporidium parvum, Cyclospora cayetanensis, and microsporidia spores. Therefore, other staining techniques may be required to complement Wheatley Trichrome Staining for a comprehensive diagnosis of intestinal parasitic infections.

The Wheatley Trichrome Stain is based on the use of two dyes that stain different components of the intestinal protozoa and the background material. The dyes are Chromotrope 2R and Light Green SF (or Fast Green FCF).

Chromotrope 2R is a red dye that stains the nuclear chromatin, chromatoid bodies, karyosomes, parasite eggs and larvae, bacteria, and ingested erythrocytes red to purple-red. This dye helps to highlight the internal structures of the protozoan cysts and trophozoites, as well as other organisms that may be present in the stool sample.

Light Green SF (or Fast Green FCF) is a green dye that stains the cytoplasm of the preserved cysts, trophozoites, and cellular constituents blue-green. This dye helps to contrast the protozoa from the background material, which also stains green but with a lighter shade. The blue-green color also helps to differentiate the protozoa from yeast cells, which stain red with Chromotrope 2R.

The Wheatley Trichrome Stain requires stool or fecal film samples that are permanently smeared on slides and fixed with Schaudinn’s solution or polyvinyl alcohol (PVA). The fixation preserves the morphology of the protozoa and prevents them from being distorted by the staining process. The staining procedure is simple and rapid, taking about 20 minutes to complete.

The Wheatley Trichrome Stain is very sensitive and can detect small protozoans that may be missed during wet mount examination. It also provides excellent detail and contrast for the identification of intestinal protozoa, such as Entamoeba histolytica, Giardia lamblia, Dientamoeba fragilis, Balantidium coli, and others.

Gomori’s trichrome stain is a staining technique that was developed by George Gomori in 1950 for the histological examination of tissues. It is based on the principle that different tissue components have different affinities for certain dyes and mordants. The stain uses a combination of chromotrope 2R (a plasma stain), fast green FCF (a connective tissue stain), and phosphotungstic acid (a mordant) in an acetic acid solution. The stain produces a color contrast between muscle fibers (red), collagen (green), nuclei (blue), and cytoplasm (purple).

The procedure for Gomori’s trichrome stain is as follows :

1. Bring sections to distilled water
2. Stain nuclei with Celestin Blue for 5 minutes
3. Rinse in distilled water
4. Stain in hematoxylin for 5 minutes
5. Wash well in running tap water for 5 minutes
6. Stain with Gomori’s stain for 15 minutes
7. Rinse with distilled water
8. Dehydrate, clear and mount.

The Gomori’s stain solution is prepared by mixing the following ingredients:

• Chromotrope 2R: 0.6 g
• Fast green FCF: 0.3 g
• Phosphotungstic acid: 0.6 g
• Glacial acetic acid: 1.0 ml
• Distilled water: 100 ml

The pH of the solution should be adjusted to 3.4 using 1 N NaOH and stored at room temperature.

Gomori’s trichrome stain is mainly used for the identification of muscle disorders, such as muscular dystrophy, inflammation, and degeneration. It can also be used to demonstrate parasites, fungi, and amyloid deposits in tissues .

Some advantages of Gomori’s trichrome stain are:

• It is a one-step staining procedure that does not require multiple solutions or rinses
• It produces a clear differentiation of tissue components with distinct colors
• It can be used on both frozen and paraffin sections

Some limitations of Gomori’s trichrome stain are:

• It may not be suitable for some types of tissues, such as nervous tissue or bone marrow
• It may not be compatible with some immunohistochemical or molecular techniques
• It may fade over time if not properly stored or mounted

To perform the Wheatley Trichrome Stain, you will need the following reagents:

• Trichrome stain solution: This is the main staining solution that contains two dyes: Chromotrope 2R and Light Green SF (or Fast Green FCF). Chromotrope 2R stains the nuclear chromatin, chromatoid bodies, karyosomes, parasite eggs and larvae, bacteria, and ingested erythrocytes red to purple-red. Light Green SF (or Fast Green FCF) stains the cytoplasm of preserved cysts, trophozoites, and cellular constituents blue-green . You can prepare this solution by mixing the following ingredients:

  • Chromotrope 2R: 0.6 g
  • Light Green SF (or Fast Green FCF): 0.3 g
  • Phosphotungstic acid: 0.7 g
  • Glacial acetic acid: 1.0 ml

  • Distilled water: 100.0 ml

    Add 1.0 ml of acetic acid to the dry components and allow it to ripen for 30 minutes at room temperature. Then add 100 ml of distilled water and the color of the solution changes to purple. Protect the solution from light and store it in a glass or plastic bottle at room temperature. The solution has a shelf life of 24 months.

• 70% ethanol plus iodine: This is used to fix and stain the PVA-preserved specimens. You can prepare a stock solution by adding iodine crystals to 70% ethanol until you obtain a dark solution (1-2 g/100 ml). To use, dilute the stock with 70% ethanol until a dark reddish-brown color or strong tea color is obtained.

• 70% ethanol: This is used to dehydrate and differentiate the specimens.

• 90% acid ethanol: This is used to remove excess stain from the specimens. You can prepare this by mixing 99.5 ml of ethanol with 0.5 ml of glacial acetic acid.

• 95% ethanol: This is used to dehydrate the specimens.

• 100% ethanol: This is used to dehydrate and clear the specimens.

• Xylene or xylene substitute: This is used to clear the specimens.

• Mounting medium (Permount): This is used to mount the coverslip on the slide.

• Immersion oil: This is used to examine the slide under oil immersion.

Make sure you label each reagent clearly and store them properly according to their specifications. You should also wear gloves and follow safety precautions when handling these reagents as they may cause eye, skin, and respiratory tract irritation.

Note: Put on hand gloves when performing the Wheatley Trichrome Stain.

• Remove the slide from the fixative (PVA or Schaudinn`s) and place it in 70% ethanol for 5 minutes.
• Place the slide in 70% ethanol plus iodine for 1 minute for fresh stool samples or 5-10 minutes for PVA-preserved air-dried samples.
• Place the slide in 70% ethanol for 5 minutes.
• Place the slide in Trichrome stain for 10 minutes.
• Place the slide in 90% acid ethanol (0.5% glacial acetic acid) for 1-3 seconds and drain the rack immediately and proceed to the next step. Do not allow the slide to remain in this solution.
• Dip the slide in 100% ethanol, this is the rinsing step.
• Place the slide in two changes of 100% ethanol for 3 minutes for each change.
• Place it in xylene or xylene substitute for 5-10 minutes.
• Mount the smear with a coverslip using a mounting medium such as Permount.
• Allow the smear to dry overnight or a minimum of 1 hour at 37°C.
• Examine the smear microscopically at 100X. Examine-in oil immersion at 200-300 fields.

Before staining the smears with Wheatley Trichrome Stain, the stool specimens need to be properly prepared. The stool preparation steps are as follows:

• Collect fresh stool samples in clean, sterile containers. Avoid contamination with urine, water, or soil.
• Place small amounts of stool (about the size of a match head) on clean glass slides using applicator sticks. Make very thin smears of the sample, spreading it evenly over an area of about 1 cm by 2 cm.
• Do not dry the smears and immediately place them in Schaudinn’s fixative for 30 minutes. Schaudinn’s fixative is a solution of mercuric chloride, potassium bichromate, and acetic acid that preserves the morphology and internal structures of the protozoa. Alternatively, you can use polyvinyl alcohol (PVA) as a fixative, which also preserves the glycogen and nuclear details of the protozoa. If you use PVA, you need to add 3 or 4 drops of it on the smeared slide and mix well. Then allow it to dry for several hours at 35° – 37°C or overnight at room temperature.
• Label the slides with patient identification and date of collection.

The stool preparation is crucial for obtaining clear and reliable results with Wheatley Trichrome Stain. If the smears are too thick, too dry, or not fixed properly, the staining quality will be compromised and the identification of protozoa will be difficult.

Note: Put on hand gloves when performing the Wheatley Trichrome Stain.

• Remove the slide from the Schaudinn’s fixative and place the slide in 70% ethanol for 5 minutes.
• Place the slide in 70% ethanol plus iodine for I minute for fresh stool samples or 5-10 minutes for PVA-preserved air-dried samples.
• Place the slide in 70% ethanol for 5 minutes.
• Place the slide in 70% ethanol for 3 minutes.
• Place the slide in Trichrome stain for 10 minutes.
• Place the slide in 90% ethanol and add acetic acid for 1-3 seconds and drain the rack immediately and proceed to the next step. Do not allow the slide to remain in this solution.
• Dip the smeared slide in 100% ethanol, this is the rinsing step.
• Place the slide in two changes of 100% ethanol for 3 minutes for each change.
• Place it in xylene for 5-10 minutes.
• Mount the smear with a coverslip using a mounting medium such as permount.
• Allow the smear to dry overnight or a minimum of 1 hour at 37°C.
• The smear is examined microscopically at 100X. Examine-in oil immersion at 200-300 fields.

After staining the fecal smears with Wheatley Trichrome Stain, the slides should be examined microscopically using the 100X objective and then under oil immersion. The stained smears should show excellent contrast and visualization of cellular details that aid in the identification of protozoa .

The typical staining reactions using the Wheatley Trichrome Stain method are as follows :

• The nuclear chromatin, chromatoid bodies, ingested erythrocytes and bacteria are purple to red-violet.
• The cytoplasm of preserved cysts and trophozoites is blue-green.
• The background material is green.

Some examples of intestinal protozoa that can be detected and identified by Wheatley Trichrome Stain are:

• Entamoeba histolytica: The trophozoites have a single nucleus with a small central karyosome and fine peripheral chromatin. The cytoplasm may contain ingested red blood cells. The cysts have one to four nuclei with the same characteristics as the trophozoites. The cytoplasm may contain chromatoid bodies with blunt ends.
• Giardia lamblia: The trophozoites have a pear-shaped body with two nuclei, four pairs of flagella and a ventral adhesive disk. The cysts have an oval shape with four nuclei arranged in a row or a cluster.
• Dientamoeba fragilis: The trophozoites have one or two nuclei with coarse chromatin granules and no peripheral chromatin. They have no cyst stage.
• Trichomonas hominis: The trophozoites have a pear-shaped body with four anterior flagella, an undulating membrane and a posterior flagellum. They have no cyst stage.

The presence of yeast cells, human cells and artifacts can also be observed by Wheatley Trichrome Stain. Yeast cells are oval or spherical structures that stain red-violet. Human cells such as red blood cells, white blood cells and epithelial cells stain red-violet with different shapes and sizes. Artifacts such as pollen grains, starch granules and fibers stain green or blue-green.

• Wheatley Trichrome Stain is not suitable for staining helminth eggs and larvae, which require other methods such as Kato-Katz or Kinyoun acid-fast stain.
• Wheatley Trichrome Stain requires high magnification and oil immersion to identify and examine protozoans, which can result in some morphological details being lost or missed .
• Wheatley Trichrome Stain cannot detect some protozoans that are too small or have a refractile wall, such as Cryptosporidium parvum, Cyclospora cayetanensis, and Microsporidia. These protozoans require special stains such as modified Ziehl-Neelsen or fluorescence microscopy.

• Wheatley Trichrome Stain can be affected by several factors that can cause poor contrast, over-staining, or under-staining of the specimens, such as fixation quality, staining time, decolorization time, and reagent quality. These factors should be carefully controlled and monitored to ensure optimal results.

  Applications of Wheatley Trichrome Stain

Wheatley Trichrome Stain is a useful technique for various purposes in parasitology and microbiology. Some of the applications are:

• For diagnosis of intestinal protozoa infections. Wheatley Trichrome Stain can detect and identify cysts and trophozoites of various protozoa such as Entamoeba histolytica, Giardia lamblia, Balantidium coli, Dientamoeba fragilis, and others . The stain provides detail and contrast that aid in the recognition of protozoan morphologies.
• For identification of parasitic morphologies. Wheatley Trichrome Stain can also differentiate between different forms of parasites such as eggs, larvae, and adult worms. The stain colors the nuclear chromatin, chromatoid bodies, karyosomes, parasite eggs and larvae, bacteria, and ingested erythrocytes red to purple-red.
• For identification of different yeast cells. Wheatley Trichrome Stain can also stain yeast cells that may be present in stool samples. The stain can help distinguish between different types of yeast such as Candida albicans, Cryptococcus neoformans, and others.
• For identification of human cells. Wheatley Trichrome Stain can also stain human cells that may be present in stool samples. The stain can help identify red blood cells, white blood cells, epithelial cells, and macrophages. The stain can also help detect inflammation, ulceration, or bleeding in the intestinal mucosa.

Wheatley Trichrome Stain is a simple and rapid staining procedure that produces uniformly stained smears of the intestinal protozoans, yeasts, artifacts, and human cells. It is a valuable tool for the diagnosis and identification of intestinal parasites and other microorganisms.

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