Iron-Hematoxylin Staining

Iron-hematoxylin staining is a traditional staining technique that is commonly used for the detection of intestinal protozoans, such as amoebae, ciliates and flagellates. It is especially useful for demonstrating the nuclear structures of these parasites, which are important for their identification and differentiation. Iron-hematoxylin staining was the stain used for most of the original morphological descriptions of intestinal protozoa found in humans .

Iron-hematoxylin staining is based on the formation of a complex between hematein, a derivative of hematoxylin, and iron salts, which acts as a mordant. The iron-hematoxylin complex is a basic dye that stains the nuclei and other acidic structures of the parasites dark purple to black, while the cytoplasm remains lightly stained or unstained. The contrast between the nuclear and cytoplasmic staining allows for easy observation and quantification of the protozoan morphology under a light microscope.

Iron-hematoxylin staining can be applied to fresh, PVA-preserved or SAF-preserved stool specimens. PVA (polyvinyl alcohol) and SAF (sodium acetate-acetic acid-formalin) are two common fixatives that are used to preserve stool specimens for parasitological examination. The advantage of using preserved specimens is that they can be stored for a longer time and transported without refrigeration. However, some modifications in the staining procedure are required for PVA-preserved and SAF-preserved specimens, such as using iodine-alcohol to remove the fixative and adding Mayer`s albumin to improve the adherence of the smear to the slide.

Iron-hematoxylin staining is a relatively simple and inexpensive technique that can be performed manually or automatically. However, it requires careful attention to the quality and preparation of the reagents, the fixation and dehydration of the specimens, and the timing and washing of the staining steps. If these factors are not controlled properly, the staining results may be distorted or inconsistent.

Iron-hematoxylin staining is one of the most widely used stains in parasitology because it provides permanent slides that can be stored and reviewed for reference. It also offers high clarity and specificity for the differentiation of intestinal protozoans, which may not be achieved by other stains such as trichrome or iodine. However, iron-hematoxylin staining also has some limitations, such as being slow and time-consuming, requiring hazardous chemicals such as mercuric chloride and xylene, and being unable to distinguish some species or genera of protozoans that have similar nuclear structures.

In this article, we will explain the principle behind iron-hematoxylin staining, describe the objectives and reagents used in this technique, outline the procedure of iron-hematoxylin staining for different types of specimens, discuss the result and interpretation of iron-hematoxylin staining, and highlight the applications, advantages and limitations of this technique. We hope that this article will help you understand and perform iron-hematoxylin staining more effectively and confidently in your laboratory practice.

Iron-hematoxylin staining is based on the formation of a complex dye called iron-hematoxylin, which is derived from the oxidation of hematoxylin by ferric ions. Hematoxylin is a natural dye extracted from the heartwood of the logwood tree. It has a purple-blue color and can bind to acidic substances such as DNA and RNA. Ferric ions act as a mordant, which is a substance that enhances the binding of the dye to the tissue components. Ferric ions also oxidize hematoxylin to hematein, which is more soluble and stable than hematoxylin.

The iron-hematoxylin complex has a dark brown to black color and can stain the nuclei and other basophilic structures of the cells and parasites. The intensity and specificity of the staining depend on several factors, such as the pH, temperature, duration, and concentration of the solutions. The staining procedure involves several steps: fixation, mordanting, staining, differentiation, dehydration, clearing, and mounting.

Fixation is the process of preserving the tissue or specimen from autolysis and decay. It also hardens the tissue and makes it more permeable to the staining reagents. In iron-hematoxylin staining, Schaudinn`s solution is used as a fixative for fresh samples, while polyvinyl alcohol (PVA) or sodium acetate-acetic acid-formalin (SAF) are used for preserved samples. Schaudinn`s solution contains mercuric chloride, which is toxic and corrosive, and must be removed by washing with alcohol before staining.

Mordanting is the process of treating the tissue or specimen with a substance that enhances the binding of the dye. In iron-hematoxylin staining, ferric ammonium sulfate (iron alum) is used as a mordant. It forms a complex with hematein called iron-hematein or lake. The mordanting step also serves as a differentiation step, which is the process of removing excess or unwanted dye from the tissue or specimen. The duration and concentration of the mordant-differentiating solution affect the contrast and clarity of the staining.

Staining is the process of applying the dye to the tissue or specimen. In iron-hematoxylin staining, hematoxylin solution is used as a stain. It contains hematoxylin dissolved in water with a small amount of acid to prevent oxidation. The stain is applied for 4 to 5 minutes to allow sufficient penetration and binding of the dye.

Differentiation is the process of removing excess or unwanted dye from the tissue or specimen. In iron-hematoxylin staining, differentiation is achieved by washing with tap water for 10 minutes. This removes any unbound or loosely bound dye and also enhances the color development by alkalizing the tissue.

Dehydration is the process of removing water from the tissue or specimen. In iron-hematoxylin staining, dehydration is achieved by immersing the slides in graded concentrations of ethanol (70%, 95%, and 100%). This prepares the tissue for clearing and mounting.

Clearing is the process of replacing the dehydrating agent with a substance that has a similar refractive index as glass. In iron-hematoxylin staining, clearing is achieved by immersing the slides in xylene for 10 minutes. This removes any traces of ethanol and makes the tissue transparent.

Mounting is the process of applying a cover slip over the slide with a suitable medium that preserves and protects the tissue. In iron-hematoxylin staining, mounting is achieved by applying permount (a synthetic resin) over the slide and placing a cover slip on top. This seals and stabilizes the stain.

The result of iron-hematoxylin staining is a permanent preparation that can be examined under a microscope at different magnifications. The nuclei and other basophilic structures of cells and parasites appear dark brown to black against a light background. The stain can be used to identify and differentiate various intestinal protozoans based on their morphology and nuclear characteristics.

Iron-hematoxylin staining is a traditional staining technique that is commonly used for the detection and identification of intestinal protozoans. The main objectives of this technique are:

• To demonstrate the presence of intestinal parasites from a given stool sample. Iron-hematoxylin staining can reveal the morphological features of protozoal cysts and trophozoites, such as nuclei, chromatoid bodies, cytoplasm, and ingested erythrocytes. These features can help to differentiate various species of protozoa, such as Entamoeba, Giardia, Dientamoeba, Cyclospora, and Cryptosporidium .
• To differentiate protozoal parasite morphologies. Iron-hematoxylin staining can provide high clarity and contrast for the observation of protozoal structures. The iron-hematoxylin compound acts as a basic dye that stains the nuclear and intracellular structures dark purple to black, while the cytoplasm stains violet . This allows for the recognition of distinctive shapes, sizes, and arrangements of protozoal nuclei and other organelles.
• To prepare permanent stained slides for reference and documentation. Iron-hematoxylin staining is a durable and stable technique that can produce long-lasting slides that can be stored and examined later. This is important for quality control, verification, and research purposes .

Iron-hematoxylin staining is a simple and effective technique that can be used with fresh or preserved stool specimens. It is adequate for routine diagnosis of intestinal protozoans and offers high resolution and specificity for their morphological identification .

The reagents used in iron-hematoxylin staining are:

• Mercuric chloride: This is a fixative that preserves the morphology of the parasites and prevents autolysis. It is also a mordant that enhances the binding of hematoxylin to the tissue components.
• Ethyl alcohol: This is a dehydrating agent that removes water from the tissue sections and prepares them for clearing and mounting.
• Hematoxylin: This is a natural dye extracted from the logwood tree that stains the nuclei and other basophilic structures blue-violet. Hematoxylin is colorless and needs to be oxidized to hematein, which is the active dye molecule.
• Iodine: This is an oxidizing agent that converts hematoxylin to hematein. It also acts as a differentiator that removes excess hematoxylin from the tissue sections.
• Alcoholic iodine: This is a mixture of iodine and ethyl alcohol that is used to treat PVA-preserved samples before staining. It removes PVA from the tissue sections and allows hematoxylin to penetrate better.
• Ferric ammonium sulfate (Iron alum): This is a mordant that forms a complex with hematein called iron-hematoxylin, which is a stable and sharp basic dye. Iron alum also acts as a differentiator that removes excess iron-hematoxylin from the tissue sections.
• Hydrochloric acid: This is an acidifier that lowers the pH of the iron-hematoxylin solution and enhances its staining ability. It also acts as a differentiator that removes excess iron-hematoxylin from the tissue sections.
• Xylene: This is a clearing agent that displaces alcohol from the tissue sections and makes them transparent for mounting.
• Mounting media: This is a substance that covers and protects the stained tissue sections on the slides. It also enhances the optical properties of the stain and prevents fading.

These reagents are used in different steps of the staining procedure, depending on whether the samples are fresh, PVA-preserved or SAF-preserved. The reagents should be prepared according to the manufacturer`s instructions or standard protocols and stored properly to ensure their quality and stability .

The procedure of iron-hematoxylin staining involves several steps that vary depending on the type of specimen (fresh, PVA-preserved or SAF-preserved). The general steps are as follows:

1. Fixation: This step is done to preserve the morphology and structure of the parasites and to prevent their degradation. For fresh specimens, Schaudinn`s solution is used as a fixative for at least 30 minutes. For PVA-preserved specimens, iodine-alcohol is used to remove excess PVA. For SAF-preserved specimens, SAF itself acts as a fixative and no additional step is required.
2. Dehydration: This step is done to remove water from the specimens and to prepare them for staining. Ethanol of different concentrations (70%, 95% and 100%) is used to dehydrate the specimens for 5 minutes each.
3. Staining: This step is done to impart color to the parasites and to make them visible under the microscope. Iron hematoxylin working solution is used as a stain for 4 to 5 minutes. The stain reacts with the mordant (ferric ammonium sulfate) to form a ferric lake (iron-hematoxylin) that binds to the nuclear structures of the parasites and stains them dark purple to black.
4. Washing: This step is done to remove excess stain and to prevent over-staining. Smoothly running tap water is used to wash the specimens for 10 minutes.
5. Clearing: This step is done to remove any residual ethanol and to make the specimens transparent. Xylene is used as a clearing agent for 5 minutes in two changes.
6. Mounting: This step is done to protect the specimens and to facilitate their examination. Permount (a mounting media) is used to cover the specimens with a coverslip.

The procedure of iron-hematoxylin staining can be summarized in the following table:

After completing the procedure, the specimens are ready to be examined under a microscope at 100x and also under oil immersion before reporting.

To perform the iron-hematoxylin staining technique, you will need to prepare the following solutions:

• Schaudinn`s solution: This is a fixative solution that contains mercuric chloride, ethyl alcohol and acetic acid. It is used to preserve the morphology and structure of the protozoa in the stool samples. To prepare Schaudinn`s solution, you will need to add a saturated solution of mercuric chloride to 80 ml of distilled water, then add 20 ml of ethyl alcohol (95%) and 3 ml of acetic acid. Mix well and store in a dark bottle at room temperature. Schaudinn`s solution is toxic and corrosive, so you should handle it with care and dispose of it properly.

• Iodine solution: This is a staining solution that contains iodine and ethyl alcohol. It is used to stain the cytoplasm of the protozoa in a brown color. To prepare iodine solution, you will need to add 2 ml of tincture of iodine (2%) to 98 ml of ethyl alcohol (95%). Mix well and store in a dark bottle at room temperature.

• The mordant-differentiating solution: This is a solution that contains iron alum (ferric ammonium sulfate), hydrochloric acid and distilled water. It is used to form a complex with hematoxylin and differentiate the nuclear structures of the protozoa in a dark purple to black color. To prepare the mordant-differentiating solution, you will need to dissolve 2 g of iron alum in 98 ml of distilled water, then add 1 ml of hydrochloric acid. Mix well and store in a dark bottle at room temperature.

• Hematoxylin solution: This is a staining solution that contains hematoxylin and distilled water. It is used to stain the nuclear structures of the protozoa in a blue color. To prepare hematoxylin solution, you will need to dissolve 0.3 g of hematoxylin in 100 ml of distilled water. Mix well and store in a dark bottle at room temperature.

Note: The iron-hematoxylin stain and the mordant-differentiating solution are stable individually for 12 months when stored at the right temperature. However, once you mix them together to form the working solution, you should use it within 7 days. You should also filter the stains periodically using a filter paper to remove any debris that may be present and always seal the container to avoid evaporation.

PVA and SAF are two types of fixatives that can be used to preserve stool specimens for iron-hematoxylin staining. PVA stands for polyvinyl alcohol, which is a liquid fixative that contains PVA plastic powder as an adhesive to glue the fecal material onto the glass slide. SAF stands for sodium acetate-acetic acid-formalin, which is a liquid medium that preserves the intestinal parasites without distorting their morphology. Both PVA and SAF samples can be stained with iron-hematoxylin, but SAF samples may produce better results .

The preparation of PVA and SAF samples for iron-hematoxylin staining involves the following steps:

• For PVA-preserved samples, the stool specimen should be mixed with PVA fixative and allowed to fix for at least 30 minutes. Then, some of the mixture should be applied to a paper towel and left to stand for 2-3 minutes to absorb the excess PVA. Next, a thin smear of the stool sample should be made on a glass slide using an applicator stick and dried in an incubator at 37°C or overnight at room temperature. Finally, iodine-alcohol solution should be added to the dry smear before staining .

• For SAF-preserved samples, the stool specimen should be mixed and strained through a wire gauze into a centrifuge tube and centrifuged for 1 minute at 500xg. Then, the supernatant should be decanted and the sediment should be resuspended in saline (0.85% NaCl) and centrifuged again for 10 minutes at 500xg. A thin smear of the sediment should be made on a glass slide coated with Mayer`s albumin and dried at room temperature for 30 minutes. Optionally, Schaudinn`s solution can be used to postfix the smear before staining .

After preparing the PVA and SAF samples, they can be stained with iron-hematoxylin following the same procedure as fresh samples.

Depending on the type of sample, different steps are required to prepare and stain the slides with iron-hematoxylin. Here are the procedures for each type of sample:

Fresh samples

• On clean microscopic glass slides, prepare thin smears of the stool samples using an applicator stick and while still wet, dip the slides into Schaudinn’s solution, for at least 30 minutes. This is called the Schaudinn’s fixation.
• Place the Schaudinn’s fixed smear in 70% ethyl alcohol for 5 minutes to remove excess fixative.
• Rinse the slide in smoothly flowing tap water at least three times.
• Place slide in iron hematoxylin working solution for 4 to 5 min. The working solution is prepared by mixing equal parts of the iron hematoxylin mordant and the iron hematoxylin stain. The working solution should be prepared fresh weekly.
• Wash slides with smooth running tap water for 10 min.
• Immerse the slides in 95% ethyl alcohol for 5 minutes.
• Place the slides in 100% ethanol for 5 minutes.
• Immerse the slides in two changes in xylene for 5 minutes for each change.
• Add a permount (Mounting Media is formulated for mounting and storing long-term slides) to the stain and cover with a coverslip.
• Examine the smear under a microscope at 100x and also under oil immersion before reporting.

PVA-preserved samples

• PVA-preserved samples should be allowed to fix for at least 30 min and mix the content with two applicator sticks.
• Pour some of the above PVA mixtures to a paper towel and allow to stand for 2-3 minutes for effective absorption of PVA.
• Apply some of the stool samples on the paper towel to the glass slides using an applicator stick and allow it to dry in an incubator at 37°C or overnight at room temperature to ensure the smear is completely dry.
• To the dry smear, add iodine-alcohol. This removes mercuric chloride from Schaudinn’s and PVA-fixed smears.
• Place in 70% ethanol for 5 minutes and wash in smooth running tap water for 10 minutes .
• Place slide in iron hematoxylin working solution for 4 to 5 min .
• Wash slides with smooth running tap water for 10 min .
• Immerse the slides in 70% ethanol for 5 minutes .
• Then immerse the slides again in 95% ethanol for another 5 minutes .
• In two changes, place the slides in 100% ethanol for 5 minutes for each change .
• Immerse the slides in two changes in xylene for 5 minutes for each change .
• Add a permount (Mounting Media is formulated for mounting and storing long-term slides) to the stain and cover with a coverslip .
• Examine the smear under a microscope at 100x and also under oil immersion before reporting .

SAF-preserved samples

• A SAF-stool sample is mixed and strained through a wire gauze to a 15ml centrifuge tube and centrifuge for 1 minute at 500xg.
• Decant the supernatant and centrifuged for 10min at 500xg and collect sediments of 0.5ml to 10ml. If necessary, adjust by repeating step 1 or by resuspending the sediment in saline (0.85% NaCl) and removing part of the suspension.
• Prepare a smear on a clean glass slide, from the sediments by adding a drop of Mayer’s albumin on the smear. Then add a drop of the SAF-preserved sample sediments and allow them to dry at room temperature for 30 minutes before staining.
• You can also postfix with Schaudinn’s solution before staining before initiating a trichrome stain procedure with 70% alcohol rinse then adding iodine- alcohol.
• After drying, place the smear in 70% alcohol.
• Place the slides in 70% ethanol-iodine solution for 5 to 10 minutes to remove mercuric chloride.
• Place slide in 70% ethyl alcohol for 5 minutes.
• Wash slide in smoothly running tap water for 10 minutes.
• Place the slides in iron hematoxylin working solution for 4 to 5 min.
• Wash the slides in smoothly running tap water for 10 minutes.
• Place the slides in 70% ethanol for 5 min.
• Then, place the slides in 95% ethanol for 5 min.
• Followed by immersing the slides in 100% ethanol in two changes for 5 minutes for each change.
• Immerse the slide in two changes of xylene for 5 minutes for each change.
• Add a permount (Mounting Media is formulated for mounting and storing long-term slides) to the stain and cover with a coverslip.
• Examine the smear under a microscope at 100x and also under oil immersion before reporting.

The result of iron-hematoxylin staining depends on the type of specimen and the parasite present. The iron-hematoxylin stain produces a dark purple to black coloration of the nuclear and intracellular structures of protozoa, while the cytoplasm is stained violet. The background material, such as erythrocytes and bacteria, is also stained dark purple to black. The contrast between the dark structures and the violet cytoplasm allows for easy identification and differentiation of protozoal morphologies .

Some examples of protozoa that can be detected by iron-hematoxylin staining are:

• Entamoeba histolytica: This parasite causes amoebic dysentery and liver abscesses in humans. The trophozoites have a single nucleus with a small, central karyosome and fine peripheral chromatin. The cysts have one to four nuclei with the same characteristics as the trophozoites. The cytoplasm may contain ingested erythrocytes.
• Giardia lamblia: This parasite causes giardiasis, a diarrheal disease in humans and animals. The trophozoites have a pear-shaped body with two nuclei, each with a central karyosome and coarse peripheral chromatin. The cytoplasm has four pairs of flagella and two median bodies. The cysts have an oval shape with four nuclei and two median bodies.
• Dientamoeba fragilis: This parasite causes dientamoebiasis, a mild to severe diarrheal disease in humans. The trophozoites have one or two nuclei, each with a large, irregular karyosome and no peripheral chromatin. The cytoplasm has numerous vacuoles and granules.
• Cryptosporidium parvum: This parasite causes cryptosporidiosis, a severe diarrheal disease in immunocompromised individuals. The oocysts are spherical and measure 4 to 6 micrometers in diameter. They contain four sporozoites that are stained dark purple by iron-hematoxylin.
• Cyclospora cayetanensis: This parasite causes cyclosporiasis, a diarrheal disease in travelers and immunocompromised individuals. The oocysts are spherical and measure 8 to 10 micrometers in diameter. They contain two sporocysts that are stained dark purple by iron-hematoxylin.

The interpretation of iron-hematoxylin staining should be done by an experienced microscopist who can recognize the distinctive features of each parasite. The identification should be confirmed by other methods, such as antigen detection or molecular techniques, especially for parasites that have similar morphology or size.

Some additional sentences to conclude the point 9 are:

Iron-hematoxylin staining is a valuable technique for the diagnosis of intestinal protozoan infections. It provides a permanent stain that can be stored and reviewed for reference. However, it also has some limitations, such as the need for proper fixation, the slow procedure time, and the interference of mercury from Schaudinn`s solution. Therefore, it is important to follow the standard protocol carefully and use quality control measures to ensure reliable results.

Iron-hematoxylin staining is a useful technique for the diagnosis and identification of intestinal protozoan parasites, such as Entamoeba histolytica, Giardia lamblia, Dientamoeba fragilis, Cyclospora cayetanensis, and Cryptosporidium parvum. These parasites can cause various gastrointestinal diseases, such as amoebiasis, giardiasis, dientamoebiasis, cyclosporiasis, and cryptosporidiosis.

Iron-hematoxylin staining can reveal the nuclear and cytoplasmic structures of the protozoan cysts and trophozoites with high clarity and contrast. The nuclei are stained dark purple to black, while the cytoplasm is stained violet. This allows the differentiation of the protozoan species based on their morphological features, such as size, shape, number of nuclei, presence of chromatoid bodies, flagella, or microvilli.

Iron-hematoxylin staining can also be used to prepare permanent slides that can be stored for reference or further examination. This is important for epidemiological studies, quality control, or confirmation of diagnosis.

Iron-hematoxylin staining can be applied to fresh, PVA-preserved, or SAF-preserved stool samples. This gives flexibility and convenience to the laboratory personnel and clinicians who collect and process the specimens.

Iron-hematoxylin staining is a simple and economical method that can be performed in most laboratories with basic equipment and reagents. It does not require complex or expensive procedures or instruments, such as immunofluorescence microscopy or polymerase chain reaction (PCR).

Iron-hematoxylin staining is a traditional and reliable method that has been used for over a century for the detection of intestinal protozoans. It is still widely used today as a routine or complementary technique for the diagnosis of parasitic infections.

Iron-hematoxylin staining is a traditional and widely used technique for the detection and identification of intestinal protozoans. It has some advantages and limitations that should be considered when choosing this method for diagnostic purposes.

Some of the advantages of iron-hematoxylin staining are:

• It produces permanent stains that can be stored and examined later for reference or quality control.
• It stains the nuclear structures of protozoans dark purple to black, which makes them stand out against the violet cytoplasm and the background. This allows for high clarity and differentiation of protozoan morphologies and species.
• It can be used with fresh, PVA-preserved or SAF-preserved specimens, which gives flexibility and convenience to the laboratory personnel.

Some of the limitations of iron-hematoxylin staining are:

• It is a time-consuming and labor-intensive procedure that requires several steps and reagents. It may take up to 24 hours to complete the staining process from fixation to mounting.
• It requires careful fixation with Schaudinn`s solution, which contains mercuric chloride, a toxic and corrosive substance that must be handled with caution and disposed of properly. If the fixation is not done properly, it may result in distorted or poor staining of the specimens.
• It may not be sensitive enough to detect low numbers or small forms of protozoans, such as microsporidia or cryptosporidia. It may also not be specific enough to distinguish some closely related species, such as Entamoeba histolytica and Entamoeba dispar.

Therefore, iron-hematoxylin staining is a useful technique for the diagnosis of intestinal protozoans, but it has some drawbacks that should be taken into account. It may be complemented or replaced by other methods, such as immunofluorescence, molecular techniques or antigen detection tests, depending on the availability, cost and accuracy of these methods.

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