Iron-Hematoxylin Staining – Principle, Procedure, Result

Iron-Hematoxylin Stain

Iron-Hematoxylin Staining is a widespread method used to study intestinal protozoans because of its stability and ability to show a high clarity protozoans nuclear structures. Each morphological character of intestinal protozoa is observed by using Iron hematoxylin stain.

Iron-Hematoxylin stain differentiates between nucleus and cytoplasm by making the intracellular and nuclear structure dark as compared to the cell cytoplasm.

Fresh specimen or polyvinyl-alcohol (PVA)-preserved specimens or sodium acetate acetic acid formalin (SAF)-preserved can be used for Iron hematoxylin staining procedure.

Iron-Hematoxylin Staining Principle

Hematoxylin is extracted from a leguminous plant, known as Haematoxylon campechianum, thus it is a natural dye. It forms hematein by oxidation  (Ripening). Oxidation is a lengthy process where crystals of Hematoxylin dissolved in water, then come in contact with the atmospheric oxygen and the increase of hydrogen peroxide with close monitoring to avoid overoxidation.  If over-oxidation occurs, it will form oxy-hematein which is not a dye.

The hematoxylin solution is a mixture of hematoxylin, hematein, oxihematein. Hematein functions as a mordant to create a lake, it undergoes a reaction with ferric ammonium sulfate and form a basic dye, ferric lake (iron-hematoxylin). Iron-hematoxylin sharply stains the structures related to recognize intestinal protozoan. For the complete fixation of smear, an  Alcoholic iodine is used.

Purposes of Iron-Hematoxylin Stain

Iron-Hematoxylin Staining is used to study the morphology of protozoal parasites and for the identification of intestinal parasites from a given stool sample.

Reagents required for Iron-Hematoxylin Stain

• Mercuric Chloride
• Distilled water
• Ethyl alcohol
• Hematoxylin
• Iodine
• Alcoholic iodine
• Ferric Ammonium Sulfate (Iron Alum)
• Hydrochloric acid

Iron-Hematoxylin Staining Procedure

Solutions preparation

Note: Iron Hematoxylin Stain and Mordant-differentiating solutions are stable individually for 12 months when stored at the right temperature. Once the working solution is prepared, let it remains stable for 7 days before using it for staining. Filtering of the stains is recommended periodically using a filter paper to remove any debris that may be present and always keep seal the container to avoid evaporation.

PVA-sample preparation

• Allow the fixation of PVA-preserved samples for 30 minutes and then add the content, mix well with applicator sticks.
• Take a clean paper towel and pour the previously prepared PVA mixtures on it and wait for 2-3 minutes, it will allow the absorption of PVA.
• Use an applicator stick to place stool samples on the paper towel to the glass slides; dry it by placing it in an incubator at 37°C or at room temperature (overnight), make sure it dries completely.
• Add iodine-alcohol on this smear.

SAF-sample preparation

• A SAF-stool sample is mixed and strained through a wire gauze to a 15ml centrifuge tube and centrifuge for 1 minute at 500xg.
• Remove the supernatant gently, centrifuge the sample for 10min at 500xg; collect sediments of 0.5ml to 10ml. If needed, set by redoing step 1 or by resuspending the sediment in saline (0.85% NaCl) and lifting part of the suspension.
• Take a clean glass slide and prepare a smear on it from the sediments; place one drop of Mayer’s albumin on the slide; place a drop of the SAF-preserved sample sediments; dry at room temperature for 30 minutes; perform staining.
• You can (NOT MUST) also postfix with Schaudinn’s solution before staining before initiating a trichrome stain procedure with 70% alcohol rinse then adding iodine- alcohol.
• Add 70% alcohol on the dry smear.

Fresh samples

1. Take a clean microscopic glass slide.
2. Take the stool samples and prepare a thin smear on the glass slide by using an applicator stick.
3. Dip the wet slide within Schaudinn’s solution and wait for 30 minutes. This process is called the Schaudinn’s fixation.
4. Place the smear in 70% alcohol.
5. Rinse the smear with flowing tap water (3 times).
6. Place the smear into an iron hematoxylin working solution and wait for 4 to 5 min.
7. Rinse the smear with running tap water for 10 min (constant stream of water into the container).
8. Dip the slides into a 95% ethyl alcohol and wait for 5 minutes.
9. Put the slides in 100% ethanol and wait for 5 minutes.
10. Dip the slides in two changes in xylene for 5 minutes for each change.
11. Place a permount (Mounting Media is formulated for mounting and storing long-term slides) to the stain and cover with a coverslip.
12. Observe the smear under a microscope using oil immersion technique.

PVA-Preserves samples

1. Take a clean microscopic glass slide.
2. Take the stool samples and prepare a thin smear on the glass slide by using an applicator stick.
3. Dip the wet slide within Schaudinn’s solution and wait for 30 minutes. This process is called the Schaudinn’s fixation.
4. For liquid specimen, add 3 to 4 drops of PVA on the slide and mix it with fecal material; spread the mixture and leave is for overnight at room temperature until it dried (Or incubate at 37 degrees centigrade for a few hours ).
5. Place the slide in iodine-alcohol.
6. Place in 70% ethanol and wait for 5 minutes.
7. Rinse the slide with running tap water for 10 minutes.
8. Place slide in iron hematoxylin working solution for 4 to 5 min.
9. Rinse the slide with smooth running tap water for 10 min.
10. Dip the slides in 70% ethanol and wait for 5 minutes.
11. Places slide in two changes of 100% ethyl alcohol for 5 minutes each.
12. Place slide in two changes of xylene for 5 minutes each.
13. Add permount to the stained area and cover with a coverslip.
14. Examine slides microscopically with the 100X objective.

Staining SAF preserved samples

1. Prior to use, make a working solution by mixing equal parts of the Iron-hematoxylin mordant and the stain (This working solution should be prepared fresh weekly)
2. Prepare a smear by mixing 1 drop of Mayer’s albumin with sediment from the SAF preserved specimen.
3. Allow slide to air dry at room temperature until smear is dry and opaque.
4. Place slide in 70% alcohol for 5 minutes.
5. Wash in container of tap water for 2 minutes.
6. Place slide in Kinyoun stain for 5 minutes.
7. Wash slide in running tap water for 1 minute.
8. Place slide in Kinyoun decolorizer for 4 minutes.
9. Wash slide in running tap water for 1 minute.
10. Place slide in Iron Hematoxylin working solution for 8 minutes.
11. Wash slide in distilled or deionized water in container for 1 minute.
12. Place slide in picric acid working solution for 3 to 5 minutes.
13. Wash slide in running tap water for 10 minutes.
14. Place slide in 70% alcohol plus ammonia for 3 minutes.
15. Place slide in 95% ethyl alcohol for 5 minutes.
16. Place slide in 100% ethyl alcohol for 5 minutes.
17. Place slide in two changes xylene or xylene substitute for 5 minutes.
18. Add permount to the stained area and cover with a coverslip.
19. Examine slides microscopically with the 100X objective. 

Result and Interpretation

• Cytoplasm of trophozoites and cysts: stain violet.
• Nuclear chromatin, chromatoid bodies, erythrocytes, and bacteria: Stain dark purple to black

Limitation of Iron-Hematoxylin Staining

1. The working should be prepared fresh on a weekly basis. A quick reliability test can be performed by adding a few drops of stain to alkaline tap water. If the mixture turns blue then the working solution is working; if the solution turns brown then a new working solution should be made.
2. Incomplete removal of mercuric chloride for Schaudinn’s and PVA-fixed smears may result in highly refractive granules that make visualization of organisms more difficult. In such instances change the 70% alcoholiodine solution.
3. This stain is not recommended for the detection or visualization of helminth eggs and larvae. Helminth eggs and larvae and Isospora Belli oocysts are best seen in wet preparations.

Quality Control of Iron-Hematoxylin Stain

Internal quality control of the working solution of Iron Hematoxylin Stain must be performed regularly on known reference organisms to ensure the performance of the stain. Include a QC slide when you use a new lot of reagents, or when you add new reagents after cleaning staining dishes. 

Storage and Shelf life

Both our Iron Hematoxylin Stain and Mordant should be stored at room temperature and protected from light. Under these conditions it has a shelf life of 52 weeks from the date of

manufacture. The diluted working solution of Iron Hematoxylin Stain has a shelf life of approximately 1 week from the date of preparation. 

Importance of Iron-Hematoxylin Staining

• To prepare permanent stains for reference.
• Used for diagnosis of parasitic infections of protozoal cysts and trophozoites in specimens.

Applications of Iron-Hematoxylin Staining

Iron-Hematoxylin staining is a commonly used staining method in histology and pathology. Some of the applications of this staining technique include:

1. Tissue and cell differentiation – Iron-hematoxylin staining is used to highlight the nuclei of cells and tissues, making it easier to distinguish different types of cells and tissues.
2. Diagnosis of diseases – Iron-hematoxylin staining can be used to help diagnose various diseases, such as cancers, by revealing changes in the structure and arrangement of cells and tissues.
3. Research and education – This staining technique is widely used in research and education to study the structure and function of cells and tissues, and to demonstrate these concepts to students.

Overall, Iron-Hematoxylin staining is a valuable tool for histologists, pathologists, and researchers to gain insights into the structure and function of cells and tissues.

Advantages of Iron-Hematoxylin Stain

1. High Contrast: Iron-hematoxylin staining provides high contrast between the stained and unstained areas, making it easier to identify and differentiate cells and tissues.
2. Versatility: This staining technique can be used on a wide range of tissues and cell types, including both hard and soft tissues.
3. High specificity: Iron-hematoxylin staining specifically stains the nuclei of cells, allowing for clear visualization of their structure and arrangement.
4. Widely used: Iron-hematoxylin staining is a widely used staining method in histology and pathology, making it a readily available and well-established technique.

Disadvantages of Iron-Hematoxylin Stain

1. Complex procedure: The staining process for Iron-hematoxylin can be time-consuming and requires multiple steps, making it more complex compared to some other staining techniques.
2. Technical difficulties: Iron-hematoxylin staining requires a good level of technical skill and experience, as the staining quality can be easily affected by variations in the procedure or by environmental factors.
3. Cost: Iron-hematoxylin staining can be more expensive than some other staining techniques due to the cost of the reagents and the time required to perform the procedure.
4. Time: Time-consuming process.
5. The result will be distorted if the fixation is not done properly.
6. Schaudinn solution contains mercury and therefore it must be removed completely before staining. it interferes with the staining reagents.

FAQ

References 

1. Spenser FM, Monroe LS. The color atlas of intestinal parasites, 2nd ed. Springfield: Charles C Thomas, 1976.
2. Yang J, Scholten. Am J Clin Path, 67:300- 4. 1977.
3. Forbes BA, Sahm DF, Weissfeld AS. Bailey and Scott’s diagnostic microbiology, 10th ed. St. Louis: Mosby, 1998.
4. Murray PR, Baron E, Pfaller M, Tenover F, Yolken. Manual of clinical microbiology, 7th ed. Washington: ASM, 1999.
5. Garcia LS. Diagnostic Medical Parasitology, 4th ed. Washingtion, DC: ASM, 2001.