Histology Staining Techniques: Principles and Procedures
Basics of Staining
Stains are chemical substances used to achieve visible color contrast in the microscopic picture of a prepared tissue. Staining involves treating tissues or cells with a series of reagents so that they acquire a color. No particles of dye are seen, and the stained element is transparent.
Purpose of Staining
- Outlines tissues and cellular components.
- Aids in the identification of tissues.
- Establishes the presence or absence of disease processes.
Dyes
Dyes are essentially aromatic benzene ring compounds or derivatives that possess the twin properties of color and the ability to bind to tissue.
Mordants
- Mordants are salts and hydroxides of metals that help in the attachment of dye to the target tissue.
- They enhance the dye’s staining capability.
- The mordant binds with the dye by covalent or coordinate bonding, forming a complex known as a “lake.”
- Pre-mordanting: The tissue is first treated with a mordant, followed by the dye.
- Meta-mordanting: A mordant in combination with the dye is used.
- Post-mordanting: The dye material is applied first, followed by the mordant.
Example: Aluminum and Hematoxylin
Hematoxylin and Eosin Staining
Hematoxylin
- Most commonly used dye in pathology laboratories for routine morphological evaluation of tissues.
- Compound hematoxylin has no capacity to act as a dye on its own.
- A mordant is used to enhance its staining capability.
- The oxidation product of hematoxylin is hematin.
Bluing
Most regressive staining with hematoxylin requires bluing. Removing excess hydrogen ions from the stain is known as bluing. Here, the soluble hemalum is converted to an insoluble form.
Bluing gives the crisp blue color to the nuclei. The tissue section is treated with an alkaline reagent, and acidic reagents are neutralized in the bluing process.
The following methods are used for bluing:
- Running tap water for several minutes.
- Treating the section with Scott’s tap water (pH 8) for 2-3 minutes.
- Ammonium hydroxide (5%) for 2-3 minutes.
- Ammonia vapor for a few seconds.
Harris’s Alum Hematoxylin
This hematoxylin is a regressive stain widely used in exfoliative cytology as a nuclear stain. Eosin is used as a cytoplasmic counterstain. Ammonium or potassium alum is used here as a mordant.
This hematoxylin takes 5 to 15 minutes in the case of regressive staining and 5 to 30 seconds in progressive staining.
Preparation of Harris’s Hematoxylin
Ingredients:
- Hematoxylin: 5 g
- Absolute alcohol: 50 ml
- Ammonium alum: 100 g
- Distilled water: 1000 ml
- Mercuric oxide: 2.5 g
- Glacial acetic acid: 40 ml
Steps:
- Dissolve 5 g of hematoxylin in 50 ml of absolute alcohol.
- Add 100 g of alum to hot water (1000 ml).
- Heat the alum solution to boil.
- Mix both solutions.
- Slowly add 2.5 g of mercuric oxide.
- Heat again until the color changes to dark purple.
- Rapidly cool the flask by dipping it in cold water.
- Add 40 ml of glacial acetic acid to the cold solution.
Eosin Counterstain
Eosin is used as a counterstain for the cytoplasm. It also stains connective tissue and matrices, imparting a rosy red to pink color.
Three types of eosin are commonly seen:
- Eosin Y
- Eosin B
- Ethyl eosin
Eosin Y is the most widely used among these three types. The term ‘Y’ indicates yellowish, while ‘B’ stands for bluish.
Eosin is soluble in both water and alcohol.
- Aqueous solution: 1% eosin in distilled water.
- Alcohol solution: 0.5% in 95% ethyl alcohol.
Washing the eosin-stained sections with tap water helps in the differentiation of the eosin staining.
Routine Hematoxylin and Eosin Staining Procedure
Reagents:
- Hematoxylin solution
- Grades of alcohol (100% and 95%)
- 1% HCl in 70% alcohol (acid alcohol)
- 1% eosin
- Xylene
Steps:
Step 1: Deparaffinization
Keep the sections in xylene for 10 minutes each, with three changes.
Step 2: Rehydration
- Absolute alcohol: 1 to 2 min
- 95% alcohol: 1 to 2 min
- 80% alcohol: 1 to 2 min
- 60% alcohol: 1 to 2 min
- Rinse in tap water
Step 3: Nuclear Staining with Hematoxylin
Harris Hematoxylin for 5 to 15 min.
Step 4: Differentiation
In the case of regressive staining, one to two dips in acid alcohol (1% HCl in 70% alcohol) are necessary for differentiation.
Step 5: Bluing
Wash by running tap water for 10 to 15 min.
Step 6: Counterstaining with Eosin
1% aqueous eosin for 2 to 3 min.
Step 7: Dehydration
- 95% alcohol: 3 min
- Absolute alcohol: 3 min
- Absolute alcohol: 5 min
Step 8: Clearing
Xylene: 5 min each in two jars.
Step 9: Mounting
Mount with DPX.
Diff-Quik Staining
Diff-Quik Stain A Composition
Ingredients:
- Methylene blue: 1.8 g
- Disodium hydrogen phosphate: 12.6 g
- Dipotassium hydrogen phosphate: 6.25 g
- Distilled water: 500 ml
Preparation Procedure for Stain A
- Dissolve methylene blue and disodium hydrogen phosphate in 50 ml of distilled water.
- Then, boil the solution in a water bath almost to dry.
- Add dipotassium hydrogen phosphate and 500 ml of distilled water.
- After 24 hours, the stain will be ready to use.
Diff-Quik Stain B Composition
Ingredients:
- Eosin: 1.8 g
- Disodium hydrogen phosphate: 12.6 g
- Dipotassium hydrogen phosphate: 6.25 g
- Distilled water: 500 ml
Preparation Procedure for Stain B
- Dissolve disodium hydrogen phosphate and dipotassium hydrogen phosphate in 500 ml of distilled water.
- Add eosin.
- After 24 hours, the stain will be ready to use.
Diff-Quik Staining Procedure
- Dry the smear properly.
- Fix the smear in methanol for 1 min.
- 6 dips in Stain B.
- Wash in water.
- 9 dips in Stain A.
- Wash in water.
- Dry, clean, and mount.
Papanicolaou Stain
Dr. George Papanicolaou, the father of cytopathology, first introduced this stain. Papanicolaou’s stain (PAP stain) is the most important stain in cytology and is used in all cervical smears and non-gynecologic exfoliative smears.
Properties of Papanicolaou Stain
- Cytoplasmic differentiation: It helps in the assessment of cellular differentiation.
- Clear nuclear details.
- Transparent staining.
- Demonstrates intracytoplasmic keratin.
Dyes Used in Papanicolaou Staining
- Hematoxylin: Harris hematoxylin for nuclear staining.
- Orange G (OG-6): For cytoplasmic counterstaining. It also stains the keratin component of the cytoplasm.
- Eosin Azure (EA): This is a polychrome stain and consists of three dyes: Eosin Y, Light Green SF Yellowish, and Bismarck Brown Y.
Principle and Basic Steps of Papanicolaou Staining
- Rehydration of the smear: Achieved with subsequent dips in graded concentrations of alcohol.
- Nuclear staining by hematoxylin: Harris hematoxylin is a good, rapid nuclear stain. Subsequent differentiation is done to remove excess hematoxylin by acid alcohol.
- Bluing: This is done by treating the smear with running tap water; alternatively, a weak alkaline solution can be used.
- Cytoplasmic staining by Orange G (OG): As OG is an alcohol-soluble dye, the smear is brought into alcohol and stained with OG.
- Cytoplasmic staining by EA: The cell cytoplasm is stained blue-green by EA.
- Dehydration: It is done by absolute alcohol.
- Clearing: Xylene.
- Mounting: By DPX mounting medium.