HISTOLOGY
LECTURE # 19
INTRODUCTION TO ROUTINE STAINING (THE H&E STAINING) Rationale: The staining process will allow us to physically be able to visualize a paraffin section slide under the microscope. The final product will stain nuclear and cytoplasmic detailsallowing for a diagnostic lecture.
Objective: Once completed this lecture, the student should be able to: a)
Describe the Difference between Hematoxylin & Eosin
b)
Describe the difference between a progressive and Regressive stain..
c)
Differentiate other routine histologic staining such as Giemsa
d)
Learn the sources of the dyes and their uses in the laboratory.
ROUTINE STAINING Staining Mechanisms Most reactions involves Physical & Chemical Reactions I. Physical Stain a) Fat Stain - the dye is absorbed and dissolved in the lipid. b) Most stains depend on the adsorption of the dye. Dye is bound to the tissue by Ionic, covalent or hydrogen bonds a) Ionic or electrostatic bonding - the dye and the substance to be dyed develop different charges and thus become attracted to each other. 1
Example: We can stain the cytoplasm by developing a positive charge on the cytoplasm proteins and a negative charge on the dye. This link is also known as a salt link. +
+
+
+
-
+
-
-
-
-
Hydrogen Bonding - happens when covalent bonded hydrogen is attracted to atoms that have a strong electromagnetic charge. Normally this happens between hydrogen and oxygen or hydrogen and nitrogen. Example: Hydrogen bonds are weak and occur naturally in water; they mat form between the dye and the water in which it is dissolved. H⎯ O⎯H
H ⏐ H⎯N⎯ H
H2O
NH3
Covalent Bonding - happens when atoms shares electrons. This is typically organic chemicals because hydrogen, oxygen and carbon form covalent bonds. Example: Hydrogen bonds are weak and occur naturally in water; they mat form between the dye and the water in which it is dissolved.
• H
•• O
••
•
••
•H
H⎯ O⎯H
••
•
Nuclear Staining It is believe that nuclear staining occurs through two different mechanisms: a) Staining done with basic positively charged dyes. b) Staining done with dyes combined with or followed by a mordant. First Mechanism: Depends on the presence of nucleic acids (DNA or RNA) to form dye salt-type unions. Second Mechanism: Happens in tissue from which the nucleic acids have been removed (decals) or tissue that are not negatively charged. 2
Cytoplasmic Staining •
It is believe that cytoplasmic staining is due primarily to proteins or to charged groups on the side chains of amino acids constituting the proteins.
•
Proteins or polymers (chains) of amino acids contain a terminal (-NH2) group on one end and a terminal carboxyl (-COOH) group on the other; in addition, amino acid side chains may have -NH2 or -COOH groups.
•
They can be either positively or negatively charged, and this charge is pH dependent and because proteins can carry either charge (+) or (-) they are said to be amphoteric.
NH2 ⏐ R ⎯ O ⎯ COOH ⏐ H Undissociated amino acid NH3+ ⏐ R ⎯ O ⎯ COOH ⏐ H
NH3+ ⏐ R ⎯ O ⎯ COO⏐ H
Basic (+)
Zwitterion (electrically neutral)
pH 1.0
pH 6.0
NH2 ⏐ R ⎯ O ⎯ COO⏐ H Acidic (-)
pH 14.0
IEP
IEP – is the point where the positive and the negative charge are equal. The IEP is approximately pH 6.0, below the pH 6.0 is the net charge is positive attracting anionic dyes. Above the pH 6.0 the net charge is negative attracting catiotic dyes. 1. Substances attracting basic dyes are called basophilic. 2. Substances attracting acidic dyes are called acidophilic. If tissue sections are placed in a solution below the pH 6.0, the cytoplasmic proteins will develop a predominance of positive charges and then will attract a negatively charged dye such as eosin.
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The Dyes •
What makes a substance a dye or colored? – The replacement of two hydrogen atoms in the benzene ring with oxygen or with another atom or group having two valency bonds instead of one resulting in the readjustment of the double bonds and the formation of a colored compound. –
Chromophore – a group that confers the property of color C=C, C=O, C=S, C=N, N=N, N=O, and NO2 The more of these compound the more intense are the colors of the dyes.
–
A benzene derivative containing chromophoric groups is called a chromogen
Chromophoric Group
O2N
OH NO2
NO2 O2N
NO2
NO2
Trinitrobenzene (Chromogen)
Picric acid (dye)
Basic dye – one in which the charge of the dye is positive (+), also known as catiotic dyes and yhe auxochrome is the amino group (NH2). Most frequently are chloride salts. Example: Crystal Violet & Safranin Acid dyes – these are dyes with a negative charge and referred to as anionic dyes. The usual anionic auxochrome is the sulfonic group (NH2-), but the carboxyl (COO-) and hydroxyl (OH-) ions are alsoanionic auxochromes. Most frequently are sodium salts. Example: Orange G and Picric Acid
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Amphoteric dyes – substance that is capable of acting either`as base or an acid, depending on the pH of the solution. Example: Hematein and lithium Carbonate Nonionic compound – incapable of electrolytic dissociation, and although they have color, they are not really dyes. Example: Lipid stains They are insoluble in water but soluble some organic solvents, and they color certain tissue components by dissolving ih them (physical staining). Natural dyes– dyes obtained from natural sources. Only a few natural dyes are used in histology, some of these have been prepared synthetically. Important Natural dyes: Carmine
Important Source for dyes: Female Cochineal insect
Orcein
lichens
Saffron
pistils of a flower
Hematoxylin
heartwood of the logwood tree
Factors affecting Dye binding: 1. pH determination 2. Increase of temperature will increase the rate of staining by increasing the diffusion of the dye molecule. 3. Increase of the dye molecule concentration 4. Other salts can decrease or increase the staining intensity of certain components of the tissue. 5. Fixation alteration Differentiation A. Progressive Staining 1. Many counterstain dyes are used in a progressive way meaning once the desired intensity is reached, the reaction is stopped. 2. Mordant dyes can also be progressively used. Mordants are substances of metal that acts as a link between the dye and the tissue. The mordant combines with the dye to create a “lake” that is usually basic. B. Regressive Staining 1. The tissue is overstained and then is differentiated or decolorized until the desired intensity is achived.
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C. Diffenrentiation Method 1. Weak acid solution for basic or catiotic dyes. Weak alkaline solution for acidic or anionic dyes. Alum Hematoxylin ---------------------------- dilute Hydrochloric acid Eosin -------------------------------------------- dilute ammonium hydroxide 2. Excessive mordant will break the tissu/mordant/dye complex. Iron Hematoxylin ---------------------------------------- Ferric Chloride 3. Oxidizing the dye to a colorless substance. Potassium permanganate --------------------------- Oxalic acid
The Nuclear Dye A. Hematoxylin 1. Is NOT a dye 2. Hematein, the oxidation product of hematoxylin, is a weak anionic dye. 3. Oxidation of hematoxylin is necessary and it may be done naturally by exposing the solution to atmospheric oxygen or by using oxidizing agents and creating a ripening situation with: Sodium iodate Mercuric chloride Potassium permanganate 4. Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten. The variety of hematoxylins available for use is based partially on choice of metal ion used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus. Hematoxylin OH
Hematein OH
O
HO
O
HO
CH2
CH2
COH C
CH2
H
• C
COH
HO
O-
CH2
H
HO
OH
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Progressive Hematoxylin (Can be used in a Regressive staining set-up). Mayer’s Delafield’s Gill’s Harris’
Xyl
Amm Water (Bluing)
Xyl
Xyl
Xyl
Water
Water
100% OH
95% OH
95% OH
Eosin
95% OH
95% OH
100% OH
95% OH
95% OH
Eosin
95% OH
95% OH
Water
Heme
Water
Water
100% OH
100% OH
100% OH
Xyl
Water
Heme
Water
Acid Alcohol
100% OH
100% OH
100% OH
Xyl
Xyl
Regressive Hematoxylin Delafields Harris’ Ehrlich’s
Xyl
Xyl
Xyl
Water
Amm Water (Bluing)
Water
Xyl
Xyl
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Stain
Delafield’s
Mordant
Ammonium Aluminum Sulfate Light & Air
Mayer’s
Pottasium Aluminum Sulfate Sodium Iodate Hematoxylin Hematoxylin
Oxidant Dye Solutions
Ehrlich’s
Weigert’s
Pottasium Aluminum Sulfate Naturally
Ferric Chloride
Ferric Chloride Hematoxylin Hematoxylin
95% Alcohol Citric Acid (to Glycerol Hydrochloric adjust pH) Acetic Acid Acid Distilled Chloral 95% Alcohol 95% Alcohol Water hydrate
The Cytoplasmic Dye B. Eosin 1. Is an acidic dye with an affinity for cytoplasmic components of the cell. 2. There are a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. 3. Eosin is much more forgiving than hematoxylin and is less of a problem in the lab. About the only problem you will see is overstaining, especially with decalcified tissues. 4. The results are various shades of pink.
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Stain
Eosin Y
Eosin Y-Phloxine B Eosin Y (1%) Eosin Y (1%) Phloxine B (1%) 95% Alcohol
95% Alcohol
Acetic Acid
Acetic Acid Shades of pink are more vivid
The H & E Staining
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The Nucleic Acid Staining I.
Fuelgen Reaction Demonstrate: DNA Fixative: Any except Bouin’s Results: reddish purple
II.
Methyl Green-Pyronin Y
Demonstrate: Differentiate between DNA & RNA Fixative: Any Results: DNA = green to blue-green RNA = Red
III.
Polychromatic Stain (Various colors) – Giemsa Stain Demonstrate: Differentiation of cells/microorganisms Fixative: Zenker’s, B-5, 10% NBF Results: Nuclei -Blue Cytoplasm - shades of pine, gray or blue Bacteria - Blue
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