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.

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 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
⏐
H2O H⎯N⎯ H 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.

•• ••
• O • •H H⎯ O⎯H
•• ••
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.
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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+ NH3+ NH2

⏐ ⏐ ⏐
R ⎯ O ⎯ COOH R ⎯ O ⎯ COO- R ⎯ O ⎯ COO-
⏐ ⏐ ⏐
H H H

Basic (+) Zwitterion Acidic (-)

(electrically neutral)

pH 1.0 pH 6.0 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
OH

O2N NO2 O2N NO2

NO2 NO2
Trinitrobenzene Picric acid
(Chromogen) (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: Important Source for dyes:

Carmine 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 Hematein
OH
OH
HO O
HO O
CH2
CH2

COH
• COH
C CH2 C CH2
H
H

HO OH
HO O-

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Progressive Hematoxylin (Can be used in a Regressive staining set-up).
Mayer’s
Delafield’s
Gill’s
Harris’

100% 95% 95%

OH OH OH
Xyl Xyl Xyl Water Heme Water Water

Amm 95% 95% 100% 100% 100% Xyl

Water Water Water Eosin OH OH OH OH OH
(Bluing)

Xyl Xyl

Regressive Hematoxylin
Delafields
Harris’
Ehrlich’s

100% 95% 95%

OH OH OH Acid
Xyl Xyl Xyl Water Heme Water
Alcohol

95% 95% 100% 100% 100%

Water Amm Xyl
Water Eosin OH OH OH OH OH
Water
(Bluing)

Xyl Xyl

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Stain Delafield’s Mayer’s Ehrlich’s Weigert’s

Mordant Ammonium Pottasium Pottasium Ferric

Aluminum Aluminum Aluminum Chloride
Sulfate Sulfate Sulfate
Oxidant Light & Air Sodium Naturally Ferric
Iodate Chloride
Dye Hematoxylin Hematoxylin Hematoxylin Hematoxylin

Solutions 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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