Principles of Staining in Histopathologic Techniques
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Introduction
Types of stain
Affinity of staining
Factors that determine selectivity of stain
Types of commonly used stains in histopathologic
techniques
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Upon completion of this chapter, the student will be
able to:
Define staining.
Discuss factors that contribute to dye-tissue affinity.
Explain factors affecting selectivity of stain.
Explain the classification of hematoxyline on the
basis of mordant used.
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Describe method of demonstration of proteins.
Describe carbohydrate stain.
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:
DNA
Proteins
Lipids and
Carbohydrates
to qualify or quantify the presence of a specific
compound.
3
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The stained slide is taken through a series of
alcohol solutions to remove the water, then
through clearing agents to a point at which a
permanent resinous substance beneath the glass
cover slip, or a plastic film, can be placed over the
section.
3
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3
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•
Affinity means the force, which binds the dye to
the tissues.
•
The word crisp is used in textile industry instead of
affinity.
•
Affinity is not a one-way procedure but a two-way
procedure as it depends on the tissue as well as
on the dye.
3
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3
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1
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Factors that can adversely affects staining of tissues:
–
Solvent-solvent interaction (Hydrophobic bonding)
–
Reagent-reagent interaction
–
Reagent-tissue interaction (Vander -Waal forces)
–
Columbic attraction
–
Hydrogen bonding
–
Covalent bonding.
3
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3
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1
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1
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There is a tendency of hydrophobic grouping or
chemicals or substances to come together even
though initially dispersed in aqueous (water) solution.
•
The water molecules are transiently held together in
clusters by hydrogen bonding.
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•
These clusters are stabilized by hydrophobic
group
•
Any process that involves breakup of the
clusters in to disorganized water molecules will
tend to occur spontaneously.
•
Phenylalanine and tryptophane side chains or
biphenyl naphthol are hydrophobic grouping.
3
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3
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1
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2
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Dye aggregation increases with concentration.
Cations or basic dyes build up in tissues where there
is high negative charge such as,
- polysaccharides in mast cell granules and
- cartilage matrix.
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This is due to dye aggregation having spectral properties
unlike those of monomeric dyes.
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There is stain-stain interaction
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There is stain-stain interaction
3
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3
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acts between dye and tissues.
•
is of short range.
Therefore, Van-deer-Walls force is strong where
close reagent contact is possible.
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Substrates and chemicals that favor Van-der-walls
bonding include:
•
Tyrosine and tryptophane residues of proteins;
•
Heterocyclic bases of nucleoproteins;
•
Halogenated dyes such as,
- rose Bengal,
- phloxine,
- enzyme substrate,
- naphthyl indoxyl system.
3
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3
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1
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4
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termed as salt link or electrostatic bond.
•
the electrostatic bonds arise from electrostatic
attraction of dissimilar ions
•
is the most widely acknowledged reagent tissue
interaction.
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The colored cation of basic dyes and tissue structures
rich in anion such as
- phosphated DNA and RNA
- carboxylated or
- sulphated mucosubstances.
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In case of columbic attraction, the amount of dye
able to enter a given tissue will depend on:
•
charge of dye,
•
magnitude of charges,
•
amount of non-electrolyte present in dye bath
•
ability of tissue to sink or swell.
3
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3
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1
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5
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is a localized bond when a hydrogen atom lies b/n
two electronegative atoms such as ,H or N .
•
It will rarely be an important source of reagent-
tissue affinity when aqueous solvents are used.
•
if dye is taken by other process, H- bonding may
subsequently contribute to affinity.
3
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Factors that determine selectivity of stain include:
•
Number and affinity of binding sites
•
Rates of reagent uptake
•
Rate of reaction
•
Rate of reagent loss.
3
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4
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The staining affinity of dye depends on:
–
the number of binding sites of the dye to tissue.
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non-ionic Sudan dye will have affinity for fat droplets, but
none for the surrounding hydrated proteins. Therefore
Sudan stains intensely fat tissues and not proteins or
carbohydrates.
5
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Selectivity of the stain by the tissue depends on the
rate of reagent uptake by the tissue.
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mucin-staining method using Alcian blue or
colloidal iron.
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nucleic acid and RNA-rich cytoplasm are also
stained using Alcian blue or colloidal iron.
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There are methods where three or more dyes
diffuse at varying rates, as a result different
structures can be stained by different dyes.
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collagen fibers stain rapidly while the muscle
fibers stain at intermediate rate
3
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Selectivity of the stain also depends on the
rate of reaction. This is because,
-
reactive stain yield colored derivatives, &
- amount of color
depend on the selective rates of reaction.
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Enzyme histochemistry provides many other
examples of reaction rates that affect selectivity.
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at low pH the hydrolysis of an organic phosphate applied to
a tissue section in a suitable incubation media will be rapid
in those parts of the tissue containing acid phosphates.
However, in structures containing alkaline phosphatase ,
the hydrolysis rate will be very slow.
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Some tissues or structures are stained and
decolorized readily while other tissues or
structures are not decolorized readily.
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•
staining of muscle striation with iron Haematoxyline and
myelin sheath with Luxol Fast Blue.
•
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The staining conditions chosen to maximize
selective affinity
.
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r
a
t
i
o
n
.
•
rate of reagent uptake, or subsequent reaction or loss of
reagent or product.
3
.
5
T
y
p
e
s
o
f
c
o
m
m
o
n
l
y
u
s
e
d
s
t
a
i
n
s
i
n
h
i
s
t
o
p
a
t
h
o
l
o
g
i
c
t
e
c
h
n
i
q
u
e
s
3
.
5
.
1
H
a
e
m
a
t
o
x
y
l
i
n
e
(
H
)
•
is the most popular & widely used histologic stain.
•
e
x
t
r
a
c
t
e
d
f
r
o
m
h
e
a
r
t
w
o
o
d
o
f
t
h
e
t
r
e
e
c
a
l
l
e
d
H
a
e
m
a
t
o
x
y
l
i
n
e
c
a
m
p
e
c
h
i
u
m
w
i
t
h
h
o
t
w
a
t
e
r
.
•
can demonstrate clearly enormous number of d/t
tissue structures.
N
a
t
u
r
a
l
o
x
i
d
a
t
i
o
n
o
f
H
a
e
m
a
t
o
x
y
l
i
n
e
n
a
t
u
r
a
l
o
x
i
d
a
t
i
o
n
(
r
i
p
e
n
i
n
g
)
•
is exposing haematoxyline to light & air and
converting it to hematin.
•
is a slow process, it may takes 3-4 months.
•
t
h
e
h
e
m
a
t
i
n
e
s
o
l
u
t
i
o
n
r
e
t
a
i
n
s
i
t
s
s
t
a
i
n
i
n
g
a
b
i
l
i
t
y
f
o
r
a
l
o
n
g
t
i
m
e
.
E
x
a
m
p
l
e
s
-Ehrlichs haematoxyline
-Delafield’s haematoxyline.
H
a
e
m
a
t
o
x
y
l
i
n
e
…
.
.
•
Haematoxyline also stains tissues that are
processed by various methods.
•
In the routine H & E stain, nuclei stain blue-black
with good intra nuclear details.
•
While Eosin stains cytoplasm and connective
tissue in varying intensity of pink, orange & red.
H
a
e
m
a
t
o
x
y
l
i
n
e
…
.
.
•
Haematoxyline has many more uses than in E &
H combination.
•
Haematoxyline by itself is not stain but
haematin, an oxidant product of Haematoxyline
is a stain & a natural dye.
P
r
o
d
u
c
t
i
o
n
o
f
h
a
e
m
a
t
i
n
f
r
o
m
H
a
e
m
a
t
o
x
y
l
i
n
e
•
Haematoxyline is extracted from heartwood or
logwood. It is precipitated by using urea.
•
Haematin is produced from Haematoxyline by two
ways.
1. Natural oxidation or ripening of Haematoxyline.
2. Chemical oxidation of Haematoxyline.
C
h
e
m
i
c
a
l
o
x
i
d
a
t
i
o
n
o
f
h
a
e
m
a
t
o
x
y
l
i
n
e
•
haematoxyline is oxidized by chemicals such
as - sodium iodate &
- Mercuric oxide.
•
Sodium is used as an oxidant in preparation of
Mayer’s haematoxyline
•
Mercuric oxide for Harris haematoxyline
C
h
e
m
i
c
a
l
o
x
i
d
a
t
i
o
n
…
…
A
d
v
a
n
t
a
g
e
s
•
Oxidize haematoxylin almost instantaneously;
•
Ready to be used after immediate preparation.
D
i
s
a
d
v
a
n
t
a
g
e
•
have shorter useful life when compared to naturally
ripened haematoxylin.
3
.
5
.
2
.
C
l
a
s
s
i
f
i
c
a
t
i
o
n
o
f
h
a
e
m
a
t
o
x
y
l
i
n
e
b
a
s
e
d
o
n
m
o
r
d
a
n
t
u
s
e
d
Classification of haematoxyline based on mordant
:
•
Alum haematoxylin;
•
Iron haematoxylin;
•
Tungsten haematoxylin;
•
Molybdenum haematoxylin;
•
Lead haematoxylin;
•
Haematoxylin with out mordant
.
3
.
5
.
2
.
1
A
l
u
m
h
a
e
m
a
t
o
x
y
l
i
n
Types of Alum haematoxylin
•
Ehrlichs haematoxylin;
•
Delafield’s haematoxylin;
•
Mayer’s haematoxylin;
•
Harris haematoxyline;
•
Coles haematoxylin;
•
Carazzi’s haematoxylin.
A
l
u
m
h
a
e
m
a
t
o
x
y
l
i
n
…
.
B
l
u
e
i
n
g
After staining with haematoxylin, the section is
treated with:- tap water or
- Scott’s tap water or
- lithium carbonate.
The red color of the nuclei becomes blue and this is
known as blueing.
A
l
u
m
h
a
e
m
a
t
o
x
y
l
i
n
…
.
.
D
i
s
a
d
v
a
n
t
a
g
e
s
o
f
a
l
u
m
h
a
e
m
a
t
o
x
y
l
i
n
•
The stain is sensitive to any subsequently applied
acid solution.
E
x
a
m
p
l
e
-
v
a
n
g
i
e
s
o
n
a
n
d
- trichrome staining.
•
The picric acid fuchsine mixture in vangieson
stain removes most of the haematoxylin so that
nucleus becomes faint and not easily seen
.
A
l
u
m
h
a
e
m
a
t
o
x
y
l
i
n
…
.
.
•
In such situation,
- Iron-mordant haematoxylin
- a combination of alum haematoxylin
- Celestine blue is used,
since Celestine blue solution is prepared in ferric
acid solution & the ferric salt in Celestine blue
solution strengthens the bond b/n the nucleus &
alum haematoxylin.
A
l
u
m
h
a
e
m
a
t
o
x
y
l
i
n
…
.
.
For routine H & E staining, the most commonly used
haematoxylin are:
•
Ehrlich’s haematoxylin
•
Harris haematoxylin
•
Mayer’s haematoxylin
•
Colles haematoxylin
•
Dalfield’s haematoxylin
E
h
r
l
i
c
h
s
h
a
e
m
a
t
o
x
y
l
i
n
E
h
r
l
i
c
h
s
h
a
e
m
a
t
o
x
y
l
i
n
•
is a naturally ripened haematoxylin
•
takes about 2-4 months to ripen.
•
Alum is used as mordant.
•
naturally ripened solution will last in bulk for years.
•
It is an excellent nuclear stain.
•
It also stains mucin and polysaccharide
•
is recommended for bone and cartilage.
E
h
r
l
i
c
h
s
h
a
e
m
a
t
o
x
y
l
i
n
U
s
e
s
o
f
E
h
r
l
i
c
h
’
s
s
t
a
i
n
•
Ehrlich’s stain stains nuclei intensely and crisply.
•
The stained sections fade much more slowly, than those
stained with other haematoxylin.
•
It is particularly useful for tissues that have been exposed to
acid.
•
is useful for bone, which has been exposed to acid for
decalcification.
•
is also valuable for tissues that have been fixed in formalin .
D
e
l
a
f
i
e
l
d
’
s
h
a
e
m
a
t
o
x
y
l
i
n
•
Delafield’s haematoxylin is a naturally ripened
haematoxylin.
•
Its longevity is similar to Ehrlich’s haematoxylin.
M
a
y
e
r
’
s
h
a
e
m
a
t
o
x
y
l
i
n
Mayer's haematoxylin
•
water is used as a solvent.
•
is not naturally ripened but artificially ripened with
sodium iodat
e.
M
a
y
e
r
'
s
…
.
.
used as:
•
Regressive stain like any other haematoxylin
•
Progressive stain particularly when nuclear counter
stain is needed to emphasize cytoplasmic
components
•
Nuclear counter stain in demonstration of glycogen
•
is used in various enzyme histochemical
techniques.
H
a
r
r
i
s
h
a
e
m
a
t
o
x
y
l
i
n
e
H
a
r
r
i
s
h
a
e
m
a
t
o
x
y
l
i
n
•
is not naturally ripened but artificially ripened with
mercuric oxide.
•
The nuclear staining deteriorates after few months
•
F
o
r
b
e
s
t
r
e
s
u
l
t
i
t
i
s
w
i
s
e
t
o
p
r
e
p
a
r
e
a
f
r
e
s
h
b
a
t
c
h
o
f
s
t
a
i
n
e
v
e
r
y
m
o
n
t
h
.
.
C
o
l
e
s
h
a
e
m
a
t
o
x
y
l
i
n
C
o
l
e
’
s
h
a
e
m
a
t
o
x
y
l
i
n
i
s
a
r
t
i
f
i
c
i
a
l
l
y
r
i
p
e
n
e
d
w
i
t
h
a
l
c
o
h
o
l
i
c
s
o
l
u
t
i
o
n
C
a
r
a
z
z
i
'
s
h
a
e
m
a
t
o
x
y
l
i
n
•
water is used as a solvent.
•
is artificially ripened with potassium iodate.
•
It can be used as progressive nuclear staining using
a short time followed by blueing in tape water
•
is suitable for pale and precise nuclear staining.
C
a
r
a
z
z
i
’
s
…
.
.
.
.
.
•
does not stain any of the cytoplasmic components
•
it is largely confided to its use with frozen section
for-urgent surgical biopsy.
•
For frozen section, it is an excellent nuclear
staining when used as double strength solution
(using one gm of haematoxylin).
3
.
5
.
2
.
2
I
r
o
n
h
a
e
m
a
t
o
x
y
l
i
n
e
Iron salts are used both as oxidizing agent and
mordant. Iron salts used are:
- Ferric chloride or
-
F
e
r
r
i
c
a
m
m
o
n
i
u
m
s
a
l
t
s
.
I
r
o
n
h
a
e
m
a
t
o
x
y
l
i
n
e
…
T
y
p
e
s
o
f
i
r
o
n
h
a
e
m
a
t
o
x
y
l
i
n
•
Weigert’s haematoxylin;
•
Heidenhain’s haematoxylin;
•
Loyez haematoxyline;
•
Verhoeff’s haematoxylin for elastic fiber.
W
e
i
g
e
r
t
’
s
h
a
e
m
a
t
o
x
y
l
i
n
weigert’s haematoxylin
•
ferric chloride is used as mordant and oxidant.
•
It is used as nuclear stain where acid solution is
applied subsequently
Example
vangienson stain.
H
e
i
d
e
n
h
a
i
n
’
s
h
a
e
m
a
t
o
x
y
l
i
n
e
H
e
i
d
e
n
h
a
i
n
’
s
h
a
e
m
a
t
o
x
y
l
i
n
•
ferric ammonium sulphate is used as oxidant and
mordant.
•
All components stain black or dark gray-black after
stained with Heidenhain’s haematoxylin,
•
then haematoxyline stain is removed progressively
from d/t tissue structures at d/t rates using iron-alum
solution.
L
o
y
e
z
h
a
e
m
a
t
o
x
y
l
i
n
L
o
y
e
z
h
a
e
m
a
t
o
x
y
l
i
n
•
Ferric ammonium chloride is used as mordant;
•
The mordant & haematoxylin are used consecutivel
•
D
i
f
f
e
r
e
n
t
i
a
t
i
o
n
i
s
d
o
n
e
b
y
u
s
i
n
g
W
e
i
g
e
r
t
’
s
h
a
e
m
a
t
o
x
y
l
i
n
•
It is used to demonstrate myelin;
•
It can be applied to paraffin, frozen,& nitrocellulose
section.
V
e
r
h
o
e
f
f
’
s
h
a
e
m
a
t
o
x
y
l
i
n
V
e
r
h
o
e
f
f
’
s
h
a
e
m
a
t
o
x
y
l
i
n
•
is used for demonstration of elastic fiber, elastic
fiber stain black.
•
The staining solution contains
- Haematoxyline,
- Lugol’s iodine, and
- 2% Ferric chloride as differentiator.
3
.
5
.
2
.
3
T
u
n
g
s
t
e
n
h
a
e
m
a
t
o
x
y
l
i
n
•
Haematoxylin can be used to make tungsten
haematoxylin.
•
potassium permanganate is used for oxidation.
•
the solution is short lived & should be used with in
24 hours.
•
The best method to make tungsten haematoxylin
is natural ripening of tungsten haematoxylin.
•
It takes some months to ripen but the staining
sol
n
is usable for many years
.
T
u
n
g
s
t
e
n
…
.
.
U
s
e
s
o
f
t
u
n
g
s
t
e
n
h
a
e
m
a
t
o
x
y
l
i
n
•
Demonstration of muscle striation, cilia,& glial fibers
•
Myelin can also be demonstrated (not satisfactory)
•
Many CNS structures are stained with PTAH.
3
.
5
.
2
.
4
M
o
l
y
b
d
e
n
u
m
h
a
e
m
a
t
o
x
y
l
i
n
M
o
l
y
b
d
e
n
u
m
h
a
e
m
a
t
o
x
y
l
i
n
•
is used rarely.
•
Molybdenum is used as mordant.
•
is used for demonstration of collagen & coarse
reticulin.
3
.
5
.
2
.
5
L
e
a
d
h
a
e
m
a
t
o
x
y
l
i
n
L
e
a
d
h
a
e
m
a
t
o
x
y
l
i
n
•
it is when haematoxylin sol
n
s incorporate lead .
•
used in the demonstration of granules & endocrine
cells of the alimentary tract and other regions.
•
Its most useful application is in the identification of
endocrine cells in tumors of doubtful origin.
3
.
5
.
2
.
6
H
a
e
m
a
t
o
x
y
l
i
n
w
i
t
h
o
u
t
m
o
r
d
a
n
t
H
a
e
m
a
t
o
x
y
l
i
n
w
i
t
h
o
u
t
m
o
r
d
a
n
t
•
Freshly used haematoxylin can be used without
mordant.
•
It has been used to demonstrate various minerals
in tissue sections.
3
.
6
E
o
s
i
n
E
o
s
i
n
•
is a xanthene dye
•
is the most suitable stain when combined with
Haematoxyline.
•
used to distinguish & differentiate the cytoplasm of d/t
types of cells.
•
Therefore, different types of cells & connective tissue
fibers can be distinguished
E
o
s
i
n
…
T
y
p
e
s
o
f
e
o
s
i
n
•
Eosin Y (eosin yellow);
•
Ethyl eosin;
•
Eosin B (eosin bluish).
E
o
s
i
n
Y
•
Most widely used eosin;
•
It is a water-soluble stain and also satisfactorily
soluble in alcohol.
E
o
s
i
n
…
Eosin solution:
•
0.5 to 1 % solution is prepared in water.
•
A crystal of thymol is added to prevent fungal
growth and the addition of little acetic acid
sharpens the staining.
E
o
s
i
n
…
U
s
e
o
f
e
o
s
i
n
s
t
a
i
n
s
E
o
s
i
n
s
t
a
i
n
s
c
a
n
b
e
u
s
e
d
a
s
d
i
f
f
e
r
e
n
t
i
a
l
s
t
a
i
n
.
•
Differentiation of eosin staining occurs with tap water.
•
Further differentiation occurs during dehydration
through alcohols.
•
The intensity of eosin staining and degree of
differentiation depend on the examining pathologist.
E
o
s
i
n
…
•
Adequate differentiation may be difficult if tissue
has been fixed in mercuric oxide.
•
Over differentiation of eosin may be continued
until only RBC & granules of eosinophils are
stained.
•
This method helps in location & identification of
eosinophils.
E
o
s
i
n
…
E
t
h
y
l
e
o
s
i
n
a
n
d
E
o
s
i
n
B
•
Ethyl eosin and Eosin B are rarely used, for example, in
Harri’s stain for Negri bodies
.
E
o
s
i
n
s
u
b
s
t
i
t
u
t
e
s
•
Certain red dyes are alternatively used as eosin. They are
phloxine, Biebrich scarlet etc.
•
But these chemicals give a more intense red color to
tissues and rarely give subtle differentiation & hence, they
are less valuable.
3
.
7
Q
u
i
c
k
h
a
e
m
a
t
o
l
o
x
y
l
i
n
a
n
d
e
o
s
i
n
s
t
a
i
n
f
o
r
u
r
g
e
n
t
b
i
o
p
s
e
s
1.
Tissue is frozen onto a chuck using aerosol spray.
2. Cut sections with cryostat of thickness from 3-6
microns.
3. Dip in neutral 10% solutions at room temperature
for 20 seconds to fix.
4. Rinse in tap water.
5. Dip in double strength Carazi’s haematoxylin to
stain.
Q
u
i
c
k
h
a
e
m
a
t
o
l
o
x
y
l
i
n
a
n
d
e
o
s
i
n
…
.
6. Dip and rinse in tap water for 10 to 20 seconds.
7. Dip in 1% aqueous eosin for 10 seconds.
8. Dip in tap and rinse.
9. Dehydrate as usual in 50% ,70%, 90%, and 100%
alcohol.
10. Mount in DPX using xylene.
3
.
8
S
t
a
i
n
i
n
g
o
f
p
r
o
t
e
i
n
s
•
Proteins are major constitutes of cells and tissues and are made up
of amino acids linked by peptide bonds.
•
Proteins in cells and tissues:
•
Proteins alone may occur in cells and tissues for example, albumin,
globulin, fibrous
•
structural proteins, enzymes, and component of complement
system;
•
•
Conjugated proteins: Proteins conjugated with other substances and
occur in tissues;
•
Lipo proteins: proteins conjugated with lipids and seen in tissues;
•
Nucleoproteins: Proteins conjugated with nucleic acid and seen in
tissues.
•
3
.
8
.
1
M
e
t
h
o
d
s
o
f
d
e
m
o
n
s
t
r
a
t
i
o
n
o
f
p
r
o
t
e
i
n
s
i
n
t
i
s
s
u
e
•
Histophysical method;
•
Amino acid histochemical method;
•
Enzyme histochemical method;
•
Immuno-fluorescent method;
•
Immuno histochemical method.
3
.
8
.
1
.
1
H
i
s
t
o
p
h
y
s
i
c
a
l
m
e
t
h
o
d
s
H
i
s
t
o
-
p
h
y
s
i
c
a
l
m
e
t
h
o
d
s
•
are based on physical configuration of their
molecules & not on their chemical composition.
•
They are also based on selective staining with
large or small molecule dyes.
E
x
a
m
p
l
e
-
t
r
i
c
h
r
o
m
e
m
e
t
h
o
d
.
H
i
s
t
o
-
p
h
y
s
i
c
a
l
m
e
t
h
o
d
s
…
…
.
Using these technique proteins present in
- collagen
- fibrin
- elastic
- reticulin
- amyloid can be demonstrated.
3
.
8
.
1
.
2
A
m
i
n
o
a
c
i
d
h
i
s
t
o
c
h
e
m
i
c
a
l
m
e
t
h
o
d
s
A
m
i
n
o
a
c
i
d
h
i
s
t
o
c
h
e
m
i
c
a
l
m
e
t
h
o
d
s
•
are based upon identification of exposed
groupings and linkages with in amino acid
molecules.
•
With this staining methods some of the amino
acids in the proteins and not the whole proteins
are demonstrated.
A
m
i
n
o
a
c
i
d
h
i
s
t
o
c
h
e
m
i
c
a
l
…
…
D
e
m
o
n
s
t
r
a
t
i
o
n
o
f
A
m
i
n
o
g
r
o
u
p
b
y
N
i
n
h
y
d
r
i
n
S
c
h
i
f
f
r
e
a
c
t
i
o
n
f
o
r
l
y
s
i
n
e
•
At neutral pH and 37
0
C, Ninhydrin reacts with
alpha aminogrops to produce aldehydes.
•
T
h
e
a
l
d
e
h
y
d
e
c
a
n
t
h
e
n
b
e
d
e
m
o
n
s
t
r
a
t
e
d
u
s
i
n
g
S
c
h
i
f
f
r
e
a
g
e
n
t
.
F
i
x
a
t
i
o
n
s
o
l
u
t
i
o
n
-Neutral formal saline or
-formaldehyde vapor
.
A
m
i
n
o
a
c
i
d
h
i
s
t
o
c
h
e
m
i
c
a
l
…
…
D
e
m
o
n
s
t
r
a
t
i
o
n
o
f
p
h
e
n
y
l
g
r
o
u
p
b
y
M
i
l
l
o
n
r
e
a
c
t
i
o
n
f
o
r
t
y
r
o
s
i
n
e
•
The section is treated with hot mercuric-sulphric-
acid nitrate mixture. A pinkish red color develops at
the site of tyrosine containing protein.
•
Tyrosine is the only amino acid, which contains the
hydroxyphenyl group.
•
This can be demonstrated histochemically.
D
e
m
o
n
s
t
r
a
t
i
o
n
o
f
p
h
e
n
y
l
g
r
o
u
p
b
y
M
i
l
l
o
n
r
e
a
c
t
i
o
n
f
o
r
t
y
r
o
s
i
n
e
•
This method can be regarded as specific for
tyrosine.
•
Tyrosine is invariably present in all tissue proteins.
•
Millon reaction is a suitable general protein
method. However, the color reaction is not strong.
F
i
x
a
t
i
v
e
:
N
e
u
t
r
a
l
f
o
r
m
a
l
i
n
,
f
o
r
m
a
l
d
e
h
y
d
e
v
a
p
o
r
S
e
c
t
i
o
n
:
P
a
r
a
f
f
i
n
,
f
i
x
e
d
c
r
y
o
s
t
a
t
,
f
r
e
e
z
d
r
i
e
d
o
r
c
o
l
l
o
d
i
n
.
A
m
i
n
o
a
c
i
d
h
i
s
t
o
c
h
e
m
i
c
a
l
m
e
t
h
o
d
s
…
.
S
t
a
i
n
i
n
g
s
o
l
u
t
i
o
n
S
o
l
u
t
i
o
n
A
:
M
e
r
c
u
r
i
c
s
u
l
p
h
a
t
e
-
s
u
l
p
h
r
i
c
a
c
i
d
m
i
x
t
u
r
e
1. Mercuric sulphate --------------------10 gm
2. Distilled water ------------------------90ml
3. Concentrated sulphric acid ----------10ml
P
r
e
p
a
r
a
t
i
o
n
o
f
m
e
r
c
u
r
i
c
s
u
l
p
h
a
t
e
-
s
u
l
p
h
r
i
c
a
c
i
d
m
i
x
t
u
r
e
1. To 90ml of Distilled water, add concentrated sulphric
acid slowly.
2. To the mixture add 10 gm of Mercuric sulphate.
A
m
i
n
o
a
c
i
d
h
i
s
t
o
c
h
e
m
i
c
a
l
m
e
t
h
o
d
s
…
.
S
o
l
u
t
i
o
n
B
:
S
o
d
i
u
m
n
i
t
r
i
t
e
s
o
l
u
t
i
o
n
Two hundred fifty mg (250 mg) of sodium nitrite is
dissolved in 10 ml of distilled water.
S
t
a
i
n
i
n
g
s
o
l
u
t
i
o
n
Five ml (5ml) of solution B is added to 50 ml of
solution A.
3
.
9
C
a
r
b
o
h
y
d
r
a
t
e
S
t
a
i
n
s
Carbohydrates in tissue include:
A.
Glycogen
B. Acid mucins
E
x
a
m
p
l
e
-
C
h
o
n
d
r
o
i
t
i
n
s
u
l
p
h
a
t
e
A
;
- Chondroitin sulphate B;
C. Neutral mucin
D. Other carbohydrates, which encompass:
E
x
a
m
p
l
e
-
C
h
i
t
i
n
;
- Starch;
C
a
r
b
o
h
y
d
r
a
t
e
S
t
a
i
n
s
…
.
.
•
Types of fixatives used depend on the type of
carbohydrate to be demonstrated. Fixatives are
least importance where starch, chitin, and cellulose
are concerned.
•
Fixatives used for glycogen include:
–
Alcohol (80%);
–
Picric acid;
–
Formalin;
–
Potassium permanganate;
–
Glutaraldehyde.
C
a
r
b
o
h
y
d
r
a
t
e
S
t
a
i
n
s
…
.
.
T
i
m
e
o
f
f
i
x
a
t
i
v
e
•
Fixative should be used promptly because there is
sharp loss of glycogen after death or removal of
tissue.
•
Ideally fixation should be carried out at 4
0
C.When
fixative solution is at room temperature, glycogen
may show artifact.
•
Freeze-drying will prevent artifact.
C
a
r
b
o
h
y
d
r
a
t
e
S
t
a
i
n
s
…
.
.
The type of fixatives will also affect the presentation
of glycogen when seen in stained solution.
E
x
a
m
p
l
e
:
–
w
h
e
n
t
i
s
s
u
e
i
s
f
i
x
e
d
i
n
f
o
r
m
a
l
i
n
g
l
y
c
o
g
e
n
i
s
s
e
e
n
a
s
f
i
n
e
g
r
a
n
u
l
e
s
.
–
when tissue is fixed in alcohol glycogen is seen as
coarse granules.
Therefore, alcohol fixative is preferred because
glycogen is seen as coarse granules.
C
a
r
b
o
h
y
d
r
a
t
e
S
t
a
i
n
s
…
.
.
•
Optimal fixative for glycogen is best accomplished
by placing the tissue as quickly as possible in to
fixative after death or after removal from the body.
•
Optimal fixatives for glycogen are:
- 80% alcohol or
-10% saturated alcohol - picric acid for 1-2
days at 4
0
C.
3
.
1
0
S
t
a
i
n
s
u
s
e
d
f
o
r
d
e
m
o
n
s
t
r
a
t
i
o
n
o
f
g
l
y
c
o
g
e
n
.
S
t
a
i
n
s
u
s
e
d
f
o
r
d
e
m
o
n
s
t
r
a
t
i
o
n
o
f
g
l
y
c
o
g
e
n
i
n
c
l
u
d
e
:
•
Haematoxylin;
•
Enzymes;
•
Periodic acid schiff (PAS);
•
Best’s carmine stain (BCS).
3
.
1
0
.
1
H
a
e
m
a
t
o
x
y
l
i
n
H
a
e
m
a
t
o
x
y
l
i
n
Glycogen stains faintly with haematoxylin and eosin
stain (H & E).
3
.
1
0
.
2
E
n
z
y
m
e
s
-
Alpha amylase,
-
Beta amylase and
-
- diastase enzymes are use to extract glycogen
from tissues.
E
n
z
y
m
e
s
…
.
.
•
Alpha amylase is extracted from pancreas,
Bacillus subtils
, and
Aspergellus ozyzenec
.
•
Beta amylase is obtained from bareley, and
sweet potato.
•
Diastase is extracted from malt.
It is cheap, contains both alpha and beta amylase.
Salivary amylase can also be used.
E
n
z
y
m
e
s
…
.
.
E
n
z
y
m
e
e
x
t
r
a
c
t
i
o
n
a
n
d
s
t
a
i
n
i
n
g
f
o
r
g
l
y
c
o
g
e
n
P
r
i
n
c
i
p
l
e
•
Test and control sections are prepared, test
sections are treated with enzyme, which extracts
glycogen, and then both test and control
sections are treated with glycogen stain.
•
The test section is negative while the control
section is positive for glycogen.
3
.
1
1
S
p
e
c
i
a
l
S
t
a
i
n
s
3
.
1
1
.
1
P
e
r
i
o
d
i
c
a
c
i
d
s
c
h
i
f
f
r
e
a
c
t
i
o
n
(
P
A
S
)
•
is useful indicator of the presence of glycogen.
•
It is a particularly useful when the technique
incorporates a diastase digestion stage.
•
Periodic acid brings about oxidation & cleavage of
carbon-to-carbon bond in glycol or their amino or
alkaline derivatives to form di-aldehydes.
P
e
r
i
o
d
i
c
a
c
i
d
s
c
h
i
f
f
r
e
a
c
t
i
o
n
(
P
A
S
)
…
•
The aldehydes react with fuchsin sulphurous acid.
•
Fuchsin sulphurous acid combines with basic
pararosaniline to form magenta colored
compound.
P
e
r
i
o
d
i
c
a
c
i
d
s
c
h
i
f
f
r
e
a
c
t
i
o
n
(
P
A
S
)
…
•
Other oxidants have been employed but they
over oxidize.
•
Hence, they have not achieved popularity of
periodic acid.
•
Such oxidants are:
-chromic acid,
-potassium permanganate (KMNO
4
) and
-lead acetate.
Procedure of PAS staining
•
Test and control sections are prepared from the
tissue to be examined.
•
The test smears are treated with enzyme
•
Both the test and control smears are stained with
suitable stains for glycogen. The control smears
stain for glycogen.
There is less need for prompt fixation of mucin.
Optimal fixatives used for mucin are less well
defined.
P
e
r
i
o
d
i
c
a
c
i
d
s
c
h
i
f
f
r
e
a
c
t
i
o
n
(
P
A
S
)
…
F
o
r
m
a
l
i
n
f
i
x
a
t
i
v
e
:
I
t
s
h
o
u
l
d
b
e
u
s
e
d
i
f
a
l
d
e
h
y
d
e
f
u
s
c
h
i
n
s
t
a
i
n
i
s
t
o
b
e
u
s
e
d
.
M
e
r
c
u
r
i
c
c
h
l
o
r
i
d
e
:
M
e
t
a
c
h
r
o
m
a
t
i
c
s
t
a
i
n
i
n
g
w
i
l
l
b
e
e
n
h
a
n
c
e
d
.
A
l
c
o
h
o
l
f
i
x
a
t
i
v
e
:
I
t
d
e
m
o
n
s
t
r
a
t
e
s
a
b
n
o
r
m
a
l
c
a
r
b
o
h
y
d
r
a
t
e
i
n
m
u
c
o
p
o
l
y
s
a
c
c
h
a
r
i
d
o
s
i
s
.
–
10 % formal alcohol: Hyaluronic acid
–
5 % trichloro acetic acid if carbohydrate in calcified
tissue should be demonstrated.
P
e
r
i
o
d
i
c
a
c
i
d
s
c
h
i
f
f
r
e
a
c
t
i
o
n
(
P
A
S
)
…
S
e
c
t
i
o
n
p
r
e
p
a
r
a
t
i
o
n
f
o
r
d
e
m
o
n
s
t
r
a
t
i
o
n
o
f
g
l
y
c
o
g
e
n
P
a
r
a
f
f
i
n
p
r
o
c
e
s
s
i
n
g
i
s
s
a
t
i
s
f
a
c
t
o
r
y
f
o
r
c
e
l
l
u
l
o
s
e
,
s
t
a
r
c
h
,
c
h
i
t
i
n
,
&
m
o
s
t
m
u
c
i
n
.
F
r
e
s
h
f
r
o
z
e
n
s
e
c
t
i
o
n
P
r
e
f
e
r
r
e
d
f
o
r
s
t
a
b
l
e
m
u
c
i
n
p
r
e
s
e
n
t
i
n
m
u
c
o
p
o
l
y
s
a
c
c
h
a
r
i
d
e
.
7
0
%
a
l
c
o
h
o
l
should be used while using paraffin embedded material.
R
o
u
t
i
n
e
c
e
l
l
u
l
o
i
d
i
n
s
o
l
u
t
i
o
n
•
Is important in fresh frozen section in order
to prevent glycogen diffusion and loss. The
test smears do not stain for glycogen
because enzyme removes glycogens from
tissues.
3
.
1
1
.
2
M
u
c
i
n
s
t
a
i
n
s
There are a variety of mucin stains, all attempting to
demonstrate one or more types of
mucopolysaccharide substances in tissues.
The types of mucopolysaccharides are as follows:
N
e
u
t
r
a
l
These can be found in glands of the GI tract and in
prostate.
They stain with PAS but not with Alcian blue,colloidal
iron, mucicarmine, or metachromatic dyes.
M
u
c
i
n
s
t
a
i
n
s
A
c
i
d
(
s
i
m
p
l
e
,
o
r
n
o
n
-
s
u
l
f
a
t
e
d
)
•
Are the typical mucins of epithelial cells containing
sialic acid.
•
They stain with PAS, Alcin blue at pH 2.5, colloidal
iron, and metachromatic dyes.
•
They resist hyaluronidase digestion.
M
u
c
i
n
s
t
a
i
n
s
A
c
i
d
(
s
i
m
p
l
e
,
m
e
s
e
n
c
h
y
m
a
l
)
•
These contain hyaluronic acid & are found in
tissue stroma.
•
They do not stain with PAS, but do stain with
Alcian blue at pH 2.5, colloidal iron, and
metachromatic dyes.
•
They digest with hyaluronic acid.
•
They can be found in sarcomas.
M
u
c
i
n
s
t
a
i
n
s
There are a variety of stains for mucin which
include:
C
o
l
l
o
i
d
a
l
i
r
o
n
:
I
r
o
n
p
a
r
t
i
c
l
e
s
a
r
e
s
t
a
b
i
l
i
z
e
d
i
n
a
m
m
o
n
i
a
a
n
d
g
l
y
c
e
r
i
n
a
n
d
a
r
e
a
t
t
r
a
c
t
e
d
t
o
a
c
i
d
m
u
c
o
p
o
l
y
s
a
c
c
h
a
r
i
d
e
s
.
•
It requires formalin fixation.
•
Phospholipids and free nucleic acids may
also stain.
M
u
c
i
n
s
t
a
i
n
s
•
The actual blue color comes from a
Prussian blue reaction.
•
Tissue can be pre-digested with
hyaluronidase to provide more specificity.
M
u
c
i
n
s
t
a
i
n
s
A
l
c
i
a
n
b
l
u
e
The pH of this stain can be adjusted to give more
specificity.
P
A
S
(
p
e
r
o
i
d
i
c
a
c
i
d
-
s
c
h
i
f
f
)
Stains glycogen as well as mucins, but tissue can be
pre-digested with diastase to remove glycogen.
M
u
c
i
c
a
r
m
i
n
e
:
V
e
r
y
s
p
e
c
i
f
i
c
f
o
r
e
p
i
t
h
e
l
i
a
l
m
u
c
i
n
s
.
M
u
c
i
n
s
t
a
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n
s
•
The mucin stain with the most specificity is
mucicarmine, but it is very insensitive, so it is not
really very useful.
•
The stain that is the most sensitive is PAS, but
you must learn how to interpret it in order to gain
specificity.
•
Colloidal iron stains are unpredictable.
.
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Alcian blue stains are simple, but have a
lot of background staining
R
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q
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1. Define staining.
2. Explain the classification of hematoxyline based
on mordant used.
3. Discuss factors that contribute to dye-tissue
affinity.
4. Elaborate the different methods of
demonstration of proteins.
5. Describe carbohydrate stain and special stains.
Bibliography
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Cheesbrough M.
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Beginning with an introduction to staining in biochemical techniques, this chapter delves into the types of stains, factors influencing selectivity, and commonly used stains in histopathology. Learning objectives include defining staining, discussing dye-tissue affinity, understanding selectivity factors, and classifying hematoxylin based on mordants. The content explores methods for protein demonstration, carbohydrate staining, and special stains. An overview of staining principles involving alcohol solutions and clearing agents is provided, along with insights on dye-tissue affinity and the two-way procedure involved.
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Presentation Transcript
CHAPTER THREE Principles of staining
Content Introduction Types of stain Affinity of staining Factors that determine selectivity of stain Types of commonly used stains in histopathologic techniques
Learning objectives Upon completion of this chapter, the student will be able to: Define staining. Discuss factors that contribute to dye-tissue affinity. Explain factors affecting selectivity of stain. Explain the classification of hematoxyline on the basis of mordant used.
Learning object Describe method of demonstration of proteins. Describe carbohydrate stain. Describe special stain.
3.1 Introduction Staining is a biochemical technique of adding a classic specific dye to a substrate: DNA Proteins Lipids and Carbohydrates to qualify or quantify the presence of a specific compound.
3.2 Types of stain Negative stain is the type of stain that does not color the tissue or cells but surrounds the tissue as a result uncolored tissue can be seen against a background color.
Types of stain Supravital staining is the stain taken by living cells. Special stain is the stain that identifies various specialized structures. Differential stain the stain uses two or more dyes that react differently.
Principle of staining The stained slide must go through the reverse process that it went through from paraffin section to water . The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass cover slip, or a plastic film, can be placed over the section.
3.3 Affinity of staining Affinity means the force, which binds the dye to the tissues. The word crisp is used in textile industry instead of affinity. Affinity is not a one-way procedure but a two-way procedure as it depends on the tissue as well as on the dye.
3.3.1 Factors contributing to dye-tissue affinity Factors that can adversely affects staining of tissues: Solvent-solvent interaction (Hydrophobic bonding) Reagent-reagent interaction Reagent-tissue interaction (Vander -Waal forces) Columbic attraction Hydrogen bonding Covalent bonding.
3.3.1.1 Solvent-solvent interaction There is a tendency of hydrophobic grouping or chemicals or substances to come together even though initially dispersed in aqueous (water) solution. The water molecules are transiently held together in clusters by hydrogen bonding.
Solvent-solvent interaction These clusters are stabilized by hydrophobic group Any process that involves breakup of the clusters in to disorganized water molecules will tend to occur spontaneously. Phenylalanine and tryptophane side chains or biphenyl naphthol are hydrophobic grouping.
3.3.1.2 Reagent-reagent interaction Dye interacts with each other and form aggregates. Dye aggregation increases with concentration. Cations or basic dyes build up in tissues where there is high negative charge such as, - polysaccharides in mast cell granules and - cartilage matrix.
Reagent-reagent Metachromatic staining This is due to dye aggregation having spectral properties unlike those of monomeric dyes. Silver impregnation technique There is stain-stain interaction. Gomer s type enzyme histochemistry There is stain-stain interaction
3.3.1.3 Dye - tissue interaction or Vander- Walls reaction Van-deer-Walls force acts between dye and tissues. is of short range. Therefore, Van-deer-Walls force is strong where close reagent contact is possible.
Van-deer-Walls force. Substrates and chemicals that favor Van-der-walls bonding include: Tyrosine and tryptophane residues of proteins; Heterocyclic bases of nucleoproteins; Halogenated dyes such as, - rose Bengal, - phloxine, - enzyme substrate, - naphthyl indoxyl system.
3.3.1.4 Columbic attraction Columbic attraction termed as salt link or electrostatic bond. the electrostatic bonds arise from electrostatic attraction of dissimilar ions is the most widely acknowledged reagent tissue interaction. Example The colored cation of basic dyes and tissue rich in anion such as - phosphated DNA and RNA - carboxylated or - sulphated mucosubstances. structures
Columbic attraction.. In case of columbic attraction, the amount of dye able to enter a given tissue will depend on: charge of dye, magnitude of charges, amount of non-electrolyte present in dye bath ability of tissue to sink or swell.
3.3.1.5 Hydrogen bonding Hydrogen bonding(H-bonding) is a localized bond when a hydrogen atom lies b/n two electronegative atoms such as ,H or N . It will rarely be an important source of reagent- tissue affinity when aqueous solvents are used. if dye is taken by other process, H- bonding may subsequently contribute to affinity.
3.4 Factors that determine selectivity ofstain Dye or stains are not taken by every part of tissue, and this is called selectivity of stain. Factors that determine selectivity of stain include: Number and affinity of binding sites Rates of reagent uptake Rate of reaction Rate of reagent loss.
3.4.1 Number and affinity of binding sites The staining affinity of dye depends on: the number of binding sites of the dye to tissue. Reagent-tissue affinities and the number of binding site vary. Example non-ionic Sudan dye will have affinity for fat droplets, but none for the surrounding hydrated proteins. Therefore Sudan stains intensely fat tissues and not proteins or carbohydrates.
5.4.2 Rate of reagent uptake by the tissue Selectivity of the stain by the tissue depends on the rate of reagent uptake by the tissue. Therefore, selectivity can be controlled by modifying the time of staining.
Rate of reagent uptake by the tissue Example short period of staining mucin-staining method using Alcian blue or colloidal iron. prolonged period of staining nucleic acid and RNA-rich cytoplasm are also stained using Alcian blue or colloidal iron.
Rate of reagent uptake There are methods where three or more dyes diffuse at varying rates, as a result different structures can be stained by different dyes. Example collagen fibers stain rapidly while the muscle fibers stain at intermediate rate
3.4.3 Rate of reaction Selectivity of the stain also depends on the rate of reaction. This is because, - reactive stain yield colored derivatives, & - amount of color depend on the selective rates of reaction. Example The periodic acid oxidation step of the periodic acid- Schiff procedure, if sufficiently prolonged, oxidize various chemical structures present in tissue.
Rate of reaction Enzyme examples of reaction rates that affect selectivity. histochemistry provides many other Example at low pH the hydrolysis of an organic phosphate applied to a tissue section in a suitable incubation media will be rapid in those parts of the tissue containing acid phosphates. However, in structures containing alkaline phosphatase , the hydrolysis rate will be very slow.
3.4.4 Rate of reagent loss Some decolorized structures are not decolorized readily. tissues or structures while are other stained tissues and or readily
Rate of reagent loss. Differentiation Is when a decolorized tissue takes up the counter stain. Example staining of muscle striation with iron Haematoxyline and myelin sheath with Luxol Fast Blue. In regressive staining all structures are first stained quite non-selectively some times penetration aid such as heat or phenol. with the assistance of Subsequently, the tissues are extracted in a solvent. The solvent extracts the dye from the tissue. And this is known as destaining.
Rate of reagent loss. The staining conditions chosen to maximize selective affinity. Basic dyes must be applied from neutral or acidic solution. the concentration of inorganic salt present in a dye bath ,called critical electrolyte concentration. rate of reagent uptake, or subsequent reaction or loss of reagent or product.
3.5 Types of commonly used stains in histopathologic techniques 3.5.1 Haematoxyline (H) is the most popular & widely used histologic stain. extracted from heartwood Haematoxyline campechium with hot water. of the tree called can demonstrate clearly enormous number of d/t tissue structures.
Natural oxidation of Haematoxyline natural oxidation(ripening) is exposing haematoxyline to light & air and converting it to hematin. is a slow process, it may takes 3-4 months. the hematine solution retains its staining ability for a long time. Examples -Ehrlichs haematoxyline -Delafield s haematoxyline.
Haematoxyline.. Haematoxyline processed by various methods. also stains tissues that are In the routine H & E stain, nuclei stain blue-black with good intra nuclear details. While Eosin stains cytoplasm and connective tissue in varying intensity of pink, orange & red.
Haematoxyline.. Haematoxyline has many more uses than in E & H combination. Haematoxyline haematin, an oxidant product of Haematoxyline is a stain & a natural dye. by itself is not stain but
Production of haematin from Haematoxyline Haematoxyline is extracted from heartwood or logwood. It is precipitated by using urea. Haematin is produced from Haematoxyline by two ways. 1. Natural oxidation or ripening of Haematoxyline. 2. Chemical oxidation of Haematoxyline.
Chemical oxidation of haematoxyline haematoxyline is oxidized by chemicals such as - sodium iodate & - Mercuric oxide. Sodium is used as an oxidant in preparation of Mayer s haematoxyline Mercuric oxide for Harris haematoxyline
Chemical oxidation Advantages Oxidize haematoxylin almost instantaneously; Ready to be used after immediate preparation. Disadvantage have shorter useful life when compared to naturally ripened haematoxylin.
3.5.2. Classification of haematoxyline based on mordant used Classification of haematoxyline based on mordant: Alum haematoxylin; Iron haematoxylin; Tungsten haematoxylin; Molybdenum haematoxylin; Lead haematoxylin; Haematoxylin with out mordant.
3. 5.2.1 Alum haematoxylin Types of Alum haematoxylin Ehrlichs haematoxylin; Delafield s haematoxylin; Mayer s haematoxylin; Harris haematoxyline; Coles haematoxylin; Carazzi s haematoxylin.
Alum haematoxylin. Blueing After staining with haematoxylin, the section is treated with:- tap water or - Scott s tap water or - lithium carbonate. The red color of the nuclei becomes blue and this is known as blueing.
Alum haematoxylin.. Disadvantages of alum haematoxylin The stain is sensitive to any subsequently applied acid solution. Example - vangieson and - trichrome staining. The picric acid fuchsine mixture in vangieson stain removes most of the haematoxylin so that nucleus becomes faint and not easily seen.
Alum haematoxylin.. In such situation, - Iron-mordant haematoxylin - a combination of alum haematoxylin - Celestine blue is used, since Celestine blue solution is prepared in ferric acid solution & the ferric salt in Celestine blue solution strengthens the bond b/n the nucleus & alum haematoxylin.
Alum haematoxylin.. For routine H & E staining, the most commonly used haematoxylin are: Ehrlich s haematoxylin Harris haematoxylin Mayer s haematoxylin Colles haematoxylin Dalfield s haematoxylin
Ehrlichs haematoxylin Ehrlichs haematoxylin is a naturally ripened haematoxylin takes about 2-4 months to ripen. Alum is used as mordant. naturally ripened solution will last in bulk for years. It is an excellent nuclear stain. It also stains mucin and polysaccharide is recommended for bone and cartilage.
Ehrlichs haematoxylin Uses of Ehrlich s stain Ehrlich s stain stains nuclei intensely and crisply. The stained sections fade much more slowly, than those stained with other haematoxylin. It is particularly useful for tissues that have been exposed to acid. is useful for bone, which has been exposed to acid for decalcification. is also valuable for tissues that have been fixed in formalin .
Delafields haematoxylin Delafield s haematoxylin is a naturally ripened haematoxylin. Its longevity is similar to Ehrlich s haematoxylin.
Mayers haematoxylin Mayer's haematoxylin water is used as a solvent. is not naturally ripened but artificially ripened with sodium iodate.
Mayer's.. used as: Regressive stain like any other haematoxylin Progressive stain particularly when nuclear counter stain is needed to emphasize cytoplasmic components Nuclear counter stain in demonstration of glycogen is used in various enzyme histochemical techniques.
Harris haematoxyline Harris haematoxylin is not naturally ripened but artificially ripened with mercuric oxide. The nuclear staining deteriorates after few months For best result it is wise to prepare a fresh batch of stain every month. .
Coles haematoxylin Cole s haematoxylin is artificially ripened with alcoholic solution Carazzi's haematoxylin water is used as a solvent. is artificially ripened with potassium iodate. It can be used as progressive nuclear staining using a short time followed by blueing in tape water is suitable for pale and precise nuclear staining.
Carazzis..... does not stain any of the cytoplasmic components it is largely confided to its use with frozen section for-urgent surgical biopsy. For frozen section, it is an excellent nuclear staining when used as double strength solution (using one gm of haematoxylin).
