STAINING
Process of applying dyes/stains on the section to see and study the architectural pattern of the tissue and
physical characteristics of cells; dye application
In staining, you need:
a. Stain – most important requirement
b. Tissue to be stained
c. Staining jar [couplin jars, slotted staining dishes] sa libro daw yan
Ano daw potek
3 STAINING CATEGORIES in general
Histological Histochemical/Histochemistry Immunohistochemical staining
a. Tissue components are demonstrated by Tissue components are studied Combination of histochemical and
direct interaction with a dye or staining through chemical reaction immunologic technique
solution (ie. There is a need to demonstrate Purpose: demonstrate the
nucleus and cytoplasm of cells); (to stain cells chemical components like fats, Instead of stains/dyes, monoclonal
& tissues) carbohydrates etc. or polyclonal antibodies are used
Ex.
b. Active tissue component is colored • Perl’s Prussian blue – for Detection of phenotypic markers
hemoglobin that are detected by antibodies
c. If you want to stain the nucleus and • Periodic acid Schiff – for i.e. monoclonal, polyclonal
cytoplasm of cells carbohydrates
• Sudan dyes – fat stain
d. Microanatomical stains (gram stain, acid- • Sudan black
fat stain); hematology (to stain blood cells – • Sudan III
wright stain); microbiology bacterial stains –
methylene blue, carbol fuchsin, crystal violet;
Specific tissue stains; blood parasites stains –
Giemsa stain
9 STAINING TECHNIQUES
Direct staining o Uses only 1 dye (tissue and dye only)
o Usually, the color of the dye is also the resulting color
o Ex. Methylene blue – resulting color is also blue
o uses aqueous or alcoholic dye solutions to produce a color
Indirect staining o Tissue component is demonstrated using a dye with the help of mordant or
accentuator (tissue, dye, mordant or accentuator)
o Uses mordant or accentuator to demonstrate tissue component
o MORDANT – substance that serves as link or bridge between the dye and the tissue
(see on the next pages)
- General example of mordants and their stain:
Potassium alum – specific example of mordant with potassium alum are:
a. Mayer’s hematoxylin stain
b. Ehrlich’s h.
c. Gill’s h.
d. Cole’s h.
Iron hematoxylin – Specifically Ferric ammonium sulfate & Ferric
ammonium chloride; Specific example of mordant with iron:
a. Heidenhain’s h. – Ferric ammonium sulfate
b. Weigert’s h. – Ferric ammonium chloride
, So pag tinanong, what are examples of stains with mordants? Ang sagot ay ehrlichs’s,
mayer’s, gill’s, weigert’s etc.
Aside from iron, we can also use COPPER as mordant = Copper hematoxylin
We can also use Phosphotungstic acid = PTAH
o Also uses ACCENTUATOR – Not part of the staining reaction; substance that ONLY
heightens the color intensity and selectivity of the stain/dye; increases the staining
power; patitingkarin nya yung kulay na matamlay.
- Ex. Of accentuators and their stain
KOH – Loeffler’s methylene blue stain
Phenol – used whenever we use carbol fuchsin and carbol thionine
Progressive staining o Involves gradual application of dye/stain to tissues; dye is applied to tissues little by
little until the desired color is achieved so there’s a sequence followed kasi nga
gradually sya inaapply eh
o Since dye is applied little by little, there is no excess dye
o Decolorization is not performed/not part of the process
o tissue elements re stained in a definite sequence.
Regressive staining o Tissue is first OVERSTAINED so there is excess dye and it is removed through
decolorization.
o DECOLORIZATION/differentiation – Selective removal of excess dye;
o Commonly used decolorizer is acid alcohol
o Rule: Use basic solution in removing acid dyes and use acid dye if the solution is basic.
o Acid alcohol is the one commonly used because it can remove both acid and basic dye
o Ex. H&E stain
Counterstaining o Involves application of a different color
o Uses another dye to produce contrast and background
o Most commonly used counterstain is eosin.
o Eosin – a red acid dye as to pH; a cytoplasmic stain.
o Nucleus of cells are ACID in nature because of nucleic acid
o Cytoplasm in nature is BASIC in nature.
o Available on 3 Forms:
1. Eosin Y – Yellowish
2. Eosin B – Bluish
3. Ethyl Eosin – Eosin S
o RULE in STAINING: if you will stain an acid structure, use a basic dye. If there is a need
to demonstrate a basic component, use acid. So opposite attracts.
Metachromatic staining o Uses a dye that will impart/produce a different color, different from the color of the
dye itself. (Stain the tissue w/ a color that is different from the stain itself, opposite of
direct staining)
o The stain/dye here will produce a different color
o Examples of metachromatic stains:
Methyl violet or crystal Cresyl blue for Safranin
violet reticulocytes
Bismarck brown Basic fuchsin Methylene blue
Thionine (also supravital Toluidine blue (also Azure A, B, C
stain) supravital stain)
Process of applying dyes/stains on the section to see and study the architectural pattern of the tissue and
physical characteristics of cells; dye application
In staining, you need:
a. Stain – most important requirement
b. Tissue to be stained
c. Staining jar [couplin jars, slotted staining dishes] sa libro daw yan
Ano daw potek
3 STAINING CATEGORIES in general
Histological Histochemical/Histochemistry Immunohistochemical staining
a. Tissue components are demonstrated by Tissue components are studied Combination of histochemical and
direct interaction with a dye or staining through chemical reaction immunologic technique
solution (ie. There is a need to demonstrate Purpose: demonstrate the
nucleus and cytoplasm of cells); (to stain cells chemical components like fats, Instead of stains/dyes, monoclonal
& tissues) carbohydrates etc. or polyclonal antibodies are used
Ex.
b. Active tissue component is colored • Perl’s Prussian blue – for Detection of phenotypic markers
hemoglobin that are detected by antibodies
c. If you want to stain the nucleus and • Periodic acid Schiff – for i.e. monoclonal, polyclonal
cytoplasm of cells carbohydrates
• Sudan dyes – fat stain
d. Microanatomical stains (gram stain, acid- • Sudan black
fat stain); hematology (to stain blood cells – • Sudan III
wright stain); microbiology bacterial stains –
methylene blue, carbol fuchsin, crystal violet;
Specific tissue stains; blood parasites stains –
Giemsa stain
9 STAINING TECHNIQUES
Direct staining o Uses only 1 dye (tissue and dye only)
o Usually, the color of the dye is also the resulting color
o Ex. Methylene blue – resulting color is also blue
o uses aqueous or alcoholic dye solutions to produce a color
Indirect staining o Tissue component is demonstrated using a dye with the help of mordant or
accentuator (tissue, dye, mordant or accentuator)
o Uses mordant or accentuator to demonstrate tissue component
o MORDANT – substance that serves as link or bridge between the dye and the tissue
(see on the next pages)
- General example of mordants and their stain:
Potassium alum – specific example of mordant with potassium alum are:
a. Mayer’s hematoxylin stain
b. Ehrlich’s h.
c. Gill’s h.
d. Cole’s h.
Iron hematoxylin – Specifically Ferric ammonium sulfate & Ferric
ammonium chloride; Specific example of mordant with iron:
a. Heidenhain’s h. – Ferric ammonium sulfate
b. Weigert’s h. – Ferric ammonium chloride
, So pag tinanong, what are examples of stains with mordants? Ang sagot ay ehrlichs’s,
mayer’s, gill’s, weigert’s etc.
Aside from iron, we can also use COPPER as mordant = Copper hematoxylin
We can also use Phosphotungstic acid = PTAH
o Also uses ACCENTUATOR – Not part of the staining reaction; substance that ONLY
heightens the color intensity and selectivity of the stain/dye; increases the staining
power; patitingkarin nya yung kulay na matamlay.
- Ex. Of accentuators and their stain
KOH – Loeffler’s methylene blue stain
Phenol – used whenever we use carbol fuchsin and carbol thionine
Progressive staining o Involves gradual application of dye/stain to tissues; dye is applied to tissues little by
little until the desired color is achieved so there’s a sequence followed kasi nga
gradually sya inaapply eh
o Since dye is applied little by little, there is no excess dye
o Decolorization is not performed/not part of the process
o tissue elements re stained in a definite sequence.
Regressive staining o Tissue is first OVERSTAINED so there is excess dye and it is removed through
decolorization.
o DECOLORIZATION/differentiation – Selective removal of excess dye;
o Commonly used decolorizer is acid alcohol
o Rule: Use basic solution in removing acid dyes and use acid dye if the solution is basic.
o Acid alcohol is the one commonly used because it can remove both acid and basic dye
o Ex. H&E stain
Counterstaining o Involves application of a different color
o Uses another dye to produce contrast and background
o Most commonly used counterstain is eosin.
o Eosin – a red acid dye as to pH; a cytoplasmic stain.
o Nucleus of cells are ACID in nature because of nucleic acid
o Cytoplasm in nature is BASIC in nature.
o Available on 3 Forms:
1. Eosin Y – Yellowish
2. Eosin B – Bluish
3. Ethyl Eosin – Eosin S
o RULE in STAINING: if you will stain an acid structure, use a basic dye. If there is a need
to demonstrate a basic component, use acid. So opposite attracts.
Metachromatic staining o Uses a dye that will impart/produce a different color, different from the color of the
dye itself. (Stain the tissue w/ a color that is different from the stain itself, opposite of
direct staining)
o The stain/dye here will produce a different color
o Examples of metachromatic stains:
Methyl violet or crystal Cresyl blue for Safranin
violet reticulocytes
Bismarck brown Basic fuchsin Methylene blue
Thionine (also supravital Toluidine blue (also Azure A, B, C
stain) supravital stain)
