HEMATOXYLIN AND EOSIN ROUTINE STAINING Flashcards
● Most widely used histological stain
HEMATOXYLIN AND EOSIN ROUTINE STAINING
serves as the basic or primary stain, or nuclear stain (purpose in routine H&E)
Hematoxylin
● First person to use paraffin wax for embedding?
Butschlii
● Natural dye
○ Derived from extraction from Heartwood of a Mexican tree
HEMATOXYLIN
First person to use hematoxylin in histology (1862)
Waldeyer
Scientific Name of Mexican tree
Hematoxylin campechianum
● May be done by exposing the substance to air and sunlight but the process is SLOW
RIPENING/OXIDATION
May be done by adding oxidizing agents such as: (5)
○ Hydrogen peroxide
○ Mercuric oxide
○ Potassium permanganate
○ Sodium perborate
○ Sodium iodate
What is the ripening agent of Harry’s hematoxylin?
Mercuric Oxide
What is the ripening agent for Erlich’s Hematoxylin?
Sodium Iodate
● Used in routine H&E
● Recommended for progressive staining of tissues but it does not mean that it cannot be used for regressive
ALUM HEMATOXYLIN
● Mordant: Potash alum
(potassium aluminum sulfate or
simply “alum”)
● Produce good nuclear stai
ALUM HEMATOXYLIN
○ Useful general purpose
hematoxylin
Harris
Examples of Alum Haematoxylin (7)
● Harris
● Ehrlich’s
● Delafield’s
● Gill’s
● Mayer’s
● Cole’s
● Carazzi
Examples of Ripening Agent (6)
● Mercuric oxide
● Natural or sodium iodate
● Natural
● Sodium iodate
● Alcoholic iodine
● Potassium iodate
Iron salts are used as oxidizing agents and mordant
IRON HEMATOXYLIN
What is the mordant in Harry’s hematoxylin?
a. Mercuric oxide
b. ALUM
c. Both
d. Neither
B. Alum
● Mordant is ferric chloride
● In combination with van
Gieson’s stain, can demonstrate
connective tissue elements and
entamoeba histolytica in sections
Weigert’s
● Standard iron hematoxylin
● For muscles/connective tissue
fibers
Weigert’s
Used for staining elastic fibers
Verhoeff/Verhöeff
● Ferric ammonium sulfate
● For mitochondria, muscle
striations, chromatin, and myelin
Heidenhain’s Hematoxylin
● Muscle striations
● Mitochondria and myelin
Heidenhain’s Hematoxylin
Used for staining myelin
Loyez
is an organelle that can be
stained with heidenhain’s hematoxylin but it can also be stained with Janus Green B
Mitochondria
is good in demonstrating
collagen
Van Gieson’s stain
Chromatin can also be stained with ______________ stain
carmine’s
TUNGSTEN HEMATOXYLIN example:
Mallory’s PTAH (Phosphotungstic
Acid Hematoxylin)
● To ripen
○ Stand in light for several
weeks or use potassium
of immediate ripening
Mallory’s PTAH (Phosphotungstic
Acid Hematoxylin)
● Used for staining
○ Muscle striation
○ Fibrin
○ Glial fiber
Mallory’s PTAH (Phosphotungstic
Acid Hematoxylin)
What stain is used to demonstrate muscle Striation?
Mallory’s PTAH
Other stains that demonstrate muscle striations?
Heidanhain’s Hematoxylin
Used for the study of spermatogenesis
COPPER HEMATOXYLIN
MOLYBDENUM HEMATOXYLIN example:
Thomas Hematoxylin
What particular part of the male reproductive system does the site of spermatogenesis or the production of sperm cell occur?
Seminiferous tubules
Used for staining:
● Collagen
● Endocrine cell granules
Thomas Hematoxylin
What other stain presents collagen?
Van Gieson Stain
LEAD HEMATOXYLIN example:
Solcia Hematoxylin
Used for staining:
● Endocrine cell granules
Solcia Hematoxylin
● In the process of H&E staining, the eosin act as a secondary stain, a counterstain, acidic stain, or cytoplasmic stain
EOSIN
● Eosin Yellowish
● Most commonly used
Eosin Y
● Eosin Bluish
● Produces a deeper red color
Eosin B
Ethyl Eosin
Eosin S
■ Temperature to fix surgical specimens (room temp)
■ Formaldehyde for 24hrs
Fixation
■ Most common dehydrating agent is alcohols
■ Should be used in ascending rates
■ We intend to infiltrate the paraffin wax unto the tissue but it is not soluble to water nor to alcohol, this we need to remove alcohol
Dehydration
■ Otherwise known as dealcoholization.
■ Makes the tissue transparent and clears the alcohol
■ Usually used is Xylene
● Which is usually miscible to
paraffin wax
Clearing
■ Temperature of paraffin oven is 55 - 60 °C or 5°C above the melting point of the wax.
Impregnation
■ We get the tissue from the melted paraffin wax, open the tissue cassette and put it in a mold to perform embedding
■ Also known as casting
■ Temp of paraffin wax is 5°C - 10°C above the melting point of the paraffin wax
■ Let the paraffin wax harden
Embedding
■ Also called as cutting or microtomy
■ Most commonly used is the rotary microtome
● Old sources: 4-6 micron of
tissue
● New sources: 3-5 micron of
tissue
○ Remember both old and
new references, choose
the closest. Depends on
what book the board
examiner uses
Sectioning
■ Remove the excess paraffin wax
■ Ideal shape is truncated pyramid or 4-sided prism
Trimming
■ Flotation water bath
● Optimal temperature is 6°C -
10°C below the melting point of
paraffin wax
■ Get a slide and apply Meyer’s Egg Albumin which is composed of;
● Egg white
● Glycerol
● Crystal sulfide (prevent growth
of mold)
■ Fish out, and place it in the paraffin oven to remove the excess paraffin wax
(deparaffinization)
■ Put it in xylene or xylol
Sectioning
CHANGES OF XYLENE OR XYLOL
○ We need to add xylene to remove the paraffin in the tissue, heat alone could not remove the
paraffin wax
Process of deparaffinization
The tissue needed water for the stain to adhere
Rehydration
DESCENDING GRADES OF ALCOHOL
Slowly adding water to the tissue
● Rehydration
When do we remove fixative artifacts and fixative treatments?
It is removed after rehydration and right before primary staining
**this artifacts can obscure the the tissue
STAIN WITH HARRIS/ EHRLICH’S/DELAFIELD’S
HEMATOXYLIN
● Primary stains
○ Alum hematoxylin, serve as the primary, basic and nuclear stain of the tissue.
○ The color of the nucleus is already ___________________
○ While the cytoplasm is still __________
● light transparent blue
● colored
ACID ALCOHOL (DIFFERENTIATOR)
● Decolorizer
○ Differentiation phase
○ The nucleus will turn _____
○ THE cytoplasm would be ______________
● RED
● COLORLESS
Blueing agents (3)
○ Ammonium hydroxide
○ Lithium carbonate
○ Scott’s tap water
AMMONIA WATER (AMMONIUM HYDROXIDE, LITHIUM CARBONATE, SCOTT’S TAP WATER)
○ Transform the color of the nucleus back to _____, Due to the secondary stain being being eosin Y, which has a pink color, and this, for differentiation, different colored stains are used in the cell (contrast)
● Cytoplasm: _______________
● BLUE
● COLORLESS
What is the expected color of the nucleus of the cell if the medtech forgot to use AMMONIA water?
RED
Eosin Y is called as (4)
○ Secondary stain
○ Acidic stain
○ Cytoplasmic stain
○ Counter stain
Stain with Eosin Y
● Color of Nucleus: _________
● Color of Cytoplasm: _______
● Color of Nucleus: still color BLUE
● Color of Cytoplasm: PALE PINK
● Ascending Grades of Alcohol: _____________
● Descending Grades of Alcohol: _____________
● Ascending Grades of Alcohol: DEHYDRATION
● Descending Grades of Alcohol: REHYDRATION
Known as CLEARING (Dealcoholization)
Xylol/ Xylene
Purpose of Xylol in the FIRST STEP of routine H&E:
DEPARAFFINIZATION
Purpose of Xylol in the LAST STEP of routine H&E:
CLEARING AGENT
Nuclei
Blue to Blue Black
Karyosome
Dark Blue
Cytoplasm, proteins in edema fluid
Pale Pink
Calcium and calcified bone
Purplish Blue
Muscle fibers
Deep Pink
● Considered as the staining method of choice for Exfoliative Cytology
PAPANICOLAOU STAINING (PAPS STAINING)
● aka Harris Hematoxylin
● Serve as nuclear stain
● Gives color to the nucleus
whatever kind of cell it is.
● stain for the nucleus of mature
superficial cells
Hematoxylin
PAPANICOLAOU STAINING (PAPS STAINING)
Fixative used:
95% Ethanol
PAPANICOLAOU STAINING (PAPS STAINING)
Three (3) Stains applied:
○ Hematoxylin
○ OG6
○ EA
● stain for the cytoplasm of the
MATURE superficial cells.
OG6 (Orange Green)
● stain for the cytoplasm of
IMMATURE vaginal cells.
○ Such as parabasal and
intermediate cells
EA 36 or 50 (Eosin Azure)
used for staining hemoglobin
Benzidine
● DNA (green fluorescence)
● RNA (red fluorescence)
Acridine Orange
Components of EA 36 or 50: (3)
○ Eosin Y
○ Light Green SF
○ Brismark Brown
● for staining amyloid in frozen
sections and platelets in blood
Crystal violet
● formed by the mixture of crystal violet, methyl violet, and dexterin
Gentian violet
● Gold standard for amyloid
demonstration
● stain for axis cylinders in
embryos
Congo Red
● used as a 4% aqueous solution
in Krajian’s method of staining
elastic tissues, amyloid, and
myelin
Congo Red
● Oldest of all stains
● stains amyloid, cellulose, starch,
carotenes, and glycogen
● widely used for removal of
mercuric fixative pigments
Iodine
● contrast stain for staining Ascaris eggs and erythrocytes
● used also as a bacterial spore
stain
Malachite Green
● used for demonstrating
mitochondria during intravital
staining (or supravital?)
Janus Green B
● used for demonstration of
neuroglia in frozen sections
Victoria Blue
● used as a substitute for carbol
fuchsin in acid-fast staining
Night Blue
● NOT real dyes
● They do NOT have auxochrome
groups.
● They give color to lipids simply
because they are more soluble in
lipid medium of the tissues than
in their medium of 70% alcohol.
Lysochromes (Oil Soluble
Dyes)
● Examples of oil soluble dyes
used for demonstration of
intracellular fats:
○ Sudan Black B
○ Sudan III
○ Sudan IV (Scharlach R)
Lysochromes (Oil Soluble
Dyes)
● PAS-positive substances =
red/magenta red
● Mucoproteins are the most
common PAS-positive
substances
Periodic Acid-Schiff Reaction
● Considered as the most reliable
and specific histochemical stain
for DNA
Fill Gel Technique
● Can observe Howell-Jolly Bodies
● Howell-Jolly Bodies: Fill Gel
Reaction (+)
Fill Gel Technique
● Best’s Carmine
● PAS (periodic acid-Schiff)
● Langhan’s Iodine Stain
Stains For Glycogen
● Used for the demonstration of
spirochetes.
Warthin–Starry Stain
● Applicable not only for muscle
striations but also for Fibrin
Phosphatase.
Mallory’s PTAH (Phosphotungstic
Acid-Hematoxylin)
Expected color:
○ Early fibrin: YELLOW
○ Old fibrin: BLUE
Lendrum’s MSB (Martius, Scarlet,
Blue)
Expected color:
○ Early fibrin:
YELLOW
Expected color:
○ Old fibrin:
Blue
PAS-positive substances =
Red/Magenta Red
