HEMATOXYLIN AND EOSIN ROUTINE STAINING Flashcards
(100 cards)
1
Q
● Most widely used histological stain
A
HEMATOXYLIN AND EOSIN ROUTINE STAINING
2
Q
serves as the basic or primary stain, or nuclear stain (purpose in routine H&E)
A
Hematoxylin
3
Q
● First person to use paraffin wax for embedding?
A
Butschlii
3
Q
● Natural dye
○ Derived from extraction from Heartwood of a Mexican tree
A
HEMATOXYLIN
4
Q
First person to use hematoxylin in histology (1862)
A
Waldeyer
4
Q
Scientific Name of Mexican tree
A
Hematoxylin campechianum
5
Q
● May be done by exposing the substance to air and sunlight but the process is SLOW
A
RIPENING/OXIDATION
6
Q
May be done by adding oxidizing agents such as: (5)
A
○ Hydrogen peroxide
○ Mercuric oxide
○ Potassium permanganate
○ Sodium perborate
○ Sodium iodate
7
Q
What is the ripening agent of Harry’s hematoxylin?
A
Mercuric Oxide
8
Q
What is the ripening agent for Erlich’s Hematoxylin?
A
Sodium Iodate
9
Q
● Used in routine H&E
● Recommended for progressive staining of tissues but it does not mean that it cannot be used for regressive
A
ALUM HEMATOXYLIN
10
Q
● Mordant: Potash alum
(potassium aluminum sulfate or
simply “alum”)
● Produce good nuclear stai
A
ALUM HEMATOXYLIN
11
Q
○ Useful general purpose
hematoxylin
A
Harris
12
Q
Examples of Alum Haematoxylin (7)
A
● Harris
● Ehrlich’s
● Delafield’s
● Gill’s
● Mayer’s
● Cole’s
● Carazzi
13
Q
Examples of Ripening Agent (6)
A
● Mercuric oxide
● Natural or sodium iodate
● Natural
● Sodium iodate
● Alcoholic iodine
● Potassium iodate
14
Q
Iron salts are used as oxidizing agents and mordant
A
IRON HEMATOXYLIN
15
Q
What is the mordant in Harry’s hematoxylin?
a. Mercuric oxide
b. ALUM
c. Both
d. Neither
A
B. Alum
16
Q
● Mordant is ferric chloride
● In combination with van
Gieson’s stain, can demonstrate
connective tissue elements and
entamoeba histolytica in sections
A
Weigert’s
17
Q
● Standard iron hematoxylin
● For muscles/connective tissue
fibers
A
Weigert’s
18
Q
Used for staining elastic fibers
A
Verhoeff/Verhöeff
18
Q
● Ferric ammonium sulfate
● For mitochondria, muscle
striations, chromatin, and myelin
A
Heidenhain’s Hematoxylin
19
Q
● Muscle striations
● Mitochondria and myelin
A
Heidenhain’s Hematoxylin
20
Q
Used for staining myelin
A
Loyez
21
Q
is an organelle that can be
stained with heidenhain’s hematoxylin but it can also be stained with Janus Green B
A
Mitochondria
21
is good in demonstrating
collagen
Van Gieson’s stain
22
Chromatin can also be stained with ______________ stain
carmine’s
23
TUNGSTEN HEMATOXYLIN example:
Mallory’s PTAH (Phosphotungstic
Acid Hematoxylin)
24
● To ripen
○ Stand in light for several
weeks or use potassium
of immediate ripening
Mallory’s PTAH (Phosphotungstic
Acid Hematoxylin)
25
● Used for staining
○ Muscle striation
○ Fibrin
○ Glial fiber
Mallory’s PTAH (Phosphotungstic
Acid Hematoxylin)
25
What stain is used to demonstrate muscle Striation?
Mallory's PTAH
26
Other stains that demonstrate muscle striations?
Heidanhain’s Hematoxylin
27
Used for the study of spermatogenesis
COPPER HEMATOXYLIN
28
MOLYBDENUM HEMATOXYLIN example:
Thomas Hematoxylin
28
What particular part of the male reproductive system does the site of spermatogenesis or the production of sperm cell occur?
Seminiferous tubules
29
Used for staining:
● Collagen
● Endocrine cell granules
Thomas Hematoxylin
30
What other stain presents collagen?
Van Gieson Stain
31
LEAD HEMATOXYLIN example:
Solcia Hematoxylin
32
Used for staining:
● Endocrine cell granules
Solcia Hematoxylin
33
● In the process of H&E staining, the eosin act as a secondary stain, a counterstain, acidic stain, or cytoplasmic stain
EOSIN
34
● Eosin Yellowish
● Most commonly used
Eosin Y
35
● Eosin Bluish
● Produces a deeper red color
Eosin B
36
Ethyl Eosin
Eosin S
37
■ Temperature to fix surgical specimens (room temp)
■ Formaldehyde for 24hrs
Fixation
38
■ 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
39
■ Otherwise known as dealcoholization.
■ Makes the tissue transparent and clears the alcohol
■ Usually used is Xylene
● Which is usually miscible to
paraffin wax
Clearing
40
■ Temperature of paraffin oven is 55 - 60 °C or 5°C above the melting point of the wax.
Impregnation
41
■ 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
42
■ 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
42
■ Remove the excess paraffin wax
■ Ideal shape is truncated pyramid or 4-sided prism
Trimming
43
■ 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
44
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
45
The tissue needed water for the stain to adhere
Rehydration
46
DESCENDING GRADES OF ALCOHOL
Slowly adding water to the tissue
● Rehydration
47
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
48
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
49
ACID ALCOHOL (DIFFERENTIATOR)
● Decolorizer
○ Differentiation phase
○ The nucleus will turn _____
○ THE cytoplasm would be ______________
● RED
● COLORLESS
50
Blueing agents (3)
○ Ammonium hydroxide
○ Lithium carbonate
○ Scott’s tap water
51
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
52
What is the expected color of the nucleus of the cell if the medtech forgot to use AMMONIA water?
RED
53
Eosin Y is called as (4)
○ Secondary stain
○ Acidic stain
○ Cytoplasmic stain
○ Counter stain
54
Stain with Eosin Y
● Color of Nucleus: _________
● Color of Cytoplasm: _______
● Color of Nucleus: still color BLUE
● Color of Cytoplasm: PALE PINK
55
● Ascending Grades of Alcohol: _____________
● Descending Grades of Alcohol: _____________
● Ascending Grades of Alcohol: DEHYDRATION
● Descending Grades of Alcohol: REHYDRATION
55
Known as CLEARING (Dealcoholization)
Xylol/ Xylene
56
Purpose of Xylol in the FIRST STEP of routine H&E:
DEPARAFFINIZATION
57
Purpose of Xylol in the LAST STEP of routine H&E:
CLEARING AGENT
58
Nuclei
Blue to Blue Black
58
Karyosome
Dark Blue
59
Cytoplasm, proteins in edema fluid
Pale Pink
60
Calcium and calcified bone
Purplish Blue
61
Muscle fibers
Deep Pink
62
● Considered as the staining method of choice for Exfoliative Cytology
PAPANICOLAOU STAINING (PAPS STAINING)
63
● 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
63
PAPANICOLAOU STAINING (PAPS STAINING)
Fixative used:
95% Ethanol
64
PAPANICOLAOU STAINING (PAPS STAINING)
Three (3) Stains applied:
○ Hematoxylin
○ OG6
○ EA
65
● stain for the cytoplasm of the
MATURE superficial cells.
OG6 (Orange Green)
65
● stain for the cytoplasm of
IMMATURE vaginal cells.
○ Such as parabasal and
intermediate cells
EA 36 or 50 (Eosin Azure)
66
used for staining hemoglobin
Benzidine
66
● DNA (green fluorescence)
● RNA (red fluorescence)
Acridine Orange
66
Components of EA 36 or 50: (3)
○ Eosin Y
○ Light Green SF
○ Brismark Brown
67
● for staining amyloid in frozen
sections and platelets in blood
Crystal violet
68
● formed by the mixture of crystal violet, methyl violet, and dexterin
Gentian violet
69
● Gold standard for amyloid
demonstration
● stain for axis cylinders in
embryos
Congo Red
70
● used as a 4% aqueous solution
in Krajian’s method of staining
elastic tissues, amyloid, and
myelin
Congo Red
71
● Oldest of all stains
● stains amyloid, cellulose, starch,
carotenes, and glycogen
● widely used for removal of
mercuric fixative pigments
Iodine
71
● contrast stain for staining Ascaris eggs and erythrocytes
● used also as a bacterial spore
stain
Malachite Green
72
● used for demonstrating
mitochondria during intravital
staining (or supravital?)
Janus Green B
73
● used for demonstration of
neuroglia in frozen sections
Victoria Blue
74
● used as a substitute for carbol
fuchsin in acid-fast staining
Night Blue
75
● 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)
76
● Examples of oil soluble dyes
used for demonstration of
intracellular fats:
○ Sudan Black B
○ Sudan III
○ Sudan IV (Scharlach R)
Lysochromes (Oil Soluble
Dyes)
77
● PAS-positive substances =
red/magenta red
● Mucoproteins are the most
common PAS-positive
substances
Periodic Acid-Schiff Reaction
78
● Considered as the most reliable
and specific histochemical stain
for DNA
Fill Gel Technique
79
● Can observe Howell-Jolly Bodies
● Howell-Jolly Bodies: Fill Gel
Reaction (+)
Fill Gel Technique
80
● Best's Carmine
● PAS (periodic acid-Schiff)
● Langhan’s Iodine Stain
Stains For Glycogen
80
● Used for the demonstration of
spirochetes.
Warthin–Starry Stain
81
● Applicable not only for muscle
striations but also for Fibrin
Phosphatase.
Mallory’s PTAH (Phosphotungstic
Acid-Hematoxylin)
82
Expected color:
○ Early fibrin: YELLOW
○ Old fibrin: BLUE
Lendrum’s MSB (Martius, Scarlet,
Blue)
83
Expected color:
○ Early fibrin:
YELLOW
84
Expected color:
○ Old fibrin:
Blue
85
PAS-positive substances =
Red/Magenta Red
