LESSON 8: STAINING Flashcards
1
Q
process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells
A
Staining
2
Q
tissues and cells display varying affinities for most dyes and stains used during the process
A
Staining
3
Q
Acidic structures—greater affinity for
Basic structures—greater affinity for
A
basic dyes
acidic dyes
4
Q
related procedure that makes use of heavy metal salts which are selectively precipitated on certain cellular and tissue components
A
Impregnation
5
Q
used for silver staining of nervous tissue and demonstration of reticulin
A
Impregnation
6
Q
Impregnation most commonly used agent:—may also be used as a staining agent
A
silver nitrate
7
Q
THREE MAJOR TYPES OF STAINING
A
- HISTOLOGICAL STAINING
- HISTOCHEMICAL STAINING/HISTOCHEMISTRY
- IMMUNOHISTOCHEMICAL STAINING
8
Q
METHODS OF STAINING
A
- DIRECT STAINING
- INDIRECT STAINING
- PROGRESSIVE STAINING
- REGRESSIVE STAINING
- METACHROMATIC STAINING
- COUNTERSTAINING
- METALLIC IMPREGNATION
- VITAL STAINING
9
Q
- VITAL STAINING:
A
INTRAVITAL STAINING
SUPRAVITAL STAINING
10
Q
tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution
A
HISTOLOGICAL STAINING
11
Q
producing coloration of the active tissue component
A
HISTOLOGICAL STAINING
12
Q
employed to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm
A
HISTOLOGICAL STAINING
13
Q
Examples: microanatomic stains, bacterial stains and specific tissue stains (muscles, CT, and neurologic stains)
A
HISTOLOGICAL STAINING
14
Q
tissue constituents are studied through chemical reactions that will permit microscopic localization of a specific tissue substance
A
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
15
Q
Examples: Perl’s Prussian blue reaction for hemoglobin, Periodic Acid Schiff staining for carbohydrates
A
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
16
Q
Enzyme histochemistry: active reagent serves as the substrate upon which the enzymes act
A
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
17
Q
final opacity of coloration produced from the substrate rather than the tissue
A
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
18
Q
combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated
A
IMMUNOHISTOCHEMICAL STAINING
19
Q
makes use of different labels: monoclonal/polyclonal, fluorescent-labeled, enzyme-labeled antibodies
A
IMMUNOHISTOCHEMICAL STAINING
20
Q
process of giving color to the sections by using aqueous or alcoholic dyes
A
- DIRECT STAINING
21
Q
process whereby action of dye is intensified by adding another reagent (MORDANT) which serves as a link/bridge between tissue and dye making staining reaction possible
A
- INDIRECT STAINING
22
Q
may be applied to tissue before staining or may be included in the staining process, or may be incorporated as part of the dye solution itself
A
MORDANT
23
Q
MORDANT Eg.
A
potassium alum with hematoxylin in Ehrlich’s hematoxylin and iron in Weigert’s hematoxylin
24
Q
not essential to the chemical union of tissue and dye
A
ACCENTUATOR
25
but merely accelerates or hastens the speed of staining reaction by increasing the staining power and selectivity of the dye
ACCENTUATOR
26
ACCENTUATOR Eg:
potassium hydroxide in Loeffler’s methylene blue and phenol in carbol thionine and carbol fuchsin
27
process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained
PROGRESSIVE STAINING
28
no decolorization or washing after the application of the dye
PROGRESSIVE STAINING
29
tissue is first over stained to obliterate cellular details, and excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained
4. REGRESSIVE STAINING
30
: selective removal of excess stain from the tissue so that a specific substance may be stained distinctly from surrounding tissues
DIFFERENTIATION/DECOLORIZATION
31
use of dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself
METACHROMATIC STAINING
32
basic dyes belonging to the thiazine and triphenylmethane groups
METACHROMATIC STAINING
33
METACHROMATIC STAINING Examples:
methyl violet or crystal violet Cresyl blue for reticulocytes Safranin Bismarck brown
34
stain tissues in color shades that are similar to the color of the dye itself
ORTHOCHROMATIC STAINING
35
application of a different color or stain to provide contrast and background to the staining of structural components to be demonstrated
COUNTERSTAINING
36
Cytoplasmic stains:
Red eosin Y, Eosin B, Phloxine B
Yellow piciric acid, orange G, Rose Bengal
Green light green SF, Lissamine green
37
Nuclear stains:
Red neutral red, safranin 0, carmine, hematoxylin
Blue methylene blue, toluidine blue, Celestine blue
38
process where specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue
METALLIC IMPREGNATION
39
agent is not absorbed by the tissue, but is held physically on the surface as a precipitate or as a reduction product
METALLIC IMPREGNATION
40
Examples: ammoniacal silver and gold chloride
METALLIC IMPREGNATION
41
reduced by argentaffin cells (melanin and intestinal glands) Gold chloride
Ammoniacal silver
42
selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle
VITAL STAINING
43
**nucleus is resistant to staining
VITAL STAINING
44
***if nuclear structures are demonstrated > death of cell already
VITAL STAINING
45
Examples: trypan blue>reticuloendothelial cells; Janus green>mitochondria
VITAL STAINING
46
done by injecting dye into any part of the body (intravenous, intraperitoneal or subcutaneous), producing specific color
INTRAVITAL STAINING
47
Examples: lithium, carmine and India ink
INTRAVITAL STAINING
48
stain living cells immediately after removal from the living body
SUPRAVITAL STAINING
49
Examples: neutral red, Janus green, trypan blue, etc.
SUPRAVITAL STAINING
50
Regressive staining due to the decolorization step using acid alcohol
H and E Staining Technique
51
H and E Staining Technique Major steps:
Deparaffinization (Step 1)
Hydration (Step 2)
Nuclear staining (Step 4)
Differentiation (Step 6)
Bluing (Step 8)
Counterstaining (Step 10)
Dehydration (Step 11)
Clearing (Step 12)
52
Nuclei:
blue to blue black
53
Karyosome:
dark blue
54
Cytoplasm, proteins in edema fluid:
pale pink
55
RBCs, eosinophilic granules, keratin:
bright orange-red
56
Basophilic cytoplasm, plasma cells, osteoblast:
purplish pink
57
Cartilage:
pink or light blue to dark blue
58
Calcium and calcified bone:
purplish blue
59
Decalcified bone matrix, collagen, osteoid:
pink
60
Muscle fibers:
deep pink
61
extracted from the core/heartwood of a Mexican tree (Hematoxylin campechianum)
HEMATOXYLIN
62
Ripening/Oxidizing: exposure to air & sunlight or to strong oxidizing agents
HEMATOXYLIN
63
Aluminum Hematoxylin:
Iron Hematoxylin:
Phosphotungstic Acid Hematoxylin:
64
Sodium iodate
Ehrlich’s
65
Tissues subjected to acid decalcification and acidic tissues
Ehrlich’s
66
Mercuric chloride
Harris’
67
Routine nuclear staining, exfoliative cytology and staining sex chromosomes
Harris’
68
Alcoholic iodine
Cole’s
69
Routine purposes, esp in sequence to Celestine blue
Cole’s
70
Sodium iodate
Mayer’s
71
Cytoplasmic glycogen
Mayer’s
72
Ferric ammonium chloride
Weigert’s
73
Muscle fibers and connective tissues
Weigert’s
74
Ferric ammonium sulfate
Heidenhain’s
75
Nuclear and cytoplasmic inclusions (chromatin, chromosomes, nucleoli, centrosomes, mitochondria)
Heidenhain’s
76
Voluntary muscle striations and myelin
Heidenhain’s
77
1% aq. phosphotungstic acid
Phosphotungstic Acid Hematoxylin
78
Nuclei, fibrin, muscle striations, myofibrils, fibroglia
Phosphotungstic Acid Hematoxylin
79
Collagen, bone, cartilage
Phosphotungstic Acid Hematoxylin
80
Paraffin, celloidin and frozen sections
Phosphotungstic Acid Hematoxylin
81
For demonstration of spermatogenesis
Copper Hematoxylin
82
Extracted from a female cochineal bug (Coccus cacti)
COCHINEAL DYES
83
Treated with alum to produce carmine
COCHINEAL DYES
84
: neuropathological studies
Carmine + Picric acid > Picrocarmine
85
: glycogen
Carmine + aluminium chloride > Best carmine
86
Vegetable dye from lichens treated with ammonia and exposed to air
ORCEIN
87
For staining elastic fibers: dark brown
ORCEIN
88
Coal tar dyes or aniline dyes
SYNTHETIC DYES
89
: produces color
Chromophores
90
: promotes color retention
Auxochromes
91
DYE:
Chromophore + auxochrome
92
ACID DYES:
BASIC DYES:
NEUTRAL DYES:
Picric acid
Methylene blue
Romanowsky
Giemsa
Irishman
93
Most sensitive
Sudan Black B
94
Stains phospholipids and neutr1al lipids (BLACK)
Sudan Black B
95
Recommended for staining neutral lipids (RED)
Sudan IV (Scharlach R)
96
First to be introduced
Sudan III
97
Fat stain for CNS tissues (ORANGE)
Sudan III
98
Other Non-Lysochrome Lipid Stains:
99
: for neutral fats and lipofuchsin (brilliant red)
Oil Red O
100
: stains lipids black
Osmic Acid
101
Most valuable for differential staining of CT & cytoplasm
Eosin
102
Connective tissues
Acid Fuchsin-Picric Acid
103
Discrimination between dead and living cells
Acridine Orange
104
DNA: green fluorescence ; RNA: red fluorescence
Acridine Orange
105
Calcium salts and sites of phosphatase activities
Acridine Red 3B
106
Acid mucopolysaccharides
Alcian Blue
107
Conterstaining of epithelial sections
Aniline Blue
108
Deep staining of acid-fast organisms, mitochondria
Basic fuschin
109
differentiation of smooth muscles
Basic fuschin With picric acid
110
: detection of aldehydes
Basic fuschin In Feulgen’s and Schiff’s reagent
111
: connective tissues, mucin and elastic tissues
Basic fuschin In Van Gieson’s
112
Hemoglobin
Benzidine
113
Contrast stain for Gram’s, acid fast and Pap’s method
Bismarck Brown
114
Used for staining diphtheria
Bismarck Brown
115
Chromatin stain for fresh materials (smear prep)
Carmine
116
With aluminum chloride > glycogen
Carmine
117
With aluminum chloride > glycogen
Carmine
118
Routine staining of fixed sections
Celestine blue
119
Axis cylinders in embryos
Congo Red
120
: elastic tissues, amyloid and myelin
Congo Red In Krajians method
121
Amyloid and platelets
Crystal violet
122
Leukocyte differentiation
Giemsa
123
Metallic impregnation
Gold sublimate
124
Oldest stain
Iodine
125
: microorganisms and fibrin
GRAM’S Iodine
126
: glycogen, amyloid and corpora amylacea
LUGOL’s Iodine
127
Mitochondria
Janus Green B
128
Ascaris eggs, erythrocytes and bacterial spore
Malachite Green
129
Chromatin
Methyl green
130
Plasma cells
Methylene blue
131
Cytologic examinations of fresh sputum for malignant cells
Methylene blue
132
Nuclei of leukocytes
Methylene violet
133
Observing cell granules and vacuoles in phagocytic cells
Neutral red
134
Carbol fuchsin substitute in acid-fast staining
Night blue
135
Elastic fibers (Taenzer Unna ? Method)
Orcein
136
Dermatological studies to demonstrate finest and most delicate skin fibers
Orcein
137
Stains fat
Osmium tetroxide
138
Demonstration of circulatory system by injection
Prussian blue
139
Fix and stain blood and glandular tissues
Rhodamine B
140
Spirochetes, reticulum and other fiber stains
Silver nitrate
141
Substitute for thionine in fresh frozen sections
Toluidine blue
142
Recommended for Nissl granules or chromophilic bodies
Toluidine blue
143
Neuroglia in frozen sections
Victoria blue
144
Magenta Red
1. Periodic Acid Schiff (PAS)
145
Stain of choice for glycogen (red)
**PAS with Diastase Control
146
Selective and specific for glycogen (bright red)
2. Best Carmine
147
Mahogany Brown
3. Langhan’s Iodine
148
Histones and protamines (green)
1. Alkaline-fast Green
149
Cystine and cysteine (blue-green)
2. Peracetic acid-Alcian blue
150
Arginine (orange-red)
3. Sakaguchi’s Test (Milton’s Reagent)
151
Most reliable and specific stain for DNA
1. Feulgen Technique
152
DNA: Red-purple
1. Feulgen Technique
153
DNA: Green or Blue-green
RNA: Rose-red
2. Methyl Green-Pyronin
154
Most widely-used fluorochrome
3. Fluorescein
155
Apple green
3. Fluorescein
156
Orange-red
4. Rhodamine
157
DNA: yellow-green
RNA: brick to orange-red
5. Acridine Orange
158
DNA: yellow
6. Acriflavine
159
1. Gomori’s Silver Impregnation
Black
160
1. Van Gieson’s
Pink or Deep red
161
2. Masson’s Trichrome
Blue
162
3. Mallory’s Aniline
Blue
163
1. Weigert’s Elastic Tissue
Dark-blue or Blue-black
164
2. Taenzer-Unna Orcein
Dark brown
165
3. Verhoeff’s
Black
166
4. Krajian’s
Bright red
167
1. Perl’s Prussian Blue
Deep blue
168
1. Masson Fontana
Black
169
1. Von Kossa’s Silver Nitrate
Black
170
1. Levaditi’s
Black
171
2. Warthin-Starry Method
Black
172
1. Wade-Fite Technique
Red
173
1. Orcein Method
Brown-black
174
also used to remove stains on the skin
1% acid alcohol
175
Other Bluing Agents:
warm tap H2O: 40-50degC, bicarbonate, potassium/sodium acetate, ammonia and Scott’s Tap Water Substitute (TWS)
176
more specific for connective tissue and epithelial mucin
Alcian blue
177
more specific for connective tissue and epithelial mucin
Alcian blue
