M1-PRINCIPLES OF STAINING Flashcards
(211 cards)
1
Q
Visual labeling of biological entities by attaching or depositing a colored marker
A
Staining
2
Q
Reagent used to generate a visible marker on tissue
A
Stain
3
Q
Tendency of a stain to transfer from solution onto tissue section
A
Affinity
4
Q
Interaction in staining involving attraction of opposite charges (e.g.+ basic dye cations to DNA anions)
A
Coulombic attraction
5
Q
Basic dye that stains the nucleus blue or purple in H&E staining
A
Hematoxylin
6
Q
Acidic dye that stains cytoplasm pink or red in H&E staining
A
Eosin
7
Q
Interaction in staining involving dipole-dipole attraction (e.g.+ elastic fiber stains)
A
Van der Waals forces
8
Q
Interaction in staining involving hydrogen atoms and electronegative atoms (e.g.+ collagen staining with Sirius red)
A
Hydrogen bonding
9
Q
Interaction in staining involving sharing of electrons and polar bonds (e.g.+ Feulgen nuclear stain+ PAS)
A
Covalent bonding
10
Q
Chemical compound that forms an insoluble+ colored precipitate with stain on tissue
A
Mordant
11
Q
Chemical that facilitates staining but is not essential to the chemical union
A
Accentuator
12
Q
Chemical used to preserve tissue structure before staining
A
Fixative
13
Q
Solution used to remove water and harden tissue for sectioning
A
Dehydrating agent
14
Q
Process that makes tissue transparent for better stain penetration
A
Clearing
15
Q
Method that uses antibodies labeled with enzymes or fluorophores to detect proteins
A
Immunohistochemical staining
16
Q
Stain that requires a mordant to bind to tissue
A
Hematoxylin
17
Q
Common fixative used in histology
A
Neutral Buffered Formalin
18
Q
Staining method that differentiates bacteria by cell wall properties
A
Gram staining
19
Q
Bacterial group that appears purple after Gram staining
A
Gram-positive bacteria
20
Q
Bacterial group that appears pink after Gram staining
A
Gram-negative bacteria
21
Q
Staining method where stain intensity increases with time
A
Progressive staining
22
Q
Staining method where excess stain is removed to achieve optimal intensity
A
Regressive staining
23
Q
Complex formed by tissue+ mordant+ and dye enabling staining
A
Tissue-mordant-dye complex
24
Q
Four basic types of human tissue
A
Epithelium+Connective tissue+Muscle tissue+Nervous tissue
25
Staining method intensified by adding a mordant
Indirect staining
26
Complex formed in indirect staining enabling color development
Tissue-mordant-dye-complex
27
Mordant used in Ehrlich's hematoxylin
Potassium alum
28
Mordant used in Weigert's hematoxylin
Iron
29
Oxidation product of hematoxylin that requires a mordant
Hematin
30
Process of giving color using aqueous or alcoholic dye
Direct staining
31
Basic dye example used in direct staining
Methylene blue
32
Interaction where hydrophobic groups cluster in water
Hydrophobic effect
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Dye type used to stain fats from aqueous solutions
Sudan dyes
34
Interaction where dye molecules attract each other
Dye-dye/reagent-reagent interactions
35
Term for tissue staining a different color than the dye itself
Metachromasia
36
Stain showing metachromasia in cartilage/connective tissue/mast cells
Azure+toluidine blue
37
Cell type with metachromatic granules stained by toluidine blue
Mast cell
38
Technique using metallic salts reduced by tissue to form deposits
Metallic impregnation
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Metals used in metallic impregnation
Gold chloride+Silver nitrate
40
Staining where the surrounding structure is stained but not the target
Negative staining
41
Stain that outlines bone canaliculi
Schmorl's picro-thionin stain
42
Reason why tissue retains stains
High affinity for tissue+Low affinity for processing fluids/mounting media
43
Basic/cationic dyes readily dissolved by lower alcohols
Crystal violet+Methylene blue
44
Acid/anionic dyes less soluble in lower alcohols
Eosin Y+Orange G
45
Non-ionic dyes soluble in dehydrating/clearing agents
Sudan stains
46
Stain that highlights hemosiderin in pulmonary hemorrhage
Perl's Prussian Blue
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Cell type that Prussian blue targets in pulmonary hemorrhage
Hemosiderin-laden macrophage
48
What causes Metachromasia in Mast cells rich in polyanions
Dye-dye aggregate formation
49
What determines why stains are not taken up into every part of the tissue?
Number and affinities of binding sites+Rate of reagent uptake+Rate of reaction+Rate of reagent loss
50
Example of stain with high affinity for fat but low affinity for proteins
Sudan dyes
51
Example of stain with high affinity for DNA
Feulgen nuclear technique
52
What charge do negatively charged acid dyes stain?
Structures carrying cationic charges
53
What factor distinguishes tissues with equal affinities and binding sites during staining?
Rate of reagent uptake
54
Example of tissue with rapid reagent uptake causing staining timing importance
Mucin with Colloidal Iron stain
55
What staining method involves staining tissue elements in a definite sequence until desired intensity?
Progressive staining
56
What does differentiation in staining rely on?
Selective affinity of dye for cellular elements
57
What factor limits coloration in PAS staining?
Short oxidation time limiting staining to fast-reacting 1,2-diol groups
58
What factor involves selective loss of stain from tissues during staining?
Rate of reagent loss
59
What staining method involves overstaining then removing excess stain to achieve desired intensity?
Regressive staining
60
What is differential staining?
Selective removal of excess stain using multiple stains to better differentiate structures
61
What chemical acts as a differentiator in differential staining?
Acid or alkaline solutions+Alcohol as differentiator for both basic and acidic dyes
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Example of a staining protocol that uses differentiation steps
Luxol Fast Blue staining with Cresyl violet counterstain
63
What does Heidenhain’s Iron Hematoxylin stain demonstrate?
Non-selective staining followed by solvent extraction removing stain from permeable structures but retaining it in impermeable structures like muscle A and Z bands
64
Why are stains retained in tissue?
High affinity for tissue elements and low affinity for processing fluids and mounting media
65
What happens if basic dyes are exposed to alcohol for too long?
They dissolve and stain may be washed out
66
What is the solubility characteristic of routine acid/anionic dyes in alcohol?
Less soluble
67
Why should Sudan stains not be exposed to dehydrating agents?
They are soluble in dehydrating and clearing agents and may be washed out
68
What does Perl’s Prussian Blue stain detect?
Hemosiderin deposition in pulmonary hemorrhage
69
What cell type is stained by Perl’s Prussian Blue in lungs?
Hemosiderin-laden macrophages
70
Selective staining of living cell constituents demonstrating cytoplasmic structures by phagocytosis or staining of pre-existing components
Vital staining
71
Stains excluded by living cells but taken up by dead cells
Vital stains
72
Staining method involving injection of dye into living animal body parts
Intravital staining
73
Common dyes used in intravital staining
Lithium carmine+India ink+Toluidine blue+Lugol’s iodine+Methylene blue+Acetic acid+Rose bengal
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Staining method examining living cells removed from organism in dilute solutions
Supravital staining
75
Common dyes used in supravital staining
New methylene blue+Brilliant cresyl blue+Neutral red+Janus green+Trypan blue
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Effect of tissue fixation on staining
Prevents loss of tissue constituents+Fixative-dependent retention and reactivity of substances
77
Fixatives that preserve lipids well
Osmium tetroxide+Dichromates
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Fixative that poorly preserves lipids
Formalin
79
Key considerations for staining procedures
Use stains compatible with fixative and embedding medium+Standardized protocols+Use controls proactively+Ensure reagent reliability
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Purpose of deparaffinization in paraffin section staining
Remove paraffin to allow stain penetration
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Solvent used for deparaffinization
Xylene
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Purpose of rehydration in staining
Replace alcohol with water to prepare tissue for aqueous stains
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Purpose of dehydration after staining
Remove water to allow mounting medium application
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Purpose of clearing in staining
Replace alcohol with xylene to make tissue transparent and miscible with mounting medium
85
Natural dye extracted from Mexican tree heartwood with nuclear staining capacity
Hematoxylin
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Natural dye extracted from female cochineal bug treated with alum
Carmine
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Vegetable dye extracted from lichens used to stain elastic fibers
Orcein
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Chemical dyes that are artificially made from coal tar and petroleum derivatives
Synthetic dyes
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Substances in dyes responsible for color production
Chromophores
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Auxiliary groups in dyes that alter shade and aid color retention
Auxochromes
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Requirement for synthetic dyes to function properly
Both chromophore and auxochrome must be present
92
Example of acid dyes where active color is in acid component
Acid fuchsin+Picric acid
93
Cell structures that have affinity for acid dyes
Basic/cationic structures like collagen+eosinophilic granules of leukocytes (acidophilic)
94
Example of basic dye
Methylene blue
95
Cell structures that have affinity for basic dyes
Acidic structures like chromatin+mucus+cartilage matrix (basophilic)
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Dyes formed by combining acid and basic dyes capable of staining cytoplasm and nucleus simultaneously
Neutral dyes
97
Examples of neutral dyes
Romanowsky dyes+Giemsa stain+Irishman’s stain
98
Common staining solution that uses hematoxylin and eosin
Hematoxylin and Eosin (H&E) stain
99
Function of hematoxylin in H&E staining
Stains cell nuclei blue/purple (basic dye staining acidic structures)
100
Function of eosin in H&E staining
Stains cytoplasm and connective tissue pink/red (acid dye staining basic structures)
101
Mordants used in hematoxylin staining
Potassium alum (Ehrlich’s hematoxylin)+Iron (Weigert’s hematoxylin)
102
Oxidation product of hematoxylin that forms colored complex
Hematin
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Natural dye extracted from Mexican tree heartwood
Hematoxylin
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Natural dye extracted from female cochineal bug
Carmine
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Vegetable dye extracted from lichens used to stain elastic fibers
Orcein
106
Composition components of eosin stain
Phloxine B+1% Eosin Y+95% alcohol+Glacial acetic acid+Crystal of thymol
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Difference between chromophore and auxochrome
Chromophore produces color+Auxochrome alters shade and retains color by donating electrons
108
Basic dyes are cationic or anionic?
Cationic (positively charged)
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Acid dyes are cationic or anionic?
Anionic (negatively charged)
110
Why do acid dyes stain basic tissue components?
Opposite charges attract (acid dye anions bind basic tissue cations)
111
Why do basic dyes stain acidic tissue components?
Opposite charges attract (basic dye cations bind acidic tissue anions)
112
Classification of hematoxylin solutions by mordant used
Alum hematoxylins+Iron hematoxylins+Tungsten hematoxylins+Molybdenum hematoxylins+Lead hematoxylins+Hematoxylins without mordant
113
Role of metal cation in mordant
Confers net positive charge to dye-mordant complex+Enables binding to anionic tissue sites
114
Mordant in alum hematoxylins
Aluminum potassium sulfate or aluminum ammonium sulfate
115
Staining type recommended for alum hematoxylins
Progressive staining+Can be used for regressive staining
116
Color change in alum hematoxylin staining
Nuclei stain red then convert to blue-black when washed in weak alkali (blueing)
117
Definition of blueing
Passing alum hematoxylin stained sections into alkaline solution to neutralize acid and form insoluble blue aluminum hematin-tissue lake
118
Common blueing solutions
Warm tap water (40-50°C)+0.05% ammonia in distilled water+Saturated lithium carbonate+Scott's tap water substitute
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Ehrlich’s hematoxylin composition
Hematoxylin 2 gm+Absolute ethyl alcohol 100 ml+Aluminum potassium sulfate 15 gm+Glycerin 100 ml+Distilled water 100 ml+Glacial acetic acid 10 ml
120
Ehrlich’s hematoxylin staining type
Regressive staining+Differentiated with 1% hydrochloric acid in 70% alcohol
121
Ehrlich’s hematoxylin staining affinity
Mucopolysaccharides like cartilage and bone cement lines
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Harris’s hematoxylin composition
Hematoxylin 1 gm+Absolute ethyl alcohol 10 ml+Ammonium/Potassium alum 20 gm+Distilled water 190 ml+Mercuric oxide 0.5 gm+Glacial acetic acid 10 ml
123
Harris’s hematoxylin staining type
Good regressive stain+Routine nuclear staining+Used in exfoliative cytology+Sex chromosome staining
124
Cole’s hematoxylin composition
Hematoxylin 1.5 gm+1% Iodine in 95% Alcohol 50 ml+Saturated aqueous ammonium alum 700 ml+Distilled water 250 ml
125
Cole’s hematoxylin use
Routine purposes+Artificially ripened with alcoholic iodine solution
126
Mayer’s hematoxylin composition
Hematoxylin 1 gm+Sodium iodate 0.2 gm+Potassium alum 50 gm+Citric acid 1 gm+Chloral hydrate 50 gm+Distilled water 1000 ml
127
Mayer’s hematoxylin staining type
Regressive and progressive staining+Nuclear counterstain+Demonstrates cytoplasmic glycogen and mucopolysaccharides
128
Unique property of Celestine Blue hemalum method
Only alum hematoxylin resistant to acid effects
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Other alum hematoxylins
Delafield’s Hematoxylin+Carazzi’s Hematoxylin+Gill’s Hematoxylin
130
Iron hematoxylins staining type
Differential or regressive staining+Use acid-alcohol as differentiator
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Iron hematoxylin mordants
Ferric chloride (Weigert’s)+Ferric ammonium sulfate (Heidenhain’s)
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Iron hematoxylin staining color
Blackish or grayish depending on differentiation
133
Iron hematoxylin special applications
Specific identification of phospholipids+Muscle fibers+Connective tissues
134
Weigert’s hematoxylin composition
Solution A: Hematoxylin 1 gm+Absolute ethyl alcohol (amount unspecified); Solution B: 30% anhydrous ferric chloride 4 ml+Concentrated hydrochloric acid 1 ml+Distilled water 100 ml
135
Weigert’s hematoxylin use
Standard iron hematoxylin stain+Recommended when preceding stains contain acid (e.g.+ Van Gieson stain)
136
Heidenhain’s hematoxylin composition
Mordant/Differentiator: Ferric ammonium sulfate 2.5 gm+Distilled water 100 ml; Hematoxylin stain: Hematoxylin 1.5 gm+95% ethyl alcohol 10 ml+Distilled water 90 ml
137
Heidenhain’s hematoxylin staining type
Regressive staining+Stains nuclear and cytoplasmic inclusions black or dark gray+Muscle striations and myelin staining
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Loyez hematoxylin mordant
Ferric ammonium sulfate+Weigert’s differentiator
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Loyez hematoxylin use
Demonstrates myelin+Applicable to paraffin+ frozen+ and cellulose sections
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Verhoeff’s hematoxylin components
2% aqueous ferric chloride (differentiator)+Lugol’s iodine
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Verhoeff’s hematoxylin staining target
Coarse elastic tissue stained black
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Tungsten hematoxylin example
Mallory’s phosphotungstic acid hematoxylin
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Mallory’s hematoxylin composition
Hematoxylin 1 gm+Phosphotungstic acid 20 gm+Distilled water 1000 ml
144
Mallory’s hematoxylin staining
Progressive+Nuclei+ fibrin+ muscle striations+ myofibrils blue+Collagen+ bone+ cartilage orange-red to deep brick-red
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Molybdenum hematoxylin mordant
Molybdic acid
146
Molybdenum hematoxylin staining
Collagen and coarse reticulin violet to black+Argentaffin cells black+Nuclei pale blue+Paneth cells orange
147
Lead hematoxylin use
Demonstration of endocrine cell granules+Localization of gastrin-secreting cells in stomach
148
Current use of lead hematoxylin
Replaced by immunohistochemistry (IHC)
149
Hematoxylin without mordant use
Demonstrates minerals such as lead+ iron+ and copper
150
Mixture of picric acid and acid fuchsin used for connective tissue demonstration
Van Gieson's stain
151
Function of acid fuchsin in Van Gieson's stain
Imparts red color to collagen fibers
152
Tissue components stained by acid fuchsin (Masson stain)
Collagen+Smooth muscle+Mitochondria
153
Role of acid fuchsin in Mallory's trichrome method
Nuclear and cytoplasmic stain
154
Fluorescent dye that differentiates dead and living cells
Acridine orange
155
Fluorescence color of DNA and RNA with acridine orange
DNA: green+RNA: red
156
Dye used to demonstrate calcium deposits and phosphatase activity
Acridine Red B
157
Water-soluble dye staining acid mucopolysaccharides
Alcian blue
158
Tissue specificity of Alcian blue
Connective tissue+Epithelial mucin
159
Dye that forms orange-red lake with calcium at pH 4.2
Alizarin Red S
160
Cytoplasmic stain used for counterstaining epithelial sections
Aniline blue
161
Stain where nuclei are deep red and cytoplasm pale red
Azocarmine
162
Dye used for deep staining of acid-fast organisms and mitochondria
Basic fuchsin
163
Reagents using basic fuchsin
Feulgen's and Schiff's reagent
164
Dye used for staining hemoglobin
Benzidine
165
Mordanted dye acting as basic dye for acidic substances
Carmalum (Mayer’s) solution
166
Oxazine dye alternative to iron hematoxylin
Celestine blue
167
Function of celestine blue
Forms strong staining lake with iron alum+Resistant to strong acid dyes
168
Dye used to identify amyloid deposits
Congo red
169
Dye staining acidic components of neuronal cytoplasm (Nissl bodies)
Cresyl violet
170
Nuclear stain used for amyloid and platelets
Crystal violet
171
Fluorescent DNA stain used with acridine orange
Ethidium bromide
172
Mixture of methylene blue and eosin for blood staining
Giemsa stain
173
Metallic impregnation stain composed of gold chloride and mercuric chloride
Gold sublimate
174
Dye used for staining amyloid+ cellulose+ starch+ carotenes and glycogen
Iodine
175
Role of Gram’s iodine in microbiology
Identify and differentiate Gram-positive and Gram-negative bacteria
176
Brown solution turning black in presence of starch
Lugol's iodine
177
Dye used to demonstrate mitochondria in intravital staining
Janus Green B
178
Weakly basic dye used as bacterial spore stain and erythrocyte counterstain
Malachite green
179
Three-color staining protocol distinguishing cells from connective tissue
Masson’s trichrome
180
Color of chromatin stained by methyl green
Green
181
Dye containing azures and methylene violet that stains acidic cell parts blue
Methylene blue
182
Process involving oxidation of methylene blue producing azures and thiazoles
Polychroming
183
Staining color of nuclei by polychrome methylene blue
Blue
184
Tissues stained reddish-violet by polychrome methylene blue
Cartilage matrix+Mucin+Mast cell granules+Connective tissues
185
Applications of methylene blue staining
Plasma cells+Cytological exams of fresh sputum for malignant cells+Bacterial diagnosis+Diphtheria diagnosis+Vital staining of nervous tissue
186
Lipophilic stain formed by boiling Nile blue with sulfuric acid
Nile red
187
Color of lipid globules stained by Nile red
Red
188
Nile red fluorescence property
Strongly fluorescent in hydrophobic environments+Yellow-gold emission for neutral lipids+Deep red for polar membrane lipids
189
Uses of Nile red
Localization and quantification of intracellular lipid droplets+Live cell staining+Environmental sensor fabrication
190
Dye used to identify neutral lipids and fatty acids in fresh smears and tissues
Oil Red O
191
Disease associated with Oil Red O staining
Multiple sclerosis (macrophages uptake lipid-rich debris)
192
Dye excellent for elastic fiber staining
Orcein
193
Selective stain for unsaturated lipids and lipoproteins such as myelin
Osmium tetroxide
194
Tissue components stained black by osmium tetroxide
Fat and myelin
195
Stain that colors glycogen+ mucin+ basement membranes+ fungi+ and intracellular carbohydrates
Periodic Acid Schiff (PAS)
196
Common negative stain for viruses+ nerves+ and polysaccharides
Phosphotungstic acid
197
Contrast stain to acid fuchsin and counterstain to crystal violet
Picric acid
198
Insoluble colored salt used to demonstrate blood and lymph vessels
Prussian blue
199
Tissues stained by Prussian blue
Hemosiderin-laden macrophages in pulmonary hemorrhage
200
Dye used with osmic acid to fix and stain blood and glandular tissues
Rhodamine B
201
Counterstain producing red nuclei and yellow collagen
Safranin
202
Solution used to identify spirochetes and reticulum fibers
Silver nitrate
203
Dye recommended for staining Nissl granules and differentiating mast cell granules
Toluidine blue
204
Connective tissue stain that colors collagen pink
Van Gieson stain
205
Dye used for neuroglia demonstration in frozen sections
Victoria blue
206
Silver reduction stain demonstrating phosphates and carbonates
Von Kossa stain
207
Stain mixture showing affinity for methylene blue+ azures+ eosin+ and complex dyes
Wright stain
208
Most sensitive oil soluble dye with affinity for phospholipids
Sudan black
209
Dye recommended for triglycerides staining but not phospholipids
Sudan IV
210
Fat stain for central nervous system tissues with lighter orange color
Sudan III
211
Chief solvents used for stains
Water+Alcohol+Aniline water+Phenol
