LECT | Staining FINALS Flashcards
1
Q
Enables one to see and study the architectural pattern of the tissue and physical characteristics and structural relationships of tissues and their cells
A
Staining/Dyeing
2
Q
Staining/Dyeing is made possible through:
A
- Capillary osmosis
- Solubility
- Absorption
- Adsorption of stains or dyes by tissues
3
Q
Alkaline structures (cytoplasm) are stained by
A
acidic stains
4
Q
Eosin stains cytoplasm color?
A
Red
5
Q
Acidic structures (nucleus) are stained
A
alkaline/basic stain
6
Q
hematoxylin stains nucleus color?
A
Blue
7
Q
Classification of Staining
A
- Histological staining
- Histochemical Staining (Histochemistry)
- Immunohistochemical Staining
8
Q
- The process whereby the tissue constituents are demonstrated in section by direct interaction with a dye or staining solution, producing coloration of the active tissue component.
- Used to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm.
A
Histological Staining
9
Q
Histological Staining examples
A
- Microanatomic stains
- Bacterial stains
- Specific tissue stains (muscles, connective tissue, neurologic stains)
10
Q
The process whereby various constituents of tissues are studied through chemical reactions that will permit microscope localization of a specific tissue substance
A
Histochemical Staining or Histochemistry
11
Q
Histochemical Staining or Histochemistry example
A
- Perl’s Prussian Blue (hemoglobin); demonstrates ferric ion (Fe3+) in tissues
- Periodic Acid Schiff (carbohydrates)
12
Q
A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope, using a wide range of monoclonal, fluorescent-labeled or enzyme- labeled antibodies.
A
Immunohistochemical Staining or Immunohistochemistry
13
Q
- It is the basis of histochemistry
- It is accomplished by controlled, specific chemical reactions designed to give a final color (staining) at the site/location of the structure of the substance in the cells or tissue
A
Specific Staining
14
Q
Specific Staining example
A
- Perl’s Prussian blue reaction – Hemosiderin
- Periodic Acid Schiff technique – Polysaccharides
15
Q
The staining of the tissue by means of simple alcoholic/aqueous solution of the dye.
A
Simple Staining/Direct Staining
16
Q
Simple Staining/Direct Staining examples
A
Methylene blue and Eosin
17
Q
The action of the dye is intensified by some other agents.
- Mordant
- Accentuator
A
Indirect Staining
18
Q
- Substance which when taken up by the tissue, helps make the in return serving as a link or bridge to make the staining reaction possible
- Combines with a dye forming a colored “lake” which combined with tissues to form an insoluble “tissue- mordant-dye complex”
- An integral part of the staining reaction itself, without which, no staining could possibly occur
A
Mordant
19
Q
Indirect staining Mordant example
A
- Potassium Alum with Hematoxylin in Erlich’s hematoxylin
- Iron in Welgert’s hematoxylin
20
Q
- Chemical substances that does not participate but merely increases or heightens the color intensity, selectively and crispness of the stain
- Differ from mordants in that they do not bind or link the tissue to the dye
A
Accentuator
21
Q
Indirect staining Accentuator example
A
- Potassium hydroxide (KOH) in Loeffler’s Methylene Blue
- Phenol in carbol thionine and carbol fuchsin
22
Q
- Staining is continued in a definite sequence until the desired intensity of coloring of the different tissue elements is attained.
- No washing/differentiation/decolorization in between is required, solely relies on the selective affinity of dyes for different cellular elements
A
Progressive Staining
23
Q
Any stain is possible as long as no differentiation is done
A
Progressive Staining
24
Q
The tissues are overstained and the excess dye is then removed until the desired intensity is obtained
A
Regressive Staining
25
Regressive Staining : 2 principle
1. Overstaining
2. Washing/ Differentiation/ Decolorization
26
Example of Regressive staining
1. Acid Fast Staining
2. Gram Staining
27
Entails the use of the specific dyes that stains tissues with a color that is different from that of the stain color itself
Metachromatic Staining
28
Metachromatic Staining is employed for staining ?
1. Cartilage
2. Epithelial mucins
3. Connective tissues
4. Amyloid
5. Mast cell granules
29
Metachromatic dye – basic dyes belonging to what group
Thizine and Triphenylmethane groups
30
Examples of Metachromatic Stain
Cresyl blue for reticulocytes
31
The application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated.
Counterstaining
32
Examples of Counterstain
Eosin ⎯ for cytoplasm
33
1. The selective staining of living cells constituents, demonstrating cytoplasmic structures by phagocytosis if dye particle (cytoplasmic phagocytosis)
2. The nucleus of the living cell is resistant to vital stains, and therefore is not demonstrated
3. Demonstration of nuclear structures during vital staining suggests permeability of the membrane by the dye, signifying the death of the cell.
Vital Staining
34
Trypan blue and Janus Green B are examples of what staining classification
Vital Staining
35
Vital stain of reticuloendothelial cells
Trypan blue
36
true vital staining of mitochondria
Janus Green B
37
Done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal, or subcutaneous) producing specific coloration of certain cells, particularly those of the RES.
Intravital Staining
38
Intravital Staining : commonly used dye
1. Lithium
2. India Ink
3. Carmine
39
Used to stain living cells immediately after the removal from the living body
Supravital Staining
40
Example of supravital stain - probably the best vital dye
Neutral RED
41
1. Makes use of heavy metals which are precipitated with selectivity of certain cellular and tissue components
2. Has its greatest application in tissue from the CNS and for the demonstration of reticulin
3. Differs from staining because it consists of an opaque black particulate precipitate
Metallic Impregnation
42
Metallic Impregnation examples
1. Silver nitrate
2. Osmium tetroxide
43
most commonly used agent for impregnation
Silver Nitrate
44
used for the demonstration of lipids
Osmium tetroxide
45
Are those obtained from plants and animal, previously utilized for dyeing of wool and cotton
Natural Dyes
46
most important and the most commonly dye used for routine histologic studies
Hematoxylin
47
1. A vegetable dye extracted from lichens which are normally colorless, but when treated with ammonia and expose to air, produce blue or violet colors.
2. Mainly used for staining Elastic fibers.
Orcein
48
An old histologic dye extracted from the female cochineal bug (coccus cacti)
Cochineal
49
A plant with orange stigmas yielding a dye
Saffron
50
Examples of natural dyes
1. Hematoxylin
2. Orcein
3. Cochineal
4. Saffron
51
Sometimes known as “coal tar dyes” since they were originally manufactured from substances that have been taken from coal tar
Synthetic Dyes
52
Synthetic Dyes consist of substances
1. Chromophores
2. Auxochromes
53
1. Greek.“color-bearers”
2. Substances capable of producing visible colors
Chromophores
54
1. Greek.“color-increasers”
2. Substances which impart to the compound property of electrolytic dissociation, thereby altering the shade of the dye, giving it the property of forming salts with another compound and ultimately retaining its color.
3. The introduction of a chromogenic group into a uncolored molecule will cause it to be colored, it will then be a chromogen, which is colored and not a dye
Auxochromes
55
Synthetic dye coloring property is attributed to the
chromophore
56
Synthetic dye, which dyeing property to the salt forming
Auxochrome
57
1. A process of selective removal of excess dye
2. If the dye is a basic one, differentiation is carried out by an acid solution
3. An alkaline medium is used for differentiation after an
acid dye
Differentiation
58
1. Natural dye is extracted from the core of heartwood of the tree Hematoxylin campechianium
2. By far the most valuable staining reagent used by the cytologist due to its powerful nuclear and active coloring agent
Hematoxylin
59
Hematin
formed by oxidation of Hematoxylin RIPENING
60
1. Usually done by exposing the substances to air and sunlight, thereby oxidizing this
2. A slow process (3-4months) but it can be accelerated by adding strong oxidizing agents
Hematoxylin
61
complex of stain and mordant is called a
“lake”
62
Mordant example of Hematoxylin
1. most commonly used mordant. Gives a blue take appearance
2. Increase the selectivity for nuclei, especially if acid is added or is used as a differentiating agent
Aluminum
63
Mordant example of hematoxylin:
caused an intense blue-black appearance when used as a mordant
Ferric salt
64
Hematoxylin: mordant example
1. Aluminum
2. Chromium
3. Iron
4. Copper
5. Tungsten/Phosphotungtic acid (PTAH)
6. Ferric salt
65
1. A red acid dye used for cytoplasm differentially
2. Commonly used as a background or contrast stain because it gives a leasing and useful contrast to nuclear stains such as hematoxylin
Eosin
66
examples of Eosin
1. Eosin Y
2. Eosin B
3. Eosin S/Ethyl Eosin
67
Most common eosin
Eosin Y
68
H and E Staining Color Reaction:
1. Cytoplasm & proteins in edema fluid
Pale pink
69
H and E Staining Color Reaction:
1. Decalcified bone
2. Osteoid
3. Collagen
Pink
70
H and E Staining Color Reaction:
Muscle fibers
Deep pink
71
H and E Staining Color Reaction:
RBC
Eosinophil granules
Keratin
Bright orange to Red
72
H and E Staining Color Reaction:
Calcium & Calcified bone
Purplish blue
73
H and E Staining Color Reaction:
1. Plasma cells
2. Osteoblast
3. Basophilic cytoplasm
Purplish pink
74
H and E Staining Color Reaction:
Karyosome
Dark blue
75
H and E Staining Color Reaction:
Nucleus
Blue to blue-black
76
H and E Staining Color Reaction:
Cartilage
Light blue to Dark blue
77
1. A specialized fixative used in frozen section which serves to localize antigen and hydrolytic enzyme
2. It is also used for preservation of lipids
Formol Calcium
78
1. It is similar with H and E technique but the differentiation stage is omitted
2. It is a progressive form of staining
Modified H and E Technique
79
Used for determining cervical cancer
Papanicolaou stain
80
Papanicolaou stain makes use of 3 stains
1. Hematoxylin
2. OG-6 (orange green)
3. Eosin Azure
81
For staining cytoplasm of mature cells.
Example: mature superficial cells
OG-6 (orange green)
82
for staining cytoplasm of immature cells.
Example: Intermediate and parabasal cells
Eosin Azure
83
Eosin Azure composition?
1. Eosin
2. Bismarck brown
3. Lithium carbonate
4. PTA
5. Light green stain (36,50,65)
84
1. It omitted the Bismarck brown dye for the EA solution
2. Sharpness of color and brilliant staining reactions are improved
Modified Pap’s Stain
85
1. Mixture of picric acid and acid fuchsin
2. for the demonstration of connective tissues, mucin and elastic tissue
Van Gieson’s (Acid fuchsin picric acid)
86
Basic acridine fluorochrome which permits discrimination between dead and living cells
Acridine Orange
87
Acridine orange gives this fluorescence color for
1. DNA
2. RNA
1. GREEN fluorescence for DNA
2. RED fluorescence for RNA
88
For calcium salts and phosphate activity
Acridine Red 3B
89
1. Stain acid mucopolysaccharide
2. More specific for connective tissue and epithelial mucin
Alcian blue
90
A cytoplasmic stain used for counterstaining of epithelial sections
Aniline blue
91
A plasma stain utilized also for deep staining of acid fast organisms mitochondria, and differentiation of smooth muscles with the use of picric acid
Basic Fuchsin
carbol fuchsin, coleman’s Feulgen, schiff’s, Mallory’s fuchsin, Aldehyde fuchsin (Gomori’s Stain)
92
Used for staining hemoglobin
Benzidine
93
1. Used for staining diphtheria
2. Used for contrast stain for gram’s technique, acid Papanicolaou method fast and Papanicolaou method
Bismarck brown
94
1. Used as a chromatin stain for fresh materials in smear preparations
2. combined with aluminum chloride to stain glycogen
Carmine
95
A mordanted dye acting as a basic dye and staining acid substances
Mayer’s Carmalum solution
96
Recommended for routine staining of fixed sections
Celestine blue
97
1. Best known as indicator
2. May be utilized as a stain for axis cylinders in embryos
3. Used for staining tissues, amyloid and myelin (Krajian’s method)
Congo Red
98
A nuclear or chromatin stain for staining amyloid in frozen sections and platelets
Crystal violet
99
mixture of crystal violet, methyl violet in frozen sections
GENTIAN VIOLET
100
Used for staining blood to differentiate leukocytes
Giemsa
101
Used for metallic impregnation, made up of gold chloride and mercuric chloride
Gold Sublimate
102
1. The oldest of all stains
2. Stains amyloid, cellulose, starch, carotenes, and glycogen.
Iodine (gram's, lugol’s)
103
Used for demonstrating mitochondria (intravital staining)
Janus Green B
104
1. Used as a contrast for staining ascaris and erythrocytes
2. Used as bacterial spore staining
Malachite Green
105
Stains chromatin green in the presence of an acid
Methyl Green
106
1. Plasma cells, fresh sputum for malignant cells, evaluation, and differentiation of bacterial organism.
2. Diphtheria diagnosis and nervous tissue vital staining
Methylene Blue
107
Used in frozen sections for rapid diagnosis
Loeffler’s Polychrome Methylene Blue
108
Coloring nuclei of leukocytes reddish-purple in presence of methylene blue
Methylene violet
109
For observing cell granules an vacuoles of phagocytic cells
Neutral red
110
Substitute for carbol fuchsin in acid- fast staining
Night Blue
111
1. An excellent stain for elastic fibers
2. Demonstrates the finest and most delicate fiber skin (dermatological studies)
Orcein
112
Used as a fixative Used to stain fats
Osmium tetroxide
113
1. Normally utilized for the manufacture of paints
2. Used as microanatomical contrast for demonstration for the circulatory system by injection (intravital staining)
Prussian blue
114
Used with osmic acid to fix and stain blood and glandular tissues
Rhodamine B
115
Used in identification of Spirochetes, reticulum and other fiber stains
Silver Nitrate
116
Recommended for staining of Nissl granules or chromophilic bodies; nuclear stain for fixed tissues
Toluidine Blue
117
Demonstration of neuroglia in frozen sections
Victoria Blue
118
RED Cytoplasmic Counterstains
1. Eosin Y
2. Eosin B
3. Phloxine B
4. Rose Bengal
119
YELLOW Cytoplasmic Counterstains
1. Picric acid
2. Orange G
120
GREEN Cytoplasmic Counterstains
1. Light green Sf
2. Lissamine green
121
RED Nuclear Counterstains
1. Neutral red
2. Safranin
3. Carmine
122
BLUE Nuclear Counterstains
1. Methylene blue
2. Toluidine blue
3. Celestine blue
4. Hematoxylin
123
What are the Lysochromes (Oil Soluble Dyes)
1. Sudan Black B (SBB)
2. Sudan IV or Scharlach R
3. Sudan III
124
1. Greatest affinity for phospholipids or neutral fats (triglycerides)
2. A more sensitive coloring agent than other lysochromes
3. Should be discarded if the brownish black color appeared
4. Demonstrates lipids that are resistant to paraffin embedding
Sudan Black B (SBB)
125
1. Recommendedforneutralfats(triglycerides)
2. Do not color phospholipids and fine lipid droplets
Sudan IV or Scharlach R
126
1. First Sudan Dye introduced into Histochemistry
2. Fat soluble
3. A good stain for the CNS
Sudan III
127
1. Used in order to promote adhesion of sections to slides
2. Spread thinly and evenly on a clean grease-free glass slide which is then gently approximated to the end of the ribbon and drawn upwards in a near vertical motion
ADHESIVES
128
Mayers’s Egg Albumin Formula:
1. Egg white (50 mL)
2. Glycerin (50 mL)
3. Thymol crystals (100 mg)
129
Dried Albumin Formula
1. Dried albumin (5g)
2. Sodium chloride (5g)
3. Distilled water (100 mL)
4. Thymol crystals (2 g)
130
1% Gelatin Formula
1. Gelatin (1g)
2. Distilled water (100 mL)
3. Glycerol (15 mL)
4. Phenol crystals (2g)
131
Gelatin Formula:
1. 1% Gelatin (5 mL)
2. 2% Formaldehyde (5 mL)
132
Starch paste Formula:
1. Powdered starch (1g)
2. Distilled water (30 ml = 10 mL cold, 20 mL boiling)
133
Readily available from outdated blood stored in blood banks, dispensed into sterile tubes of 0.5 ml each
Plasma
134
1. 0.1% aqueous detergent solution which is further diluted 1:10 with distilled water prior to use (final dilution: 0.01%)
2. Used as section adhesive in immunochemistry
Poly-L-Lysine
135
Very useful in cytology particularly for cytospin preparation if proteinaceous or bloody material
3-APES (3-aminopropyltriethoxysilane)
136
1. A syrupy fluid applied between the section and the coverslip after staining setting the section firmly, preventing the movement of the coverslip
2. Necessary to protect the stained section from physical injury and from bleaching or deterioration of the stain as a result of oxidation
Mounting
137
In mounting, for best results with stained tissue sections, they must be impregnated by an transparent medium having a refractive index close to that of
91.518
138
2 groups of mounting media
1. Aqueous / Water Soluble Mountants
2. Resinous Mountants
139
Designed to mount water-miscible preparations directly water in cases where the stain is removed or decolorized with alcohol or xylene
Aqueous / Water Soluble Mountants
140
1. Low refractive index
2. Good only for temporary mounting
Water
141
Mountant:
1. Also used as preservative
2. Has high index of refraction
Glycerin
142
Glycerin RI
RI: 1.47
143
Mountant:
1. Does Not solidify upon storage
Farrant’s medium (Gum syrup)
144
Farrant’s medium (Gum syrup) RI
RI: 1.43
145
Used for methylene blue-stained nerve preparations and as general purpose aqueous mountant
Apathy’s medium
146
Recommended for mounting frozen sections from water or paraffin sections which require dehydration and clearing
Brun’s fluid
147
Examples of Aqueous / Water Soluble Mountants
1. Water
2. Glycine
3. Farrant’s medium or gum syrup
4. Apathy’s medium
5. Brun’s fluid
148
Used for preparations that have been dehydrated and cleared in xylene or toluene and are recommended for majority of staining methods.
Resinous Mountants
149
Examples of Resinous Mountants
1. Canada Balsam
2. DPX
3. XAM
4. Clarite
150
1. natural resin extracted from the Canadian Tree (Abus balsamea)
2. Recommended for whole mounts and for thick sections because it does not shrink
3. Miscible with xylene and is quite expensive
Canada Balsam
151
Canada Balsam RI
RI: 1.524
152
Mountant recommended for small tissue sections
DPX
153
DPX RI
RI: 1.532
154
Synthetic resin mixture in xylene in pale yellow or colorless solution
XAM
155
XAM RI
RI: 1.52
156
Synthetic resin which is soluble in xylene (usually diluted to 60% with xylene)
Clarite
157
Clarite RI
RI: 1.544
158
2 mounting media most commonly used today
1. Eukitt
2. Entellan
