MODULE 5 - Staining & Mounting Flashcards
1
Q
Process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of cells
A
staining
2
Q
Tissue components are demonstrated by direct interaction with a dye or staining solution
A
Histological stain
3
Q
used for chemical component/reaction
A
Histochemical stain
4
Q
Active tissue component is colored
A
Histological stain
5
Q
Examples of histological stains
A
Micro-anatomical stains
Bacterial stains
Specific tissue stains
6
Q
Uses antibodies instead of stains
A
Immunohistochemical stain
7
Q
Perl’s Prussian blue reaction is for
A
hemoglobin
8
Q
Periodic acid Schiff is for
A
Carbohydrates
9
Q
Examples of histochemical stains
A
Perl’s prussian blue
Periodic and schiff
10
Q
Detection of phenotypic markers that are detected by antibodies
A
immunohistochemical stain
11
Q
Examples of immunohistochemical staining
A
monoclonal
polyclonal
12
Q
uses aqueous or alcoholic dye solutions to produce a color
A
Direct staining
13
Q
Uses only one dye, usual color is the resulting color
A
direct staining
14
Q
Example of direct staining
A
methylene blue
15
Q
Uses mordant/accentuator
A
Indirect staining
16
Q
Link/bridge between dye and tissue
A
mordant
17
Q
Examples of mordant and solutions that contains them
A
Potassium alum- Meyer’s hematoxylin
Iron (Ferric ammonium chloride)- Weigert’s hematoxylin
18
Q
What is accentuator
A
It heightens the color intensity and selectivity
Increases staining power
19
Q
Examples of accentuator and solutions that contains them
A
KOH- Loeffler’s methylene blue
Phenol- carbol fuschin or carbol thionine
20
Q
tissue elements re stained in a definite sequence
gradual application of dyes
No excess dye No decolorization
A
Progressive staining
21
Q
Tissue is overstained
A
Regressive staining
22
Q
Excess dye in regressive staining are removed by
A
Decolorization/differentiation
23
Q
Example of regressive staining
A
Acid alcohol- can remove both acid and basic dyes
24
Q
staining tissue with a color that is different from the stain itself
A
Metachromatic staining
25
Examples of metachromatic staining
```
Methyl violet or crystal violet
Cresyl blue for reticulocytes
Safranin
Bismarck brown
Basic fuschin
Methylene blue
Thionine
Toluidine blue
Azure A,B,C
```
26
involves application of a different color to produce contrast background
Counterstaining
27
Example of counterstain and explain its use
Eosin- red acid dye for cytoplasmic stain
28
Forms of eosin
✓ Eosin Y- yellow
✓ Bluish- eosin b
✓ Ethyl Eosin- eosin s
29
tissue elements are demonstrated NOT by stains but by colorless solutions of metallic salts
Metallic impregnation
30
When applied to tissues, it is not absorbed and can only leave black deposits on surface
Metallic impregnation
31
Examples of metallic impregnation
Ammoniacal silver
Silver nitrate
Gold chloride
32
Demonstrates living cells, fresh and viable cells.
Vital staining
33
2 types of vital staining
Intravital
| Supravital
34
Dye is injected to any part of the body
Intravital
35
Examples of intravital stains
Lithium
Carmine
India ink
36
stain is applied immediately to tissue after its removal from living body
Supravital
37
Best vital stain
Neutral red
38
Supravital stain for mitochondria
Janus green
39
Examples of supravital stain
```
Neutral red- best vital stain
Janus green- for mitochondria
Trypan blue
Nile blue
Toluidine blue
Thionine
```
40
Most common method utilized for microanatomical studies of tissues
Rountine H&E
41
H&E stain is what type of stain
Regressive staining
42
Steps in H&E staining
1. Initial xylene bath- for further deparaffinization
2. Descending grades of alcohol- for hydration
3. Hematoxylin- primary dye, nuclear stain-basic dye
4. Acid alcohol- decolorizer
5. Ammonia water- bluing agent
6. Eosin- counterstain, cytoplasmic stain-acid dye
7. Ascending grades of alcohol- for dehydration
8. Final xylene bath- for clearing prior to mounting
43
Enumerate H&E results
Nuclei- blue to blue black
Karyosome- deep blue
Cytoplasm- pale pink
RBCs, eosinophilic granules, keratin- bright orange red
Calcium and decalcified bones- purplish blue
Decalcified bone matrix, osteoid, collagen- pink
Muscle tissue- deep pink
44
Staining Methods for FROZEN SECTION
H&E
Thionine
Polychrome methylene blue
Alcohol pinacyanol method
45
– derived from plants and animals
natural dyes
46
Examples of natural dyes
Hematoxylin
Cochineal dyes
Orcein
Saffron
47
Hematoxylin is a stain
| True or False
False
48
active coloring agent of hematoxylin
Hematane
49
Hematoxylin is formed from the oxidation of hematoxylin either by
ripening (Exposure to sunlight)
| adding oxidizing agents
50
Oxidizing agents in hematoxylin
```
Hydrogen peroxide
Mercuric peroxide
Sodium iodate
Sodium perborate
Potassium permanganate
```
51
Hematoxylin is used in combination with
alum
iron
chromium
copper salts
52
ALUM HEMATOXYLIN uses _______ as mordant
potassium alum
53
Ripening agent of Ehrlich's hematoxylin
Sodium iodate
54
Ripening agent of Harris hematoxylin
Mercuric oxide- for exfoliative cytology and sec chromosomes
55
Ripening agent of Cole's hematoxylin
Alcoholic iodine
56
Ripening agent of Mayer's hematoxylin
Sodium iodate
Contains citric acid and chloral hydrate as preservative
preferred for immunohistochem
57
Ripening agent of Delafield's hematoxylin
Glycerol- to stabilize oxidation process
| Smells like wine, color purple-red shade
58
Ripening agent of Gill's hematoxylin
sodium iodate
| used to stain mucin in goblet's cell
59
Ripening agent of Carazzi's hematoxylin
Potassium iodate
60
– in general for photomicrography
IRON HEMATOXYLIN
61
Weigert’s Hematoxylin is for?
| and what is its component/mordant
Muscle fibers and CT
| Ferric ammonium chloride- both mordant and ripening agent
62
Heidenhain’s Hematoxylin is for?
| and what is its component/mordant
nucleus and cytoplasm (mitochondria)
| Ferric ammonium sulfate- both mordant and ripening agent
63
COPPER HEMATOXYLIN is used for
spermatogenesis
64
TUNGSTEN HEMATOXYLIN contains
phosphotungstic acid
65
LEAD HEMATOXYLIN is for
demo of granules of endocrine cells of alimentary tract
66
MOLYBDENUM HEMATOXYLIN is for
demo of collagen, reticulin and argentaffin cells
67
extracted from bug treated with alum to produce carmine
Cochineal dyes
68
Cochineal dyes is treated with alum to produce
carmine
69
Cochineal dyes with picric acid will produce
picro carmine- for neuropathological studies
70
Cochineal dyes with aluminum chloride will produce
Bescarmine- glycogen
71
– vegetable dye extracted from lichens and is used for ______
Orcein
| staining elastic fibers
72
vegetable dye extracted from lichens to be used as pH indicator because of poor staining property
Litmus
73
Synthetic dyes also known as
Coar tal/aniline dyes
74
2 components of Synthetic dyes
1. Chromophore- coloring property
| 2. Auxochrome- dyeing propert, to retain the imparted color
75
what is lysochrome dyes
also known as Oil soluble dyes, used for FATS
| It has no auxochrome
76
Most sensitive soluble dye
Sudan black B
77
Sudan IV also known as and what is its use
Sharlach R
| Neutral lipids staining them red
78
First sudan dye to be introduced in histochemistry
Sudan III
79
Sudan III is good for
fat stain for CNS tissues
80
Sudan Black B, Sudan IV and Sudan III are considered what type of stain
Histochemical stain
81
Other examples of histological stains
```
Wrights
Giemsa
Methylene blue
Crystal violet
Carbol fuschin
```
82
Accentuator is a part of staining reaction
| True or False
False
83
Components of acid alcohol
HCL + ethanol
84
What is orthochromatic staining
Tissue stained with same shade/hue as the dye
85
– uses aqueous or alcoholic dye solutions to produce a color
Direct Staining
86
Metachromatic staining is used to stain what structures
Cartilage
Amyloid
Mast cell granules
Epithelial mucins
87
2 examples of staining jar
couplin jar
| slotted staining dishes
88
Stains under Alum hematoxylin
Gills
Ehrlichs
Meyers
Coles
89
Process of applying a mounting medium between the stained tissue section and the coverslip
mounting
90
Reasons for doing mounting
permanent safekeeping
easy handling and storage
prevent bleaching of sections
91
Mounting medium should have a refractive index close to that of the slide which is
1.518
92
What is resinous mountant
dehydrated and cleared by xylene
| permanent
93
What is aqueous mountant
for water miscible preparation
| semi-permanent, temporary
94
Canada balsam's refractive index is
1.524
95
Routine mountant
Canada balsam
96
Canada balsam is extracted from
Abus Balsamea
97
Disadvantage of using canada balsam
Darkens with age
| causes gradual fading of stains
98
Enumerate types of resinous mountant and their refractive index
Canada balsam- 1.524
DPX- 1.532
XAM- 1.52
CLARITE- .544
99
Glycerin jelly's aka ______ and refractive index
Kaiser's
| 1.47
100
Farrant's aka ______ and refractive index
Gum arabic
| 1.43
101
Apathy's refractive index
1.52
| for methylene blue stained nerve preparations
102
Brun's is used for
frozen sections directly from water
103
Water as mountant
not used because of its low refractive index
| easily evaporates and cannot examine those under IOI
104
Ringing is mandatory
| True or False
False
105
Sealing margins of coverslip
ringing
106
Ringing is done to
prevent escape of fluid
prevent evaporation
prevent sticking of slides upon preparation
immobilize coverslip
107
Type of ringing medium
Kronig cement
| durofix
108
Color that eosin stains
pink
109
color that hematoxylin stains
blue
110
examples of NEUTRAL / AMPHOTERIC DYE
Wrights
Irishman
Giemsa
Leishmann
111
Process of coating slide with thin celloidin solutions. Done if sections will be subjected to strong alkali or acid solutions and for tissues containing glycogen for demonstration
COLLODIONIZATION
112
Solvents Used for Stains
Water
Alcohol (methyl or ethyl)
Aniline water
Phenol
113
Steps in RE-STAINING of OLD SECTIONS
1. immerse in xylene for 24 hrs
2. remover coverslip
3. immerse in xylene for 30 mins
4. immerse in 0.5% potassium permanganate for 5-10 mins. rinse with tapwater
5. immerse in 5% oxalic acid
6. re-stain
114
Years of retention for Clinical pathology laboratory reports
10 yrs
115
Years of retention for Autopsy forensic reports
indefinitely
116
Years of retention for Surgical pathology and bone marrow Reports
10 yrs
117
Years of retention for Cytogenetics report
20 yrs
118
Years of retention for Pathology /Bone marrow slides
10 yrs
119
Years of retention for Pathology blocks
10 yrs
120
Years of retention for Cytogenetic slides
3 yrs
121
Years of retention for Cytogenetic diagnostic images
20 yrs
122
it governs the contrast between the cellular detail and the background, and also the transparency of the observed sample against the bright field of the microscope.
refractive index
123
The mounting media must always have an RI _____ than the mounted sample to impart more transparency.
higher
124
The slide may then be incubated at __________ after mounting, to harden the medium.
37 degC
| 12-24 hrs
125
You can use immersion oil on an uncovered slide
| True of False
False
126
Oil can be applied, but do not attempt to wipe it off until the mounting medium is cured for how long?
at least overnight or in a hot plate or oven for 1 hr
127
Do not store mounted slides vertically for ______ if cured at room temperature.
2 days
128
Excessive mounting medium will cause it to ooze out of the sides of the cover glass, and should be carefully wiped with a fine cloth moistened with ____________.
xylene
129
Excessive blotting will
dry up the section, causing shrinkage and cracking of the specimen.
130
If the section has to be remounted, the cover glass may be removed by soaking in
xylene
131
Excess xylene, if not removed, will
mix with the mountant and form bubbles on the slide.
132
Too little mounting medium may
| What is the remedy
cause improper setting of the coverslip or formation of bubbles on the section
teased out by gently pressing on the cover glass with a pointed forceps or mounting needle.
133
Setting may be hastened in
hot oven at 50°C for 2 hours.
134
basic aniline dyes should be mounted in
non-acid containing mountants
135
Prussian blue reaction should be mounted in
non-reducing media
136
a paper label should contain
patient's name
section number
staining method used
137
used for mounting sections from distilled water when the stains would be decolorized or removed by alcohol and xylene as would be the case with most of the fat stains (Sudan methods) or for metachromatic staining of amyloid.
AQUEOUS MOUNTING MEDIA
138
glycerin jelly or gum arabic used to
solidify the medium
139
glycerol used to
cracking and drying of the preparation
140
sugar is used to
increase the refractive-index
141
usually regarded as the standard mountant for fat stains.
glycerin
142
standard mounting medium used when dehydration and clearing with xylene cannot be made
glycerin jelly
143
recommended as an alternative for glycerine jelly.
Polyvinyl alcohol
144
has the highest index of refraction
Pure glycerin
145
used as a mountant in immunofluorescence microscopy
Polyvinyl alcohol
146
The mountant is not set in the desired amount of hardness and therefore requires "ringing"
glycerin jelly
147
does not solidify upon storage and therefore does not need to be heated before use.
gum arabic medium
148
general purpose aqueous mountant
apathy's
149
not compatible with normal histological stains.
von apathy's
150
It is one of the most useful aqueous mountants for fluorescent microscopy, being virtually nonfluorescent.
apathy's
151
Canada balsam can be made neutral or acid by adding excess amounts of
calcium carbonate
| salicylic acid
152
It is a transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard consistency without granulation
Canada balsam
153
substitute for xylene in canada balsam that may be less ofa health hazard but may cause other problems such as slow hardening and premature darkening.
histomount
154
may be substituted for xylene as a solvent in canada balsam. The medium can only be neutralized temporarily since the mixture becomes acidic and changes into a brown color upon storage.
Benzene
155
may be added to canada balsam to maintain its neutral reaction
calcium carbonate chips
156
Canada balsam is recommended for
whole mounts and thick sections
157
This is a resinous medium recommended for small tissue sections but not for whole mounts because of shrinkage produced on drying
DPX
158
It has a greater advantage over Canada balsam in that slides can be cleaned of excess mountant simply by stripping it off after cutting around the edge of coverslip
DPX
159
generally preferred over D.P.X
Clarite
160
generally suitable for all enzymatic label/chromogen combinations and fluorescent labels.
Aqueous mounting medium
161
quenches the staining intensity.
glycerol
162
exposure to excitation light of most fluorescent labels which results in diminished staining
photo bleaching
163
may form when the mounting media is too thin, and as it dries air is sucked in under the coverslip.
bubbles
164
Contamination of clearing agents or cover slipping media may
produce a bubbled appearance under the microscope.
