CLEARING/IMPREGNATION/EMBEDDING Flashcards
1
Q
Removal of dehydrating agent from the tissues and replacing it by a solvent
A
Clearing
2
Q
Removal of dehydrating agent from the tissues and replacing it by a solvent
A
Clearing
3
Q
Produce transparent and translucent tissue
A
Clearing
4
Q
True or false
- not all dealcoholizing agents act as clearing agent
A
True
5
Q
When is clearing in embedding done
A
Done after dehydration and before infiltration
6
Q
Agents for clearing in embedding
A
- xylene
- toluene
- Dioxane
- chloroform
7
Q
When is clearing in mounting done
A
Done after staining and before mounting
8
Q
Agents of mounting media
A
- xylene
- toluene
- terpineol
- carbol - xylene
9
Q
An ideal clearing agent should be miscible with
A
Alcohol and paraffin wax
10
Q
Most commonly and rapidly used clearing agent
A
Xylene/ xylol
11
Q
Duration of xylene
A
15-30 mins or 30 mins - 1 hr
12
Q
Excellent clearing agent but tends to make tissues excessively hard and brittle
A
Xylene/xylol
13
Q
Can be used for celloidin sections
A
Xylene
14
Q
Unsuitable for brain and lymph nodes
A
Xylene
15
Q
Turns milky when dehydration is not complete
A
Xylene
16
Q
Rapid agent and highly inflammable; doesn’t make tissues hard and brittle but may cause considerable shrinkage
A
Benzene
17
Q
Excessive exposure to this may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in aplastic anemia
A
Benzene
18
Q
Duration of benzene
A
15-60 mins
19
Q
Similar to xylene but does not harden tissues nearly so much
A
Toluene/toloul
20
Q
Slower than xylene or benzene, it is not carcinogenic but emits toxic fumes
A
Toluene
21
Q
Duration of toulene or toluol
A
1-2 hours
22
Q
For nervous tissues, lymph nodes, and embryos
A
Chloroform
23
Q
Best for large specimens and tough tissues
A
Chloroform
24
Q
Little shrinkage and doss not harden tissues excessively; not flammable
A
Chloroform
25
Slow and doesn't make tissues transparent, it is toxic to the liver on prolonged inhalation
Chloroform
26
Tissues tend to float in chloroform, how to avoid this
Wrap the tissues with absorbent cotton gauze to facilitate sinking of the section in solution
27
Recommended for CNS, smooth muscles, and skin
Cedarwood oil
28
Used to clear both paraffin and celloidin sections during the embedding process
Cedarwood oil
29
Duration of clearing using Cedarwood oil
2-3 days
30
Slow clearing (2-3 days) with minimal shrinkage
Cedarwood oil
31
Must be followed by immersion in xylene or benzene to remove oil from tissues, and must be filtered prior to using
Cedarwood oil
32
Turns milky on prolonged storage
Cedarwood oil
33
Tissue floats in cedarwood oil, what is the remedy
Superimpose with absolute alcohol
34
This is not normally utilized as a routine agent but it is recommended for clearing embryos, insects, and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening
Aniline oil
35
It has the ability to clear 70% alcohol without excessive tissue shrinkage and hardening
Aniline oil
36
Slow, may be adultered, removes aniline dyes
Clove oil
37
Aniline dyes are removed, and celloidin is dissolved
Clove oil
38
Similar to chloroform
CCL4
39
Fill all natural cavities, spaces and interstices of the tissue with a firm consistency
Impregnation or infiltration
40
Impregnated tissues are placed into a precisely arranged position in a mold containing a medium which is the allowed to solidify
Embedding, blocking, or casting
41
Simplest, most common and best embedding medium used for routine tissue processing
Paraffin wax
42
Shrinks about 10% on cooling
Paraffin wax
43
Melting point for normal routine work
56C
44
At what temp should the paraffin oven adjusted
2-5C higher
45
Temperature adjustment if the lab temp is 20-24C
54-58C
46
Temperature adjustment if lab temp is 15-18C
50-54C
47
Not recommended for fatty tissues
Paraffin wax
48
T/f: paraffin wax allows storage for an indefinite period
T
49
Paraffin wax disadvantages
*Overheating = brittle tissue
* Prolongation= excessive shrinkage and hardening
* Inadequate infiltration= soft and shrunken
50
At least 4 changes of wax are required at 15 mins interval in order to ensure complete removal of clearing agent from the tissue
Manual processing
51
2-3 changes of wax are required to remove the clearing agent and properly impregnate the specimen with constant agitation
Automatic processing
52
Temp of wax bath for automatic processing
At least 3C greater than MP
53
MP
Melting point
54
Negative atmospheric pressure
Vacuum embedding
55
Negative atmospheric pressure of vacuum embedding
400-500 mmHg
56
Embedding that is Recommended for urgent biopsies and delicate tissues
Vacuum embedding
57
Temp of vacuum embedding above mp of wax
2-4C
58
Advantage: effects of heat are prevented
Vacuum embedding
59
Substitute for paraffin wax
1. Paraplast
2. Embeddol
3. Ester wax
4. Water soluble waxes ( 38-41 or 46-56)
60
Melting point of paraplast
56-57C
61
Highly purified paraffin and synthetic plastic polymers with a melting point of 56-57C
Paraplast
62
Hastens the removal of air bubbles and clearing agent from the tissue block
Vacuum embedding
63
Urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen, cns
Vacuum embedding
64
With vacuum embedding, the time required for complete impregnation is reduced by ___ of the normal time required for tissue processing
25-75%
65
More elastic and resilient and doesn't crumble
Paraplast
66
Better ribboning with ease and doesn't require cooling
Paraplast
67
Better ribboning with ease and doesn't require cooling
Paraplast
68
At what temp should the paraffin oven must be maintained to be used for impregnated
2-5C above melting point
69
Water must therefore be removed by heating the wax to ____ thereby raising its melting point
100-105C
70
Mp of embeddol
56-58C
71
Synthetic wax substitutes similar to paraplast
Embeddol
72
Less brittle and less compressible than paraplast
Embeddol
73
Two types of embeddol
- bioloid
- tissue mat
74
Semisynthetic wax recommended for embedding eyes
Bioloid
75
Product of paraffin, containing rubber, with the same property as paraplast
Tissue mat
76
Has a lower mp, but it is harder than paraffin
Ester wax
77
Mp of ester wax
46-48C
78
It is not soluble in water, but is soluble in 95% ethyl alcohol and can be used for impregnation without prior clearing of the tissue
Ester wax
79
Examples of heavy duty microtome
Sliding or sledge type
80
How many changes of ester wax are required to ensure complete tissue impregnation
3-4 changes
81
Plastic polymers, mostly polyethylene glycols
Water soluble waxes
82
Mp of water soluble waxes
- 38-41C
- 46-56C
83
Most commonly used water soluble waxes
Carbowax
84
Polyethylene glycol containing 18 or more carbon atoms, which appears solid at RT
Carbowax
85
- Does not require dehydration and clearing
- does not remove neutral fats and lipids
- suitable for enzyme srudies
Carbowax
86
Carbowax processing
*70% - 90% - 100% (2x) at 56C
* Blocking at 50C
87
What should be added to water to reduce tissue distortion and promote flattening and floating out of sections
- add soap to water or using 10% polyethylene glycol 900
88
Highly soluble in water and hygroscopic; cannot be floated in water
Carbowax
89
Purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse
Celloidin or collodion
90
Two methods for celloidin impregnation for tissue
- wet celloidin method
- dry celloidin method
91
For bones, teeth, large brain scetions and whole organs
Wet method
92
Evaporating solvent ( until no fingerprint may be observed)
Wet method
93
In what solvent should method be stored and used as a lubricant
70-80% alcohol
94
Celloidin embedding that is for the whole eye sectioning
Dry method
95
In what reagent should the dry method be stored
Gilson's mixture
96
Components of gilson's mixture
Chloroform and cedarwood oil
97
Types of plastic/resin embedding medium
1. Epoxy
2. Polyster
3. Acrylic
98
Types of epoxy
-Araldite base (bisphenol)
-glycerol (epon)
- cyclohexane dioxide (spurr)
99
Slowest type of epoxy
Araldite base (bisphenol)
100
Fastest type of epoxy
Cyclohexene dioxide (spurr)
101
Toxic component of epoxy that is carcinogenic
VCD (vinyl cyclohexane dioxide)
102
Plastic embedding medium for EM
Polyster
103
Plastic embedding medium for high resolution LM
Acrylic
104
2 applications of acrylic
*GMA - Glycol methacrylate
*MMA - Methyl methacrylate
105
Application of acrylic that is hydrophilic and allows wide stain selection
Glycol methacrylate
106
Application of acrylic that is for hardness, for dense tissues
Methyl methacrylate
107
Rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies
Gelatin impregnation
108
For delicate specimens, histochemical and enzyme studies, and for frozen sections
Gelatin embedding
109
Why is gelatyaccompanied with phenol?
10% gelatin - 10% - 20% - (+1% phenol)
Gelatin embedding
110
How many times should paraffin wax be used
2 times
111
At what temp is water removed by heating paraffin
100-105C
112
At what temp are tissues submerged in two or more changes of melted paraffin wax in the paraffin oven or incubator
55-60C
113
When the tissue is to be cleared directly from water (no dealcoholization, it merely improves the RI)
Glycerin, Gum syrup and Brun's sltn
114
Slow, used in double embedding
Methyl benzoate
115
Dehydrating and clearing agent
Dioxane
116
For large pieces of tissues and embryonic materials
Amyl acetate
