CHAPTER 11 IMPREGNATION AND EMBEDDING Flashcards
1
Q
is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen.
A
Impregnation (Infiltration)
2
Q
This allows easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components.
A
Impregnation (Infiltration)
3
Q
is the process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.
A
Embedding (Casting or Blocking)
4
Q
The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes is known as an
A
Embedding Medium.
5
Q
There are generally four types of impregnation and embedding medium, namely:
A
- Paraffin wax
- Celloidin (collodion)
- Gelatin
- Plastic
6
Q
is the simplest, most common and best embedding medium used for routine tissue processing.
A
Paraffin
7
Q
wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
A
Paraffin
8
Q
It is solid at room temperature but melts at temperatures up to about 65°C or 70°C.
A
Paraffin
9
Q
can be purchased with melting points at different temperatures, the most common for histological use being about 56°C to 58°C.
A
Paraffin
10
Q
- Thin individual serial sections may be cut with ease from the majority of tissues without distortion.
A
PARAFFIN WAX IMPREGNATION
11
Q
- The process is very rapid, allowing sections to be prepared within 24 hours.
A
PARAFFIN WAX IMPREGNATION
12
Q
- Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction.
A
PARAFFIN WAX IMPREGNATION
13
Q
- Because formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, they are an important resource for historical studies in medicine.
A
PARAFFIN WAX IMPREGNATION
14
Q
- Many staining procedures are permitted with good results.
A
PARAFFIN WAX IMPREGNATION
15
Q
- Overheated paraffin makes the specimen brittle.
A
PARAFFIN WAX IMPREGNATION
16
Q
- Prolonged impregnation will cause excessive tissue shrinkage and hardening, making the cutting of sections difficult.
A
PARAFFIN WAX IMPREGNATION
17
Q
- Inadequate impregnation will promote retention of the clearing agent. Tissues become soft and shrunken, and tissue blocks crumble when sectioned and break up when floated out in a water bath.
A
PARAFFIN WAX IMPREGNATION
18
Q
- Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes, need long immersion for proper support; otherwise, they will crumble on sectioning. Prolonged immersion in paraffin, on the other hand, is not advisable.
A
PARAFFIN WAX IMPREGNATION
19
Q
- Paraffin processing is not recommended for fatty tissues. The dehydrating and clearing agents used in the process dissolve and remove fat from the tissues.
A
PARAFFIN WAX IMPREGNATION
20
Q
The traditional advice with paraffin wax is to use this about [?] above its melting point.
A
2°C
21
Q
depends upon the molecular weight of the components and the ambient temperature.
A
Wax hardness (viscosity)
22
Q
To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above
A
60°C or 65°C.
23
Q
Paraffin wax is traditionally marketed by its melting points which range from
A
39°C to 68°C.
24
Q
depends upon the crystal morphology of the embedding medium.
A
Tissue-wax adhesion
25
After being completely cleared, the tissue is submerged in two or more changes of melted paraffin wax, either in a paraffin oven or in an incubator which has been regulated at
55-60°C.
26
The duration and number of changes required for thorough impregnation of tissue depends on:
Size and type of tissues: Longer time is required for thicker tissues.
Use of vacuum imbedding: Vacuum reduces the time required for complete impregnation.
Clearing agent employed
27
Common waxes have melting points of
45°C, 52°C, 56°C and 58°C.
28
The [?] is normally used for routine work. In a laboratory with temperature ranging from [?], paraffin wax with a melting point of [?] is indicated.
56°C wax
20-24°C
54-58°C
29
If the laboratory temperature is between [?], the melting point of wax to be used should be between [?].
15-18°C
50 and 54°C
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There are three ways by which paraffin wax impregnation and embedding of tissues may be performed:
1. By manual processing
2. By automatic processing
3. By vacuum embedding
31
1. Manual Processing:
Fixation:
10% Buffered Formalin 24 hours
Dehydration:
70% Alcohol 6 hours
95% Alcohol 12 hours
100%Alcohol 2 hours
100% Alcohol 1 hour
100%Alcohol 1 hour
Clearing:
Xylene or Toluene 1 hour
Xylene or Toluene 1 hour
Impregnation:
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Embedding:
Paraffin wax 3 hours
32
At least four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent from the tissue.
Manual Processing
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The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to insure complete embedding or casting of tissue.
Manual Processing
34
This method makes use of an automatic tissue processing machine (i.e., Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues. This results in a more rapid diagnosis with less technicality.
Automatic Processing
35
Usually, only 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen. This is made possible due to constant tissue agitation which accelerates and improves tissue penetration giving rise to more consistent results.
Automatic Processing
36
The first [?] bath should be discarded, and the others moved down, so that the final bath has fresh [?] after two complete processing runs of loads of at least three-quarters capacity.
100% ethanol
37
The clearing agent and the dilute ethanols should be changed at least
once a week.
38
Wax bath thermostats should be set at least [?] above the melting point of the wax, and timing should be checked when loading the machine, especially if the machine is equipped with a delay mechanism.
3 degrees
39
involves wax impregnation under negative atmospheric pressure inside an embedding oven.
Vacuum Embedding
40
involves wax impregnation under negative atmospheric pressure inside an embedding oven.
Vacuum Embedding
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It reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening.
Vacuum Embedding
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It facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination.
Vacuum Embedding
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hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of thetissue.
Vacuum
44
This technique is particularly recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen and central nervous system.
Vacuum Embedding
45
requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to 760 mm Hg is applied using a water or mechanical pump.
Vacuum infiltration
46
Of the three methods of [?] gives the fastest result.
paraffin wax impregnation, vacuum impregnation
47
are easily removed from the tissues
Benzene and xylene
48
are more difficult to remove and require more frequent wax changes.
chloroform and cedarwood oil
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Addition of [?] may hasten displacement of cedarwood oil with less tissue shrinkage.
benzene
50
Since prolonged treatment in melted paraffin causes shrinkage and hardening of tissues, making cutting difficult, the tissue should not be left in the paraffin oven for more than
4 hours.
51
Tissues become increasingly harder and more brittle as they are heated. Infiltration in overheated paraffin (above [?]) will also produce shrinkage and hardening of tissues and destroy lymphoid tissues completely. To avoid this, the paraffin oven must be maintained at a temperature [?] above the melting point of paraffin to be used for impregnation.
60°C
2 to 5°C
52
Fresh wax should be filtered before use in a wax oven at a temperature [?] higher than its melting point.
2°C
53
Water must therefore be removed by heating the wax to [?], thereby raising its melting point.
100 -105°C
54
SUBSTITUTES FOR PARAFFIN WAX:
55
is a mixture of highly purified paraffin and synthetic plastic polymers, with a melting point of 56-57°C.
Paraplast
56
It is more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily with the same result as in double embedding.
Paraplast
57
Blocks obtained are more uniform than with any other medium, with better ribboning of sections.
Paraplast
58
Serial sections may be cut with ease, without cooling the tissue block, thereby preventing the formation of ice crystal artefacts.
Paraplast
59
No deposit is left on the slide after staining, and no special processing schedule is required.
Paraplast
60
It is soluble in common clearing agents and follows the same time schedule for paraffin impregnation, and does not tend to crack like other paraffin wax substitutes.
Paraplast
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Generally, [?] with a melting point of 56 to 58oC is recommended.
Paraplast
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During the winter, 54 to 56oC Paraplast may be used if the tissue is cut in a cool room. During the summer it may be necessary to use 60 to 63oC, although this is to be avoided if possible in order to not to "cook" the tissue. "Cooked" tissue does not section well or, if it does, it does not stain well and most details are destroyed.
Paraplast
63
is synthetic wax substitute similar to Paraplast with a melting point of 56-58°C.
Embeddol
64
It is less brittle and less compressible than Paraplast.
Embeddol
65
is a semisynthetic wax recommended for embedding eyes.
Bio/aid
66
is a product of paraffin, containing rubber, with the same property as Paraplast.
Tissue Mat
67
has a lower melting point (46-48°C), but it is harder than paraffin.
Ester Wax
68
It is not soluble in water, but is soluble in 95% Ethyl Alcohol and other clearing agents; hence, it can be used for impregnation without prior clearing of the tissue.
Ester Wax
69
may be used as clearing agents, if indicated.
Cellosolve (ethylene glycol monoethyl ether) or xylene
70
are plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C.
Water Soluble Waxes
71
are incorporated in the majority of proprietary histological paraffin wax blends presently available to improve adhesion, hardness and plasticity.
Polymer waxes
72
The most commonly used Polymer wax is [?], a polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature.
Carbowax
73
It is soluble in and miscible with water; hence does not require dehydration and clearing of the tissue.
Carbowax
74
added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.
Dimethyl sulphoxide (DMSO)
75
scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration.
DMSO
76
is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo.
Celloidin (Collodion)
77
It is supplied in thin (2%), medium (4%) or thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol.
Celloidin (Collodion)
78
This is used mainly for preparing soft tissue sections of mixed consistency such as eyes and brain.
Celloidin (Collodion)
79
No heat is required, and the resultant block has a rubbery consistency which gives good support to the tissues.
Celloidin (Collodion)
80
Disadvantages include inability to cut thin sections, storage of blocks in alcohol and speed of technique (which can take several weeks or months).
Celloidin (Collodion)
81
Two methods are used for celloidin impregnation of tissue:
Wet Celloidin Method
Dry Celloidin Method
82
- is recommended for bones, teeth, large brain sections and whole organs.
Wet Celloidin Method
83
is preferred for processing of whole eye sections.
Dry Celloidin Method
84
is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly. Because of this, many workers prefer L.V.N. to the ordinary celloidin for impregnation and embedding.
Low Viscosity Nitrocellulose (L.V.N.)
85
It forms a harder tissue block and makes cutting of thinner sections possible.
Low Viscosity Nitrocellulose (L.V.N.)
86
The tendency of tissues to crack may be prevented by adding plasticizers (e.g. oleum ricini or castor oil) when embedding chrome-mordanted tissues.
Low Viscosity Nitrocellulose (L.V.N.)
87
Paraffin embedded tissues are arranged at the bottom of the mold together with their proper labels and immersed in melted paraffin at a temperature between [?] above its melting point and then cooled rapidly in a refrigerator at [?] or immersed in cold water to solidify.
5-10°C
-5°C
88
The process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining, is known as
Orientation.
89
Several types of Blocking-out Molds may be used:
1. Leuckhart’s Embedding Mold
2. Compound Embedding Unit
3. Plastic Embedding Rings and Base Mold
4. Disposable Embedding Molds
90
- consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen .
Leuckhart’s Embedding Mold
91
It is recommended for routine use, although, too slow and cumbersome for use in a busy laboratory.
Leuckhart’s Embedding Mold
92
is made up of a series of interlocking plates resting on a flat metal base, forming several compartments.
Compound Embedding Unit
93
-consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting.
Plastic Embedding Rings and Base Mold
94
disposable thin plastic embedding molds, available in 3 different sizes, are simply peeled off one at a time, as soon as the wax has solidified, giving perfect even block without trimming.
Peel-Away
95
It may be placed directly in the chuck or block holder of the microtome.
Peel-Away
96
-such as those used in ordinary refrigerators may be recommended for busy routine laboratories.
Plastic Ice Trays
97
are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks.
Paper Boats
98
They have the advantage of being cheap and easy to make.
Paper Boats
99
They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks.
Paper Boats
100
used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs like the eye, since the delicate layers of the eyeball are difficult to keep intact when other media are used.
Celloidin or Nitrocellulose Embedding Method
101
is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded.
Double-Embedding
102
This is used to facilitate cutting of large blocks of dense firm tissues like the brain.
Double-Embedding
103
They are also recommended for making small sections of celloidin blocks.
Double-Embedding
104
The introduction of plastic resin embedding media has provided superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone and for high resolution light microscopy of tissue sections thinner than the usual [?], such as renal biopsies andbone marrow biopsies.
4-6 μm
105
Plastics are classified into [?], based on their chemical composition.
epoxy, polyester, or acrylic
106
are made up of a carefully balanced mixture of epoxy plastic, catalysts and accelerators.
Epoxy embedding plastics
107
Three types of epoxy plastics are used in microscopy, i.e., those based on either bisphenol A (?), or glycerol (?), or cyclohexene dioxide (?).
Araldite
Epon
Spurr
108
Infiltration by [?] is slow, partly because the epoxy plastic itself is a large molecule.
Araldite
109
have a lower viscosity but are often sold as mixtures of isomers.
glycerol-based epoxy plastics (Epon)
110
can be obtained pure, have very low viscosity, and infiltrate fastest.
Cyclohexene dioxide-based plastics (Spurr)
111
were originally introduced for electron microscopy in the mid- 1950s, but have been superseded by more superior epoxides, and are now seldom used.
Polyester plastics
112
are made up of esters of acrylic or methacrylic acid, and are used extensively for light microscopy.
Acrylic plastics
113
has proved to be a popular embedding medium for light microscopy because it is extremely hydrophilic, allowing many staining methods to be applied, yet tough enough when dehydrated to section well on most microtomes.
Polyglycol methacrylate (GMA)
114
The polar water soluble, 2-hydroxyethyl methacrylate, commonly known as [?], has found an increasing number of applications for the embedding of biological tissue for transmission electron microscopy (TEM), for the preservation and observation of fine structure not previously subjected to solvent dehydration.
"glycol methacrylate", or GMA
115
forms only non-crosslinked straight chains on polymerization and therefore requires no hardener.
GMA
116
is added to the plastic as a catalyst that decomposes to form phenyl radicals acting as an active site for the polymerization of acrylics.
Benzoyl peroxide
117
Acrylic plastics based on [?] are also widely used because of its hardness as the ideal embedding medium for undecalcified bone and is widely used for bone histomorphometry and bone marrow hematopathology.
methyl methacrylate (MMA)
