Infiltration and Embedding Flashcards
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
Infiltration/ Impregnation
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.
Embedding
What are the 4 types of infiltration and embedding medium?
- Paraffin wax
- Celloidin (collodion)
- Gelatin
- Plastic
- simplest, most common, and best embedding medium used for routine tissue processing
- a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils
- solid at room temperature but melts at temperatures up to about 65°C or 70°C
Paraffin wax
the most common melting point at different temperatures for histological use of paraffin wax
56°C - 58°C
The traditional advice with paraffin wax
use about 2°C above its melting point
To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to
60°C or 65°C
Paraffin wax is traditionally marketed by its melting points which range from
39°C to 68°C
What happens when the paraffin was overheated?
brittle
What happens if prolonged impregnation of paraffin wax?
tissue shrinkage and hardening
Tissues become soft and shrunken, and tissue blocks crumble when sectioned and break up when floated out in a water bath.
Inadequate impregnation that promote retention of the clearing
agent
In a laboratory with temperature ranging from 20-24°C, paraffin wax with a melting point of______ is indicated. If the laboratory temperature is between 15-18°C, the melting point of wax to be used should be between _______
54-58°C
50 and 54°C
3 ways by which paraffin wax infiltration and embedding of tissues may be performed
- manual processing
- automatic processing
- vacuum embedding
at least 4 changes of wax are required at 15-minute interval and then the specimen is immersed in another fresh solution of melted paraffin for approximately 3 hours
Manual Processing
One example of automatic tissue processing machine
Elliot Bench-Type Processor
Presence of any odor in the clearing agent during final paraffin wax bath in automatic processing indicates
paraffin wax needs to be changed
the most critical stage of tissue processing
dehydration
Wax bath thermostats should be set at least ______ the melting point of the wax
3 degrees above
- involves wax impregnation under negative atmospheric pressure inside an embedding oven
- 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
With vacuum embedding, the time required for complete impregnation is reduced by _________ of the normal time required for tissue processing
25% -75%
In vacuum embedding oven, the vacuum chamber is enclosed in a thermostatically controlled water-jacket, usually maintained at a temperature of _____ the melting point of the wax
2-4°C above
In vacuum embedding oven, the degree of the vacuum should not exceed
500 mm. Hg
This is provided to prevent water from being sucked back into the trap bottle and vacuum chamber when the water or suction pump is closed
stopcock
Out of the 3 methods of paraffin wax impregnation, which one gives the fastest result?
Vacuum Impregnation
infiltration in overheated paraffin (above 60°C) will
produce shrinkage and hardening of tissues and destroy lymphoid tissues completely
To avoid shrinkage, hardening of tissues, and destroying lymphoid tissues completely due to infiltration in overheated paraffin _______
the paraffin oven must be maintained at a temperature 2 to 5°C above the melting point of paraffin to be used for impregnation
Example of a coarse filter paper
Green’s No. 904
When wax has been reused, some amount of water inevitably is mixed with it, how can you remove the water?
heating the wax to 100-105°C, thereby raising its melting point
Substitute for Paraffin wax
- Paraplast
- Embeddol
- Ester wax
- Water soluble waxes
- Dimethyl sulphoxide
- a mixture of highly purified paraffin and synthetic plastic polymers, with a melting point of 56-57°C.
- more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily
Paraplast
- synthetic wax substitute similar to Paraplast with a melting point of 56-58°C.
- less brittle and less compressible than Paraplast
Embeddol
a semisynthetic wax recommended for embedding eyes
Bio/aid
a product of paraffin, containing rubber, with the same property as Paraplast.
Tissue Mat
- has a lower melting point (46-48°C), but it is harder than paraffin
- not soluble in water, soluble in 95% ethyl alcohol, hence can be used for impregnation without prior clearing of tissue
Ester wax
microtome used for ester wax-impregnated tissue
sliding or sledge type microtome
- plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C
Water soluble waxes
- a polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature
- It is soluble in and miscible with water; hence does not require dehydration and clearing of the tissue
- Cytologic details are excellently preserved
carbowax
Routine processing of carbowax
70% for 30 minutes
90% for 45 minutes
100% for 1 hour
100% for 1 hour
temperature at 56°C
What will reduce tissue distortion and promote flattening and “floating out” of sections?
Adding soap to water or using 10% Polyethylene Glycol 900
- added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning
- scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration.
Dimethyl sulphoxide (DMSO)
Some individuals who handle DMSO-paraffin wax may experience
unpleasant and annoying oyster or garlic taste caused by DMSO metabolites
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)
2 methods for celloidin impregnation
- Wet Celloidin Method
- Dry Celloidin Method
celloidin method recommended for bones, teeth, large brain sections and whole organs
Wet Celloidin Method
Celloidin method preferred for processing of whole eye sections
Dry Celloidin Method
made up of equal parts of chloroform and cedarwood oil
Gilson’s mixture
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
Low Viscosity Nitrocellulose (L.V.N.)
examples of plasticizers
oleum ricini
castor oil
- rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies
- used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut.
- water-soluble, and does not require dehydration and clearing
- has a low melting point and does not cause over-hardening of tissues by heating.
GELATIN
serves to prevent the growth of molds in gelatin impregnation
1% phenol
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
Orientation
4 Types of Blocking-out Molds
Leuckhart’s Embedding Mold
Compound Embedding Unit
Plastic Embedding Rings and Base Mold
Disposable Embedding Molds
an embedding mold which 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
an embedding mold that is made up of a series of interlocking plates resting on a flat metal base, forming several compartments. It has the advantage of embedding more specimens at a time, thereby reducing the time needed for blocking.
Compound Embedding Unit
an embedding bold that is 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
3 types of disposable embedding molds
Peel-away
Plastic Ice Trays
Paper Boats
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. It may be placed directly in the chuck or block holder of the microtome.
Peel-Away
such as those used in ordinary refrigerators may be recommended for busy routine laboratories. Each compartment may be utilized for embedding one tissue block, which may then be removed by bending once the wax has solidified or by smearing the inner mold with glycerin or liquid paraffin before embedding
Plastic Ice Trays
- are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks
- They have the advantage of being cheap and easy to make.
- They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks.
Paper Boats
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
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 (obsolete)
Double-Embedding
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 4-6 µm, such as renal biopsies and bone marrow biopsies
PLASTIC (RESIN) EMBEDDING
3 types of Epoxy
- Bisphenol A (Araldite) - slow
- Glycerol (Epon) - low viscosity
- Cyclohexene dioxide (Spurr) - very low viscosity
- for electron microscopy
- most widely applied
- carcinogenic due to vinyl cyclohexane dioxide (VCD)
Epoxy
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
made up of esters of acrylic or methacrylic acid, and
are used extensively for light microscopy
Acrylic plastics
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)
- 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.
- forms only non-crosslinked straight chains on polymerization and therefore requires no hardener
glycol methacrylate (GMA)
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
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)
