Infiltration & Embedding Flashcards
Used for bones and brain specimens
Paraplast
A substitute for paraffin wax that is used for infiltrating eye specimens?
Bioloid
Product of paraffin with rubber
Tissue mat
Eliminates dehydration and clearing process
Carbowax
Uses 70-80% alcohol for storage
Wet Celloidin Method
Melting point of 56-58 °C
Embeddol
Method of paraffin wax infiltration that uses Autotechnicon
Automatic Processing
Most rapid method of paraffin wax infiltration
Vacuum Embedding
Removes clearing agent and fills up cavities
Infiltration
Requires 4 changes of wax at 15 minutes interval in order to insure complete removal of the clearing agent from the tissue
Manual Processing
T or F. Prolonged process of paraffin wax infiltration method may cause excessive shrinkage and tissue hardening
True
Melting point for routine work
56°C
a.k.a. COLLODION
Celloidin Infiltration
Harder than paraffin and requires heavy duty type of microtome (e.g. sliding or sledge type microtome)
Ester wax
Melting point 46-48°C
Ester wax
Water soluble wax
Carbowax
Another form of celloidin soluble in equal concentration of ether and alcohol; highly explosive
Low Viscosity Nitrocellulose (L.V.N.)
Mixture of highly purified paraffin and synthetic plastic polymer
Paraplast
Combination of chloroform and cedarwood oil
Components of Gilson’s mixture
Prevents growth of molds
1% phenol
Substitue for paraffin wax that is used for enzyme histochemistry
Carbowax
Remedy when Carbowax is used?
Add soap to water or 10% polyethylene glycol 900
Commonly used embedding medium
Paraffin wax
Tissue infiltrated with this tends to crack
Low Viscosity Nitrocellulose (L.V.N.)
What to add in Low Viscosity Nitrocellulose (L.V.N.) to prevent from tissues in cracking?
Add Plasticizers: OLEUM RICINI & CASTOR OIL
Uses Gilson’s Mixture for storage
Dry celloidin
Methods of Celloidin Infiltration for infiltrating eye specimens
Dry Celloidin
Methods of Celloidin Infiltration for infiltrating bones, large brain sections, teeth, and whole organs
Wet Celloidin Method
Difficult to float out when this is used
Carbowax
No longer requires clearing, only dehydration
Ester wax
Concentration is 4-6% for 5-7 days
Medium Celloidin
Consist of 2 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
Recommended only for frozen sections and histochemical and enzyme studies
Gelatin Infiltration
Concentration is 2-4% for 5-7 days
Thin Celloidin
Used if there is need to be embed more than 1
specimen
Compound embedding unit
It is equipped with a warm plate to manage the impregnated specimen, and a cold plate at -5°C for rapid solidification of the block
Tissue Tek
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 molds
Cutting off excess paraffin wax to form a 4-sided prism
Trimming
Concentration is 8-12% for 3-5 days
Thick Celloidin
A disposable mold that can produce perfect block even without trimming
Peel-Away
Temperature of melted paraffin used for embedding
5-10° above its melting point
This is done when we infiltrate using celloidin then embedding using paraffin wax
Double-Embedding
T or F. Inadequate process in paraffin wax infiltration method may cause retention of clearing agent
True
It is more rapid because it is heat generated, constant agitation (make the processing time shorter)
Automatic Processing
Recommended for urgent biopsies, for delicate tissues such as lungs, brain, connective tissues, decalcified bones, eyes, spleen and CNS
Vacuum Embedding
Carried out using paraffin wax infiltration and an oven
Manual Processing
NOT FOR fatty tissue because we need to apply heat (fats will dissolved)
Paraffin Wax Infiltration Method/Paraffin processing
It is soluble to 95% ethyl alcohol
Ester wax
For tissue with large and hallow cavities that tends to collapse
Celloidin Infiltration Method
Does not add heat so the process is slow
Nitrocellulose Method (Low Viscosity Nitrocellulose)
Tissue for processing should be 2-3mm thick
Gelatin Infiltration Method
Embedding for EM
Plastic (Resins) Embedding
T or F. To allow solidification of block either place the preparation inside the refrigerator at -5° or immerse it in cold water
True
This is recommended for busy routine laboratories
Plastic ice trays
For extremely hard tissues
Plastic (Resins) Embedding
Are made up of esters of acrylic or methacrylic acid, and are used extensively for light microscopy (i.e.,MMA,GMA)
Acrylic plastics
Made up of a carefully balanced mixture of epoxy plastics, catalysts and accelerators
Epoxy Embedding Plastics
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
Impregnation (Infiltration)
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
Embedding (Casting or Blocking)
It is solid at room temperature but melts at temperatures up to about 65°C or 70°C
Paraffin Wax
3 ways by which paraffin wax impregnation and embedding of tissues may be performed
- By manual processing
- By automatic processing
- By vacuum embedding
Involves wax impregnation under negative atmospheric pressure inside an embedding oven
Vacuum Embedding
Melting point of 56-57°C
Paraplast
Synthetic wax substitute similar to Paraplast
Embeddol
Substance added to proprietary blends of plastic polymer paraffin waxes
Dimethyl sulphoxide (DMSO)
Are plastic polymers, mostly polyethylene glycols
Water Soluble Waxes
T or F. During the winter, 54 to 56oC Paraplast may be used if the tissue is cut in a cool room.
True
A polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature
Carbowax
Reduces infiltration times and facilitates thin sectioning
Dimethyl sulphoxide (DMSO)
No heat is required, and the resultant block has a rubbery consistency which gives good support to the tissues
Celloidin Impregnation
Is more explosive than celloidin and should therefore be handled with care
Low Viscosity Nitrocellulose (L.V.N.)
Is rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies
Gelatin Impregnation
Volume should be at least 25 times the volume of the tissue
Gelatin Impregnation
Is made up of a series of interlocking plates resting on a flat metal base, forming several compartments
Compound embedding unit
A 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
Peel-Away
Are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks
Paper Boats
Can be used to control the rate or evaporation of the solvent
Bell jars
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
Recommended for making small sections of celloidin blocks
Double-Embedding
Can be obtained pure, have very low viscosity, and infiltrate fastest
Cyclohexene dioxide-based plastics (Spurr)
Cause sensitization if absorbed by skin or inhalation
Epoxy resins
The polar water soluble, 2-hydroxyethyl methacrylate, is commonly known as
“Glycol methacrylate”, or 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)
Can easily and completely be removed from tissue sections, which results in superior staining characteristics and excellent morphological detail.
Methyl methacrylate (MMA)
Infiltration is slow, partly because the epoxy plastic itself is a large molecule
Bisphenol A (Araldite)
Has a lower viscosity but are often sold as mixtures of isomers
Glycerol (Epon)
Is a Low Viscosity mixture which provides rapid infiltration of tissues
Spurr’s Resin
This resin is compatible with ethanol so no change to propylene oxide is needed prior to infiltration
Spurr’s Resin
Polymerization at 60°C is recommended in
Spurr’s Resin
They are hydrophobic and subsequent oxidation by peroxide to correct this may produce tissue damage (disadvantage)
Epoxy plastics
The components of many epoxy plastics are toxic and one of its components is known to be carcinogenic
Vinyl cyclohexane dioxide (VCD)
They have the advantage of being cheap and easy to make
Paper boats
Is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils
Paraffin wax
It facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination
Vacuum Embedding
It is less brittle and less compressible than Paraplast
Embeddol
It is 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
Gelatin Impregnation
It is water-soluble, and does not require dehydration and clearing, although fixatives (such as 10% formalin) should still be washed out by running water whenever indicated
Gelatin Impregnation
It has a low melting point and does not cause over-hardening of tissues by heating
Gelatin Impregnation
Each compartment may be utilized for embedding one tissue block, which may then be removed by bending the plastic tray once the wax has solidified or by smearing the inner mold with glycerin or liquid paraffin before embedding
Plastic Ice Trays
They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks
Paper Boats
Can be cut with glass or diamond knives
Thin sections of polymerized GMA
Does not need to be water-free and indeed it works best with at least some water present
Glycol methacrylate (GMA)
It forms only non-crosslinked straight chains on polymerization and therefore requires no hardener
Glycol methacrylate (GMA)
T or F. Ideally, an infiltrating and embedding medium should be expensive.
False. Inexpensive
T or F. Ideally, an infiltrating and embedding medium should be soluble in processing fluids.
True
T or F. Ideally, an infiltrating and embedding medium should be heterogenous.
False. Homogenous
T or F. Ideally, an infiltrating and embedding medium should be suitable for sectioning and ribboning.
True
T or F. Ideally, an infiltrating and embedding medium should be easy to handle.
True
T or F. Ideally, an infiltrating and embedding medium should be non-toxic, translucent or transparent; colorless and odorless.
True
T or F. Ideally, an infiltrating and embedding medium should be stable and capable of flattening after ribboning.
True
T or F. Ideally, an infiltrating and embedding medium should be molten between 30°C and 60°C.
True
The simplest, most common and best embedding medium used for routine tissue processing
Paraffin
What temperature of the wax is used when the laboratory temperature is between 15-18°C?
The melting point of wax to be used should be between 50 and 54°C
What temperature of the wax is used when the laboratory temperature is ranging from 20-24°C?
Paraffin wax with a melting point of 54-58°C
Only 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen (what process)
Automatic Processing
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
Consists of a flat-bottomed heavy brass chamber covered with a heavy glass lid resting on a wide and thick rubber valve which produces an airtight seal when the chamber is being used
Vacuum Embedding Oven
Temperature is maintained at 2°C to 4°C above the MP of the wax
Vacuum Embedding
T or F. Wax that has been trimmed away from the impregnated tissue may be melted and filtered for future use, with a coarse filter paper (e.g. Green’s No. 904)
True
Paraffin wax are only used how many times?
2
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
T or F. During the summer it may be necessary to use 60 to 63°C, although this is to be avoided if possible in order to not to “cook” the tissue.
True
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
T or F. In celloidin, disadvantages include inability to cut thin sections, storage of blocks in alcohol and speed of technique (which can take several weeks or months)
True
What are the 2 methods to allow solidification of block?
a) Place the preparation inside the ref at -5°
b) Immerse it in cold water
Product of embedding?
TISSUE BLOCK
What embedding uses High Resolution Light Microscopy (tissue sections thinner than the usual 4-6 μm, such as renal biopsies a n d bone marrow biopsies)
Plastic (Resins) Embedding
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
