Impregnation and Embedding Flashcards
Process whereby the clearing agent is completely removed from the tissue & replaced by a medium that will completely fill all the tissue cavities, thereby giving a firm consistency to the specimen
Impregnation
_________ (Casting or Blocking) 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
Characteristics of an Ideal infiltrating and embedding medium
- soluble in processing fluids
- suitable for sectioning and ribboning
- molten between 30°C and 60°C
- translucent or transparent; colorless
- stable
- homogeneous
- capable of flattening after ribboning
- non-toxic
- odorless
- easy to handle
- inexpensive
Tissue Impregnation
- Paraffin wax Impregnation
- Celloidin Impregnation
- Gelatin Impregnation
- Plastic
• _________ is the simplest, most common and best embedding medium used for routine tissue processing.
• __________ is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
• It is solid at room temperature but melts at temperatures up to about 65°C or 70°C.
Paraffin Wax Impregnation
Paraffin wax advantages:
- Thin individual serial sections may be cut
with ease - The process is very rapid
- Tissue blocks and unstained mounted
sections may be stored in an indefinite
period of time - Formalin-fixed, paraffin-embedded
tissues may be stored indefinitely at room
temperature, - Many staining procedures are permitted
with good results.
Paraffin wax disadvantages:
- Overheated - brittle.
- Prolonged impregnation - tissue shrinkage and hardening
- Inadequate impregnation will promote retention of the clearing agent. - Tissues become soft and shrunken,
- Tissues that are difficult to infiltrate, need long immersion for proper support
- Paraffin processing is not recommended for fatty tissues.
• Common waxes have melting points of:
• _____________________________.
• ______ wax is normally used for routine work.
• melting point of ________ - In a laboratory with temperature ranging from _______.
• Between ___________. If the laboratory temperature is between _________.
• Hard tissues require wax with a higher melting point than soft tissues.
- Temperature
- 45°C, 52°C, 56°C and 58°C
- 56°C
- 54-58°C
- 20-24°C
- 50 and 54°C
- 15-18°C
Three ways by which Paraffin wax impregnation and embedding may be performed
- Manual Processing
- Automatic Processing
- Vacuum Embedding
Manual Processing
• 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.
• The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to insure complete embedding or casting of tissue.
- Fixation : 10% Buffered Formalin
- Dehydration : 70% Alcohol
- 95% Alcohol
- 100% Alcohol
- 100% Alcohol
- 100% Alcohol
- Clearing : Xylene or Toluene
- Impregnation : Paraffin wax
- Embedding : Paraffin wax
- 24 hours
- 6 hours
- 12 hours
- 2 hours
- 1 hour
- 1 hour
- 1 hour (2x)
- 15 minutes (4x)
- 3 hours
Automatic Processing
• This method makes use of an automatic tissue
processing machine (i.e., Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues.
• This results in a more rapid diagnosis with less
technicality.
• Usually, only 2- 3 changes of wax are required to remove the clearing agent
- 10% Buffered Formalin
- 10% Buffered Formalin
- 70% Alcohol
- Absolute Alcohol (1)
- Absolute Alcohol (2)
- Absolute Alcohol (3)
- Xylene or Toluene (1)
- Xylene or Toluene (2)
- Benzene (1)
- Benzene (2)
- Chloroform (1)
- Chloroform (2)
- Chloroform (3)
- Wax (1)
- Wax (2)
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 1 hr.
- 1 hr.
- 1 hr.
- 1 hr.
- ## 1 hr.-
-
-
- - 2 hrs.
- 3 hrs.
- 10% Buffered Formalin
- 10% Buffered Formalin
- 70% Alcohol
- Absolute Alcohol (1)
- Absolute Alcohol (2)
- Absolute Alcohol (3)
- Xylene or Toluene (1)
- Xylene or Toluene (2)
- Benzene (1)
- Benzene (2)
- Chloroform (1)
- Chloroform (2)
- Chloroform (3)
- Wax (1)
- Wax (2)
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 3 hrs.
- 3 hrs.
- ## 3 hrs.-
- 30 mins.
- ## 1 hr.-
- - 3 hrs.
- 3 hrs.
- 10% Buffered Formalin
- 10% Buffered Formalin
- 70% Alcohol
- Absolute Alcohol (1)
- Absolute Alcohol (2)
- Absolute Alcohol (3)
- Xylene or Toluene (1)
- Xylene or Toluene (2)
- Benzene (1)
- Benzene (2)
- Chloroform (1)
- Chloroform (2)
- Chloroform (3)
- Wax (1)
- Wax (2)
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 2 hrs.
- ## --
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 2 hrs.
- 2 hrs.
• Changed of solutions will depends on the number and sizes of the tissues processed.
• Dehydrating fluids should be changed frequently
• The clearing agent and the dilute ethanols should be changed at least once a week.
• To avoid spillage, fluid and wax containers must be filled to the appropriate level and correctly located in the machine.
• Wax accumulating on any surface or beaker leads must be remove
• Wax bath thermostats should be set at least 3 degrees above the melting point of the wax, and timing should be checked
Precautions with Automatic Processing
• ____________ involves wax impregnation under negative atmospheric pressure inside an embedding oven.
• It reduces the time when tissues are subjected to high temperatures
• It facilitates complete removal of transition solvents, and prolongs the
life of wax by reducing solvent contamination.
• ________ hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue.
• This technique is particularly recommended for urgent biopsies
• With ___________, the time required for complete impregnation is reduced by 25% -75% of the normal time required for tissue processing
Vacuum Embedding
• The tissue should not be left in the paraffin oven for more than 4 hours.
• The paraffin oven must be maintained at a temperature 2 to 5°C above the melting point of paraffin to be used for impregnation.
• Paraffin wax must be pure
• Fresh wax should be filtered before use
• Paraffin wax may be used only twice
• When using an automatic tissue processing machine, wax usually becomes admixed with the clearing agent
• For fixed knife microtomes, a relatively hard wax with a higher melting point is recommended.
Practical Considerations
Factors Affecting Paraffin Wax Impregnation
• Nature and size of the tissues to be processed
• Type of clearing agents to be used
• Benzene and Xylene - easily removed
• Chloroform and cedarwood oil - difficult to remove
Substitutes for Paraffin Wax
- Paraplast
- Ester wax
- Mixture of highly purified paraffin and synthetic plastic polymers
- Melting point of 56-57°C
Paraplast
Paraplast
- Embeddol
- Bioloid
- Tissue mat
semisynthetic recommended for embedding eye
Bioloid
melting point 56-58°C
Embeddol
product of paraffin, containing rubber, same
property as paraplast
Tissue mat
- Has lower melting point 46-48°C
- Harder than paraffin
- Not soluble in water but soluble in 95%EA, can be used for impregnation without prior clearing of tissue - ( Cellosolve or xylene)
Ester wax
Mostly polyethylene glycols
Melting point 38-42°C or 45-56°C
Water Soluble waxes
– most common (hydroscopic) it appears solid at RT.
- does not require dehydration and clearing
- The tissues are fixed, washed out - transferred directly to the melted carbowax
4 changes of carbowax for routine processing, one each in 70%, 90%, and 2x in 100% , at a Temp of 56°C at 30 minutes 45 minutes and 1 hour (with agitation)
- suitable for many enzyme histochemical studies
*** tissue sections are difficult to float and mount
Carbowax
added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.
Dimethyl sulphoxide (DMSO)
is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse
Celloidin (Collodion)
Celloidin Impregnation Advantages:
- It permits cutting of tissue sections which are thicker than in paraffin wax, and is recommended for processing of neurological tissues.
- Its rubbery consistency allows tissue blocks that are either very hard
- Dense tissues which are hard to infiltrate (e.g. bones and brain) and specimens which tend to collapse easily due to air spaces (e.g. eyes) are supported better.
- It does not require heat during processing.
Celloidin Impregnation Disadvantages:
- Celloidin impregnation is very slow (lasting for several days or weeks).
- Very thin sections (less than I 0 μ) are difficult to cut.
- Serial sections are difficult to prepare.
- Vapor of the ether solvent is very flammable
- Photomicrographs are difficult to obtain.
- It is very volatile
2 Methods for Celloidin Impregnation
- Wet Celloidin Method
- Dry Celloidin Method
is recommended for bones, teeth, large brain sections and whole organs. The tissue is then placed in thin celloidin (2-4%) for 5-7 days, transferred to medium celloidin (4-6%) for another 5-7 days, drained off and poured with thick celloidin (8-12%) until the specimen has become impregnated, usually between 3-5 days. The tissue block is then stored in 70-80% alcohol until ready for cutting.
Wet Celloidin Method
is preferred for processing of whole eye sections. The principle and procedure of this method is similar to wet celloidin method,
except that 70% alcohol is not used for storage before cutting. Instead, Gilson’s mixture, made up of equal parts of chloroform and cedarwood oil, is added to the celloidin block before hardening, to make the tissue transparent.
Dry Celloidin Method
• ________________ is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity.
• It forms a harder tissue block and makes cutting of thinner sections
• possible.
• The tendency of tissues to crack may be prevented by adding plasticizers (e.g.oleum ricini or castor oil) when embedding chrome-mordanted tissues.
• is more explosive than celloidin
- Nitrocellulose Method
- 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.
It is used as an embedding medium for delicate specimens and frozen tissue sections
Gelatin impregnation
After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify.
Embedding
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
Embedding Molds
- Leuckhart’s embedding mold
- Compound embedding Unit
- Plastic embedding rings & base molds
- Tissue tek - Disposable embedding molds
- Peel-away
- Plastic Ice trays
- Paper Boats
OTHER EMBEDDING METHODS
- Celloidin or Nitrocellulose Embedding Method
- Double-Embedding
used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs.
- Tissues were embedded in shallow tins of enamel pans which covered by sheetts of weighted glass
- Bell jars were used to control the rate of the evaporation of the solvent
Celloidin or Nitrocellulose Embedding Method
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.
- This is used to facilitate cutting of large blocks of dense firm tissues like the brain.
- They are also recommended for making small sections of celloidin blocks.
Double-Embedding
The introduction of ____________ 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 4-6 μm, such as
renal biopsies a n d bone marrow biopsies.
Plastics are classified into epoxy, polyester, or acrylic, based on their chemical composition.
PLASTIC (RESIN) EMBEDDING
• made up of balanced mixture of epoxy plastic, catalysts and accelerat
Epoxy embedding plastic
Three types are used in microscopy
- bisphenol A (Araldite)
- glycerol (Epon)
- cyclohexene dioxide (Spurr)
slow infiltration
bisphenol A (Araldite)
lower viscosity
glycerol (Epon)
have very low viscosity, and infiltrates
faster
cyclohexene dioxide (Spurr)
were originally introduced for EM in the mid-1950s, but have been superseded by more
superior epoxides, and are now seldom used.
Polyester plastic
made up of esters of acrylic or
merthacrylic acid, and are used
extensively for light microscopy
Acrylic plastics
