IMPREGNATION & EMBEDDING Flashcards
This 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
Impregnation is also called …
Infiltration
It is the process by which the impgregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify
Embedding
Embedding is also called …
casting/blocking
Embedding and infiltrating mediums should be soluble in processing fluids
true or false
true
embedding and infiltrating mediums must be molten between what temperatures?
30C and 60C
What is the medium used to infiltrate the tissue and is usually the same medium utilized for impregnation
embedding medium
What are the 4 types of impregnation and embedding mediums?
PPCG
- PARAFFIN WAX
- PLASTIC
- CELLOIDIN
- GELATIN
Celloidin is also called …
collodion
This is the simplest, most common, and best embedding medium
paraffin wax
this is used for routine tissue processing
paraffin wax
This is a polycrystalline mixture of solid hydrocarbons produced during refining of coal and mineral oils
Paraffin
Paraffin melts at what temperatures?
65C to 70C
This is solid at room temperature
paraffin
Paraffin wax at 56C to 58c are the most commonly used for what?
histological use
Paraffin, at its melting point, tends to be what?
Slightly viscous
Viscosity decreases as the temperature increases
true or false
true
The traditional advice with paraffin wax is to use this at what temperature?
2C above its melting point
Wax hardness (viscosity) of paraffin depends on what?
- molecular weight of the components
- ambient temperature
To decrease viscosity and improve filtration, what do technologists do?
they increase the temperature
For paraffin, to decrease viscosity and improve infiltration of the tissue, the technologists often increase the temperature to what temperature?
to above 60C ot 65C
High molecular weight mixtures melt at lower temperatures than waxes comprised of lower molecular weight fractions
true or false
false
higher temperatures
High molecular weight mixtures melt at lower temperatures than waxes comprised of lower molecular weight fractions
true or false
false
higher temperatures
Paraffin wax is traditionally marketed by its melting points which range from what?
39C to 68C
Tissu-wax adhesion depnds upon the what?
crystal morphology of the embedding medium
These provides better physical support for specimens through close packing
Small, uniform sized crystals
Small, uniform sized crystals provide better physicaly support for specimens through what?
through close packing
Crystalline morphology of paraffin eax can be altered by incoportating what?
additives
The incoporation of additives result in a less brittle, more heterogeneous wax with good cutting characteristics
true or false
false
less brittle, more homogeneous, with good cutting characteristics
The incoporation of additives result in a less brittle, more heterogeneous wax with good cutting characteristics
true or false
false
less brittle, more homogeneous, with good cutting characteristics
There is consequently more deformation during thin sectioning
true or false
false
less deformation
There is consequently more deformation during thin sectioning
true or false
false
less deformation
This does not appreciably affect crystal size
setting temperature
They are an important source for historical studies in medicine
formalin-fixed, paraffin-embedded tissues
What are the types of Microtomes?
FRRUCS
- Freezing
- rotary
- rocking
- ultrathin
- Cryostat
- Sliding
It is an example of a heavy duty microtome
Sliding
What are the 3 general methods for infiltration?
- Paraffin
- Celloidin
- Gelatin
What are the 3 methods under paraffin?
- Manual
- Automatic
- Vacuum
What are the substitutes for Paraffin wax?
- Paraplast
- Embeddol
- Ester wax
- Bioloid
- Tissue mat
- Carbowax
This is a method used for celloidin impregnation that is recommended for the bones, brain, teeth, and whole organs
Wet celloidin method
This is a method used for celloidin impregnation that is recommended for the eyes
dry celloidin method
How many percent celloidin does the THIN CELLOIDIN contain?
2-4 %
For how many days must the THIN CELLOIDIN be left at?
5-7 days
How many percent celloidin does the MEDIUM CELLOIDIN contain?
4-6% celloidin
For how long must the medium celloidin be immersed in 4-6% celloidin?
5-7 days
What percent of celloidin must THICK CELLOIDIN contain?
8-12% celloidin
For how long must the THICK CELLOIDIN be immersed in 8-12% celloidin?
3-5 days
f the specimen that you are processing have bigger spaces of cavities and the specimen is bigger, IT WILL MAKE THE PROCESS LONGER.
true or false
true
This is an equipment that is used for cutting tissues.
Microtome
This has a melting point of 46-48C
Ester wax
This is recommended for enzyme studies
Carbowax
It is hygroscopic or water-soluble
Carbowax
It is a substitute for paraffin that is recommended for eye specimens
Bioloid
It is a substitute for paraffin that is recommended for bones and brain specimen
Paraplast
What is the melting point of Paraplast?
56-57C
It is a substitute for paraffin wax that has a melting point of 56-58C
Embeddol
This method is under negative atmospheric pressure inside the oven
Vacuum method
This is the routine infiltration method
Paraffin wax infiltration
What are the 2 types of autotechnicon?
- Tissue type transfer
- Fluid transfer type
The fluid transfer type is also called …
Also called as Enclosed Type
The Tissue Transfer Type is also known as the …
Dip and Dunk type
What are the parts of an Autotech?
STEC10
- Spring loaded plunger
- Transfer arm
- Electric clock
- Cover plate
- 10-12 1L capacity beakers
The first 2 beakers of an autotechnicon contain what?
FORMALIN
Beaker’s 3, 4, 5 of an autotechnicon contains what?
ALCOHOL in INCREASING concentration
Beaker’s 6, 7, 8 of an autotechnicon contains what?
XYLENE, 3 changes of xylene
Beaker’s 9 and 10 of an autotechnicon contains what?
contains PARAFFIN WAX
Also called as Timing Disk
Electric clock
It determines the duration of the process
Electric clock
It facilitates automatic transfer of tissue/s from one reagent to another
Transfer arm
Its purpose is to cover all the beakers
Cover plate
The opening and closing function of the cover plate is AUTOMATIC and done by what?
Spring loaded plunger
This facilitates the removal of the cover plate
Spring loaded plunger
This is the type of autotechnicon wherein the tissue/s stays within the chamber where the reagent for each process is pump in and out
Fluid transfer type
What are the 2 advantages of the Fluid Transfer type?
- Drying of tissues is prevented
- Vapors are vented through filters
Paraffin wax is compatible with many stains
true or false
true
What must be the approximate temperature in the manual processing?
2-5℃ higher than the wax melting point
This is the cause of specimen brittleness
Overheated paraffin
This will cause excessive tissue shrinkage and hardening, making the cutting of sections difficult
Prolonged impregnation
This will promote retention of the clearing agen
Inadequate impregnation
What happens to the tissues in cases of inadequate impregnation?
tissues become soft and shrunken
What happens to tissue blocks in cases of inadequate impregnation?
tissue blocks crumble when sectioned and break up when floated out in a water bath
What are examples of tissues that are difficult to infiltrate?
BBET
- Bones
- brain
- Eyes
- teeth
Tissues that are difficult to infiltrate require what?
long immersion for proper support
What will happen to these tissues that are difficult to infiltrate if they do not undergo long immersion?
they will crumble on sectioning
Prolonged immersion in paraffin is not advisable
true or false
true
Paraffin processing is recommeneded for fatty tissues
true or false
false
not recommended
Paraffin processing is recommeneded for fatty tissues
true or false
false
not recommended
What does dehydrating and clearing agents used in infiltration do to the fat from the tissues?
they dissolve and remove fat from tissues
After being completely cleared, the tissue is submerged in two or more changes of melted paraffin wax, what are the two approached to do this?
either:
- paraffin oven
- in an incubator which has been regulated at 55-60C
The duration and number of changes required for thorough impregnation of tissues depends on what?
STUC
- Size and type of tissues
- Use of vaccum embedding
- Clearing agent employed
Longer time is required for thicker tissues
true or false
true
This reduces the time required for complete impregnation
vacuum
What are the melting points of common waxes?
45, 52, 56, 58
This temperature is normally ised for routine work
56C
If a laboratory is at the temperature of 20-24C, what is the melting point of the paraffin wax?
54-58C
If the laboratory temperature is between, 15-18C, the melting point of wax to be used must be between what?
50-54C
Soft tissues require wax with a higher melting point than hard tissues
true or false
false
hard tissues require wax with a higher melting point
Soft tissues require wax with a higher melting point than hard tissues
true or false
false
hard tissues require wax with a higher melting point
What are the three ways by which paraffin wax impregnation and embedding of tissues may be performed?
- Manual processing
- Automatic processing
- Vacuum embedding
How many changes of wax are required at how many minutes intervals must be done in Manual Processing?
At least four changes of wax are required at 15 minutes intervals
After the 4th change of wax in manual processing, the specimen is then immersed in another fresh solution of melted paraffin for approximately how many hours?
3 hours
The thickness of the specimen for manual processing must be how many millimeters?
3 mm thick
This method makes use of an automatic tissue processing machine (i.e., Autotechnicon)
Automatic processing
This method fixes, dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues
Automatic processing
What is the machine used or automatic processing?
Autotechnicon
This results in a more rapid diagnosis with less technicality
Automatic processing
How many changes of wax is required to remove the clearing agent and properly impregante specimens in automatic processing?
2-3 changes
This accelerates and improves tissue penetration giving rise to more consistent results
constant tissue agitation
One example of automatic tissue processing machine
Elliott Bench-Type Processor
This machine is mounted on rollers to permit the turning of platforms and easy access t beakers and wax baths
Elliott Bench-Type Processor
The Elliott Bench-Type Processor makes use of how many inidividuals steps?
12
The frequency with which fluids are changed depends on what?
depends on the number of sizes of the tissues processed
This indicates that the paraffin wax needs to be changed
The presence of any odor in the clearing agent during the final paraffin wax bath
Dehydrating fluids should be changed frequently
true or false
true
The first 100% ethanol bath should be discarded
true or false
true
The clearing agent and the dilute ethanols should be changed at least twice a week
true or false
false
once a week
The clearing agent and the dilute ethanols should be changed at least twice a week
true or false
false
once a week
The clearing agent and the dilute ethanols should be changed at least twice a week
true or false
false
once a week
It reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening
Vacuum embedding
It facilitates complete removal of transition solvents, and prolongs the life of wax by REDUCING SOLVENT CONTAMINATION
Vacuum embedding
This hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue
vacuum
This technique is recommended for urgent biopsies and for delicate tissues
Vacuum embedding
What are the delicate tissues for vacuum embedding?
- brain
- CNS
- Connective tissues
- eyes
- lung
- spleen
- decalcified bones
What are the delicate tissues for vacuum embedding?
- brain
- CNS
- Connective tissues
- eyes
- lung
- spleen
- decalcified bones
The addition of this may hasten displacement of cedarwood oil with less tissue shrinkage
Addition of benzene
Benzene and xylene are easily removed from the tissues while chlorofrom and cedarwood oil are difficult to remove and require more frequent wax changes
true or false
true
Larger and densner tissue blocks usually require longer periods and more frequent changes of wax
true or false
true
This depend upon the nature and size of the tissues to be processed, and type of clearing agents to be used
total impregnation time
Of the three methods of paraffin wax impregnation, which gives the fastest result?
Vacuum
The tissue should not be left in the paraffin oven for more than how many hours?
for more than 4 hours
Tissues become increasingly harder and more brittle as they are heated
true or false
true
Infiltration in overheated paraffin (above 60C) will produce what?
produce shrinkage and hardening of tissues, and destroy lymphoid tissues completely.
Infiltration in overheated paraffin (above 60C) will produce what?
produce shrinkage and hardening of tissues, and destroy lymphoid tissues completely.
To avoid the shrinkage and hardening of tissues and from the lymphoid tissues from being destroyed, the paraffin oven must be maintained at what temperature?
2 to 5C above the melting point of paraffin
Paraffin wax must be pure
True or false
true
Fresh wax should be filtered after use in a wax oven at a temperature 2C higher than its melting point
true or false
false
filtered before use
Fresh wax should be filtered after use in a wax oven at a temperature 2C higher than its melting point
true or false
false
filtered before use
Wax that has been trimmed away from th eimpregnated tissue may bmelted and filtered for future use, using a what?
coarse filter paper
Example of coarse filter paper
Green’s No. 904
Water must therefore be removed by heating the wax to what temperature?
100-105C
Paraffin wax may only be used thrice, after which, fresh wax must be utilixed
true or false
false
may only be used twice
When using an automatic tissue processing machine, wax usually becomes ADMIXED with the dehydrating agent, especially in the last beaker; hence, water must be discarded
true or false
false
admixed w clearing agent; FIRST BEAKER
A relatively hard wax with a higher melting point is recommended for this microtome
fixed knife mimcrotome
Paraplast is a mixture of what?
highly purified paraffin + synthetic plastic polymers
Paraplast has melting point of 56-57C
TRUE OR FALSE
TRUE
It is a substitute that is more elastic and resilient than paraffin wax
paraplast
This permits the large dense tissue blocks such as bones and brains to be cut easily with the same result as in double embedding
paraplast
Blocks obtained in this paraffin substitute are more uniform than with any other medium, with better ribboning of sections
paraplast
It is soluble in common clearing agent and follows the same time schedule for paraffin impregnation
paraplast
does not tend to crack like other paraffin wax substitutes
paraplast
During the winter, paraplast may be used at what temperature if the tisue is cut in a cool room?
54-56C
During the summer, paraplast may be at what temperature?
60-63C
During the summer, paraplast may be at what temperature?
60-63C
These kinds of tissues do not section well or, if it does, it does not stain well and most details are destroued
“cooked” tissues
It is a synthetic wax substitute similar to paraplast with a melting point of 56-58C
Embeddol
This is a semisynthetic wax recommended for embedding eyes
Bioloid
This is a product of paraffin, containing rubber, with the same property as paraplast
tissue mat
This substitute has a lower melting point of 46-48C
ester wax
This substitute is not soluble in water
ester wax
ester wax is soluble in what?
95% ethyl alcohol and other clearing agents
this substitute can be used for impregnation without prior clearing of the tissue
ester wax
Cellosolve is also called ..
ethylene glycol monoethyl ether
These may be used as clearing agents, if indicated
cellosolve and xylene
In such instances, removal of the clearing agent must be GRADUAL, that is, the tissue must be placced in a solution containing what? for how many hours? before finally transferring it to pure wax.
equal proportion of CLEARING AGENT and ESTER WAX FOR 3-6 HOURS
How many chages are required to ensure complete tissue impregnation in ester wax?
3-4 changes of wax
Sectioning of ester wax-impregnated tissues should be done on a what?
on a heavy duty microtome
example of heavy duty microtome
- sliding
- sledge-type microtome
heavy duty microtome should be used in sectioning of ester wax-impregnated tissue due to what?
due to relative hardnes of wax
These are plastic polymers, mostly polyethylene glycols
water soluble waxes
Water soluble waxes have melting points of 38-42 or 45-56C
true or false
true
These are incorporatedinthe majority of proprietary histological paraffin wax blengds presently available to improve ADHESION, HARDNESS and PLASTICITY.
Polymer waxes
The most commonly used water souble wax
carbowax
Carbowax is a polyethylene glycol containin how many carbon atins, which appears solid at room temp.
18 or more carbon atoms
It is soluble in and miscible with water; hence dosnot require dehydration and clearing of the tissue
carbowax
it does not remove neutral fats and lipids
carbowax
This technique is suitable for many enzyme histochemical studies
carbowax
Cytologic details are excellently preserved
carbowax
How many changes of wax is required in carbowax?
4 changes
how are the four changes of wax in carbowax?
- once each in 70% and 90%
- 2 times in 100% concentration
- at a temperature of 56C, at 30 mins, 45 mins, and 1 hour (with agitation), respectively
Specimens are embedded in fresh carbowax at what temperature? and rapidly cooled in a refrigerator
50C
Carbowax is very easily dissolved in water due to what?
due to its hydroscopic nature
This will reduce tissue distortion and promote flattening and “floating out” of sections
adding soap to water or using 10% polyethylene glycol 900
This is added to proprietary blengds of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning
Dimethyl sulphoxide (DMSO)
DMSO scanges residual transition solvent and probably alters tissue permeability by what?
by substitutingfor our removing bound water thus improving infiltration
Some individuals who handle DMSO-paraffin wax may experience what?
unpleasant and annoying oyster or garlic taste probably caused by DMSO metabolites
This is a purified form of nitrocellulose soluble in many solvents
celloidin
this is suitable for specimens with large hollow cavities which tend to collapse
colloidon
This is used mainly for preparing soft tissue sections of mixed consistency such as eyes and brain
Celloidin
No heat is required, and the resultant block has a rbbery consistency which gives good support to tissues
Collodion
This recommended for processing of meurological tissues
celloidin
This is used in the dry celloidin technique to help soften the brittle layers
cedarwood oil
This type of celloidinis recommended for bones, teeth, large brain sections and whole organs
wet celloidin method
This celloidin method is preferred for processing whole eye sections
Dry celloidin method
This is made up of equal parts of chloroform and cedarwood oil, and is added to celloidin block before hardening, to make tissue transparent
Gilson’s mixture
This method does not make use of alcohol due to the presence of cedarwood oil in the block
dry method
This is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentration and still penetrate tissues rapidly
Low Viscosity Nitrocellulose
many workers prefer L.V.N to the ordinary cellidin and makes cutting of thinner sections possible
true or false
true
It forms a harder tissue block and makes cutting of thinner sections possible
LVN
How can the tendency of tissues to crack be prevented?
by adding plasticizers
example of plasticizers
- oleum ricini
- castor oil
example of plasticizers
- oleum ricini
- castor oil
LVN is more explosive than celloidin
true or false
true
this must be kept tightly covered and protected from sunlight to avoid evaporation of alcohol
LVN
THIS SERVES TO PREVENT THE GROWTH OF MOLDS
1% phenol
the volume of impregnating medium should be at least 20 times the volume of the tissue in gelatin impregnation
true or false
false
at least 25 times
the volume of impregnating medium should be at least 20 times the volume of the tissue in gelatin impregnation
true or false
false
at least 25 times
What are the several types of blocking out molds?
- Leuckhart’s embedding mold
- Compound embedding unit
- Plastic embedding rings and base mold
- Disposable embedding molds
What are the kinds of disposable embedding molds?
- peel away
- plastic ice trays
- paper boats
