MODULE 3 - Infiltration/Impregnation Flashcards
Main reason why we do Infiltration
To remove clearing agents from tissues
2nd reason why we do Infiltration
to fill up cavities and tissue spaces
to give firm consistency for easy cutting
The most commonly used and rapid infiltration method
Paraffin wax
Advantages of using paraffin wax infiltration method
For serial sections
Compatible for any staining procedure
Prolonged process using paraffin wax will cause
tissue shrinkage and hardening
Inadequate process when using paraffin wax will cause
retention of clearing agents
Paraffin wax infiltration method is not used for ______.
fatty tissues
Melting point for routine work in paraffin infiltration method
56 degrees celsius
This method of paraffin wax needs autotechnicon and is more rapid than manual
automatic
This method of paraffin wax needs an oven to keep the wax melted
manual
How many changes of wax is needed in manual paraffin wax method? In what interval?
4 changes
15 minutes interval
This paraffin wax infiltration method is more rapid than manual
Automatic
Automatic can cause shorter processing time because of these variables
heat and agitation
Vacuum is under _________ and is the most ___________.
negative atmospheric pressure in the oven
RAPID of all paraffin wax infiltration method
Vacuum is recommended for?
urgent biopsies
lungs, brain, eyes, spleen and CNS
Enumerate different substitutes for paraffin wax
Paraplast Embeddol Bioloid Ester wax Tissue mat Carbowax
It is used for eye specimens
bioloid
it is a mixture of highly purified paraffin and synthetic plastic polymers
paraplast
Melting point of paraplast
56-57 degrees Celsius
Paraplast is used for
bones and brains
it is similar to paraplast but has a melting point of 56-57 degrees Celsius
embeddol
wax with a much lower melting point
ester wax
what is the melting point of ester wax
46-48 degrees
it is harder than paraffin and thus requires ____________
ester wax, heavy duty microtome
ester wax is soluble to?
and what is the effect of this on processes?
alcohol
it will not require clearing process, only dehydration
Product of paraffin with rubber
tissue mat
– a water soluble wax for enzyme histochemistry
Carbowax
Carbowax advantage and disadvantage?
it will not require both dehydration and clearing process
tissues will not be able to float out
Remedy for carbowax
Soap to water
10% polyethylene glycol 900
Celloidin is also known as
Collodion
Celloidin is soluble in
alcohol and ether
Celloidin is a purified form of
nitrocellulose
How many percent of celloidin solutions is dissolved in ____________?
4.8%
dissolved in equal parts of alcohol and ether
Celloidin is used for
Large and hollow specimens that tends to collapse
Disadvantage of using celloidin
Does not require heat, VERY slow, thin sections,
sections for photomicrography are difficult to obtain
Process of infiltration that does not require heat
Celloidin and gelatin
In methods of cellodin infiltration, enumerate the concentration and duration for thin, medium and thick specimens
Thin- 2-4% , 3-5 days
Medium- 4-6% 3-5 days
Thick- 8-12% for 5-7 days
Wet celloidin is used for
bones, brain, teeth and whole organs
What should be used for storage of wet celloidin method
70% alcohol
Dry celloidin is used for
Eye specimen
What should be used for dry celloidin storage?
Gilson’s method- chloroform + cedarwood oil
Disadvantage of using LVN
highly explosive
cracking of tissues
Advantage of using LVN
produces harder tissue block
thinner sections to cut
To prevent cracking of tissues in LVN what should be done
add plasticizers
Oleum recini and castor oil
Type of infiltration method that is RARELY used
Gelatin
Steps in doing gelation method
1st step- put specimen in container 10% gelatin and 1% phenol for 24 hrs
2nd step- put in 20% gelatin and 1% phenol for 12 hrs
3rd step- put in 20% gelatin and 1% phenol and refrigerate until impregnation and embedding is complete
What should be added to gelatin method in order to prevent formation of molds?
phenol
Gelatin method is ___________ and is used for ______________.
water-soluble
frozen sections and enzyme histochemistry
in gelatin infiltration method, tissue for processing should not be _______
2-3 mm thick
EMBEDDING is also known as
CASTING / BLOCKING
Placing infiltrated tissue in a mold containing the embedding medium which is allowed to solidify A. Fixation B. Dehydration C. Clearing D. Embedding
D
Requirements for embedding
Embedding mold, medium and infiltrated tissue
Temperature of melted paraffin used for embedding
5-10 degrees above its melting point
To allow solidification of block in embedding, what can be done?
- put in -5 degrees refrigerator
2. put in cold water
cutting off excess paraffin wax to form a four-sided prism
trimming
What is double-embedding
different infiltration and embedding medium.
Ex. infiltrate- celloidin, embedding- paraffin wax
Enumerate the non-disposable of embedding molds
Leuckhart’s
Compoung embedding unit
Plastic embedding ring and base molds
Tissue tek
Enumerate the disposable embedding molds
Peel-away
Paper boat
Plastic ice tray
Type of embedding mold that – consist of 2 L- shaped strips of heavy brass or metal
Leuckhart’s
What does peel-away molds do
produce perfect blocks without trimming
Type of Embedding mold that is economical and easy to prepare
paper boat
disadvantage and advantage of using leukhart’s
need to assemble
can produce different sizes of tissue blocks
What should be done when using plastic ice trays
need to smear the inner wall with glycerin for easy for removal of tissue blocks
PLASTIC (RESINS) Embedding is used for
EM, high resolution for LM
For extremely hard tissues, what can be used
Expoxy, Polyester and Acrylic
Enumerate chemicals under expoxy
Bisphenol
Glycerol
Cyclohexane dioxide
Type of plastic (resins) embedding that is rarely used and for EM
Polyester
Enumerate chemicals under Acrylic
MMA and GMA
the most critical stage of tissue processing
dehydration
It facilitates complete removal of transition solvents, and
prolongs the life of wax by reducing solvent contamination
Vacuum Embedding
The degree of the vacuum should not exceed
500 mm
the tissue should not be left in the paraffin oven for ________
more than 4 hours.
Carbowax technique is suitable for
enzyme histochemical studies
carbowax is _______ in nature
hygroscopic
Celloidin 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, teeth and for large tissue sections of the whole embryo.
recommended for processing of neurological tissues.
celloidin
It is used as an embedding medium for delicate specimens and frozen
tissue sections
gelatin
Usually, embedding medium is miscible with water
True or False
False
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.
Generally speaking, the surface of the section to be cut should be placed __________ to the bottom of the mold in which it is oriented.
parallel
made up of a series of interlocking plates resting on a flat metal base, forming several compartments
Compound Embedding Unit
Blocks produced are even, with parallel sides, and with a fairly shaped initial setting of the wax.
Leuckhart’s Embedding Mold
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
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
This is used to facilitate cutting of large blocks of dense firm tissues like the brain
Double-Embedding
Radicals can be produced spontaneously by
heat or light
widely used for bone histomorphometry and bone marrow
hematopathology.
Acrylic plastics based on methyl methacrylate (MMA)
type of embedding that causes almost complete loss of
enzyme activity and protein antigenicity in the tissues
mma
