Impregnation Flashcards
Enumerate advantages of impregnation (3)
✨ Easier handling
✨ Firm consistency of the specimen
✨ Cutting of suitably thin section w/o damage/distortion tothe tissue and cellular components
Types of tissue impregnation and embedding media (4)
✨Paraffin wax
✨Celloidin (collodion)
✨Gelatin
✨Plastic
The simplest, most common and best embedding medium used for routine tissue processing
Paraffin
✨how many times will you submerged a tissue in a melted paraffin wax after it has been completely cleared?
Follow-up Qs:
✨ what instrument/s will you use?
✨ regulated temp of the instrument?
✨ two or more changes
✨ paraffin oven; incubator
✨ 55-60 degrees C
Melting point of waxes (4)
✨ 45 C
✨ 52 C
✨ 56 C
✨ 58 C
temperature of wax normally used for routine work?
✨ 56 C
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
Impregnation (infiltration)
lab temp ranging from 20-24 C, what is the temp range of the paraffin wax?
54-58 C
lab temp ranging from 15-18 C, what is the temp range of the paraffin wax?
50-54 C
3 methods of paraffin wax impregnation and embedding
✨ Manual processing
✨ Automatic processing
✨ Vacuum embedding
For manual processing, how many mins is required in order to ensure complete removal of the clearing agent from the tissue?
15 mins (at least 4 changes)
For manual processing how many hours is needed to ensure complete embedding or casting of tissue?
3 hrs
this method makes use of an automatic tissue processing machine w/c fixes, dehydrates, clears and infiltrates tissues?
example of this machine?
✨ automatic processing
✨ Autotechnicon; Elliot bench-type processor
how many processing steps in automatic processing?
✨ 12 individual processing steps
in automatic processing, how many glass beakers are present? and what is their capacity?
✨ ten 1-liter glass beakers
moves the tissues from one processing reagent to another
transfer arm
what is the temperature of wax bath thermostats?
✨ Atleast 3°C above the melting point of the wax
process which involves the wax impregnation under negative atmospheric pressure inside an embedding oven to hasten removal of air bubbles and clearing agent to promote more rapid wax penetration of the tissue
Vacuum embedding
vacuum pressure
✨ not more than 500 mmHg (400-450 mmHg)
in vacuum embedding, what example of organ undergoes wax impregnation and removal of any residual air bubbles?
Lungs
Vacuum embedding is recommmended for (2)?
1) Urgent biopsies
2) Delicate tissues such as:
✨lung
✨brain
✨ CT
✨ decalcified bones
✨ eyes
✨spleen
✨ CNS
Factors that affects the total impregnation time (3)
✨Nature of the tissue
✨ size of the tissue
✨ type of clearing agent used
benzene and xylene are _____ removed from the tissues
easily
chloroform and cedarwood oil are _____ to remove from tissues
difficult
addition of this clearing agent may hasten displacement of cedarwood oil will less tissue shrinkage
benzene
Prolonged treatment in melted paraffin causes ______ and ______ tissues
shrinkage; hardening
Infiltration in ________ (temp?) will also produce shrinkage and hardening of tissues and destroy ________ (what kind of tissue?) completely.
✨overheated paraffin ( above 60°C )
✨lymphoid tissues
what is the temperature range of paraffin to be used for impregnation?
2 to 5°C about the melting point of the paraffin
paraffin wax must be _____
pure
how many times can you use a paraffin wax?
twice
temp needed in heating a wax to remove water?
100-105°C
substitutes for paraffin wax
✨paraplast
✨ester wax
✨water soluble waxes
mixture of highly purified paraffin and synthetic plastic polymers
paraplast
melting point of paraplast
56-57°C
substitute to paraplast
embeddol
melting pount of embeddol
56-58°C
a semisynthetic wax recommended for embedding eyes
bioloid
Product of paraffin containing rubber
Tissue mat
melting point of ester wax
46-48 °C
Harder than paraffin
ester wax
ester wax is soluble in ?
95% ethanol and other clearing agents (cellosolve and xylene)
can be used for impregnation without clearing of the tissue
ester wax
when using ester wax, how many changes of wax is required to ensure complete tissue impregnation?
3-4 changes
how many hrs is needed in immersing the tissue in a solution containing equal proportion of clearing agent and ester wax before tranferring to pure wax?
3-6 hrs
These are mostly polyethylene glycols
Water soluble waxes
melting point of water soluble waxes
38-42°C or 45-56°C
A polyethylene glycol containing 18 or more carbon atoms, appears solid at room temp
Carbowax
the reason why carbowax does not require dehydration and clearing of the tissue?
It is soluble in and miscible with water
Reduces tissue distortion and promote flattening and “floating out” of sections
soap; 10% polyethylene glycol 900
A purified form of nitrocellulose soluble in many solvents
Celloidin
Uses of celloidion
- for specimens w/ large hollow cavities w/c tend to collapse
- for hard & dense tissues (bones,teeth)
- large tissue sections of the whole embryo
Two methods for celloidin impregnation
1) Wet celloidin method
2) Dry celloidin method
example of heavy duty microtome in sectioning ester wax
sliding or sledge type microtome
type of celloidin method that is recommended for bones, teeth, large brain sections, and whole organs
Wet CM
type of celloidin method preferred for processing of whole eye sections
Dry CM
tissue block in Wet cm is stored in ____ to avoid dehydration and tissue shrinkage ?
70-80% alcohol
tissue block in Dry cm is stored in _____?
Gilson’s mixture (chloroform & cedar wood oil)
Another form of celloidin soluble in equal concentration of ether and alcohol w/ a lower viscosity
Low Viscosity Nitrocellulose (LVN)
Forms a harder tissue block and makes cutting of thinner sections possible
LVN
Example of plasticizers that prevents the tendency of tissues to crack when embedding chrome-mordanted tissues using LVN
- oleum ricini
- castor oil
type of impregnation used when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies
Gelatin impregnation
Thickness of tissue used in gelatin impregnation
Act as a tissue softener and prevents the growth of molds
1% phenol