LEC 10 - CLEARING Flashcards
Transition step between dehydration and infiltration with the
embedding medium
CLEARING
Process whereby alcohol is removed from the tissue and
replaced with a substance that will dissolve the wax with
which the tissue is impregnated (paraffin) or the medium on
which the tissue is to be mounted (Canada Balsam)
clearing
Most commonly used clearing agents are
xylene, dioxane, chloroform and cedarwood oil
The clearing agent will make microscopic tissue
preparations __due to their high index refraction
transparent
__ is often used as an
indication of the effectiveness or completeness of the
clearing process
Optical clarity or transparency
purpose of clearing
transition between dehydration and infiltration
make the tissue transparent
remove a substantial amount of fat from the tissue
Characteristics of a Good Clearing Agent
miscible with alcohol to promote rapid removal for the dehydrating agent
should be miscible with and easily removed by melted paraffin wax
should not produce excessive shrinkage, hardening or damage of tissue
should not dissolve out aniline dyes
should not evaporate quickly in a water bath
should make tissues transparent
choice of clearing agent depends upon
the type of tissues to be process, and the type of processing to be undertaken
the processor system to be used
intended processing conditions such as temperature, vaccum, and pressure
safety factors
cost and convenience
speedy removal of dehydrating agent
ease of removal by molten paraffin wax
minimal tissue damage
factors affecting clearing agent
clearing fluids with a low boiling point are generally more readily replaced by melted paraffin, although chloroform which has a lower boiling point than xylene in fact takes longer than the latter to clear
viscosity also affects the speed of penetration of clearing agent
prolonged exposure to most clearing agents causes the tissues to become brittle and therefore more difficult to cut
xylene is also called as
xylol
clearing time of xylene
30 mns to 1hr
used for clearing, both for embedding and mounting procedures
xylene (xylol )
also used in cover slipping, in cleaning tissue processors as solvent to remove synthetic immersion oil from the microscope objective in recycling of used slides
xylene (xylol )
most rapid clearing agent, suitable for urgent biopsies
xylene
xylene is applicable to tissues with maximum __ thichness
5 mm
can xylene extract aniline dye?
no
can xylene be used in celloidin section?
yes, it does nit dissolve celloidin
this evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and embedding
xylene
is xylene expensive?
nope
xylene
highly flammable and should be appropriately stored
true
if xylene is used for more than 3 hrs, it will make the tissues __
excessively hard and brittle
what type of tissue is xylene not applicable for as it can cause hardening and shrinkage to them
nervous tissue and lymph nodes
xylene can iritate
eyes, nose, respiratory tract
xylene will become milky if an incomplete __ is immersed in it
dehydration
xylene can be absorbed through the skin and cause
___
dermatitis
at high concentration, it is toxic and narcotic
xylene
keep container tightly closed to prevent xylene from
subliming and entering the atmosphere
only __ tools may be used to handle xylene
non sparking
it is highly recommended that all chemical be stored BELOW eye level so cracking or leaking of containers are immediately visible and there is less potential chemicals falling unto lab workers when pulling from shelves
t or f
true
transport chemicals in closed containers, in the BIGGEST amount possible, and use aids such as carts, chemical transport carriers, etc.
false, must be smallest amount possible
clearing time of toluene
1-2 hrs
better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene
toluene
toluene is can be used as substitute for __
xylene and benzene
toluene is miscible with both
absolute alcohol and paraffin
acts fairly rapidly and is recommended for routine purposes
toluene
tissues do not become excessively hard and brittle even in left for 24 hrs
toluene
clears overnight and is not carcinogenic
toluene
relatively slower than xylene and benzene
toluene
tends to acidify in a partially filled vessel
toluene
highly concentrated solutions of toluene will emit __that are toxic upon prolonged exposure
fumes
which one is more expensive
toluene or xylene
toluene
clearing time of benzene
15-60 mns
preferred as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly
benzene
rapid acting, recommended for urgent biopsies (15-60 mns ) and routine purposes
benzene
volatilizes rapidly in paraffin oven and is therefore easily eliminated from the tissues
benzene
this clearing agent does not make tissues hard and brittle but it causes minimum shrinkage
benzenei
makes tissues transparent and clears overnight
benzene
is benzene flammable?
yes
if section is left in benzene for a long time, what will happen?
considerable tissue shrinkage may be observed
excessive exposure to benzene is __ and __ to human
toxic and carcinogenic
benzene can damage the bone marrow resulting in __
aplastic anemia
chloroform clearing time is
6-24 hrs
this clearing agent is suitable for large tissue specimens
chloroform
chloroform can clear tissue blocks up to ____ cm
1 cm
recommended for routine work (6-24 hrs )
chloroform
recommended for tough tissues, nervous tissues, lymph nodes and embryos
chloroform
why do chloroform is recommended for tough tissues, nervous tissues, and lymph nodes and embryos?
it causes minimum shrinkage and hardening or the tissues
relatively toxic to the ___ after prolonged inhalation of chloroform
liver
wax impregnation after chloroform clearing is relatively __
fast or slow
slow
this clearing agent does not make tissues transparent
chloroform
this clearing agent is not very volatile in paraffin oven
chloroform
difficult to remove from paraffin sections because it is not very volatile
chloroform
may produce considerable deterioration of the wax
chloroform
its vapor may attack the rubber seal used in vacuum impregnating bath
chloroform
this clearing agent’s complete clearing is difficult to evaluate
chloroform
tissues tend to float in this clearing agent
chloroform
tissue floating in chloroform is avoided by ___
wrapping the tissues with absorbent cotton gauze to facilitate sinking of the section in solution
clearing time of cedarwood oil
2-3 days
used to clear both paraffin and celloidin sections during embedding process
cedarwood oil
recommended for central nervous system tissues and cytological studies
cedarwood oilve
cedarwood oil is miscible wth ___ alcohol which it removed readily
96%
how many days can cedarwood oil clear celloidin
5-6 days
tissues can be left in cedar wood oil indefinitely without considerable damage and distortion
true or false
true
is cedarwood oil can be dissolve in aniline dyes
nope
this does not harden the tissues and does not interfere too seriously with paraffin penetration if it is not completely removed
cedarwood oil
can cedarwood oil make tissues transparent?
yes
this clearing agent often improves cutting of the section
cedarwood oil
due to its slow capability as a clearing agent, it is not recommended for routine purposes
cedar wood oil
becomes milky upon prolonged storage and should be filtered before use
cedar wood oil
hard to eliminate from the tissues in paraffin bath, making the wax impregnation very slow
cedarwood oil
since cedarwood oil is hard to be replaced by the impregnation wax, we can hasten it by
transferring the specimen from oil to benzene for 30 mns before finally placing the tissue in wax
this clearing agent’s quality is not always uniform and good
cedarwood oil
in this clearing agent, tissues initially float before gradually staying to bottom as clearing proceeds
cedarwood oil
this clearing agent may fry out the tissue before it is completeley cleared
cedarwood oil
how to prevent the frying out of tissue caused by cedar wood oil
this can be prevented by superimposing absolute alcohol on the surface of the clearing agent. Once saturated, the specimen should then be transferred to a fresh solution of cedarwood oil
a cedarwood oil that has been previously used to clear acetic-alcohol fixed tissues may produce what
crystals with a melting point of approximately 35*C and therefore interfere with adequate clearing of tissue
remedy in the crystals formed because of cedarwood oil used in acetic-alcohol fixed tissues
the solution must be heated to 200*C in order to dissolve crystals and restore the solution to its normal state
do tissues become translucent if cleared with cedar wood oil
no
is cedarwood oil as clearing agent expensive?
very expensive
is aniline oil used as a routine clearing agent
no
recommended for clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening
aniline oil
the only advantage of clove oil
causes minimum shrinkage of tissues
clove oil
its quality is not guaranteed due to its tendency to become __
adulterated
wax impregnation after clearing with oil is
slow and difficult
effect of clove oil to tissues, aniline dyes, and celloidin
tissues become brittle
aniline dyes are removed
celloidin is dissolved
unsuitable for routine clearing purposes
clove oil
its properties are similar to chloroform
carbon tetrachloride
it produces considerable tissue hardening and dangerous to inhale on prolonged exposure due to its highly toxic effects
carbon tetrachloride
relatively toxic to liver as well after prolonged inhalation aside from chloroform
carbon tetrachloride
can carbon tetrachloride make tissues transparent?
nope
CARBON TETRACHLORIDE
easy to remove from paraffin sections because it is very volatile
false
ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation
tetrahydrofuran
non toxic but has offensive odor and should be used in a well-ventilated room
tetrahydrofuran
slow acting clearing agents that can be used when double embedding techniques aree required
methyl benzoate and methyl salicylate
clearing agent for frozen section and no de alcoholization is involved in the process
glycerin and gum syrup
dehydration time if 10% formol saline is used
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % -
alcohol 90 % -
absolute alcohol 1
abs alcohol 2
abs alcohol 3
dehydration time if 10% formol saline is used
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % - 3-12 hr
alcohol 90 % - 1-2 hr
absolute alcohol 1 - 1-2 hr
abs alcohol 2 - 1 - 2 hr
abs alcohol 3 - 1-2 hr `
dehydration time if zenker’s or helly’s fluid is used
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % -
alcohol 90 % -
absolute alcohol 1
abs alcohol 2
abs alcohol 3
running water - 1-12 hrs
alcohol 30 % - 1-6 hrs
alcohol 50 % - 1-6 hrs
alcohol 70 % - 1-6 hrs
alcohol 90 % - 1-6 hrs
absolute alcohol 1 - 1-2 hrs
abs alcohol 2 - 1-2 hrs
abs alcohol 3 - 1-2 hrs
dehydration time if bouin’s fluid is used
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % -
alcohol 90 % -
absolute alcohol 1
abs alcohol 2
abs alcohol 3
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % - 3-12 hrs
alcohol 90 % - 3-12 hrs
absolute alcohol 1 - 1-2 hrs
abs alcohol 2 - 1-2 hrs
abs alcohol 3 - 1-2 hrs
dehydration time if susa, carnoy or formol sublimate is used
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % -
alcohol 90 % -
absolute alcohol 1
abs alcohol 2
abs alcohol 3
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % -
alcohol 90 % - 1-6 hrs
absolute alcohol 1 - 1-2 hrs
abs alcohol 2 - 1-2 hrs
abs alcohol 3 - 1-2 hrs
dehydration time if flemming’s fluid is used
running water -
alcohol 30 % -
alcohol 50 % -
alcohol 70 % -
alcohol 90 % -
absolute alcohol 1
abs alcohol 2
abs alcohol 3
running water - 1-12 hrs
alcohol 30 % - 30mns - hrs
alcohol 50 % - 30 mns - 3 hrs
alcohol 70 % - 30 mns - 3 hrs
alcohol 90 % - 1-3 hrs
absolute alcohol 1 - 1-2 hrs
abs alcohol 2 - 1-2 hrs
abs alcohol 3 - 1-2 hrs
