LEC 10 - CLEARING Flashcards by CHARLENE SAN JOSE

Transition step between dehydration and infiltration with the
embedding medium

CLEARING

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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

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Most commonly used clearing agents are

xylene, dioxane, chloroform and cedarwood oil

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The clearing agent will make microscopic tissue
preparations __due to their high index refraction

transparent

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__ is often used as an
indication of the effectiveness or completeness of the
clearing process

Optical clarity or transparency

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purpose of clearing

transition between dehydration and infiltration
make the tissue transparent
remove a substantial amount of fat from the tissue

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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

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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

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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

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xylene is also called as

xylol

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clearing time of xylene

30 mns to 1hr

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used for clearing, both for embedding and mounting procedures

xylene (xylol )

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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 )

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most rapid clearing agent, suitable for urgent biopsies

xylene

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xylene is applicable to tissues with maximum __ thichness

5 mm

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can xylene extract aniline dye?

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can xylene be used in celloidin section?

yes, it does nit dissolve celloidin

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this evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and embedding

xylene

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is xylene expensive?

nope

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xylene

highly flammable and should be appropriately stored

true

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if xylene is used for more than 3 hrs, it will make the tissues __

excessively hard and brittle

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what type of tissue is xylene not applicable for as it can cause hardening and shrinkage to them

nervous tissue and lymph nodes

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xylene can iritate

eyes, nose, respiratory tract

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xylene will become milky if an incomplete __ is immersed in it

dehydration

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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

is cedarwood oil as clearing agent expensive?

very expensive

is aniline oil used as a routine clearing agent

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