Dehydration Flashcards by John Lesther Alonsagay

Fixation > Decalcification > ___

Dehydration

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Removal of fixative and water from the tissue

Dehydration

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Dehydrating time must be ___ as much as possible

brief

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Tissue to fixative ration is?

1:10

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Most fixatives are ___ solutions

aqueous

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Most dehydrating agents are ___ that bring about some shrinkage and extraction of cell components

strong organic solvents

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To minimuze thrse effects dehydrating agents are used in a graded series for ___, and water is gradually replaced so that violent osmotic changes do no produce distortions

graded series for short periods of time

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Characteristics of ideal dehydrating solution

● Rapid action with minimal tissue shrinkage and
distortion
● Should not evaporate fast
○ If it evaporates the tendency is the water
will be retained. Water will not be included
in the evaporation process
● Able to dehydrate even fatty tissues
○ Fatty tissues contain liquid particles that
are difficult to evaporate
● Should not hardened tissues excessively
● Should not remove stains
○ Dehydrating agent is capable of removing
stains we cannot appreciate the cellular
details of our tissue sample
● Non-toxic and not a fire hazard

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But every dehydrating agent is used the amount in each step shoyuld not be less than __ times the volume of the tisse in order o ensure complete penetration of the tissue by the dehydratingb agent

10 times

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Done in ascending grades to avoid distortion of tissue

Alcohol

70% ROH > 90% ROH > 100% ROH

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For delicate tissues start at __%

30%

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For delicate tissues start at 30%:

○ for thin and fragile tissues.
○ If it’s exposed to higher concentrations
of alcohol it causes shrinkage,hardening and brittlen

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__*C will hasten dehydratiuon rate for urgent exams

37*C

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To ensure complete dehydration, add at least ___ deep layer of ___ at bottom of container and cover with filter paper

1/4, anhydrous Cu2SO4

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___ of coper sulfate crystals windicates full saturation of dehydrating fluids with water does alcohol must be change with a fresh solution

Bluing

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Storage of tissue in ___ alcohol can be
done but not for a long time since it may
interfere with the staining properties of
specimens.

70%-80%

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best dehydrant because it is fast acting, mixes
with water and many organic solvents, and
penetrates tissues easily

Ethanol 78.3*C

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not poisonous and not very expensive

Ethanol 78.3*C

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clear colorless and flammable

Ethanol 78.3*C

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Toxic
Blood and smear prep

Methanol

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It is the alcohol recommended for routine dehydration of tissues

Ethanol 78.3*C

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It is a toxic dehydrating agent

Primarily employedw for blood and tissure films and smearing preperations

Methanol

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Slow
Plant and animal microtechnique

Butyl Alcohol 117.3*C

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It is a slow dehydrating agent producing less shrinkage hardening than ethyl alcohol

Butyl Alcohol 117.7*C

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Recommended for tissues which do not require rapid processing

Butyl alcohol 117.7*C

70% ethanol 90% ethanol 100% ethanol 100% ethanol 100% ethanol 100% ethanol

70% ethanol - 15 min 90% ethanol - 15 min 100% ethanol - 15 min 100% ethanol - 15 min 100% ethanol - 30 min 100% ethanol - 45 min

fast the hydrant

Acetone

Time for acetone

30 mins to 2 hrs

Highly flammable, evaporates fast

acetone

not recommended for routine dehydration purposes

Acetone

Boiling point of acetone

56*C

Is a clear colorless fluid that mixes with water ethanol and most organic solvents

Acetone

It is more miscible with epoxy resins than alcohol but is highly flammable and requires considerable care in handling

Acetone

It is a rapid in actipon but penetrates tissues poorly and causes brittleness and tissues that are placed in acetone for long period of time

Acetone

Because of considerable tissue shrinkage produced, it is not recommended for routine dehydration purposes

Acetone

Diethylene dioxide

Dioxane 4-Cellosolve

Dehydrating and clearing agent

Dioxane 4-Cellosolve

Graupner pure dioxane > paraffin

Dioxane 4-Cellosolve

Weisenberger's method

Wrapping tissue in gauze bag and suspended in a bottle containing dioxane with anhydrous calcium oxide or quicklime

Weisenberger's method

Water is displaced from the tissue by dioxane and intern absorbed by calcium oxide or quicklime

Weisenberger's method

Wesenberger's method dehydration period ranges from

3-24 hours

Expensive and extremely dangerous

Dioxane 4-cellosolve

It is an excellent dehydrating clearing agent and readily miscible in water, melted paraffin, alcohol and xylol.

Dioxane 4-Cellosolve

It produces less tissue shrinkage as compared to alcohol dehydration

Dioxane 4-Cellosolve

Tissues can be left in this reagent for long periods of time without affecting the consistency or staining properties of the specimen

Dioxane 4-Cellosolve

Because dioxane is miscible with both water and paraffin tissues may be placed directly into the solution after washing out

Dioxane 4-Cellosolve

it's vapor produces a cumulative and highly toxic action in man; hence it should not be used routinely

Dioxane 4-Cellosolve

Tissues which have been treated with the chromate fixative should be thoroughly washed in running tap water prior to treatment with dioxin in order to remove the chromate.

Weisenberger's method

Weisenberger's method example:

Regaud's or Moller's fluid

Ethylene glycol monomethyl ether

Cellosolve

Rapid prolonged storage does not cause distortion to tissues

Cellosolve

Toxic inhalation, skin irritant, and combustible at __ to __ F

110 to 120F

Alternative of cellosolve:

proylene glycol ethers

This dehydrates rapidly

Cellosolve

Tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months without producing hardening or distortion

Cellosolve

Soluble in alcohol water after beans in chloroform acetone and xylene

Triethyl Phosphate

Produces minimum shrinkage

Triethyl Phosphate

Not commonly available in the market

Triethyl phosphate

● Dehydrant and clearing agent ● Dissolves fats ● Staining procedures or improved ● Eye and skin irritant (conjunctival irritation)

Tetrahydrofuran (THF)

It is a reagent that both dehydrates and clears tissues

Tetrahydrofuran

Since it is miscible in both water and paraffin it can dissolve many substances including fats and is in self miscible with lower alcohols after chloroform acetone benzene and xylene

Tetrahydrofuran

It may be used for demixing, clearing, and dehydrating paraffin sections before and after staining

Tetrahydrofuran

it causes less shrinkage and easier cutting at sections with fewer artifacts

Tetrahydrofuran

It does not dissolve out aniline dyes, in fact most staining procedures give improved results with tetrahydrofuran

Tetrahydrofuran

Additives to dehydrating agents

1. 4% Phenol 2. Glycerol/alcohol mixture

dehydrating solvent

Ethanol

Transition fluid

Propylene oxide

solubilizes lipids

Ethanol

completely miscible with embedding resins and because of it's low viscosity, it can infiltrate tissues readily and reduced the viscosity of embedding resin mixtures

PROPYLENE OXIDE

a good substitute for propylene oxide

Acetonitrile

it is reported to be non-carcinogenic less toxic and not as flammable as propylene oxide

Acetonitrile

polymerization and the resulting cured resins have excellent cutting quality and beam stability

Acetonitrile

an excellent dehydrating agent whose use does not necessitate modification of current techniques

Acetonitrile

low solubility of phospholipids ⇒ limits the loss of membrane lipids ⇒ a better preservation of tissue features.

Acetonitrile

it is also used as a dehydrating agent for cells prepared for Scanning Electron Microscopy

Acetonitrile

solutions utilized to make dehydration possible

Dehydrating agents

magpino-pino/ puro

Maceration

Universal solvent

Tertiary butanol 82.7 *C

May be used in staining series as a dehydrating agent

Tertiary butanol 82.7*C

Excellent substitute for ethanol

Isopropanol 82.3*C

Less shringkage and hardening than ethanol

Isopropanol 82.3*C

No government restrictions

Isopropanol 82.3*C

Sufficiently water-free to use in place of absolute ethanol

Isopropanol 82.3*C

Lillie considers it the best all around substitute fore ethyl alcohol

Isopropanol 82.3*C

Miscible with 90% alcohol, toluene and xylene

Pentanol 128*C

Dissolves paraffin wax

Pentanol 128*C