LEC 9 - DEHYDRATION Flashcards by CHARLENE SAN JOSE

How well did you know this?

Not at all

Perfectly

dehydration is a Process of removing intercellular and extracellular ______
from the tissue following fixation and prior to wax impregnation

water

How well did you know this?

Not at all

Perfectly

is the removal of water by EVAPORATION from a solid,
semi-solid or liquid

Drying

How well did you know this?

Not at all

Perfectly

Solid tissues should never be allowed to air dry

true or false

true

How well did you know this?

Not at all

Perfectly

involves slow substitution of the water in the
tissue with an organic solvent

Dehydration

How well did you know this?

Not at all

Perfectly

Most dehydrating agents are strong organic solvents that bring about some ___ and ___ of cell components

shrinkage and extraction

How well did you know this?

Not at all

Perfectly

how to prevent the shrinkage and extraction of cell components in dehydration caused by being a strong organic solvent

To minimize these effects, dehydrating agents are used in a graded series for short periods of time, and water is gradually replaced so that violent osmotic changes do not produce distortions

How well did you know this?

Not at all

Perfectly

CHARACTERISTICS OF AN IDEAL DEHYDRATING
SOLUTION

➔ It must dehydrate rapidly without producing considerable shrinkage or distortion of tissues
➔ Not evaporate very fast
➔ Be able to dehydrate fatty tissues
➔ It should not harden the tissue excessively
➔ It should not remove stains
➔ Not toxic to the body
➔ Not be a fire hazard

How well did you know this?

Not at all

Perfectly

general rule in dehydration

, whatever dehydrating agent is used, the
amount in each step should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution

It is also important to keep the dehydration times as brief as possible to minimize the risk of extracting cellular components

How well did you know this?

Not at all

Perfectly

FACTORS TO CONSIDER IN DEHYDRATION

size and nature of the tissue: 30%
type of fixative used
temperature -
ratio

How well did you know this?

Not at all

Perfectly

temperature for dehydration that hastens the dehydration time and is used specially for tissue sections that require urgent examination

37*C

How well did you know this?

Not at all

Perfectly

an example of process wherein we are using 37*C for faster dehydration time

fragmentary biopsies

How well did you know this?

Not at all

Perfectly

perfect ratio for dehydration

not less than 10x the tissue sample

How well did you know this?

Not at all

Perfectly

To ensure complete dehydration:

A layer of anhydrous copper sulfate, about ¼ inch deep is placed in the bottom of the container and covered with filter
paper

How well did you know this?

Not at all

Perfectly

what is the purpose of anhydrous copper sulfate in dehydration

This will accelerate dehydration by removing water from the dehydrating fluid

How well did you know this?

Not at all

Perfectly

Blue discoloration of copper sulfate crystals =

full saturation of dehydrating fluids with water

How well did you know this?

Not at all

Perfectly

example of alcohol as a dehydrating agents

ethyl alcohol (ethanol)
methyl
butyl tertiary
isoprophyl
pentanol (arnyl alcohol)

How well did you know this?

Not at all

Perfectly

boiling point of ethanol

78.3 * C

How well did you know this?

Not at all

Perfectly

can ethanol and wax mix?

they can’t

How well did you know this?

Not at all

Perfectly

in using ethanol

Advisable to remove water gently and allow the tissue to slowly adjust to its removal
○ The more delicate the tissue, the more gently this should be done, but there is no hard and fast rule

true or false

true

How well did you know this?

Not at all

Perfectly

How well did you know this?

Not at all

Perfectly

is ethanol toxic?

How well did you know this?

Not at all

Perfectly

ethanol is miscible in all proportions with water

t or f

true

How well did you know this?

Not at all

Perfectly

used For routine dehydration of tissue

ethanol

How well did you know this?

Not at all

Perfectly

best dehydrating agent (fast acting)

ethanol

For delicate tissues, particularly for embryonic tissues, dehydration starting with ___ethanol is recommended

30%

can we use ethanol in eyes and embryos

yes

can ethanol Mixes with water and many organic solvents and is Reliable

yes

Appears to cause less extraction of cellular components in general than other agents

ethanol

● Penetrates tissues easily ● Not poisonous ● Inexpensive and easily obtained

ethanol

Long periods in absolute ethanol will cause

excessive shrinkage and hardening

ethanol Extracts __ from sections

methylene blue and other thiazine dyes

ethanol Extracts more lipids than __

acetone

Primarily employed for blood and tissue films and for smear preparation

methyl alcohol

Boiling point of butyl alcohol

117.7 *C

Utilized in plant and animal micro-technique ● Slow dehydrating agent

butyl alcohol

advantage of butyl alcohol

● Less shrinkage and hardening than with ethyl ● Excellent for slow processing ● Miscible with paraffin

can butanol and wax mix>

yes

DISADVANTAGES of butyl alcohol

● Odorous ● Slow-acting ● Long periods of infiltration necessary ● Dehydrating power low

Boiling point of tertiary butanol

82.8 * C

Universal solvent

tertiary butanol

act as dehydrating and clearing agent

tertiary butanol

May be used in staining series as a dehydrating agent

tertiary butanol

Mixes with water, ethanol, xylene and paraffin in all

tertiary butanol

DISADVANTAGES of tertiary butanol

● Odorous ● More expensive than butanol ● Primary infiltration must be done in half tertiary butanol and half paraffin, prior to paraffin impregnation ● Reagent tends to solidify at room temperature or below 25°C

boiling point of ISOPROPYL ALCO

82.3*C

no government restriction on its use

isopropyl alcohol

● Sufficiently water-free to use in place of absolute ethanol

isopropyl alcohol

“The best all-around substance for ethyl alcohol”

isopropyl alcohol

Less expensive than tax-free alcohol

isopropyl alcohol

Cannot be used in the celloidin technique since nitrocellulose is insoluble in it

isopropyl

Cannot be used for preparing staining solutions, since dyes are not soluble in it

isopropyl

Boiling point of pentanol

Boiling point: 128°C

pentanol is also called

arnyl alcohol

pentanol is miscible in what substances

Miscible with 90% alcohol, toluene and xylene

this alcohol dissolves paraffin wax

pentanol (arnyl alcohol )

● Toxic ● Cannot be used in poorly ventilate rooms ● Not miscible with water

pentanol (arnyl alcohol )

the strength of the initial alcohol required will depend upon the ___

size and nature of each tissue and fixative used

A very concentrated solution (above 80%) makes tissues

hard, brittle and difficult to cut

85-95% of alcohol concentration is liable to produce____

shrinkage and hardening of tissues leading to distortion

above 80% of alcohol concentration is liable to produce____

hard, brittle, and difficult to cut

95% - absolute of alcohol concentration is liable to produce____

hard tissue surface but the deeper parts are unpenetrated

result of 95% -absolute used of alcohol at starter will caused in tissue impregnation and cutting

UNEQUAL impregnation of tissues with consequently POOR CUTTING of section

how to avoid the consequences of using 95%-absolute alcohol as a starter

To avoid this, 70% or lower concentrations of alcohol, gradually increased to 95% are used

Prolonged storage in lower concentration (below 70%)tends to __the tissue

macerate

DEHYDRATION SEQUENCE FOR SPECIMENS (NOT MORE THAN 4MM THICK)

1. 70% ethanol, 15 minutes 2. 90% ethanol, 15 minutes 3. 100% ethanol, 15 minutes 4. 100% ethanol, 15 minutes 5. 100% ethanol, 30 minutes 6. 100% ethanol, 45 minute

boiling point of acetone

56*C

Cheap, rapid acting dehydrating agent utilized for most urgent biopsies

acetone

acetone Dehydrates in __ to __ hours

½ to 2

is acetone Clear, colorless

yes

can acetone Mixes with water, ethanol and most organic solvents

yes

More miscible with EPOXY RESINS than alcohol but highly flammable and extremely volatile fluid

acetone

Limited only to small pieces of tissues due to its extreme volatility and inflammability

acetone

Rapid in action but penetrates tissues poorly and causes brittleness in tissues that are prolonged dehydrate

acetone

Most lipids are removed

acetone

Produces considerable tissue shrinkage * Not recommended for routine dehydration purposes

acetone

Does not extract methylene blue and other dyes from stained sections

acetone

* May cause less shrinkage of specimen than ethanol * Not reactive with OsO4 remaining in specimen * Miscible with most embedding resins

acetone

do acetone Requires a clearing agent

yes

volume needed for acetone

must be 20 times the tissue

best processing of acetone needs _

Best processing requires a graded series of a mixture of acetone and xylene before one can go into paraffin

* Needs good ventilation * Evaporates rapidly * Flammable

acetone

* Absolute acetone is easily contaminated with water, resulting in __

complete dehydration

substances that are only soluble in dilute solutions of acetone

Uranyl acetate and phosphotungstic acid

Refractive index of dioxane (diethylene dioxide)

1.42

boiling point of dioxane (diethylene dioxide)

101.5*C

Excellent dehydrating and clearing agent

Dioxane (Diethylene Dioxide)

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

dioxane (diethylene dioxide )

Tissue sections dehydrated with dioxane tend to __poorly

ribbon poorly

Universal solvent – it dehydrates and clears

Dioxane (Diethylene Dioxide)

Costs about four times more than absolute alcohol

Dioxane (Diethylene Dioxide)

Graupner’s Method (Time Schedule for Dehydration with Dioxane)

* (1st) Pure dioxane solution = 1 hour * (2nd) Pure dioxane solution = 1hour * (3rd) Pure dioxane solution = 2 hours * 1st Paraffin wax = 15 minutes * 2nd Paraffin wax = 45 minutes * 3rd Paraffin Wax = 2 hours * Embed in mold and in cool water

Tissue is wrapped in gauze bag suspended in dioxane solution and a little anhydrous calcium oxide

Weiseberger’s Method

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

Weiseberger’s Method

dehydration time using Weiseberger’s Method

3-24 hrs

Tissue fixed in chromate fixative, e.g.,____ fluid must be washed in running tap water prior to treatment with dioxane in order to remove the chromate

Regaud’s or Moller’s

* Boiling point of cellosolve

156.4*C

The tissue may be transferred from water or normal saline directly to __and stored in it for months without producing hardening or distortion

cellosolve

Ethylene glycol ether is combustible at what temperature

110°F and 120*F

Following exposure, the reproductive, fetal, urinary and blood systems are particularly vulnerable to their toxic side effects

Cellosolve (Ethylene Glycol Monoethyl Ether)

Boiling point pf triethyl phosphate

215*C

Removes water very readily

triethyl phosphate

Used to dehydrate sections and smear

triethyl phosphate

It both dehydrates and clears tissues since it is miscible in water and paraffin

Tetrahydrofuran (THF)

Can dissolve many substances including fats and is in itself miscible with lower alcohols, ether, chloroform, acetone, benzene and xylene

Tetrahydrofuran (THF)

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

Tetrahydrofuran (THF)

It does not dissolve aniline dyes

tetrahydrofuran

Eye and skin irritant, and prolonged exposure (up to 6 months) may cause conjunctival irritation

Tetrahydrofuran (THF)

Solvents of mounting media

tetrahydrofuran

Better results than most universal solvents

tetrahydrofuran

Added to each 95% ethanol baths as part of dehydration process

4% PHENOL

i t acts as a softener for hard tissues

GLYCEROL ALCOHOL MIXTURE OR IN “MOLLIFLEX”

Tissue processing for ___is commonly accomplished using ethanol as a dehydrating solvent and propylene oxide as a transition fluid

transmission electron microscopy (TEM)

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol as a dehydrating solvent and ____ as a transition fluid

propylene oxide

__is a good substitute for propylene oxide

Acetonitrile

is acetonitrile carcinogenic?

this DA for electron microscopy that Does not interfere with epoxy polymerization

acetonitrile

Resulting cured resins have excellent cutting quality and beam stability

acetonitrile

Also used as a dehydrating agent for cells prepared for scanning electron microscopy (SEM)

acetonitrile