Dehydration (part 1)

Dehydration

This process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation

Dehydrating Agent

Solutions utilized to make dehydration possible

Drying

__ is the removal of water by evaporation from a solid, semi-solid or liquid.

NEVER

Solid tissues should _____ be allowed to air dry.

Dehydration

low substitution of the water in the tissue with an organic solvent.

graded series

To minimize shrinkage effects, dehydrating agents are used in a ____ for short periods of time

strong organic solvents

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

Water

____ is gradually replaced so that violent osmotic changes do not produce distortions.

True (ideal dehydrating solution)

T/F: It should dehydrate rapidly without producing considerable shrinkage or distortion of tissues.

False (fast)

T/F: It should not evaporate very slow.

False (it should)

T/F: It should not be able to dehydrate even fatty tissues.

True

T/F: It should not harden tissues excessively.

False (not)

T/F: It should remove stains.

True

T/F: It should not be toxic to the body.

True

T/F: It should not be a fire hazard.

less than 10 times the volume of the tissue

As a general rule, whatever dehydrating agent is used, the amount in each step should not be ____ in order to ensure complete penetration of the tissue by the dehydrating solution.

anhydrous fluid

Almost any water miscible, ____ can be used as a dehydrating agent providing that it does not damage the tissue proteins and is also miscible with the fluids to be used subsequently

Alcohol

Commonly Used Dehydrating Agents Are:

Al_oh_l is the most common.

Acetone

Commonly Used Dehydrating Agents Are:

A_et_ne

Dioxane

Commonly Used Dehydrating Agents Are:

Di_x_ne

Cellosolve

Commonly Used Dehydrating Agents Are:

Cel_o_ol_e

Triethyl phosphate

Commonly Used Dehydrating Agents Are:

T_ie_hy_ p_osp_a_e

Tetrahydrofuran

Commonly Used Dehydrating Agents Are:

Te_ra_y_rof_ra_

Ethyl alcohol (ethanol)

The alcohol recommended for routine dehydration of tissues. It is a clear, colorless, flammable fluid.

Ethyl alcohol (ethanol)

It is considered to be the best dehydrating agent because it is fast-acting,

Ethyl alcohol (ethanol)

It is not poisonous and not very expensive.

Methyl alcohol

toxic dehydrating agent, primarily employed for blood and tissue films and for smear preparations.

Butyl alcohol

which is utilized in plant and animal micro-techniques, is a slow dehydrating agent, producing less shrinkage and hardening than ethyl alcohol and is recommended for tissues which do not require rapid processing.

not advisable

It is ____ ____ to transfer fixed tissues directly from water or aqueous fixative directly into absolute ethanol.

advisable

It is ____ to remove water gently and allow the tissue to slowly adjust to its removal.

distort the appearance

transfer fixed tissues directly from water will cause rapid removal of water which can ____ of more delicate cells and structures.

more gently

The more delicate the tissue, the ____ this should be done, but there is no hard and fast rule.

70, 95, 100

dehydration starts by placing the fixed specimen in __% ethyl alcohol in water, progressing through __% ethyl alcohol to __% ethyl alcohol.

30

For delicate tissues, particularly embryonic tissues, dehydration starting with __% ethanol is recommended.

NEVER/NO

Under what circumstances should a formalin- fixed tissue be transferred directly to higher grades of alcohol (e.g. 85-95% alcohol)

Concentrated

____ alcohols (95% or absolute) tend to harden only the surface of the tissue while the deeper parts are not completely penetrated.

poor cutting of sections.

This will result in a relatively unequal impregnation of tissue with

consequently ____.

size, and nature of each tissue and fixative used

The strength of initial alcohol required in each concentration will depend upon the ____.

lower

Generally, smaller and more delicate tissues require ____ concentrations and shorter intervals bet ween changes of succeeding ascending grades of alcohol.

concentrated solution

A very ____ (above 80%) makes tissues hard, brittle and difficult to cut.

macerate

Prolonged storage in lower concentrations of alcohol (below 70%) tends to ____ the tissue.

70-80

The tissue may be stored in __-__% alcohol, although not for very long periods of time, since this may later interfere with the staining properties of the specimen.

100 (ethanol and wax don't mix)

Although the tissue reaches the final stage of dehydration in __% ethanol, it's not possible to proceed straight to wax embedding

True

T/F: Ethanol and wax don't mix!

clearing

refers to the property of the solvents used- -when they have a relatively high refractive index and when tissue is immersed in them, the tissue becomes transparent and clear.

1-2 hrs

For tissue preparation, __-__ hours in each solution should be adequate.

shorter

Tissues with a high water content such as embryo tissue would require a much ____ time.

100

To ensure complete removal of water during dehydration, use at least two changes of __% ethanol of at least one half hour each.

95 or 100, 2 hrs

Never leave tissues in __ or __% ethanol more than a total of __ hours or the tissues will harden

70

Tissues can be stored in __% ethanol at any time during an interruption in the routine.

3-12 hrs

10% Formol-Saline:

Alcohol 70%

3-12 hrs

10% Formol-Saline:

Alcohol 90%

1-2 hrs

10% Formol-Saline:

Absolute alcohol (1-3)

1-12 hrs

Zenker/Helly's:

Running water

1-6 hrs

Zenker/Helly's:

Alcohol 30%-50%-70%-90%

1-2 hrs

Zenker/Helly's:

Absolute Alcohol (1-3)

3-12 hrs

Bouin's Fluid:

Alcohol 70%-90%

1-2 hrs

Bouin's Fluid:

Absolute Alcohol (1-3)

1-6 hrs

Susa Cornoy/Formol Sublimate:

Alcohol 90%

1-2 hrs

Susa Cornoy/Formol Sublimate:

Absolute Alcohol (1-3)

1-12 hrs

Flemming's Fluid:

Running water

1/2-3 hrs

Flemming's Fluid:

Alcohol 30%-50%-70%

1-3 hrs

Flemming's Fluid:

Alcohol 90%

1-2 hrs

Flemming's Fluid:

Absolute Alcohol (1-3)

4mm

typical dehydration sequence for specimens not more than _mm thick

15 minutes

How many minutes for:

1. 70% ethanol

2. 90% ethanol

3. 100% ethanol

4. 100% ethanol

30 mins

How many minutes for:

5. 100 minutes

45 mins

How many minutes for:

6. 100% ethanol

37°C

A temperature of __°C will hasten dehydration time and is especially used for tissue sections that require urgent examinations such as fragmentary biopsies.

anhydrous copper sulfate, about 1/4 inch

To insure complete dehydration, a layer of ____, about ____ inch deep is placed in the bottom of the container and covered with filter paper.

blue

A ____ discoloration of copper sulfate crystals will indicate full saturation of dehydrating fluids with water.