M4: dehydration, decalcification, clearing

50-80%

The human body would contain roughly about — water depending in age of person

Dehydration

To remove the fixative and water from the specimen; replace them with dehydrating fluid in preparation for infiltration

Dehydration

Process of removing intracellular and extracellular water from tissue following fixation and prior to wax infiltration

10:1

Dehydrating agent to tissue ratio

Dehydrating agent

Soltns used to facilitate removal of water in tissues

To prevent violent osmotic changes to tissues that may produce distortions

To prevent shrinkage and extraction of cell components brought by strong organic solvents

Why do we use ascending grades of alcohol in dehydration

70%, 95%, 100%

Initial dehydration concentration for routine tissue processing

30% onwards

Alcohol concentration for delicate tissues

Should NOT harden tissues excessively

Should NOT remove stains

Should NOT be toxic to the body

Should NOT be fire hazard

Should not evaporate very fast

Characteristics of an ideal dehydrating soltn

Ethanol/alcohol

acetone

Dioxane

Triethyl phosphate

Tetrahydrofuran

Cellosolve

Commonly ised dehydrating egnts

ethanol/ethyl alcohol

Recommended for routine dehydration of tissues.

Considered as the best agent because its fast acting, mixes with water and organic solvents and penetrates easily

Ethanol

A type of alcohol that is non poisonous, flammable and not expensive

Hard, brittle, difficult to cut

> 80% alcohol causes tissues to be

Intefering with staining

Prolonged storage of tissue and dehydrating agents results to

Methanol/ methyl alcohol

An alcohol dehydrating agent Primarily employed for blood and tissue films and for smear preparations.

Toxic dehydrating agent.

Butanol / butyl alcohol

A dehydrating agent Utilized in plant and animal microtechniques and is Recommended for tissues which do not require rapid processing.

Butanol (butyl alcohol)

It has a rancid or sweet odor

Butanol

A type of alcohol that produces less shrinkage and hardening than ethanol

Tertiary butanol

A type of alcohol that is universal solvent that mixes with water, ethanol, xylene, and paraffin.

Odorous; tends to solidify at room temp or below 25C

isopropanol (isopropyl alcohol)

Its an excellent substitute for ethanol as it provides less shrinkage and hardening

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

Isopropanol (isopropyl alcohol)

Best clearing agents for microwave technique

Pentanol

A type of dehydrating agents Miscible with 90% alcohol, toluene, and xylene And Dissolves paraffin wax

Toxic; cannot be used in poorly ventilated rooms

Cheap, rapid acting, dehydrating agent

Penetrates tissue poorly, causes brittleness in tissues upon prolonged exposure

Advantage and disadvantage of acetone

Acetone

30 mins to 2 hours

used for urgent biopsies (time)

excellent universal solvent

Ribbon poorly, expensive, extremely dangerous

Advantage and disadvantage of dioxane

Dioxane

tissues can be left for long period of time without affecting the staining qualities or consistency of the specimen.

Diethyl dioxide

Dioxane is also called as

Ethylene glycol monoethyl ether

Cellosole is also called a s

rapid dehydrating agent

expensive and rapidly absorbs water from air, and is combustible at 100-120 F, highly toxic

advantage and disadvantage of cellosolve

Triethylphosphate

A type of dehydrating agent that Removes water very readily and produces very little distortion and hardening of tissue.

Used to dehydrate sections and smears following certain stains and produces minimum shrinkage

alcohol, water, ether, benzene,

chloroform, acetone, and xylene

Triethyl phosphate is soluble in

Universal solvent and can dissolve many substance like fats, less shrinkage

Toxic if ingested or inhaled

Advantage and disadvantage of tetrahydrofuran

Water and paraffin

Tetrahydrofuran is miscible in

Tetrahydrofuran

It has offensive ether like odor

ETHANOL

PROPYLENE OXIDE

ACETONITRILE

Dehydrating agents for EM

Ethanol

Solubilizes lipids

Propylene oxide

Miscible with embedding resins and it can infiltrate tissues readily and reduce the viscosity of embedding resin mixtures.

Acetonitrile

Good substitute for propylene oxide.

Excellent dehydrating agent.

Low solubility of phospholipids

Anhydrous copper sulfate

what is added to ensure complete dehydration

Anhydrous copper sulfate

speeds up the process of dehydration as it removes water from the dehydrating fluid color change from white to blue indicates the need to replace the dehydrating fluid with a fresh solution

Bones

Teeth

Tuberculous organs

Atherosclerotic blood vessels

Spx for decalcifications

20:1

Decalcifying agent to tissue ratio

Decalcification

Removal of Ca ions or lime salts from a bone oR calcified tissue following fixation and prior to infiltration

Surface decalcification

What to do when unsuspected mineral deposits are found in soft tissues

Remove tissue block w/ partially decalcified bone from chuck

Place face down on a pad of cotton/ gauze w/ 10% HCL for 1 hr

Rinse to remove corrosive acid and re section

Enumerate surface decalcification

Acid (strong or weak)

Ion exchange resin

Chelating agents

Electrical ionization

Three main types of decalcifying agents

Aq 10% nitric acid

Formol nitric acid

Perenyi’s fluid

Phloroglucin Nitric acid

What are the different types of nitric acid

Aq Nitric acid soltn 10%

12-24 hrs

A type of nitric acid used for urgent biopsies ( time) and is good for nuclear staining with minimal distortion

It also imparts yellow color due to formation of nitrous acid

Perenyi’s fluid

2-7 days

Both a decalcifying agent and tissue softener and is not used for urgent buopsies (time)

Phloroglucin nitric acid

12-24 hrs

Most rapid decalcifying agent and is recommended for urgent work (time)

Hydrochloric acid

Inferior compared to nitric acid in its role as a decalcifying agent.

Slower action and areater distortion of Tissue produces

Formol nitric acid

1-3 days

A type of strong acid that must be used inside a fume hood and is rapid (time)

HCl

Recommended for surface decalcification of tissue blocks.

Von ebner’s fluid

A type of HCL that is recommended for teeth and bine and is moderately rapid

Formic acid

Trichloroacetic acid

Chromic acid

Citric acid citrate buffer solution

Weak organic acids

Formic acid

Moderate acting decalcifying agent.

Produces better nuclear staining with less tissue distortion.

Recommended for routine decalcification of postmortem Research tissues

2-7 days

Days Staining for formic acid

Trichloroacetic acid

4-8 days

Permits good nuclear staining.

Does not require washing out.

Weak decalcifying agent; not recommended for urgent examinations.

It is also both a decalcifying agent and fixative and stains for how many days

Trichloroacetic acid

Used for small spicules of bine

Chromic acid

May be used both as fixative and decalcifying agent.

Maybe used for decalcifying minute bone spicules.

Nuclear staining with hematoxylin is inhibited.

CITRIC ACID-CITRATE BUFFER SOLUTION

Permits excellent nuclear staining and cytoplasmic staining.

Does not produce cell or tissue distortion.

Action is too slow for routine purposes

Chelating agents

Substances which are Combined with calcium ions to form weakly dissociated complexes and facilitate removal of calcium salt.

EDTA

Most common chelating agents

Ph at 7-7.6

Optimum binding capacity of EDTA

Damages alkaline sensitive protein linkages

A ph of >8 causes what to tissue

Chelating agent will not bind well to calcium

A ph of <5 causes what

Versene

Commercial name for edta

1-3 weeks

Small specimen for EDTA (weeks)

6-8 weeks

Dense spx for edta (weeks)

Ammonium form of polystyrene resin

-hastens decalcification by removing Cazt ions from decalcifying solution with formic acid (increased solubility)

-not for strong mineral acids

. Concentration

• Fluid access

• Temperature

• Agitation

• Size & consistency

Factors affecting rate of decalcification

Ectrophoresis

• cationic CA2 are attracted to cathide

• - they are removed by makjg use of an e,ectrical charge

Heat

Electrolytic reaction

How to shorten decalcification by electrophoresis

88% formic acid

What does electrophoresis use

Physical test

Chemical test

Radiographic test

Methods to check if end point of decalcification has been reached

Physical test

method in checking decalcification that may be subjective, vague or inaccurate

Chemical test

Method to check end point of decalcification that is simple, convenient and reliable

Discarded fluid

In chemical test, what is tested

Ammonium hydroxide

Ammonium oxalate

30 mins

Red: blue lithmus paper → neutraize with — (Red - blue, then add — stand for —

Radiographic test

Type of method to check end point of decalcification that is MOST ideal, sensitive, and reliable BUT very expensive.

it is Not recommended for mercuric chloride-fixed tissues due to radio-opacity which will interfere wit

Neutralize acid

Rinse

Store

What to do after decalcification

saturated lithium carbonate

5-10% aq. Sodium bicarbonate

What is used to neutralize acid

Formol saline containing 15% sucrose

PBS with 15-20% sucrose

Frozen section (4C)

What is used to store tissues after decal

Tissue softener

What should be used if hard tissues are damaged with microtome knives

12-24 hrs

Selected oortions for tissue softener are left in the fluid for —

Perenyi’s fluid

4% aq phenol soltn

Molliflex

2% HCl

1% HCl in 70% alcohol

Examples of tissue softeners

Molliflex

Produces soapy and swollen effect on tissues

Clearing

Process of removing the dehydrating fluid from tissue to replace with a soltn miscible with wax

Remove alcohol

Impart clarity

Eliminate fat

Function of clearing agents

10:1

Clearing agents to tissue ratio

Should be miscible with alcohol

Should not evaporate quickly

Make tissue translarent

Characteristics of good clearing agents

Xylene

Most widely used clearing agents during tissue processing and dewaxing agents during staining. It is also cost effective and rapid acting

30 mins

1 hr

If tissues are <5mm thick, xylene clears tissues for about — to —

Toluene

Substitute for xylene or benzene and is fairly rapid (time) it does not make tissue brittle or hard even after its left for 24 hrs.

Toluene

It acidifies in partially filled vessel; fumes are toxic upon prolonged exposure

Benzene

Used to be popular clearing agents but is carcinogenicand is highly rapid (time)

Chloroform

6-24 hrs

Slower than xykene (time) but causes less brittleness

Usually used for nervous tissues, lymph nodes,

Embryos, and large tissues

It does not make tissue transparent

Cedarwood oil

2-3 days, 5-6 days if celloidin

Used to clear both baraffin nd celloidin section. It req 2 changes

Its extremely slow (time), milky upon prolonged storage, difficult to achieve uniform quality

Aniline oil

Recommended for clearing embryos, insects, and very delicate specimens

Clove oil

Causes minimum shrinkage of tissues

Expensive, unsuitable for routine use, infiltration is also slow and difficult after clearing, tissues become brittle, aniline dyes are removes, celloidin is dissolved