DECALCIFICATION - DEHYDRATION - CLEARING Flashcards
● Not commonly done in the Histopath Laboratory
● Only certain hard human tissues need decalcification
(bone, teeth, nails)
● Same ratio in fixation and decalcification
DECALCIFICATION
● High concentrations and greater amounts of fluid will increase the speed of the process
● More concentrated acid solutions decalcify bone more rapidly but may cause more harm to the tissue.
DECALCIFICATION
FD CIETS SML
Fixation → Dehydration → Clearing → Impregnation, → Embedding → Trimming → Sectioning → Staining → Mounting → Labeling
● Heat can hasten _____________
but can also damage the tissue
decalcification
Recommended ratio of fluid to tissue volume for decalcification
20:1
Ideal time required for Decalcification
24-48 hours
● Dense bone tissues usually
require up to ___ days or longer
in order to complete the
process.
14 days
At ___C impaired nuclear staining of Van Gieson’s stain for collagen fibers.
At 37 C
Optimum temperature = Decalcification
Room temp (18-30°C)
At __ C = tissue will undergo
complete digestion within
24-48 hours.
55 C
DECALCIFYING AGENTS (4)
● Acids
● Chelating Agents
● Ion Exchange Resins
● Electrical Ionization
● Most commonly used, but not the best decalcifying agent
Nitric Acid
○ EDTA (Ethylene Diamine Tetraacetic Acid)
○ Used as an anticoagulant to bind calcium
Chelating Agents
2 examples of Nitric Acid
○ Perenyi’s Fluid
○ Phloroglucin-Nitric Acid
- functions as both decalcifying agent and tissue softener
Perenyi’s Fluid
most rapid decalcifying agent
Phloroglucin-Nitric Acid
- best general decalcifying agent
5% Formic Acid
● Both a fixative and decalcifying agent
● Recommended for small pieces of bones and teeth
Formic Acid
Example of Hydrochloric Acid
Von Ebner’s Fluid
What is the best general decalcifying agent?
5% Formic acid
recommended for teeth and small pieces of bones
Von Ebner’s Fluid
What kind of acid makes up Von Ebner’s?
Hydrochloric acid
What is the most commonly used decalcifying agent?
Nitric Acid
3 WAYS TO MEASURE DECALCIFICATION
- Physical/Mechanical Test
- X-ray/Radiological Method
- Chemical Method
● Inaccurate since it is very subjective
● Commonly used in schools since it is cheap.
Physical/Mechanical Test
● Done by touching or bending tissue with the fingers
● Alternate method: pricking the
tissue with a fine needle or a probe
Physical/Mechanical Test
● For unduly hard tissues that may damage the microtome knives
● Utilized to soften tough tissue
TISSUE SOFTENERS
● Most ideal, Most Sensitive, Most
reliable method.
● The best way to measure the extent of decalcification
X-ray/Radiological Method
● Very Expensive; not commonly done
● Uses X-ray to measure the extent of decalcification
X-ray/Radiological Method
● Calcium Oxalate test
● Simple, reliable, recommended for routine purposes
Chemical Method
● Must not be skipped so that the next step, Impregnation (Paraffin Wax), to completely fill the spaces in between the specimen.
- Water and Paraffin Wax does not mix
DEHYDRATION
(4) Examples of TISSUE SOFTENERS
● 4% aq. Phenol.
● Molliflex
● 2% HCl
● 1% HCl in 70% alcohol
● We want the impregnating medium to be able to penetrate
the spaces between the organ and tissue
● ___________ fluids are generally used in increasing strengths (all the aqueous tissue fluids are removed but with little disruption to the tissue due to diffusion currents)
DEHYDRATION
● Aims to remove fixative and water from the tissue and replacing them with dehydrating fruit in preparation for impregnation
DEHYDRATION
● Used in increasing strengths or
ascending grades
○ In order for the tissue to not be
distorted
○ 70% Alcohol → 80% Alcohol →
90% Alcohol → 100% Alcohol (2x)
Alcohol
● MOST COMMONLY USED DEHYDRATING AGENT
● Also used for fixation
Alcohol
For routine dehydration of
tissues. BEST DEHYDRATING
AGENT
Ethyl Alcohol
● Used for blood and tissue films
Methyl Alcohol
● Used for plant and animal microtechniques
Butyl Alcohol
● Less shrinkage and hardening than ethanol
Isopropyl Alcohol
● Denatured Alcohol
● Ethanol + Small amount of Methanol
Industrial Methylated Spirit
● Both Fixative and Dehydrating Agent
● Also used for diffusible enzymes, brain (for rabies diagnosis)
Acetone
● Sometimes, instead of using 100% again at the end of dehydration, ___________ can be used.
Acetone
● Both dehydrating and clearing agent at the same time
● Diethylene Dioxide and
Tetrahydrofuran
(6) COMMONLY USED DEHYDRATING AGENTS
- Alcohol
- Acetone
- Dioxane
- Tetrahydrofuran (THF)
- Cellosolve (Ethylene Glycol Monoethyl Ether)
- Triethyl Phosphate
● Acts as a tissue softener when added with 95% ethanol
● If the tissue is already soft, no need to add this
4% Phenol
ADDITIVES TO DEHYDRATING AGENTS (2)
- 4% Phenol
- Anhydrous Copper Sulfate
● BOTH dehydrating and indicator of H2O (water) content (100% ETOH)
Anhydrous Copper Sulfate
Anhydrous Copper Sulfate
● If copper sulfate is dry = ______ in color
● Has water/Hydrated = ________ in color (incomplete dehydration)
White
Blue
A typical dehydration sequence for specimens NOT more than 4 mm thick would be:
- 70% ethanol - 15 min
- 90% ethanol - 15 min
- 100% ethanol - 15 min
- 100% ethanol - 15 min
- 100% ethanol - 30 min
- 100% ethanol - 45 min
NOTE AND RECALL:
Higher concentration of alcohol = water concentration will decrease (slowly removing the water from interstitial spaces so tissues will not wrinkle)
After you remove the water, alcohol is now inside the interstitial spaces after the dehydration process
What is the Best General Decalcifying Agent?
5% Formic Acid
What is considered to be the Best General Tissue Fixative?
10% Buffered Neutral Formalin
What is the Best Way to Measure the Extent of Decalcification?
X-Ray or Radiological Method
What is the Best Dehydrating Agent?
Alcohol
Most commonly used Impregnating Medium?
Paraffin Wax
■ Not soluble to water
■ Not soluble to alcohol
■ Needs to be the one to fill the spaces in the specimens so that when it is cut in the microtome, there will be no empty holes.
Paraffin Wax
Aka: DEALCOHOLIZATION
CLEARING
● Making the tissues transparent
● Process of replacing the dehydrating fluid with a fluid that
is miscible with BOTH the dehydrating fluid and the
impregnating/embedding medium
CLEARING
CLEARING AGENTS SUITABLE FOR ROUTINE USE:
● Most commonly used
● Most rapid
Xylene/Xylol
● Some of the other uses:
○ Removes alcohol from tissue
section, fat, and paraffin
○ For cleaning tissue processors
Xylene/Xylol
If xylene turns MILKY =
___________ DEHYDRATION
INCOMPLETE DEHYDRATION
● Toxic to the liver after prolonged inhalation and it does not make the tissues transparent
Chloroform
● Used for TOUGH tissue
specimens
Chloroform
especially recommended for CNS tissues and cytological studies,
particularly of smooth muscles and skin
Cedarwood oil and clove oil
Can be utilized to replace Xylene
because it is not as toxic and is
cheaper
Citrus Fruits Oils
Recommended for clearing
embryos, insects, and very
delicate specimens
Aniline Oil
Why do we need to remove the alcohol?
The paraffin wax that will be utilized later on is not soluble to alcohol
Aka: INFILTRATION
IMPREGNATION
● Process of replacing the clearing agent with the infiltrating medium
● The medium used to infiltrate the tissue is usually the same medium used for embedding
IMPREGNATION
FOUR TYPES OF TISSUE IMPREGNATION AND EMBEDDING MEDIA
1 Paraffin
2 Celloidin (Collodion)
3 Gelatin
4 Plastic
● The man who introduced paraffin wax embedding:
Butschlii
● Simplest, most common and the BEST infiltrating/embedding medium
● NOT recommended for fatty tissues
PARAFFIN
Temperature of paraffin oven =
55-60 C
Paraffin oven must be maintained at a temperature
2-5 C above the MP (Melting Point) of the paraffin wax
● MP: 56-57 C
● Mixture of highly purified paraffin and synthetic plastic polymers
● More elastic and resilient than
paraffin
● SPECIMEN: For large dense tissue blocks such as bones and brain
Paraplast
● MP: 56-58 C
● Less brittle and less compressible than paraplast
Embeddol
● Recommended for embedding
eyes
Bioloid
A product of paraffin, containing
rubber, with the same property as paraplast
Tissue Mat
● MP: 46-48 C
● Harder than paraffin
● NOT soluble in water
● Soluble in 95% ETOH (ethyl alcohol) and other clearing agents
● Can be used for impregnation
without prior clearing of the tissue
○ as it is soluble in ETOH
Ester Wax
● MP: 38-42 C or 45-56 C
● Mostly polyethylene glycols
○ No need to perform dehydration and clearing
Water-soluble waxes
Suitable for many enzyme
histochemical studies
Carbowax
● Purified form of nitrocellulose
CELLOIDIN (COLLODION)
● Suitable for specimens:
○ With large hollow cavities
○ Hard and dense tissues (bones and teeth)
○ Large tissue sections of the whole embryo
CELLOIDIN (COLLODION)
2 METHODS FOR CELLOIDIN IMPREGNATION
- Wet celloidin
- Dry celloidin
Recommended for bones, teeth, large brain sections, and whole organs/organs with large cavities
Wet celloidin
Preferred for processing whole eye sections
Dry celloidin
Name of the other impregnating medium that may also be for the eye section?
Bioloid
Fixative For Embryos
Bouin’s solution
Impregnation Medium
For Embryos
Celloidin
Impregnating Medium
For Eyes
Bioloid or Celloidin
● Share the same characteristic as the carbowax
● Rarely used except when dehydration is to be avoided
● Used when tissues are for histochemical and enzyme
studies
GELATIN
● Embedding medium for delicate specimens and frozen
tissue sections
○ When we are using frozen tissues usually it is fresh meaning there is still a water inside
■ Even though there is still water inside the gelatin can still pass through
GELATIN
● Water-soluble impregnating medium
○ Not a wax
○ No need for dehydration
Gelatin
Why are we allowed not to do dehydration if we intend to use gelatin as our impregnating
medium?
Because gelatin is a water soluble
impregnating medium
Gelatin → Water-Soluble Impregnating Medium
Why do we remove water?
○ To prepare the tissue into the paraffin wax impregnation
○ Paraffin wax is not soluble to water
What is the impregnating medium that we can use for impregnating frozen sections?
Gelatin
Used for Electron Microscopic Studies
PLASTIC / RESIN
PLASTIC / RESIN are CLASSIFIED INTO 3 categories
1 Epoxy
2 Polyester
3 Acrylic
Brand name: Bisphenol A
Araldite
Brand name: Glycerol
Epon
Brand name: Cyclohexene dioxide
Spurr
Most commonly used routine tissue impregnation medium
Paraffin wax
Impregnation medium for large dense tissues like bones, brain and whole organs (embryo
Celloidin
Impregnation medium for
frozen sections
Gelatin
Impregnation medium for
electron microscopic studies
Plastic / Resin
Since Epon is a glycerol, and a
glycerol is a form of epoxy, Epon
therefore is used for _______
microscopic studies
Electron
What is the use/purpose of EPON?
Utilize for electron microscopic studies
■ One of the impregnating medium to utilize for that purpose
■ A kind of glycerol epoxy plastic
impregnating medium
■ A brand name of glycerol epoxy
plastic impregnating medium
