HISTOPATHOLOGY LABORATORY •

TECHNIQUE
Representative
HISTOLOGY areas are biopsied
• Study of normal tissues in a wedge
HISTOPATHOLOGY fashion.
• Margins should
• Study of abnormal, diseased tissues extend into normal
• It is performed by examining a thin tissue tissue on the deep
section under light microscope surface.
• Necrotic tissue
• Also called as microscopic anatomy should be avoided.
HISTOTECHNIQUE • A narrow deep
• Consists of a number of procedures that specimen is better
allow visualization of tissue and cell than a broad
shallow one.
microscopic features and recognize
• removal of the
specific microscopic structural changes of whole organ
diseases. • implies the
HISTOTECHNOLOGY complete removal
of the lesion
• Art and science performed by the medical INDICATIONS
technologist/histotechnologist to produce a • Should be
tissue section of good quality that will employed with
small lesions less
enable the pathologist to diagnose the
than 1 cm.
presence or absence of a disease or • The lesion on
malignancy EXCISIONAL BIOPSY clinical exam
appears benign.
• When complete
SPECIMENS SUBMITTED FOR excision with a
HISTOPATHOLOGICAL EXAMINATION margin of normal
tissue is possible
dead source, systemic
without mutilation.
dissection of a dead body
AUTOPSY TECHNIQUES
to determine the actual
cause of death. The entire lesion with 2 to 3
mm of normal appearing
BIOPSY/SURGICAL
from living source tissue surrounding the
SPECIMENS lesion is excised.
• removal of a part involves the use of a
of a whole organ special type of needle by
• If a lesion is large which tissues can be taken
or has different NEEDLE BIOPSY from an organ without
characteristics in making an incision.
various locations
more than one involves the use of
area are needed ordinary syringe and
to be sampled needle to aspirate from a
ASPIRATIONAL BIOPSY cyst to find out the nature
INDICATIONS of its contents.
• Size and
INCISIONAL BIOPSY limitations SMEAR
• Hazardous involves scraping of cells
BIOPSY/EXFOLIATIVE
locations of the from lining epithelium
CYTOLOGY
lesions
• Great suspicion of
malignancy

DISADVANTAGES
Crush, splits and
hemorrhage are the
artifacts most frequently
found in incisional biopsy
IN HANDLING PATHOLOGIC SPECIMENS FOR may lead to
HISTOTECHNIQUE PROCESSING, THE mislabeled
specimens
FOLLOWING CRITERIA ARE OBSERVED: causing
1. The specimen important
cannot be patient
accepted in assessment and
the pathology treatment
laboratory confusion.
unless formal
request has • considered as
been properly the most critical
accomplished step in tissue
. processing,
2. Immediately The following are to be must be done
upon receipt recorded in the logbook: adequately.
of the • Patient’s whole name • Done to avoid
specimen, a • Birthday autolysis
laboratory • Age and sex 2. FIXATION PRIMARY GOAL
number is • Address to preserve tissue
assigned and • Civil status morphology
recorded in • Tentative diagnosis (if SECONDARY GOAL
the logbook there’s any)
to harden the tissue for
with proper • Examination desired
easier cutting, to protect
checking to • Name of the the tissue from the
avoid requesting Physician remaining tissue
confusion and
processing steps
possible
• procedure
interchanges.
whereby
calcium or lime
3. Any specimens which are not in good condition
salts are
must reported to the pathologist as soon as
removed from
possible.
tissues, not all
4. The pathologist must be notified when a
specimens will
specimen is received for the proper description
undergo this
and other procedures before fixation.
step.
5. Careful processing must be done with great
• Should be done
easy by the technologist.
after fixation
6. All infectious specimens, such as tuberculosis and before
specimens, should be placed in an antiseptic
3. DECALCIFICATION impregnation
solution such as formalin and Lysol. GOAL
7. As soon as the medical technologist receives
• to ensure &
the specimen, he should check the pertinent
facilitate normal
information written on the patient’s request
cutting of
form. He has to assign a specimen number and
sections
enter it in the corresponding logbook.
• to prevent
obscuring the
microanatomic
detail of such
STEPS IN ROUTINE HISTOTECHNIQUE/TISSUE sections by bone
PROCESSING dust and other
• is one of the first cellular debris
important steps 4. DEHYDRATION process of gradual
in all removal of intracellular
histopathologic and extracellular waste,
techniques. Its water from the tissue
purpose is to 5. CLEARING process of removing the
identify properly alcohol used as a
all specimens dehydrating fluid from the
without writing tissue and is replaced
1. NUMBERING the name of the with a substance that will
patient on the dissolve the wax, should
accompanying not be more than 10x the
tag. volume of the specimen.
• Extra care 6. IMPREGNATION/ process whereby the
should be INFILTRATION clearing agent is
practiced when completely removed
assigning from the tissue and is
specimen replaced by a medium
number since it that will completely fill
 all the tissue cavities,
should be 25x the volume
TISSUE PROCESSING STEPS
of the specimen (FIXATION-INFILTRATION)
process by which the FIXATION
impregnated tissue is Primary Goal: to preserve the morphology and chemical
placed into a precisely integrity of the tissue as close to the original as possible.
arranged position in a Secondary Goal:
mold. 1.) to harden the tissue for easier cutting
2.) to protect the tissue from trauma of further handling
ORIENTATION 2 TYPES OF FIXATIVE:
precise positioning of the ADDITIVE:
7. EMBEDDING tissue section inside the The chemical component of the fixative becomes part of
molder the tissue.
MOLDING Ex: Mercuric fixatives, Formalin
NON-ADDITIVE:
provides shape to the
The chemical component of the fixative does not become
tissue sample part of the tissue but
BLOCKING alters the tissue component. Ex: Alcohol fixatives
rapid solidification of FACTORS INVOLVED IN FIXATION
tissue block 1. Hydrogen ion concentration
CASTING 2. Temperature
removal of tissue block 3. Thickness of sections
from molder 4. Osmolality
TRIMMING 5. Concentration
removal of excess 6. Duration of fixation
paraffin wax from the AMOUNT OF FIXATIVE
mold should be 20x the volume of the specimen (15-20:1 ratio)
8. MICROTOMY/ Electron Microscopy: 20x the volume of the specimen
Osmium tetroxide: 5-10x the volume of the specimen
SECTIONING procedure whereby the Museum preparations: not less than 50x the volume of
processed tissue is the specimen
trimmed and cut FACTORS THAT WILL RETARD/PROLONG FIXATION TIME
uniformly into to thin 1. Size and Thickness: the larger, the longer time.
slices or sections to 2. Cold temperature
facilitate study under the 3. Presence of Mucus and blood: wash with Normal
microscope Saline
If the specimen is Human Brain, wash with
Ringer’s Lactate
9. STAINING
FACTORS THAT WILL ACCELERATE FIXATION TIME
1. Size and Thickness: the smaller, the shorter time
process of applying dyes 2. Agitation: Continuous mixing
on the sections to see 3. Heat and Pressure: 37-56 degree celcius
and study the FACTORS IN TO CONSIDER IN CHOOSING THE
architectural pattern of APPROPRIATE FIXATIVE
the tissue and cells that 1. The need for immediate examination
make up the whole tissue 2. The type of tissue to be processed
3. The tissue structure to be studied
4. The type of stain to be used
EFFECTS OF FIXATIVE IN GENERAL
10. MOUNTING
1. Harden the tissue
procedure whereby the 2. Prevent bacterial growth
processed tissue is 3. Reduce the risk of infection
placed in a slide 4. Increase optical differentiation of cells and tissue
containing adhesives,
mounting medium and
cover slip to protect the
tissue section.

11. LABELLING

done to facilitate
proper and correct
specimen
identification.
 FIXATIVES ALDEHYDE FIXATIVES
contains only one • 10%
SIMPLE FIXATIVE Formaldehyde:
substance
COMPOUND FIXATIVE contains more than one commonly used
Thermal coagulation of for mailing
proteins, usually use for specimen, with
HEAT FIXATION 1mm/hr
bacterial smears and
frozen sections. penetration rate.
• Acts as both • Fumes are
Fixative and irritating to the
Dehydrating eyes and Nasal
FORMALDEHYDE
agent. Should be mucosa. may
in Ice cold cause dermatitis.
temperature ( -5 • Precipitation of
to 4 degC) white
• Used for the paraformaldehyde
preservation of is possible.
Lipase and
Phosphatase, 37-40% Formaldehyde:
ACETONE diluted form and is also
Enzyme studies.
• Used small pieces known as 100% formalin.
of tissues and for fixes Sputum specimen,
Brain specimen used for Microincineration
GENDRE’S FLUID
for the diagnosis Technique.
of RABIES.
• Dissolves Fats, • used for Central
Evaporates easily Nervous System,
and FLAMMABLE. Fats and Enzymes
• Diluted with
10% FORMOL SALINE
Sodium Chloride,
• Acts as both Used generally for
Fixative and Post-mortem
Decalcifying tissues.
agent. • contains 100%
TRICHLOROACETIC ACID formalin and
• Poor penetrating
fluid, often Mercuric chloride.
incorporated in FORMOL CORROSIVE • recommended for
compound fixative. Lipids,
• for Nucleoproteins and Phospholipids and
Nuclear chromatin and Neutral fats.
Cytoplasmic recommended for Electron
differentiation. Micrsocopy and Enzyme
GLACIAL ACETIC ACID • Often used in Histochemistry, made up of
conjuction with other 2 formaldehyde residues,
fixatives. SOLIDIFIES linked by 3 carbon chains
GLUTARALDEHYDE
at 17degC. 2.5%solution recommended
for small tissue fragments
ALCOHOL FIXATIVES 4% solution recommended
• causes Glycogen polarization, rapidly denatures for larger tissues with
and precipitates proteins. thickness less than 4mm
• Acts as both Fixative and Dehydrating agent. KARNOVSKY’S
Used for Electron
Ideal for Small tissue fragments PARAFORMALDEHYDE-
Microscopy and
for fixing Wet and Dry GLUTARALDEHYDE AND
Immunohistochemistry
preparations, smears ACLROLEIN
METHYL ALCOHOL METALLIC FIXATIVES
(blood smears and BM
tissues) fixatives used for Acid
ETHYL ALCOHOL for Blood and Tissue films LEAD FIXATIVES Mucopolysaccharide and
ISOPROPYL ALCOHOL for Touch Preparations Connective tissue mucin
• Most rapid alcohol Highly corrosive to skin and
fixative, for urgent mucus membrane.
biopsies CHROMIC ACID:
(Chromosomes, • fixative used for
CARNOY’S Carbohydrates
Lymph glands)
• Also used for the 3% POTASSIUM
CHROMATE FIXATIVES
diagnosis of DICHROMATE:
Rabies. • preserves lipids
Compatible with FUELGEN. and mitochondria
Used for REGAUD’S/MOLLER’S:
NEWCOMER’S • used for
Mucopolysaccharide and
Nuclear proteins. Chromatin,
 Mitochondria, POST-CHROMATIZATION
Golgi bodies, RBC form of secondary fixation using any CHROMATE
cont., colloid WASHING OUT: removing of excess fixatives
tissues. 1. Tap water: removes excess Osmic acid,
ORTH’S FLUID: chromates (Kelly’s, Zenker’s and Flemmings
• used for early soln., Formalin)
degenerative 2. 50-70% alcohol: wash out excess picric acid
processes and 3. Alcoholic Iodine: removes excess mercuric fix.
tissue necrosis.
• used for fixation of
Rickettsia and
other DEHYDRATION
microorganisms. DEHYDRATION
MERCURIC FIXATIVES • The amount of the dehydrating fluid should be
• most common metallic fixative. Highly Toxic fluid. NOT LESS THAN 10X THE VOLUME of the
• excellent for Tissue photography and Trichrome specimen.
staining. • Starts by placing the fixed tissue in 70% alcohol
contains Glacial Acetic to ASCENDING GRADES of alcohol.
ZENKER’S FLUID Acid. For Liver, Spleen, CT • ASCENDING GRADES: DEHYDRATION
fibers and Nuclei. • DESCENDING GRADES: HYDRATION
for pituitary glands, bone ALCOHOL
ZENKER’S FORMOL/
marrow and blood • used for ROUTINE tissue processing, used in
HELLEY’S
containing organs increasing concentration to avoid distortion.
for Tumor biopsies of the • for smaller and delicate tissues, low
HEIDENHAIN’S SUSA
Skin. concentration of alcohol can MACERATE the
contains anhydrous sodium tissue.
B5 FIXATIVE acetate, for Bone marrow • Longer storage in 70-80% may affect the process
specimen of staining, Hastens at 37degC
PICRIC ACID FIXATIVES • ANHYDROUS COPPER SULFATE: used as an
• Imparts Yellow color (treat with Acid Lithium indicator that will accelerate the process.
Carbonate). Can cause cell shrinkage. • Removes the water from the agent.
• Can act as both Fixative and Stain for small tissues. • Turns to BLUE when it its fully/completely
• recommended for dehydrated.
fixation of • used for routine
Pituitary biopsies, dehydration of
Embryo and urgent biopsies,
Endometrial NON-TOXIC but,
curetting FLAMMABLE.
BOUIN’S FLUID
• NOT SUITABLE ETHYL ALCOHOL • BEST
FOR KIDNEY DEHYDRATING
SPECIMEN. agent, FAST
• Abolishes Fuelgen ACTING suitable
reaction. for FATTY
TISSUES
BRASIL’S ALCOHOLIC Excellent fixative for • Toxic dehydrating
PICROFORMOL Glycogen agent, used for
OSMIUM TETROXIDE/OSMIC ACID METHYL ALCOHOL Blood and Tissue
used for Electron Microscopy, Myelin and Pheripheral smear
nerves specimen. preparations.
most common chrome • Dehydrating
osmium acetic acid fixative. agent suitable for
FLEMMING’S BUTYL ALCOHOL
Excellent for NUCLEAR Plants and
structures Animals.
FLEMMING’S WITHOUT for CYTOPLASMIC • Dehydrating agent
ACETIC ACID structures. substitute for Ethanol
MICROWAVE TECHNIQUE ISOPROPANOL and Xylene.
• Accelerates FIXATION, DECALCIFICATION and • Used in Microwave
STAINING. technique.
• Physical ageant with similar mechanism to oven, ACETONE
vacuum and agitation. : Evaporates easily, Can cause TISSUE SHRINKAGE,
• maybe used for neurochemical substances in RAPID ACTING, suitable for small pieces of tissue
brain like ACETYLCHOLINE. DIOXANE
SECONDARY FIXATION: • Excellent Dehydrating and Clearing agent. The tissue
placing an already Fixed Tissue in a second fixative in can be left for long period of time.
order to: FIXATIVE. • Toxic in man, TISSUE MAY TEND TO FORM
• Facilitate and improve demonstration of RIBBON POORLY.
particular substances 2 METHODS:
• Make special staining techniques possible • GRAUPNER’S: involves 3 changes of pure Dioxane
• Ensure further and complete hardening and soln. and 3 changes of Paraffin wax.
preservation of tissues.
• WEISEBERGER’S: The tissue is wrapped in a gauze CEDARWOOD OIL
bag. Calcium Oxide absorbs the water. • used to clear both PARAFFIN and CELLOIDIN
ETHYLYNE GLYCOL/CELLOSOLVE sections during embedding.
• dehydrates rapidly but is COMBUSTIBLE at 110- • recommended for cytological studies. (Smooth
120degF. muscles of skin and Central Nervous System)
• Removes ANILINE DYES, toxic by inhalation, skin CLOVE OIL
contact and ingestion. • tissues tends to become ADULTERATED.
• Prolong exposure can be toxic to fetal and • when used, the wax impregnation is slow and
reproductive system. difficult
TETRAHYDROFURAN • tissue becomes brittle, Removes Aniline dye,
• used for atheromatous arteries and hard dissolves Celloidin
collagenous specimen. ANILINE OIL
• Acts as both Dehydrating and Clearing fluid. May • not normally utilizes as a clearing agent.
cause Dizziness and Nausea. • recommended for EMBRYOS, INSECTS and other
TRIETHYL PHOSPHATE DELICATE specimens.
• removes water readily and produces minimum CARBON TETRACHLORIDE
shrinkage. • properties and disadvantages are similar with
• used to dehydrate sections and smears following chloroform.
certain stains. • Dangerous to inhale due to its toxicity.
• produces little distortion and hardening of tissue. METHYL BENZOATE
CARBOXYL • slow acting, used when DOUBLE EMBEDDING
acts as both Dehydrating and Clearing agent. TECHNIQUE is required.
OTHER CLEARING AGENTS:
• Oil of Bergamot: for Skin and Smooth Muscles
CLEARING • Oil of Origanum: for Skin
CLEARING • Oil of Wintergren: for Delicate tissue
• REMOVES ALCOHOL and MAKE THE TISSUE • Carbon disulfide: for Smooth Muscle
TRANSPARENT prior to infiltration • Carbon Xylene: for Friable tissue
• Clearing agent must be miscible with: • Terpineol: for Eyes
o DEHYDRATING FLUID • Phenol: for Smooth Muscle
o INFILTRATING MEDIUM • Limonil: Substitute for Xylene
o MOUNTING MEDIUM • High Test Aviation Lead Free Gasoline
FACTORS AFFECTING CLEARING: o Excellent clearing agent
1. Viscosity: affects PENETRATION o Produces LESS SHRINKAGE.
2. Boiling point: low boiling pt. replaced by wax • Creosole: for Celloidin section
3. Prolonged exposure to most clearing agents. • Carboxylol: Dehydrating and Clearing agent.
FROZEN SECTIONS CLEARING AGENT MISCIBLE WITH ABSOLUTE ALCOHOL
Glycerin and Gum syrup is used. No dealcoholization is • Xylene (miscible with paraffin)
involved • Toluene (miscible with paraffin)
XYLENE • Benzene
• Chloroform
• most rapid clearing agent for urgent biopsies.
Miscible with absolute alcohol and Paraffin. • Carbon tetrachloride
• Can be used for celloidin sections. However, the
fluid evaporates quickly in paraffin oven.
• Not suitable for NERVOUS TISSUE and LYMPH DECALCIFICATION
NODES. DECALCIFICATION
• Disadvantage is it is CARCINOGENIC. • Procedure whereby calcium ions or lime salts
TOLUENE/TOLUOL are removed from the tissue
substitute for XYLENE or BENZENE. Tends to acidify in • Done to ensure and facilitate the normal cutting
partially filled vessel. of sections, prevent obscuring the
microanatomical detail
• Best reagent for EMBEDDING process, • of such sections and other cellular debris.
Penetrates and clears rapidly for urgent Calcified bones/tissues are usually cut into small
biopsies. pieces.
• Does not make the tissue hard and brittle but • Both decalcification and processing depends on
causes minimum shrinkage. bone thickness. Ideal thickness: 1-3mm
• May damage the Bone Marrow of man in prolong METHODS OF DECALCIFICATION
exposure. May cause APLASTIC ANEMIA. NITRIC ACID:
• Highly Inflammable Most commonly used decalcifying
CHLOROFORM agent. The FASTEST used so far.
• Slower in action compared with Xylene. DOES • used for
NOT MAKE THE TISSUE TRANSPARENT urgent
• Used for Thicker tissue block and large tissue Use of Acid biopsies,
specimen. Fast acting.
10% Aqueous
• Recommended for TOUGH TISSUES (SKIN, • Recommend
Nitric Acid
FIBROID and DECALCIFIED Tissues) ed for
• Tissues may tend to float (wrap the tissue in a HEAVILY
gauze), may also attack the rubber seal. CORTICAL
• Not inflammable but toxic to LIVER. BONES.
 • Can damage when
tissue prolong
stainability, decalcificati
Imparts on.
YELLOW • Nuclear
color with staining is
Nitrous Acid. poor.
• Rapid Acting
for urgent
biopsies.
• Yellow HYDROCHLORIC ACID
imparted by used for Surface decalcification of
nitric acid blocks and small pieces of bones.
formation • permits
impair relatively GOOD
staining CYTOLOGIC
reaction of STAINING.
the cell • DOES NOT
• Neutralizes REQUIRE
VON EBNER’S
the tissue WASHING OUT
FLUID
with 5% before
FORMOL Sodium dehydration.
NITRIC ACID Sulfate and • - Recommended
wash in for TEETH and
running SMALL PIECES
water. OF BONES.
• Addition of • produces
0.1% Urea to BETTER
pure NUCLEAR
concentratio STAINING and
n nitric acid less tissue
can also distortion.
disappear • Safer to handle
the for routine
• the tissue decalcification of
discoloratio POST-MORTEM
n. research tissues
FORMIC ACID
• : composed • recommended
of NITRIC for Research and
ACID + Autopsy,
CHROMIC Cartilage and
ACID + Bone Marrow
ETHYL • Addition of
ALCOHOL CITRATE
• Recommend accelerates
ed for decalcification
routine time by chelating
purposes, calcium.
PERENYI’S DECALCIFY TRICHLOROACETIC ACID:
FLUID and SOFTEN Permits good nuclear staining, weak
the tissue. agent
• When used, SULFUROUS ACID:
Nuclear and Very weak decalcifying agent. Used
Cytoplasmic only for minute pieces of bones
staining is CHROMIC ACID:
good. • may be used as both fixative
• Slow and decalcifying agent
decalcifying • recommended for minute bone
agent for spicules
DENSE • considered as an
BONES. environmental toxin,
• MOST RAPID carcinogenic.
so far. • corossive to skin and mucus
Recommend membrane.
ed for urgent CITRIC ACID CITRATE BUFFER:
PHLOROGLUCI
biopsies. • Weak decalcifying agent.
N NITRIC ACID
• may cause • Uses chloroform as
EXTREME preservative
TISSUE
DISTORTION
 Substances which combines with To remove acids: 30mins. Washing with Running tap
calcium ions and other salts like iron water (small) 1-4hours (larger specimen)
and magnesium deposits. FACTORS INFLUENCING RATE OF DECALCIFICATION
• recommended More concentrated:
CONCETRATION and VOLUME
for detailed Rapid but harmful
microscopic Hastens
srudies. decalcification nut
HEAT
• Potent increases damaging
anticoagulant but effects
WEAK AGENT. Fluid to tissue ratio:
Excellent for STRUCTURE, TEMPERATURE
20:1
Electron Influences fluid
Microscopy exchange,
Use of • Binds with aaccelerates the rate
Chelating calcium to form MECHANICAL AGITATION
of diffusion and
Agent EDTA/ weakly speeds up the
VERSENE dissociated process.
complex. TISSUE SOFTENERS
• Inactivates 1. Molliflex: 1%HCL in 70%Alcohol
ALKALINE 2. Perenyi’s Fluid: 2%HCL in 70% Alcohol
PHOSPHATASE 3. Lendrums method: 4% Aqueous phenol
(Add Magnesium
Chloride)
• For
specimens:
small IMPREGNATION/INFILTRATION
1-3 weeks IMPREGNATION/INFILTRATION
• For dense • Process whereby the clearing agent used from
tissues: 6-8 the tissue is removed and fill all cavities and
weeks spaces.
• hastens decalcification by • Provides a firm consistency to the specimen to
removing calcium ions from allow easier handling and cutting of suitably
FORMIC ACID containing decal. • thin section without any damage or distortion.
agent. • Clearing agent EASILY REMOVED:
Ion Exchange benzene and xylene
• the amount of volume should be
Resin • Clearing agent DIFFICULT TO REMOVE:
20-30x the volume of the specimen.
1-14days duration chloroform and cedarwood oil
• uses Ammonium form Polystyrene PARAFFIN WAX IMPREGNATION
resin. X-ray measures the process. • Most commonly used, rapid acting.
• (MOST RAPID METHOD) • Allows cutting serial sections and compatible
• process whereby positively with many staining procedure
charged ions are attracted to a • not recommended for fatty tissues
negative electrode and • Prolonged process may lead to excessive
subsequently hardening of the tissue Tissue Shrinkage
Electrolytic • Remove from the decalcifying • Inadequate process may case retention of the
Method agent. clearing agent used
/Electrophores • Satisfactory for small bone Melting Point for
56°
is fragments ROUTINE WORK
• Principle is similar to chelating If the laboratory
agents, utilizes electricity and temperature is between 54-58°
dependent upon a supply of 20° - 24°
direct current remove calcium If the laboratory
deposits. temperature is between 50-54°
MEASURING EXTENT OF DECALCIFICATION: 15° - 18°
Inaccurate, damages METHODS OF PARAFFIN WAX IMPREGNATION
Physical Method/Mechanical A. MANUAL:
the tissue
• Most Sensitive • makes use of a PARAFFIN OVEN (2-5degC higher
and Reliable than the Melting Point) or 55-60° temperature.
• can detect even • Involves 4 changes of paraffin wax
smallest amount B. AUTOMATIC:
X-ray or Radiologic Method • makes us of autotechnicon
of calcium
• if calcium is still • Usually 2-3 changes of wax for more rapid
present, it will diagnosis and less technicalities.
appear OPAQUE. C. VACCUM:
involves using of • Infiltration under negative atmospheric pressure
Ammonium Oxalate • Recommended for urgent biopsiesand delicate
Chemical Method - Turbidity indicates tissues: Lung, Connective Tissue, Brain
incomplete Decalcified bones, Eyes, Spleen, Central Nervous
decalcification System
Post Decalcification: procedure that involves washing the
tissue with LITHIUM CARBONATE.
 SUBSTITUTE FOR PARRAFIN WAX • combination of
• Mixture of highly GILSON’S MIXTURE Chlorofom and
purified paraffin Cedarwood Oil
and synthetic GELATIN IMPREGNATION
plastic polymer • Rarely used for frozen tissues.
PARAPLAST (MP: 56-57°) • For bones and • Recommended for histochemical and enzyme
brain specimen studies, water soluble
• With better • Tissue for processing should be 2-3mm; add 1%
ribboning of phenol to prevent molds
sections USE OF PLASTIC RESIN
• Similar to • Provides superior results for light microscopy
paraplast but less • For hard tissues like uncalcified bones
EMBEDDOL (MP: 56-58°)
brittle and less • For for high resolution of light microscopy of
compressible tissue sections thinner than 4-6u
• Harder than • For renal biopsies, bone marrow biopsies
paraffin. component are toxic
• Makes use of Bisphenol A (Araldite): Slow
heavy duty acting for it is a LARGE
ESTER WAX (MP: 46-48°)
microtome. MOLECULE
• Eliminates Glycerol based (Epon):
Process of EPOXY
Have lower viscosity
clearing. Cyclohexine dioxide
• semi-synthetic wax (Spurr): Have very low
BIOLOID recommended for viscosity but can infiltrate
embedding eyes faster
• product of paraffin Originally introduced for
TISSUE MAT with rubber POLYESTER Electron Microscopy in
1950’s
WATER SOLUBLE WAX: CARBOWAX Made up of esthers of
(MP: 38-42degC or 45-56°) acrylic or methacyclic acid.
• For thicker sections, recommended for neurological Used for light microscopy
tissues Polyglycol methacrylate:
• Eliminates process of dehydration and clearing, Hydrophillic, Allows many
recommended for enzymes and histochem studies. ACRYLIC staining method
Cytologic pictures are preserved but carbowax is very Methyl methacrylate:
easily to be dissolved in water. “very hygroscopic“or Ideal medium for
water soluble Uncalcified bones and
CELLOIDIN IMPREGNATION other hard tissues.
• A purified form of nitrocellulose. For tissues with
large hollow cavities that
• Tends to collapse (teeth, bones, brain, eyes), for
hard and dense tissues
• 2% (for thin), 4% (thicker), and 8% (more thicker)
medium solutions dissolved in equal parts of
ether and alcohol. Very slow acting
• Serial section: difficult to cut; photomicrograph:
difficult to obtain
METHODS
• recommended for
bones and brain
specimen. Uses
70% alcohol
(storage)
• 2-4% for 5-7days
transferred to
WET CELLOIDIN medium celloidin
of 4-6% for
another 5-7days
• Drained off and
poured with thick
celloidin of 8-12%
usually for 3-5
days.
• Same as wet but
does not involve
70% alcohol before
DRY CELLOIDIN
cutting
• recommended for
EYE specimen.