PROSTATIC SECRETIONS

3 specimens:
 Voided urine before prostatic
massage
 Prostatic secretions during
EXFOLIATIVE massage (in smears)
 Urine after massage

CYTOLOGY GASTRIC SECRETIONS AND

Non-Gynecologic Specimens ASPIRATES
 Respiratory tract Specimens Specimen Types
 Sputum  Gastric Lavage
 Bronchoalveolar  Gastric Brush
Lavage/Bronchial  FNA (for submucosal lesions)
washings  Uncommon
 Bronchial Washings  Fasting
 Prostatic Secretions SEROUS FLUIDS
 Gastric Secretions and  Heparin: prevent clot
Aspirates
 Serous Fluids URINE
 Urine Specimen Type
 Breast Secretions  Voided Urine
 Catheterized Urine
SPUTUM  Washings from bladder or urinary
 Atleast 3 consecutive morning pelvis
sputum specimens
Twice collection
 Deep cough
 Early morning
 Saccomano’s Fixative
 Late afternoon
 Satisfactory Specimen: presence
 Atleast 50 ml volume each
of alveolar macrophage
BREAST SECRETIONS
BAL
 Spontaneous secretions are
 AIDS
smeared or swabbed
 Pneumocystis jiroveci
 Fixed immediately
BRONCHIAL BRUSHINGS
 Satisfactory specimen: bronchial
epithelial cells

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Valdez, P.L.A
 HISTO- SURGICAL PATH 10
(AND BONE
MARROW
TECHNIQUES REPORTS
Histotechniques CYTOGENETICS 20
 Histotech: Fixation – Staining REPORTS
 Patho: Gross section and cutting,
Microscopic Examination
SPECIMENS RETENTION
Q A and DOCUMENTATION SERUM/OTHER 48 HRS
 Histopath Reports BODY FLUIDS
 Surgical pathology report ROUTINE BLD 7 DAYS
 Cytopathology report SMEARS
 Autopsy report PATHO/BONE 10 YRS
 Signatories MARROW
SLIDES
 Request forms
PATHO BLOCKS 10 YRS
 Result forms
MICROBIO 7 DAYS
 Specimen Handling SMEARS
 Ensure if fixed first BB 7 DAYSPOST
 Check correct label DONOR/RECIPI TRANSFUS
 Storage ENT ION
 Specimen (organs) SPECIMENS
 Tissue blocks CYTOGEN SLIDES 3 YRS
 Slides CYTOGEN 20 YRS
DIAGNOSTIC
Records Retention IMAGES
Requisitions 2
QC 2 F-ixation
Instrument 2 D-ecalcification
Maintenance
C-learing
Donor/Recipients Indefinitely
I-infiltration/Impregnation
Records
E-mbedding
Employee 10
T-rimming
Signature/Initials
S-ection cutting
QC 5
S-taining
M-ounting
Reports RETENTION
L-abeling
CLIN PATH LAB 2 YRS
REPORTS
FIXATION
AUTOPSY INDEFINITELY
FORENSIC  Preservation
REPORTS  20:1

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Valdez, P.L.A
Effects  Chon: neutral buffered formol
 Chon denaturation saline/formaldehyde vapor
 Trauma resistance  Electron xcopy: osmium
 Staining is strongly influenced tetroxide and glutaraldehyde
 Prevent bacterial decomposition
 Hardening of soft and fragile FIXATION CAN BE ENHANCED
RETARDED BY:
tissues
BY:
 Better microscopic examination
SIZE AND SIZE AND
 Safe handling and processing
THICKNESS THICKNESS
2 Mechanisms COLD AGITATION
Additive fixation TEMPERATUR
 The chemical constituent of the E
fixative is taken in and becomes PRESENCE OF TEMPERATUR
MUCUS, FATS E (37-56C)
part of the tissue
AND BLOOD
Non-additive fixation
 The fixing agent is not taken in, DECALCIFICATION
but changes the tissue  Bone
composition and stabilizes the  Teeth
tissue by removing the bound  Teratomas with bony structures
water attached to hydrogen bonds  Tuberculosis foci
of certain GRPS within the chon  Arteries calcified by atheroma
molecule
Forms of calcium salts
Main factors  CA urates
 PH  CA phosphates
 Temp  CA fluoride
 Thickness of section osmolality
 Calcium oxalate
 Concentration
 Duration of fixation

General Considerations
After fixation, before fixation
Types of tissues
 Use of hackjaw, jig saw, fret saw
 Lipids: mercuric
 Ideal time: 24-48 hrs to
chloride/K2Cr2O7
decalcify/14 days or more (big
 Phospholipids: baker’s formal spx)
calcium  Volume of reagent tissue: 20:1
 CHOLESTEROL: Digitonin  Optimum temp: RT (18-30c)
 Cho: Alcoholic fixatives
(Rossman’s fluid) Heat

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Valdez, P.L.A
  Accelerates demineralization, Sulphurous Acid
promotes destructive action of Chromic Acid
acids on matrix  Nuclear STG with hematoxylin
 55 C: complete digestion within is inhibited
24-48 hrs
Citric acid-citrate buffer solution (ph
 37 C: impair nuclear staining
4.5-5.5)
Phloroglucin-nitric acid
Routine Techniques  Most rapid decalcifying agent
 Acids should be changed once or  Complete decal cannot be
2x a day until decalcify is done determined by chemical means
 Poor nuclear STG
DECALCIFYING AGENTS
Perenyi’s fluid
Nitric acid
 Composition: 0.5% chromic acid,
 Inhibits nuclear stains
10% nitric acid, absolute ROH
 If very concentrated, destroys
 For calcified arteries and thyroid
tissues
glands
 USU 5-10%
 Slow for dense bones
HNO3 Formulations
 De castro’s fluid: for silver CHELATING AGENTS
impregnation of nerve fibers EDTA
 10% aqueous nitric acid: 12-24  Commercial name: versene
hrs  Decalcifier and water softener
 Formol nitric acid: 1-3 days  Very slow, not for routine nor
 Perenyi’s Fluid (with chromic urgent purposes
acid): 2-7 days
 Phloroglucin nitric acid: 12-24 hrs ION EXCHANGE RESIN
 Vol: 20-30x the volume of tissue
Formic acid  Measured by physical/radiological
 Better nuclear staining methods
 Recommended for routine decal
of post-mortem research tissues Electrophoresis/Electrical Ionization
 Not for urgent specimens  30-35C
 Safer to handle than HNO3/HCL  90% formic acid and concentrated
 Formic acid-sodium citrate HCL
 Recommendation for autopsy  For small bone fragments
specimens How to test for complete Decal:
HCL  Physical/Mechanical
Von Ebner’s Fluid  X-ray or Radiological
 Good cytologic STG  Chemical
 5 ml of used decalcifying agent
Trichloroacetic Acid

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Valdez, P.L.A
  Alkalinize with NH3 water  Tissue may be taken directly
 Add 0.5ml saturated aqueous into dioxane (except chromate
NH4 oxalate (1% sodium fixatives)
oxalate), stand for 15-30mins  Graupner’s Method:
1. Pure dioxane:1hr
DEHYDRATION 2. Pure dioxane: 1hr
 Increasing strengths 3. Pure dioxane: 2hrs
 Reagent Volume: not less that 10x 4. Paraffin wax: 15minutes
the tissue volume 5. Paraffin wax: 45minutes
 Reagent used must have high 6. Paraffin wax: 2hrs
affinity with water  Triethyl phosphate
 THF (Tetrahydrofuran)
DEHYDRATING AGENTS
 6 months: prolonged exposure
 Ethanol
causes conjunctival irritation
 Fast-acting
 Less shrinkage
 Not expensive
 Easier cutting of sections with
 Miscible with water
fewer artifacts
 Not poisonous
 With offensive odor
 Methanol  Does not dissolve aniline dyes
 Blood smear and tissue films
 Butyl alcohol Clearing/Dealcoholization
 Plant and animal  Tissue may become transparent
 Isopropyl alcohol and translucent
 Substitute to ethanol but
expensive Applications
 Acetone (cheapest)  Clearing in embedding
 Hardens tissue more than  Clearing in mounting
ethanol does  Xylene, toluene, terpineol,
 Both a fixative and dehydrating carbol xylenes
agent  For the purpose of making the
 For most urgent biopsies tissues transparent
 Cellosolve (ethylene glycol CLEARING AGENTS
monoethyl ether Xylol/Xylene
 Combustible at 110-120
 Excellent but makes tissues hard
degrees F
and brittle if cleared longer than 3
 Tissues may be stored for
hours
months without distortion
 Suitable for urgent biopsies
 Dehydrates rapidly and is not
 Does not extract aniline dye
harmful to the tissues
 Miscible with cannada balsam
 Dioxane
 Readily miscible with water,
alcohol, paraffin wax and xylol

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Valdez, P.L.A
  Unsuitable for brain and lymph  CNS tissues and cytological
nodes turns milky when studies
dehydration is not complete
Clove oil
 Highly flammable
 Minimum shrinkage of tissues
 Most rapid and commonly used
 15-30 mins or 30mins-1hr Carbon tetrachloride
 Toxic
Benzene
 Doesn’t make tissues hard and THF (Dioxane)
brittle 
 Cause considerable shrinkage
Amyl Acetate
 Highly flammable
 Large pieces of tissues and
 Miscible with absolute alcohol
embryonic materials
 Carcinogenic- aplastic anemia
(bone marrow damage) Terpineol (Artificial oil of Lilac)
 Urgent biopsies  Can be substitute for cedarwood
 15-60mins oil
Toluene/Toluol Methyl benzoate/Salicylate
 1-2hrs clearing time  Slow
 Not carcinogenic  Double embedding
 Emits toxic fumes  Expensive
Chloroform Glycerin, Gum syrup and brun’s
 Causes little shrinkage solution
 Does not harden tissues that  For tissue to be cleared directly
much from water
 Not flammable Carbo-Xylene
 Not volatile in paraffin oven  Used for materials that are difficult
 Toxic to the liver on prolonged to clean
inhalation
Cedarwood oil Others
 Slow: 2-3 days  Oil of bergamot
 Quality is not good  Phenol in alcohol
 Turns milky on prolonged storage  creosote
 Tissue tends to float
 May form crystals with acetic- Newer Clearing Agents
alcohol fixed tissues  Based on limonene
 Melting point: 35c  Clearite
 Heated: 200c

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Valdez, P.L.A
IMPREGNATION AND Methods of paraffin impregnation
Automatic processing
EMBEDDING  Wax bath: atleast 6c higher than
Impregnation the wax
 To set tissues to firm consistency  Uses automatic tissue processor
to allow the cutting
Manual processing
 Volume of medium: 25x the
volume of tissue  Requires for changes for 15 mins
 Melting point: 56c Vacuum embedding
Embedding Media  400-500 millimeters of mercury
 Materials used to infiltrate,  2-4c above mp of wax
support and enclose the tissue Precautions in infiltration
spx  Paraffin wax must be pure and
Characteristics use only twice
 Crystallization  Water must be removed by
 Evaporation of the solvent heating paraffin to 100-105c
 Polymerization  Xylene and benzene are easily
removed while chloroform and
Types of Embedding cedarwood oil are difficult to
Paraffin Embedding remove
 Shrinks about 10% on cooling Substitutes for Paraffin Wax
 Melting point for normal routine Paraplast
work: 56-58c  Better ribboning with ease
 Simplest and most common  Doesn’t require cooling
ADVANTAGES:  Embeddol
 Rapid  MP: 56-58%
 Allows storage for an indefinite  Less brittle
period Bioloid
 Permits many staining procedures  For eyes, thin walled structures
DISADVANTAGES: Tissue mat
 Overheating may result to brittle  Has rubber (from paraffin)
tissues
 Prolongation may cause to Ester wax
excessive shrinkage and  Mp: 46-48c
hardening  Soluble in 95% ethanol and other
 Inadequate infiltration may result clearing agents
for a soft tissue Water soluble waxes
 It is not recommended for fat  Polyethylene glycol or carbol wax

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Valdez, P.L.A
  It does not require dehydration  Rubbery consistency (no
and clearing distortion)
 It does not remove neutral fats  Does not require heat
and lipids
TYPES
 Suitable for enzyme studies
1. Wet method
EMBEDDING  Evaporate solvent
 Store in 70-80% alcohol
Orientation
 Bones, teeth, brain sections
1. Arranging the tissues in the mold
and whole organs
2. Fixing the tissue block on the
2. Dry method
microtome
 No need to be stored in 70%
3. Arranging the tissue ribbons on
alcohol
the slide
 Store in gilson’s mixture
4. Arranging the tissues in the mold
(Chloroform and cedarwood
5. Fixing the tissue block on the
oil)
microtome
 Whole eye section
6. Arranging the tissue ribbons on
the slide Gelatin Embedding
7.  Water soluble
 Low mp and does not overharden
TYPES OF EMBEDDING MOLDS  10% then 20% then 20% + phenol
1. Leuckhart’s embedding mold-  Delicate specimens
routine use (histochemical and enzyme
2. Compound E unit-several studies, frozen section)
compartments  Volume: 25x the tissue volume
3. Disposable molds
 Peel-away Plastic/Resin Embedding Medium
 Plastic ice trays Epoxy
 Paper boats  Araldite base (bisphenol): slowest
4. Plastic embedding rings and  Glycerol base (epon)
base molds
 Cyclohexene dioxide (spur):
TISSUE-TEK SYSTEM fastest
 Stainless steel in which the tissue  Used for hard tissues
block is embedded
Disadvantages:
Celloidin/Collodion embedding  Hydrophobic: tissue damage
 Purified pyroxylin nitrocellulose  Reduce antigenicity: not for
immunochemical studies
Advantage  Sensitization: skin and inhalation
 Less shrinkage and distortion  Contains toxic components
 Slow process (days to weeks)

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Valdez, P.L.A
Polyster to the next step in the tissue
 For electron microscopy processing.
 Decalcification is the procedure
Acrylic whereby calcium or time salts are
 Use for high resolution light removed from tissues (most
microscopy especially bones and teeth)
following fixation. This is usually
Glycol Methacrylate (GMA) carried out by the use of chemical
 Hydrophilic: allows wide stain agents, either with acids to form
selection soluble calcium salts, or with
chelating agents that bind to
Methyl Methacrylate (MMA)
calcium ions.
 Hardness: for dense tissues  Decalcification should be done
Benzoyl Peroxide after fixation and before
Added as a catalyst (for drying) impregnation, to ensure and
facilitate the normal cutting of
 Decomposes and act as active
sections and to prevent obscuring
site for polymerization of acrylic the microanatomic detail of such
ADVANTAGES OF PLASTIC sections by bone dust and other
 Permits ultrathin microscopy cellular debris. Inadequate
decalcification may result in poor
 Little distortion of tissues
cutting of hard tissues and
 Can be stored indefinitely
damage to the knife edge during
 sectioning.
 Bones and calcified tissues (such
ACTIVITY 7 as tuberculous organs and
DECALCIFICATION arteriosclerotic vessels) are
usually cut into small pieces with a
 After fixation, selected pieces of fine fret-saw and trimmed with a
tissues are taken from the hand razor to permit complete
specimen, properly labeled and penetration of the decalcifying
identified, and then subjected to solutions. With minimal surface
the subsequent steps of damage and tissue distortion.
processing. There are, however, Selected pieces of tissues are
certain specimens e.g. bones, taken from the teeth by a sharp
teeth and other calcified tissues razor blade usually when it has
like tuberculous lungs, which been either partially or completely
contain some amount of calcium decalcified.
that is apt to interfere with the  Rapidly decalcifying agent must
accurate evaluation and be capable of removing calcium
examination of histologic sections. salts from tissue completely
Hence, one must see to it that without adversely affecting any
such extraneous materials have subsequent staining. This is
been removed before proceeding especially noticeable in cell nuclei
due to failure of nuclear chromatin

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Valdez, P.L.A
 to take up hematoxylin and other determining if a tissue has been
basic dyes as readily as soft tissue completely decalcified or not.
that has not been exposed to acid  An alternate method of evaluating
solutions or decalcifiers. tissues mechanically is by pricking
 A good decalcifying agent must be the tissue with a fine needle or a
capable of removing calcium salts probe. This method is apt to
from tissues completely without produce needle tract artifacts and
producing considerable destroy important cellular details.
destruction of cells and tissue Pricking, slicing, bending or
components and without squeezing tissue can disrupt soft
adversely affecting the staining tumor from the bone or cause
capacity of the cell, particularly of false positive microfractures of
the nucleus. Calcium may be fine trabeculae, leading to a
removed by any one of the potential misdiagnosis. Aside from
following agents: this disadvantages, small calcified
foci may not even be detected.
1. Acids
2. Chelating agents 2. X-Ray or Radiological Method
3. Ion exchange resins
 This is a very expensive although
4. Electrical ionization the most ideal, most sensitive and
(electrophoresis) most reliable method of
determining extent of
Measuring Extent of Decalcification decalcification due to its ability to
detect even the smallest focus of
Frequent monitoring is required to ensure calcium which appears opaque in
that bone tissue is taken from the acid an X-ray plate. After rinsing the
solution as soon as all calcium is acid decalcifying agent from the
removed from the specimen, and before sample; decalcified bone is placed
the tissue becomes completely on waterproof polyethylene sheet
macerated. There are three ways by on top of the X-ray film, exposed
which the extent of decalcification may to X-ray at a setting at
be measured, namely: approximately 1 minute, 30 kV,
and left in place until film is
1. Physical or Mechanical Test developed and examined for
calcifications.
 This is done by touching and  It is not recommended for
bending the tissue with the fingers mercuric chloride-fixed tissues
to determine the consistency of due to the latter’s characteristic
tissues. Decalcified tissues radio-opacity which will interfere
usually have diminished with the correct interpretation of
consistency and are softer to the plate.
touch. This is, however, a very
vague and inaccurate way of 3. Chemical Method (calcium oxalate
test)

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Valdez, P.L.A
  If chemical method of
 This is a simple, reliable and determination is to be done, the
convenient method recommended decalcifying agent should be
for routine purposes, to detect the prepared with distilled water,
presence of calcium in the since false positive readings may
decalcifying solution. This method be produced by the calcium ions
involves the detection of calcium present in tap water. It is
in acid solutions by precipitation of unsuitable for solutions containing
insoluble calcium hydroxide or over 10% acid, although these
calcium oxalate. could be diluted and result in a
 Solutions: Ammonium less sensitive test.
hydroxide, concentrated
 Saturated aqueous ammonium ACTIVITY 8
oxalate
 The decalcifying fluid is usually
DEHYDRATION
changed every 24-48 hours and
 As soon as tissue has been fixed, and
the chemical test is performed on
the bones and teeth have been
the discarded fluid. A piece of blue decalcified, it is necessary to remove
litmus paper is added to a test the fixative and water from the tissue
tube containing 5 ml of the and replace them with dehydrating
discarded decalcifying agent (the fluid in preparation for impregnation.
litmus paper will turn red due to  A process of water removal from the
the acidity of the fluid). Strong tissue. Dehydration of tissues is the
ammonia is then added drop by important process because of the
drop until the fluid is neutralized paraffin, in which the tissues are
(this can be detected by the embedded, is not miscible with water
change in color of the litmus paper and does not penetrate the tissue
from red to blue, indicating effectively.
alkalinity). The presence of  Tissues are generally soft after
fixation and thus for satisfactory
cloudiness indicates that there is staining and the right degree of
still calcium found in the solution. hardening, the tissue is treated with
The tissue is then immersed in a the dehydrating agent.
new solution of decalcifying agent.  The ideal technique of tissue
 If the solution remains cleat after dehydration is by passing the
neutralization with concentrated specimens in tissue cassettes in a
ammonia, 0.5 ml of saturated series of ascending concentrations of
aqueous solution of ammonium hydrophilic or water miscible fluids,
oxalate is added and the solution progressively replacing the free
is allowed to stand for 30 minutes. water. Graded alcohol as 70%, 80%,
Cloudiness will signify incomplete 90% and then Absolute alcohol is the
commonly used dehydrating
calcium removal; hence, the need reagents and known as dilution
for further decalcification. If the dehydration technique. This method
solution remains clear after 30 prevents the mechanical damage to
minutes, decalcification is delicate intracellular structures,
considered to be complete. which may occur due to the rapid

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Valdez, P.L.A
 passage of water from the cells to the embedding process are xylene,
dehydrating medium. dioxane, chloroform, and
 As a general rule, whatever cedarwood.
dehydrating agent is used, the  When used after the tissue section
amount in each stage should not be has been stained, the clearing
less than 10 times the volume of the
agent will make microscopic
tissue in order to ensure complete
penetration of the tissue by the tissue preparations transparent
dehydrating solution. Commonly due to their high index of
used dehydrating agents are: refraction. Aside from removing
1. Alcohol alcohol, a clearing agent must
2. Acetone also be miscible with Canada
balsam and other resins that are
3. Dioxane 4-cellosolve used for mounting sections. The
4. Triethyl phosphate most commonly used clearing
5. tetrahydrofuran agent for this purpose is xylene.
 Glycerin and gum syrup are used
ACTIVITY 9 when the tissue is to be cleared
directly from water, as in a frozen
CLEARING section. No de-alcoholization is
 Clearing (de-alcoholization) is the involved in the process. The
process where alcohol or a clearing agents merely improve
dehydrating agent is removed the refractive index of the tissue.
from the tissue and replaced with  Because of the high refractive
a substance that will dissolve the indices of most reagents used for
wax with which the tissue is to be de-alcoholization, tissues,
impregnated (e.g. paraffin) or the particularly embryos and
medium on which the tissue is to parasites become transparent so
be mounted (e.g. Canada that the internal structures
balsam). When the dehydrating become visible to the naked eye.
agent has been entirely replaced Due to this property of making
by the solvent, the tissue has a tissues transparent, solutions
translucent appearance; hence, utilized for alcohol removal are
the use of the term “clearing also called “clearing agents”. Not
agent”. all clearing agents, however,
 Few dehydrating agents are exhibit this property.
miscible with paraffin wax. When  Most clearing agents are
used after alcohol dehydration, flammable liquids that warrant
the clearing agent serves to mix considerable caution in their use,
with alcohol and removes it from and the histotechnologist should
the tissue. It should also be be aware of the large quantities
miscible with paraffin in order to used in routine processing, so that
facilitate the penetration of this the safest method of use and
embedding medium. The most storage can be adopted. Clearing
commonly used clearing agents agents with a low boiling point are
for de-alcoholization in the generally more readily replaced

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Valdez, P.L.A
 by melted paraffin, although 3. Gelatin
chloroform which has a lower 4. Plastic
boiling point than xylene in fact  After impregnation, the tissue is
takes longer than the latter to placed into a mold containing the
clear. Viscosity also affects the embedding medium and this
speed of penetration of the medium is allowed to solidify.
clearing agent. Prolonged Paraffin embedded tissues are
exposure to most clearing agents arranged at the bottom of the mold
causes the tissue to become together with their proper labels
brittle and therefore more difficult and immersed in melted paraffin
to cut. at a temperature between 5-100 C
above its melting point and then
ACTIVITY 10 cooled rapidly in a refrigerator at -
IMPREGNATION AND 50C or immersed in cold water to
solidify. This will allow the
EMBEDDING hardening of tissues, giving them
a firmer consistency and better
 Impregnation (infiltration) is the support, thereby facilitating the
process whereby the clearing cutting of sections.
agent is completely removed from
the tissue and replaced by a  The process by which a tissue is
medium that will completely fill all arranged in precise positions in
the tissue cavities, thereby giving the mold during embedding, on
a firm consistency to the the microtome before cutting, and
specimen, and allowing easier on the slide before staining, is
handling and cutting of suitably known as Orientation. Generally
thin sections without any damage speaking, the surface of the
or distortion to the tissue and its section to be cut should be placed
cellular components. parallel to the bottom of the mold
 Embedding (casting or blocking) in which it is oriented.
is the process by which the
impregnated tissue is placed into
a precisely arranged position in a ACTIVITY 11
mold containing a medium which
is then allowed to solidify. MICROTOMY
 The medium used to infiltrate the
tissue is usually the same medium  The process by which processed
utilized for impregnation, and for tissue, most commonly a paraffin
general purposes is known as an embedded tissue, is trimmed and
Embedding Medium. cut into uniformly thin slices or
 There are generally four types of “sections” to facilitate studies
tissue impregnation and under the microscope is known as
embedding media, namely: Microtomy. The basic instrument
1. Paraffin wax used is a microtome that is
2. Celloidin (collodion) capable of cutting a section at a

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Valdez, P.L.A
 predetermined thickness by
sliding the block into a cutting tool,  Once the wax has solidified, the
usually a steel knife or blade, wax block is removed from the
which is fixed and attached to the mold, the identification number is
machine. noted and the excess wax is cut
 Whatever the type of microtome is off from the block to expose the
used, the principle remains tissue surface in preparation for
essentially the same, that is, a actual cutting. This procedure is
spring -balanced teeth or pawl is known as TRIMMING. Only thin
brought into contact with, and slices are taken out at a time to
turns a ratchet feed wheel prevent the block from cracking.
connected to a micrometer screw,  The sides, top and bottom of the
which is in turn rotated, moving tissue block are trimmed until
the tissue block at a perfectly level and all sides are
predetermined distance towards parallel, almost to the edge of the
the knife for cutting sections at tissue. After a course trimming, a
uniform thickness. heated spatula is held between
 Sharpening and polishing are no the tissue block and the block
longer common practice in most holder until the wax begins to melt.
modern laboratories because of The spatula is withdrawn and the
the availability of disposable block is gently pressed into
knives that are cheaper to use position. The block is allowed to
than conventional steel knives. harden for cutting properly by
They have a sharp cutting edge facing them down in ice cold water
that can cut 2-4 μ thick sections or refrigerator for 5-10 minutes.
with ease. Some microtome
manufacturers have also now B. Celloidin
incorporated a disposable blade
holder in place of a knife holder.  Celloidin sections are usually cut
Magnetic knives are also now between 10-15 μ in thickness. The
available that can attach to some blocks are trimmed in the same
blade holders and are particularly manner as in paraffin blocks, but
suitable for use in the cryostat. they do not require hardening by
chilling before cutting. The
ACTIVITY 12 sections are usually cut with the
sliding microtome.
PARAFFIN SECTIONS  To avoid dehydration and
shrinkage, sections are usually
Sectioning is a process whereby tissues
cut by the Wet Method, both the
are cut into uniformly thin slices or
sections and the block being kept
“sections” with the aid of microtome, to
moist with 70% alcohol during
facilitate the studies under the
cutting. Sections tend to roll up
microscope.
during cutting, and moistening the
block and section with alcohol by
A. Paraffin Sections
means of a camel hair brush will

Good luck, future RMT! <3

Valdez, P.L.A
 serve to flatten the sections on the
knife.
 Celloidin sections do not come off
in ribbons and have to be
collected into 70% alcohol
immediately. They are then stored
in the same solution in jars with
tightly fitting lids. And finally
mounted on to slides after they
have been stained.

Good luck, future RMT! <3

Valdez, P.L.A