TYPES OF FIXATIVES • No formalin pigment • DisBlled water – 100

ml
a. Based on composi9on • For glycogen • 40% formalin – 15 ml
• Picric acid 1N 95%
SIMPLE FIXATIVES COMPOUND FIXATIVES Gender’s fluid alcohol – 80 ml
Formaldehyde Bouin’s fluid • Glacial aceBc acid – 0.5
Picric Acid Formol saline ml
Osmium tetroxide Zenker’s fluid
FORMALIN FIXATION
b. Based on ac9on
Advantages Disadvantages
HISTOCHEMICAL MICROANATOMICAL CYTOLOGIC Rapid penetraBon Irritant to the nose, eyes, mucous membranes
Formaldehyde Bouin’s fluid Champy’s fluid Easy availability & cheap FormaBon of precipitate of paraformaldehyde
Glutaraldehyde 10% formalin Glacial aceBc acid Does not overharden the Bssue FormaBon of black formalin pigment, acid
Vapor fixaBve Zenker’s fluid Alcohol formaldehyde hemaBn
Formol calcium Formol saline Fixes lipids for frozen secBons
Heidenhain’s susa Carnoy’s fluid Ideal for mailing
Helly’s fluid Clarke’s fluid
Rossman’s fluid Newcomer’s fluid GLUTARALDEHYDE FIXATION
Flemming’s fluid
Advantages Disadvantages
c. Based on chemical nature FormaBon of more cross linkages with beaer Expensive
preservaBon of cellular & fluid proteins
PHYSICAL OXIDIZING Resists acid hydrolysis Less stable
ALDEHYDES COAGULANTS MISCELLANEOUS Causes less shrinkage than formalin Penetrates Bssue more slowly from formalin
AGENTS AGENTS
Osmium More pleasant & less irritant Inferior formalin for PAS saBn
Heat Formaldehyde Methyl alcohol Picric acid Does not corrode metal
tetroxide
Microwave Acrolein Ethyl alcohol Mercuric chloride Does not cause dermaBBs
Glutaraldehyde AceBc acid
METALLIC FIXATIVES
FORMALIN-BASED FIXATIVES
a. Mercuric Fixa9ves
FIXATIVE DESCRIPTION COMPOSITION
FIXATIVE DESCRIPTION COMPOSITION
• For CNS material • 40% formalin – 100 ml
• Maintains color – museum • NaCl – 8.5 Gm Zenker’s fluid • For fixing small pieces of • HgCl2 – 50 gm
fixaBve • DisBlled water – 900
liver, spleen, connecBve • Potassium dichromate –
10% Formal Saline Bssue fibers & nuclei 25 gm
• Very safe ml
• Viral inclusions (Negri • Sodium sulphate – 10
• Slow & liable for shrinkage
bodies) gm
during dehydraBon
• DisBlled water – 1000
• For rou9ne post-mortem • 40% formalin – 100 ml
ml
material • Saturated HgCl2 – 900
• Glacial aceBc acid – 50
Formol Sublimate • Cytologic details & RBCs are ml
ml
well preserved
• No hardening/shrinkage Zenker-formol (Helly’s SoluBon) • For fixing pituitary gland, • Zenker’s fixaBve
bone marrow, spleen, liver (mercuric chloride stock
• For lipid fixa9on • 40% formalin – 10 ml
Formal Calcium soluBon
• Have a near neutral pH • Ca+acetate – 2 Gm
 • Formalin
Haidenhain’s Susa • For fixing tumor biopsies • HgCl2
esp. skin • Glacial aceBc acid
formalin
B-5 FixaBve • For fixing bone marrow • HgCl2
samples • Sodium acetate
• DisBlled water
 • Forms protein picrates
b. Chromate Fixa9ves • Bouin’s fluid
- For fixaBon of embryos & pituitary biopsies
Fixa9ve Descrip9on - ComposiBon:
Chromic Acid Preserves carbohydrates, precipitates proteins § 1.2% aqueous picric acid – 75 ml
Potassium dichromate Preserves mitochondria § Formalin – 25 ml
Regaud’s (Moller’s fluid) Used for mitochondria, mitoBc figures & golgi § Glacial aceBc acid – 5 ml
bodies
ComposiBon: ALCOHOL FIXATIVES
• Potassium dichromate • rapidly denatures proteins by destroying hydrogen bonds
• Formalin
Orth’s fluid Demonstrates rickeasiae, Bssue necrosis Alcohol Descrip9on
ComposiBon: Methyl alcohol • For fixing dry & wet smears, blood smears and bone marrow
• Potassium dichromate Isopropyl alcohol • For touch preparaBon
• Formalin Ethyl alcohol • Used in histochemistry for enzyme studies
• Sodium sulfate Carnoy’s fluid • For fixing chromosomes & urgent biopsies; most rapid fixaBve
• ComposiBon:
c. Lead Fixa9ves - absolute alcohol
- chloroform
MERCURIC FIXATIVES - glacial aceBc acid
Newcomer’s fluid
ADVANTAGES DISADVANTAGES
• Beaer staining of nuclei and connecBve • Corrodes the metals ACETONE
Bssue • for the study of water diffusible enzyme (lipases & phosphatases)
• Cytoplasmic staining –enhanced with acidic • Lysis of RBC & removes much iron from • for fixing brain 9ssues for rabies cases
dyes. hemosiderin
• used at cold temperature (-5C – 4C)
• Nuclear chromaBn shown in detail • Deteriorates rapidly
• PreservaBon of details for photography. • Causes marked shrinkage
• Best results with metachromaBc stain • Reduces the amount of demonstrable HEAT FIXATION
glycogen • Ether saline (0.85%) or 10% formal saline is used.
• B5 fixaBve is frequently used for bone • Slow penetraBon • 20 to 40 ml is heated below the boiling point then the Bssue slice (3 to 5mm thick) is placed in
marrow,spleen, lymph nodes and other hot fluid & heaBng is conBnued for 1 min unBl Bssue floats to the surface.
hematopoeBc Bssue • Aler this it is cooled quickly in water & mounted on microtome.
• Tissues become hard & briale
• FormaBon of Diffuse black granules in
Bssues DECALCIFICATION
• Radiopaque: preclude use of x-rays to - The removal of calcium ions from a bone or calcified 9ssue through a histological process
determine and point of calcificaBon
that makes them flexible and easier to cut
- It adjusts the hard substance of bones to the solness of paraffin embedding medium
CHROMATE FIXATIVES - OpBonal step in Bssue processing
- It is done only if calcium and lime slats are present in the processed Bssue.
ADVANTAGES DISADVANTAGES
• For demonstraBon of chromaffin Bssues • Prolonged fixaBon in chromate – Bleach all Bone – the main object of decalcificaBon in a surgical pathology laboratory
(eg: Adrenal medulla, mitochondria, Golgi Bssues pigments (melanin)
- Teeth, calcified tumors, calcified heart valves
apparatus, mitoBc figures & RBC’s)
• Best for preserving phospholipids • Glycogen preservaBon is poor Principle of Decalcifica9on

Picric Acid Fixa9ve

 Strong mineral acids, such as 10% hydrogen chloride, or weak organic acids such as 5-10% - Is rapid in acBon; produces minimum distorBon of Bssues, and
formic acid, for soluble calcium salts in an ion exchange that moves calcium into the decalcifying soluBon good nuclear staining
- Disadvantage: prolonged decalcificaBon = Bssue distorBon
- The same final effect makes EDTA an ideal chelaBng agent that sequesters metallic ions, d. Formol-Nitric Acid
including calcium, in aqueous soluBons - 1-3 days – decalcifying Bme
- More concentrated acid soluBons decalcify bone more rapidly but are more harmful to - ComposiBon:
the Bssue § Concentrated nitric acid, formaldehyde, disBlled
- High concentraBons and greater amount of fluid will increase the speed of process water
Formic Acid - 2-7 days – decalcifying 9me
Recommended raBo of fluid to Bssue volume for decalcificaBon = 20:1 - Is a moderate-acBng decalcifying agent which produces beaer
nuclear staining with less Bssue distorBon
- Heat will serve to hasten decalcificaBon but it also increases the damaging effects on - Safer to handle compared to nitric acid or hydrochloric acid
Bssues - For rou9ne decalcifica9on of postmortem research 9ssues
§ 37˚C – impaired nuclear staining of Van Gieson’s stain for collagen fibers - For small pieces of bones and teeth
§ 55˚C – Bssue will undergo complete digesBon within 24-48 hours - Is both fixaBve and decalcifying agent
§ Opium temperature – room temperature (18-30˚C) Hydrochloric acid - it is inferior compared to nitric acid in its role as decalcifying agent
- 24-48 hours – ideal Bme required for decalcifying Bssue because of its slower acBon and greater distorBon of Bssues
- Dense bone Bssues – require up to 14 days or longer to complete the process produced
- for surface decalcificaBon of the Bssue blocks
DECALCIFYING AGENTS - disadvantage: extent of decalcificaBon cannot be measured by
- Used to remove calcium and lime salts following fixaBon chemical tesBng
- Must be capable of removing calcium without producing considerable Bssue destrucBon - Von Ebner’s fluid – for teeth and small pieces of bones
TitrochloroaceBc Acid - 4-8 days – decalcifying Bme
TYPES OF DECALCIFYING AGENTS - Weak decalcifying agent, not used for dense Bssues
1. Acid decalcifying agents - Suitable only for small spicules of bone
2. ChelaBng Agents (EDTA - Permits good nuclear staining
3. Ion exchange resins (ammonium form of polystyrene resin) - Do not require washing out (90% alcohol)
4. Electrical ionizaBon (electrophoresis) Sulfurous Acid - Very weak decalcifying agent, suitable for minute pieces of bone
Chromic Acid - Both fixaBve and decalcifying agent
ACID DECALCIFYING AGENTS (Flemming’s fluid) - May be used for decalcifying minute bone spicules
- Disadvantage: it is an environmental toxin (corrosive and
AGENT DESCRIPTION carcinogenic)
Nitric Acid - Most common & fastest decalcifying agent used Citric Acid - 6 days – decalcifying Bme
- Usually combined with formaldehyde or alcohol - Permits excellent nuclear and cytoplasmic staining
- Disadvantage: progressive Bssue damage - Doesn’t produce cell distorBon
a. Perenyi’s fluid - Disadvantage: too slow for rouBne process
- acts as both Bssue solener and decalcifying agent Others:
- for rouBne process Ion exchange resin - Hastens decalcificaBon by removing calcium ions from formic-acid
- composiBon: containing decalcifying soluBons
§ nitric acid - A layer of the ion resin (1/2 thick) is spread over the boaom of the
§ chromic acid container to be used and the specimen is placed over the top. The
§ absolute ethyl alcohol decalcifying agents is then added, usually 20-30 Bmes the volume
b. Phloroglucin-Nitric Acid of the Bssue. The Bssue is allowed to stay in the soluBon for 1-14
- 12-24 hours – decalcifying Bme days
- Most rapid decalcifying agent - Advantages:
- Disadvantage: poor nuclear staining - Cellular detail is well preserved
c. Aqueous Nitric Acid Solu9on 10% - Daily washing of soluBons is eliminated
 - Permits excellent staining results and decalcificaBon is - Is done on the discarded fluid
hastened - A piece of blue litmus paper is added to a test tube
- Disadvantages: containing 5ml of the discarded decalcifying agent
- It is very slow and causes slight Bssue hardening - Strong ammonia is then added drop by drop unBl the fluid
Electrophoresis - A process whereby posiBvely charged calcium ions are aaracted to is neutralized
the negaBve electrode and removed from the decalcifying soluBon - Presence of cloudiness indicates that there is sBll calcium in
- Same principle as with chelaBng agents, but this process applies the soluBon
electricity to remove the calcium ions - The Bssue is then immersed in a new soluBon of
- For small bone fragments decalcifying agent
- For processing a limited number of specimens at a Bme - Ammonium oxalate can be used to ensure decalcificaBon
- Good cytologic and histologic details are not always preserved in - Cloudiness aler applicaBon of ammonium oxalate will
Bssues signify incomplete decalcificaBon
Microwave Oven - Used olen for Bssue processing but there are studies describing its - Simple, reliable, and recommended for rouBne process
DecalcificaBon use in decalcificaBon of bone or teeth
- Faster than rouBne decalcificaBon TISSUE SOFTENER
- Does not adversely affect cell morphology - For unduly hard Bssues that may damage the microtome knives
- Uniformity of the thickness of the Bssue is a strict requirement a. 4% aq. Phenol
b. Mollifex
c. 2% HCl
d. 1% HCl in 70% alcohol
e. Perenyi’s fluid – both decalcifying agent and 9ssue socener
FACTORS INFLUENCING DECALCIFICATION
1. Concentra9on DEHYDRATION
- 20:1, the recommended volume to Bssue raBo - The process of removing intracellular and extracellular fluid water from the Bssue in preparaBon for
2. Fluid access wax infiltraBon
- Fresh decalcifier must have access to all surfaces of the specimen DEHYDRATING AGENTS
- May be hastened by suspending the Bssue in decalcifying agent for beaer access 1. Alcohol – most common
3. Size and consistency - Ethyl Alcohol – for rouBne dehydraBon
- Increased size = longer periods of decalcificaBon - Tissue is passed in ascending grade/concentraBon of alcohol
4. Agita9on - 70% to 90% to 100% (absolute alcohol)
- Gentle agitaBon may increase the rate of decalcificaBon 2. Acetone – for urgent biopsies
5. Temperature 3. Dioxane
- Increased temperature will hasten decalcificaBon but will also increase the damaging effects 4. Cellosolve
of the acids on Bssue 5. Tetrahydrofuran

EXTENT OF DECALCIFICATION CLEARING

Physical/Mechanical Test
X-ray or Radiological Method
Chemical Method
- The process of removing alcohol from the Bssue
- Requires manipulaBon, bending, probing, trimming of the - Also known as de-alcoholizaBon
specimen to feel for remaining calcified areas
- Generally considered to be inaccurate CLEARING AGENTS
- Mechanical damage can occur
- Pricking of Bssues may produce false posiBve 1. Xylene- most common
microfractures 2. Toluene- subsBtute for xylene
- Very expensive 3. Benzene- for urgent biopsies
- Most ideal, sensiBve, and reliable 4. Chloroform- for tough Bssues
- Not recommended for mercuric chloride fixed Bssues 5. Cedarwood oil - CNS
- The decalcifying agent is usually changed every 24-28 hours 6. Aniline oil- clearing embryos
 7. Clove oil PROCEDURE:
8. Carbon- tetrachloride
9. tetrahydrofuran Aler clearing, the Bssue is submerged in two or more changes of melted paraffin wax, in an
10. Methyl benzoate incubator/paraffin oven.
- Temperature must be between 55-60˚C
INFILTRATION/IMPREGNATION - Most waxes in commercial lab use have melBng points of 45˚C, 52˚C, 56˚C and 58˚C
- the process of removing the clearing agent (xylene) completely out from the Bssue and replaced by - The 56˚C is used normally for rouBne work.
a medium that will completely fill all the Bssue caviBes
- allows easier curng and handling of Bssue secBons - Melt in paraffin oven or an incubator
- Oven or incubator should be regulated at 55-60˚C
Why is Xylene removed? - Common waxes melBng points:
- Xylene can cause Bssue distorBon if exposed for too long in the Bssue • 45˚C
- It gives room for the impregnaBng media to enter the Bssue • 52˚C
• 56˚C – most common
Why is the Bssue impregnated? • 58˚C
- ImpregnaBon and embedding media will fill all Bssue caviBes completely.
- Gives a firm consistency to the specimen. LABOARATORY TEMP AND WAX MELTING POINTS
- This will allow easier handling and curng of suitably thin secBons without any damage or distorBon - In a laboratory having an internal temperature of:
to the Bssue and its cellular components. • 20-24˚C – paraffin wax melBng point 54-58˚C
• 15-18˚C – paraffin wax melBng point 50-54˚C

ADVANTAGES DISADVANTAGES TISSUE CHARACTERISTICS

Serial secBons are cut with ease and majority Overheated paraffin makes Bssue briale - Hard Bssues – wax with higher melBng points
w/o undue distorBon - Sol Bssues – wax with lower melBng points
Very rapid, allows secBons to be prepared within Prolonged impregnaBon will cause Bssue
24 hours shrinkage 3 WAYS OF IMPREGNATING AND EMBEDDING OF TISSUES
Tissue blocks and unstained can be stored in Inadequate impregnaBon promotes clearing
paraffin for an indefinite period of Bme agent retenBon (Bssues become sol and Manual Processing - 4 changes of wax for 15 minutes each interval
shrunken, crumble and break up) Automa9c Processing - makes use of an automaBc processing machine
Many staining procedures are permiaed Tissues difficult to infiltrate need long immersion - fixes, dehydrates, clears, and infiltrates Bssues automaBcally
for proper transport - decreases the Bme and labor needed during the process
Nucleic acids may be recovered from them Not recommended for faay Bssues - 2-3 changes of wax are required to remove the clearing agent
decades aler fixaBon - Autotechnicon
• Has 12 processing steps
• Has 10 1L glass beakers
TYPES OF IMPREGNATING AND EMBEDDING MEDIA • 2 thermostaBcally wax baths
• Transfer arm (facilitates clock schedules)
A. Paraffin Wax Vacuum Processing - Involves wax impregnaBon under negaBve atmospheric pressure
B. Celloidin inside an embedding oven
C. GelaBn - Reduces the Bme when Bssues are subjected to high temperature,
D. PlasBc minimizing heat-induced Bssue hardening
- Facilitates complete removal of transiBon solvents
PARAFFIN WAX IMPREGNATION - Prolongs the life of wax by reducing solvent contaminaBon
- The simplest, most common and best embedding medium used for rouBne processing - Hastens the removal of air bubbles and clearing agent from the
- Paraffin Wax – a polycrystalline mixture of solid hydrocarbons produced during the refining of coal Bssue block, promoBng a more rapid wax penetraBon of the Bssue
and mineral oils. It is solid at room temperature but melts at temperature up to 65˚C or 70˚C - Recommended for urgent biopsies
- For delicate Bssues such as lung, brain, connecBve Bssue, bone, eyes
 - Time is reduced by 25%-75% of the normal Bme required - Can be used for impregnaBon without prior clearing of the Bssue
- Pressure should not exceed 500 mm hg - It is used with heavy duty microtome such as sliding and sledge
microtome
PRECAUTIONS IN PARAFFIN WAX IMPREGNATION Water Soluble Wax - MelBng point: 38˚C-42˚C or 45˚C-56˚C
- Polyethylene glycol
1. Over exposure of the Bssues with the paraffin can cause Bssue hardening and shrinkage, - Carbowax
2. Overheated paraffin wax (60C above) will cause also hardening and Bssue shrinkage. (2-5C) • Most commonly used
above melBng point. • Miscible and soluble water
3. Paraffin wax must be pure, free from dust, water droplets and other unwanted maaer. • Does not require dehydraBon and clearing
• Does not remove neutral fats and lipids
FACTORS AFFECTING PARAFFIN WAX IMPREGNATION • Suitable for enzyme histochemical studies
• Cytological details are preserved
1. Nature and size of the Bssues • Easily dissolved in water
• Larger and denser Bssue blocks = longer period of Bme and frequent changes of wax • Difficult to float out mount due to its extreme solubility in
2. Type of clearing agents water, dehydraBng and clearing agents
• Benzene and Xylene are easily removed • Adding soap or 10% polyethylene glycol to water reduce Bssue
• Chloroform and Cedarwood oil are difficult to remove and require frequent wax changes distorBon and promote flaaening and “floaBng out” of secBons
3. Paraffin wax must be pure and free from:
a. Dust CELLOIDIN IMPREGNATION
b. Water droplets - Purified form of nitrocellulose
c. Other foreign materials - Suitable for large hollow caviBes, hard and dense Bssues such as bones and teeth
4. Paraffin wax may be used twice only - For large secBons of embryo
5. Fixed microtomes, a relaBvely hard wax with a higher melBng point is recommended
6. Heavier microtome knives require harder paraffin wax than lighter ones ADVANTAGES DISADVANTAGES
Permits curng which are thicker than in paraffin Very slow
SUBSTITUTES FOR PARAFFIN WAX wax
Its rubbery consistency allows the Bssue to be cut Very thin secBons (10 um below) are difficult
Paraplast - MelBng point: 56˚C-57˚C w/o undue distorBon to cut
- More elasBc and resilient than paraffin wax Dense Bssues w/c are hard to infiltrate are Vapor of ether are very inflammable
- Highly purified and syntheBc plasBc polymers supported beaer
- For large dense Bssue blocks (bones and brain) It does not require heat during processing Photomicrographs are difficult to obtain
- Prevents ice crystals formaBon Very volaBle
- No deposits aler staining
- Soluble in common clearing agents 2 METHODS OF CELLOIDIN IMPREGNATION
- Same Bme schedule as paraffin
- Embeddol Method Descrip9on
• MelBng point: 56˚C-58˚C Wet Celloidin - Recommended for bones, teeth, large brain secBons and whole
• Synthe9c wax, less briale organs
- Bioloid - When ball of the fingers leaves no mark on the surfaces of the
• Semisynthe9c wax; for embedding eyes Bssue block the processed is considered to be complete
- Tissue Mat
Dry Celloidin - Preferred for whole eye secBons
• Product of paraffin, containing rubber w/ same property as
- Uses Gilson’s mixture (made up of equal parts of chloroform and
paraplast
cedarwood oil)
Ester Wax - MelBng point: 46˚C-48˚C - Does not require alcohol due to the presence of cedarwood oil in
- Harder than paraffin the bock
- Not soluble in wat
Others:
- Soluble in 95% ethyl alcohol and other clearing agents
Nitrocellulose Method - Another form of celloidin
 - w/ lower viscosity allowing penetraBon of the Bssue rapidly throughout fixaBon, dehydraBon, clearing, and
- makes curng of thinner secBons possible wax impregnaBon
GelaBn ImpregnaBon - rarely used except when dehydraBon is to be avoided or the Disposable Embedding Molds a. Peel-Away
Bssues are subjected histochemical and enzyme studies • Disposable thin plasBc embedding molds,
- prevents fragmentaBon of Bssues when used in frozen secBon and available in 3 different sizes
as embedding medium • Are simply peeled off one at a Bme as soon as
- soluble to water the wax has solidified
- does not require dehydraBon and clearing • Gives even block without trimming
- Bssues should not be more than 2-3 mm thick • May be placed directly in the shuck or block
- 1% phenol – prevents the growth of molds holder of the microtome
- ImpregnaBng medium is 25 Bmes the value of the Bssues b. PlasBc Ice Tray
• Such as those used in ordinary refrigerators
EMBEDDING may be recommended for busy rouBne
- The Bssue is placed in a mold containing the embedding medium and is allowed to solidify laboratories
- Paraffin wax – 3 hours • Each compartment may be uBlized for
- Ideally the embedding medium should match the Bssue type in strength and hardness embedding one Bssue block, which may then
- Paraffin embedded Bssues are arranged at the boaom of the mold together with their proper labels be removed by bending the plasBc tray once
- immersed in melted paraffin at a temperature between 5˚C-10˚C above its melBng point the wax has solidified or by smearing the inner
- It is cooled rapidly at -5˚C in a refrigerator or immersed in cold water to solidify to allow Bssue mold with glycerin or liquid paraffin before
hardening embedding
• Giving them a firmer consistency and beaer support, facilitaBng curng of the secBons c. Paper Boat
- OrientaBon: process by which Bssue is arranged in precise posiBons in the mold during embedding • UBlized for embedding celloidin blocks but are
• The most important step in infiltraBon and embedding equally useful for paraffin wax blocks
- The surface of the secBon to be cut should be placed parallel to the boaom of the mold • Cheap and easy to make
• Provide easy and accurate idenBficaBon of
TYPES OF BLOCKING-OUT MOLDS specimens, avoiding confusion and interchange
of Bssue blocks
TYPE DESCRIPTION
L-mould (Leuckhart’s Embedding Mould) - Consists of two L-shaped strips of heavy brass or MICROTOMY
metal arranged on a flat metal place which can be - Process by which a processed Bssue, most commonly a paraffin embedded Bssue, is trimmed and
moved to adjust the size of the mold to the size of the cut into uniformly thin slices known as secBons to facilitate studies under the microscope
specimen
Compound Embedding Unit - Made up of series of interlocking plates resBng on a Microtome
flat metal base forming several compartments - Capable of curng a secBon at a predetermined thickness by sliding the block into a curng tool,
- Advantage: can embed more specimens at a Bme, usually a steel knife, glass and diamond blade, which is fixed and aaached to the machine
reducing the Bme needed for blocking
PlasBc Embedding Ring and Base Mold - Consists of special stainless steel base mold fiaed ESSENTIAL PARTS OF A MICROTOME
with a plasBc embedded ring, which will serve as the
block holder during curng 1. Block holder – where Bssues are being held in posiBon
- Tissue Tek 2. Knife Carrier and Knife – for actual curng of Bssue secBons
• Equipped with a warm plate to manage the 3. Pawl, Ratchet Feed Wheel and Adjustment Screws – to line up the Bssue block with the knife,
impregnated specimen and a cold plate at -5˚C adjusBng the thickness of the Bssues for successive secBons
for rapid solidificaBon of the block
• Consists of a white plasBc casseae mold with 5 KINDS OF MICROTOME
detachable, perforated stainless hinge and
snap-on lid, used to hold the Bssue specimen MICROTOME DESCRIPTION
Rocking Microtome - Used for curng secBons of large blocks of paraffin embedded Bssues
(Paldwell Trefall 1881) - The simplest among the different types
- Tissue is cut in 10-12 um thick
- Available in two sizes, small and large blocks of paraffin Bssues
- TheoreBcally not recommended for serial secBons
- Not favored in most laboratories because of the restricBons in size
- Difficult to orient the block
- Consists of:
• Heavy base
• Two arms
• Lower arm – resBng on pivots and supporBng column and
aaached the micrometer screw at the base of which is
found the ratchet wheel
• Upper arm – carrying the block holder on one end by
means of screw, connected to a lever by a piece of nylon
thread
Rotary Microtome - Used for curng secBons embedded in paraffin
(Minot 1885-1886) - Most common type used in rouBne and research laboratories
- Operated by rotaBon of the flywheel, causing reciprocal moBon of
the knife over the block
- Thickness of secBon is regulated by ratchet feed wheel
- Heavier and more stable than a rocking microtome
- More complex in design and construcBon, more expensive
- May also be used in curng large blocks of Bssues
- The knife is placed in a blade-up posiBon which is dangerous
- Heavier knife, less vibraBon
- Pawl – used to come in contact with the ratchet wheel which in turn
rotates the micrometer screw
- Adjustment Screws – make the block parallel to the microtome knife
at all planes
- Electrically Driven Rotary Microtome
• Can be ideally used when to produce ribbons for serial secBons
Sliding Microtome - Used for curng celloidin embedded secBons
(Adams 1789) - Knifes can be set at an angle for curng celloidin
- For curng extremely hard and rough Bssue blocks
- The most dangerous type of microtome due to movable exposed
knife
- Two types:
a. Base Sledge Microtome
• Consists of 2 movable pillars holding the adjustable knife
clamps, allowing the knife to be set at an angle for curng
celloidin secBons
• The chunk or block holder can be moved forwards and
backwards under the knife
• Favored for hard and large blocks secBoning MICROTOME KNIVES
• SecBons are cut flat, allowing excellent serial secBons
b. Standard Sliding Microtome
• The block remains staBonary while knife is moved forwards
and backwards
• Mainly developed for curng celloidin embedded Bssue
blocks
• More dangerous because of the movable knife
Freezing Microtome - Used for curng unembedded frozen secBons
(Queckea 1848) - Useful in rapid diagnosis and sensiBve Bssue consBtuents that are
damaged or destroyed by heat
- Used to cut
• Undehydrated thin to semi-thin Bssues of fresh, frozen Bssues
when rapid diagnosis is required
• Histological demonstraBon of fats
• For certain neurological structures
- The stage for block holder is hollow and perforated around its
perimeter
- w/ flexible lead pipe thru CO2 passes from a cylinder
- lever operated valve allows the release of rapid and intermiaent burst
of CO2
- a cooling device for lowering the temperature of the knife to facilitate
secBoning
- gives the best result and is the most used
Cryostat/Cold - A refrigerated apparatus used in fresh Bssue microtomy
Microtome - Consists of a rotatory microtome, kept in cold chamber
- Maintained temperature at: -5˚C- -30˚C or -20˚C average
- Used for rapid preparaBon of urgent Bssue biopsies for intraoperaBve
diagnosis
- For curng secBons for electron microscope
- Its thermostat is capable of freezing fresh Bssues within 2-3 minutes
- Provides secBons for fresh Bssue examinaBon, esp. Fluorescent
AnBbody Staining Techniques or histochemical enzymes studies
- Olen housed in the frozen secBon room close to the operaBng room
to allow direct consultaBon between the surgeon and pathologist
Ultrathin Microtome - Used for curng Bssues in preparaBon for electron microscopy
- Equipped with a glass or gem diamond knife used to cut very thin
secBons (60-100 mm)
- Examined using Transmission Electron Microscope
- Size: 0.5 micra thin
- Knife: fragments of broken plate glass
- Specimens must be:
• Small
• Fixed in osmium tetroxide
• embedded in plasBc
Plane Concave Knife Biconcave Knife Plane Wedge Knife
25 mm length 120 mm length 100 mm length
One side of the knife is flat, while
the other side is concave
For celloidin embedded Bssues
Both sides are concave Both sides are straight
For paraffin embedded Bssues For frozen secBons and
extremely heard specimens
in paraffin