MLSBIO107: HISTOPATHOLOGIC TECHNIQUES

LABORATORY
TRANSCRIBED BY: COMIA, MAUI GWYCYLL D.
BLOCKING TO LABELLING

BLOCKING TO LABELING • OCT (Optimal Cutting Temperature Compound) –

recommended.
• Tissue block – 2-4 mm
• Tissue section can be cut from 5-10 um.

BLOCKING
• A step that goes hand in hand with embedding if individual
mold is used because blocks are produced after
solidification.
• If a big mold tray is used, use a sharp knife to separate
one tissue from one another. CRYOSTAT

TRIMMING SECTIONING
• Remove excess wax. MICROTOMY
• To form a TRUNCATED Pyramid (Prism)
• At least 2mm of wax should surround the tissue block. 3 ESSENTIAL PARTS
1. Block Holder: aka “CHUCK” - where the tissue is
held in position.
2. Knife Carrier and Knife - for the actual cutting of
tissue sections.
3. Pawl, Ratchet Feed Wheel and Adjustment
Screws - line up the tissue block properly and adjust
proper tissue thickness.

5 KINDS OF MICROTOMES
1. Rocking Microtome (Paldwell Trefall) 1881
o Simplest Microtome
SECTIONING o Can cut 10-12 um tissue sections
• Embedded tissue is trimmed and cut into uniformly thin o For large paraffin-embedded blocks; Not for serial
slices using a microtome. sections
2. Rotary Microtome (Minot) 1885-1886
o Routinely used microtome
TYPES OF SECTION
o Cutting of paraffin-embedded tissues
• Paraffin sections - 4-6 um sections: most commonly
o Excellent serial sections
used.
3. Sliding Microtome (Adams) 1789
• Celloidin Sections – 10-15 um sections - ultrathin 0.5 –
o For celloidin sections and hard rough tissue block
1.5 um
o Two Types:
• Frozen sections (Cryostat) - varying 10 um - cryostat ▪ Base Sledge
consists of rotary microtome ▪ Standard Sliding
o Rapid diagnosis 4. FREEZING MICROTOME (Queckett) 1848
o For enzyme histochemistry o For rush frozen sections (cold knife procedures
o Demonstration of soluble substances o CO2 is used as a propellant.
o Immunofluorescent and immunocytochemical o *Cryostat or Cold Microtome
staining ▪ for rush sections; now commonly used.
o Specialized silver stains ▪ Refrigerated (-5 to -30oC) ave (-20oC)
▪ inside is a ROTARY MICROTOME
TWO METHODS OF FROZEN SECTIONS 5. ULTRATHIN MICROTOME
1. Cold knife – old method o Cutting sections as thin as 0.5um
o Use of conventional freezing microtome (carbon o Electron Microscopy
dioxide) o Tissues are usually embedded in Plastic Resin-hard
o Tissue block: 3-5 mm o Special Knife: Diamond Knife (not easily dull; its
o Dew line – point in which sections may be cut a 10 sharp)
um.
2. Cryostat - common method MICROTOME KNIVES
o Optimum temperature: -18 to -20C 3 BASIC TYPES
1. PLANE CONCAVE (25mm)
MOUNTING o Less concave side - for celloidin
• Use of synthetic water-soluble glycols and resins o More concave side - for paraffin

1
 MLSBIO107: HISTOPATHOLOGIC TECHNIQUES
LABORATORY
TRANSCRIBED BY: COMIA, MAUI GWYCYLL D.
BLOCKING TO LABELLING

o For base sledge, rotary or rocking microtome. o Found in the tapered edge of the knife.
2. BICONCAVE KNIFE (120mm) • Bevel Angle – angle formed between the cutting edge
o Both sides are concave o 27–32degrees
o Paraffin sections • Cutting Angle - 15degrees(optimum)
o Rotary Microtome o There is maximum penetration of tissue and less
3. PLANE WEDGE (100mm) distortion.
o For frozen sections or very hard tissues. • Clearance Angle - 0.15degrees
o Base Sledge or sliding microtome. • Angle formed between the surface of the block and the
cutting edge of the knife.
• Knife Materials
o Disposable knives
o Stainless Steel
o Tungsten Carbide
o Glass knives - “Ralph Knives” for ultramicrotomy
o Diamond Knives – cutting resin sections.
o Steel Knives

HONING VS STROPING

HONING STROPING
• “Knife sharpening” • “Polishing”
• From heel to toe 20- • Removal of burrs
30times • From toe to heel
• Removal of Nicks • Use Paddle Strop
(irregularities of knife) Made of horse leather.
• Hones (8inch x 3inch) • Strops should be oiled
o Belgium Yellow on the back (DON’T
(Best Result) USE MINERAL OIL)
o Arkansas - more • 40-120 double strokes
polishing effect
o Fine carborundum
(badly nicked
knives)
• Lubricants (soapy water,
mineral oil, clove oil,
xylene or liquid paraffin)
• Knife Sharpeners
o Mechanical Knife
Sharpener (use of
vibrating frosted
glass plate)
o Flat Circular Glass
Plate with
Powdered
Aluminum Oxide

• Bevel - cutting facet

2
 MLSBIO107: HISTOPATHOLOGIC TECHNIQUES
LABORATORY
TRANSCRIBED BY: COMIA, MAUI GWYCYLL D.
BLOCKING TO LABELLING
OTHER EQUIPMENT USED IN SECTIONING
• Floatation Water Bath
o Temp:6-10̊ClowerthanMPofWax(45-50°C)
o Colored Enamel Black to easily visualize tissue
sections.
o Diameter - 11inch.
o Height - 4inch;2Lcapacity
• Drying Oven
o Temp: 5C Higher than MP of wax
• Forceps
• Clean Slides - 76x25mm thick; Frosted
FISHING OUT
• Place the ribbon of sections into the water bath with a
black base for several minutes.
• Separate the ribbon of sections into individual sections
using forceps.
• Start fishing out each section using a slide (lightly
smeared with adhesive)
• Immerse the slide vertically.
• Fish-out section
• Gently lift the slide vertically.
FISHING
TISSUE ADHESIVES
1. Albumin (Mayer’s Egg Albumin)
o Preservative is added to prevent putrefaction
o Glycerol is also added to increase viscosity and
prevent complete drying.
o Background staining may be detected due to its
uptake of dyes.
o Sources: Egg albumin, Bovine, Human albumin
2. Gelatin - Provides firmer attachment then albumin.
o Has to be gently heated before use to melt the
gelatin.
o Added to the floatation water bath.
o Disadvantage: Stains with many dyes.
3. Cellulose - in the form of 1% Methylcellulose
o Advantage: Not staining to any appreciable extent
with commonly used in stains of histochemical
reagents
o Poly-L-lysine - Use as a general-purpose
o Section adhesive
o *No production of background staining
4. Sodium Silicate - commercial syrup = 1:10 dilution
o Had strong adhesive properties.
o Advantages: Little tendency to staining with the most
dyes; not affected by the use of mild alkaline
solutions
o Disadvantages: Blackening in some silver
impregnation techniques, in some reticulin methods,
and red staining in methyl green pyronin technique
5. Resins - Greatest adhesion
o Araldite- made of epoxy resins.
▪ Diluted 1:10 with acetone.
▪ Little affected by most fluids in any treatment of
sections.
SPECIAL PROCESSING TECHNIQUES
• For Histochemical Evaluation
A. Freeze Drying
o Preservation by rapid freezing (quenching) at 160̊C
o Desiccation by transferring the frozen tissue in a
vacuum at -40̊C (Sublimation)
o 2mm tissue thickness
o Expensive and Time Consuming
B. Freeze Substitution - similar to freeze drying except
the vacuum procedure.
o Makes use of Rossman’s Formula or in 1% Acetone
and Dehydrated in Absolute Alcohol
o Routine Paraffin Wax Impregnation and Embedding
is done.
STAINING
• Application of dyes on tissue sections to study the
architectural patterns and physical characteristics of cells.
• Different tissues and cells have varying affinities for most
dyes and stains.
• AFFINITY:
o Acidic (nucleus) = basic stains
o Basic (cytoplasm) = acidic stains
MOUNTING
• Use of a medium and a coverslip to facilitate the handling,
storage, and protection of the tissue section.
• Mounting Medium is used (syrupy fluid to prevent
movement of the coverslip; protects and prevents
scratches to the tissue section)
• A Mounting Medium should have a refractive index close
to that of glass 1.518.
KINDS OF MOUNTING MEDIUM
A. Aqueous
• solidify the medium: glycerol (prevent cracking)
• usually for lipids because resinous media contain xylene
which may dissolve fats.
o Water (Temporary) - cheapest, evaporate easily
o Glycerin (Semi-permanent) sections are kept
preserve for years if sealed with paraffin 1.47
o Farrant’s Medium (Gum Arabic Medium) 1.43;
Arsenic trioxide (subs of sodium Merthiolate for
preservation)
o Apathy’s Medium (Methylene Blue Stained Nerve
Preparations)1.52
o Brun’s Fluid -Mounting Frozen Sections from Water
B. Resinous - longer storage
o Canada Balsam - (Canadian tree. A bus Balsamea)
transparent and colorless but darens and oxidizes
with ages; for whole mounts and thick sections.
Refractive Index: 1.524
o DPX - small tissue sections; dries rapidly 1.532
o XAM - synthetic resin mixture dissolved in xylene
1.52
o Clarite - synthetic resin; preferred over DPX 1.544
o Permount, HSR, Clearmount
3
 MLSBIO107: HISTOPATHOLOGIC TECHNIQUES
LABORATORY
TRANSCRIBED BY: COMIA, MAUI GWYCYLL D.
BLOCKING TO LABELLING

RINGING
• Sealing of Margins of the Coverslip to prevent escape of
fluid and evaporation; prevents sticking of slides.

KINDS OF RINGING MEDIA

• Kronig Cement
o (2 Parts Paraffin mixed with 4-9 parts Powdered
Colophonium Resin)
• Durofix (Cellulose Adhesives)

LABELING
• Process of Indicating the year and Specimen Number on
one end of the prepared slide for proper identification.