HISOPATH LAB SECTIONING AND • Most simple

STAINING • Used for cutting serial sections,

regarded as the simplest among
Sectioning – process of cutting tissues
the different types of microtome
into the thin sizes under the microscope
• Used to cut small and large
Microtome – equipment used for paraffin blocks
cutting tissues • Thickness of sections produced is
10-12 mm
Types of Tissue Sections

A. Paraffin- (paper boat) width 4-6 2. Rotary microtome

mm • Developed by Minot
B. Celloidin – 10-15 mm • Most common type
C. Frozen - 5-10 mm • Commonly used specifically for
- Cryostat 4 mm cutting embedded tissues for
- Rapid diagnosis for enzyme routine and research
histochem laboratories.
- Demonstration of soluble • Allows excellent serial sections to
substance be cut
- Immunofluorescent – • Knife and block holder move in an
immunocytochemical upward & vertical direction
staining; specialized silver • Thickness of section produced is
stains. 4-6 mm (same as paraffin)
- • Cut paraffin embedded tissue
PARTS OF MICROTOME 3. Sliding microtome
• Invented by Adams
a. Block holder or Chuck- where • Most dangerous- expose to knife
the tissue block holds on • Used for cutting celloidin
(position) embedded tissues
b. Knife & knife carrier- actual • Thickness of sections produced is
cutting of tissues 7-9 mm
c. Pawl/ Ratchet feed wheel – line
up the tissues in proper Types:
arrangement; essential for cell
1. Base sledge – less dangerous,
block
movable part is block holder
d. Micrometer- regulate the
2. Standard sliding- where the
thickness/ thinnest sections
knife is movable part.
during cutting
e. Rotating wheel – to move 4. Freezing Microtome
mechanically the procedure
• Developed by Queckett
TYPES OF MICROTOME • Used for cutting unembedded
(CUTTING ENGINE IN frozen section and unhydrated
HISTOPATH ) tissues
1. Rocking microtome/ • Thickness of sections produced
Cambridge is 10-15 mm
• Invented by Trefall
 • Used for rapid diagnosis and to 2. Isopentane- liquid at room
demonstrate fats temp
• Uses intermittent burst of CO2 3. Aerosol spray (cryokwik)
to freeze block holder and 4. Carbon dioxide- often used
eventually the tissue when using freezing microtome
• Cutting tissue with heat Freon 2.2 high thermal
sensitive conductivity
• For demonstration of fats and TYPES OF MICROTOME KNIFE
other neurological structures
1. Plane concave
5. Ultrathin microtome (EM)
• Length of 25 mm
• Used for EM for cutting tissue • One side is flat and the other
• Thickness produced is 0.5 mm side is concave
• Can produce more thinner than • Flat side is used for cutting
rotary microtome celloidin while the more concave
side is for cutting embedded
paraffin tissues
6. Cryostat/cold microtome 2. Biconcave
• Used for cutting fresh tissues, an • Has a length of 120 ml
apparatus capable of freezing • Both sides are concave used for
(refrigerated apparatus) the cutting paraffin embedded tissue
tissue up to a certain degree of on rotary microtome
hardness to facilitate sectioning. 3. Plane wedge
• Temperature of chamber is -5 to • 100 mm in length
-30 C, average is -20 C • Both sides are flat, used for
• This can freeze tissue up to 2-3 cutting celloidin, hard tissue
mins • Recommended for frozen section
• Thickness of section is 4 mm SHARPENING OF MICROTOME
• Used for open surgery procedure. KNIVES
• For fluorescent antibody staining
or histochemical studies A. Honing
• Process of sharpening a knife by
Methods for preparing Frozen grinding the cutting edge either
sections on a stone or an abrasive
1. Cold knife procedure- need to compound
have freezing microtome, used • Direction: heel to toe
freezing agent; tissue block 3-5 • Material used for honing is hone
mm; drew line: point in wc PURPOSE OF HONING
section may be cut at 10 mm
2. Cold microtome - To remove gross mix blemishes
procedure/cryostat method • Number of strokes required 20 to
30 each side to grind cutting
FREEZING AGENT: edge to acquire an even edge
1. Liquid nitrogen- histochem. • After honing, wipe off oil from
Most rapid knife with tip with xylene
 2 stages: • Number of strokes: 40-120
A. Coarse honing- removal of gross double strokes
blemishes • Do not use mineral oil, it causes
B. Honing proper-grinding cutting blister
edge
Clearance angle ---- eto ang gusto ko
TYPES OF HONES: lang malaman nyo
1. Belgium yellow- manual • 0-15”
sharpening of nicked edge, the • Angle between the tissue block
type of that usually gives the and the cutting facets of the
best results. knife.
2. Arkansas- artificial stone with • actual
more polishing effect than
Belgium yellow.
3. Fine carborundum- artificial
Wedge angle
stone with much coarser bite,
used for badly nicked knives, • 15”
requires finishing using one or • Angle formed by the sides of the
two other hones knife
4. Plates glass- excellent substitute
for stone, finely powdered Bevel angle
Aluminum oxide made into • 27-32”
pastel with water • Angle formed between the
*Lubricants used for home- soapy cutting edges
water, castor oil, clove oil, xylene, Ribbon- end product of sectioning
and liquid paraffin (MINERAL OIL
can’t be used-metal containing)

B. STROPPING CHARACTERISTICS OF GOOD

RIBBON
• Process of cutting edge of a knife
on a leather or canvas after • Thin and transparent
honing • Easily separated from one
• Direction: toe to heel another
• Use paddle strop made of horse • Does not wrinkle or fold when
leather floated out
• Strops are usually treated with • No irregularities
oil • Uniform in thickness
• Material: horse leather, leather, • Continuous
strop requires oiling before they
REMOVAL OF RIBBONS FROM
are used, oil used is castor oil or
MICROTOME
vegetable oil
• Purpose: to polish and sharpen • Camel hair brush
the cutting edge and remove • Pair of unbladed forceps
burrs and irregularities formed • Finger
during honing • Unbladed spatula
FLOAT- OUT BATH EXAMPLES OF ADHESIVE:

• Thermostatically controlled bath A. Mayer’s egg albumin – most

used specifically to prevent commonly used because it is
wrinkling or folding of ribbons, to easy to prepare and inexpensive.
flatten the ribbons. Prepared by mixing equal
• 45-50C temp amount of egg white, glycerin,
• 10% lower of the melting point of and thymol – prevent growth of
the paraffin molds.
• 46 C – lower than 10 for paraffin B. Dried albumin- dried albumin,
• Used to remove wrinkle/ to NaCl, thymol crystals.
flattened ribbons C. Starch paste- starch, 1N HCl
• Temp 6-10 % lower than wax and thymol crystals
melting point 45-50C D. Gelatin- Gelatin, glycerol, phenol
crystals
PURPOSE OF FLOAT- OUT BATH E. Pooled serum or plasma-
F. Gelatin- formaldehyde mixture
• To flatten the ribbon (stretch
G. 1 percent gelatin
• To prepare the tissue for
H. Plasma
mounting
I. Poly-L- Lysine- immunochem
• To remove the irregularities J. APES (3-
• To select the perfect ribboned aminopropylthriethoxysilane)-
tissue from others cytology
• Fishing out, orientation formed K. Sodium silicate- commercially
DRYING OF SLIDES- syrup 1:10 and dilution with
strong adhesive property.
- Leaving slides in 37C
incubator overnight DEPARAFFINIZATION- removal of
- Placing an oven 50-60C 2 hrs paraffin from tissues one properly fixed
- Drying using hot plate 45- on the slide carried out by:
55C 30-45 minutes ➢ Immersion of slides on xylene
FISING OUT bath’
➢ Placing slides inside the oven
- Removal of ribbons from the ➢ Heating slide over a flame using
float out bath an alcohol lamp
- Temp paraffin over 55-56C
/2-5 degree higher than wax
melting point STAINING

ADHESIVES – promote • Process of applying dyes or

adhesion/attachment of ribbons to stains to tissue to produce
the glass slides and to prevent affinity, contrast, differentiation,
detachment of section from the slide and better microscopic
during staining. visualization.
• Carried out to promote optical
*Excess adhesive can cause differentiation and identification
overstaining* proteinaceous of cells and tissue components.
• Purpose of staining is to 4. Plasma dye- for color reagent
recognize the tissue elements used as extracellular stain
and color reaction. (Azocarmine, azothionine)

CHROMOGEN- Imparts color to the STAINING METHODS:

tissue temporarily
1. Vital staining- used for staining
DYE- Imparts color to the tissue living cells that are visible.
permanently a. Intravital staining (LIC)- living
cells done by injecting dye in
AUXOCHROME- responsible for other part of animal. ( Lithium,
transferring the color to a substance Carmine, India ink)
or a material in a process known as b. Supravital staining (JNT) –
dyeing. living cells done immediately
CHROMOPHORE – any chemical after removal of living body;
growth wc gives specific color to reticulocytes (Neutral red-best
compound and unites auxochrome vital stain, Janus green- for
group to form a dye that becomes mitochondria, Nile blue, Trypan
permanent. blue, Toluidine blue, Thionine)
2. Direct staining- Process of
CLASSIFICATION OF DYES staining tissue using simple soln
A. According to chemical dyes; “Substantive staining” ;
composition color of dye is also the resulting
1. Natural- obtained from plants color. (methylene blue)
and animals (hematoxylin, - Uses aqueous or alcoholic dye
orcein, saffron, cochineal dyes) soln to produce a color
2. Synthetic- artificial dyes, 3. Indirect staining- process of
manufactures from coal tar, staining tissues with the help of
sometimes referred as “coal tar mordant and accentuator;
dyes” (Aniline, eosin, crystal “adjective staining”
violet, gentian violet) Mordant- link to make staining
B. According to reaction reactions possible. May part or the
1. Basic dyes- active coloring agent staining technique or may be added
is base; have affinity for acid to the dye itself
components; nuclear stain
(hematoxylin, methylene blue) STAIN MORDANT
2. Acid dye – active coloring agent Ehrlich’s Potassium
is an acid; affinity for basic hematoxylin/Meyer’s alum
components; said to be hematoxylin
cytoplasmic stain (picric acid, Weigert’s Iron (ferric
hematoxylin ammonium
eosin yellow, alcoholic eosin)
chloride)
3. Neutral dye- mixture of acid and
basic stain; soluble in alcohol
but insol. In water; capable of Accentuator- only accelerates or
staining nucleus and cytoplasm hasten the speed of staining reation
( giemsa stain, Leishman’s, by heightening the color intensity or
Romanowsky, Wright’s) increase selectivity of the dye
 STAIN ACCENTUATOR 8. Special Staining- used to demo
Loeffler’s Potassium special feature of tissue ( sudan
Methylene blue hydroxide black- for fats)
Carbon Phenol 9. Metallic Impregnation- Tissue
fuchsin elements demo not by stains but
Carbol Phenol by colorization of metallic salt
thionine - Agent that is applied is not
4. Progressive – ascending staining absorbed by tissue and will leave
method because tissue are black deposits (Gold chloride and
stained until the desired color is Silver nitrate, ammoniacal silver)
reach; differentiation and
decolorization is not required SOLVENTS USED FOR STAINS
- The stain is gradually little
1. Distilled H20
by little (no excess dye and
2. Alcohol (methyl and ethyl)
disadvantage).
3. Aniline H2O
5. Regressive staining- descending
4. Phenol (0.5 %- 5%)
method because tissue are first
overstained and the excess is HEMATOXYLIN
selectively removed; Diff and
decolo is required; • Natural dye derived from the
- DECOLORIZATION- selective heartwood of a Mexican tree
removal of excess dye. (Acid (Hematoxylin campechianum)
alcohol- can remove acid and
base) • The main color agent is hematein
- Basic dye- excess acid which is formed by the oxidation
6. Metachromatic staining- stain of the hematoxylin in a process
used produces different colors; known refining.
method applied or employed by • Refined: Exposing the material
staining cartilage, CT, epithelial from air and sunlight
mucins, mast granules and • H202 and Sodium iodate- hasten
amyloids.
HEMATOXYLIN- EOSIN STAINING
Methyl violet, Bismarck brown, Basic
fuchsin, Crystal violet, methylene blue, • MOST COMMONLY USED
STAINING METHOD
Azura A, B, C, Cresyl blue, Thionine,
Safranin, Toluidine blue • It is regressive staining method

7. Counterstaining- application of Techniques in H&E staining

diff colors or stain to provide 1. Initial xylene bath to further
contrast and background (Eosin- deparaffinize
counterstain hematoxylin) 2. Descending grades of alcohol for
-eosin (red acid dye /cytoplasmic hydration so tissue will able to
stain) absorb the stain.
Forms: 3. Hematoxylin for nuclear
- Eosin Y 4. Wash with water to the excess
- Bluish stain
- Ethyl eosin or eosin f
 5. Acid alcohol to decolorize/ 4. Should not dry quickly
differentiator 5. Should not cause shrinkage or
6. Ammonia water is the bluing distortions of tissue’
agent used to intensify color of 6. Should set hard to facilitate
nucleus permanent mounting of sections
7. Wash with water – remove the
Techniques:
excess of ammonia water
8. Eosin-secondary stain/ 1. Clean the slide, excess xylene
counterstain for cytoplasm should be wiped off
9. Ascending grades for alcohol- 2. Place 2-3 drops of mountant on
dehydrate the section and allow it to run
10. last xylene bath- clearing agent down the full length of section
prior to mounting 3. Place the coverslip onto the
sections. Orientation is
H & E STAINING RESULT
necessary
• Nuclei- blue to blue black 4. Remove air bubbles by applying
• Karyosome- Dark blue gentle pressure.
• Cytoplasm- pale pink 5. Clean excess medium with
• RBC, Eosinophilic granules, xylene and water.
keratin- bright orange red 37C for 12-24 hrs 30c for 2 hrs
• Calcium and decalcified bones- CLASSIFICATION OF MOUNTING
purplish blue MEDIA
• Decalcified bone matrix, collagen
and osteoid- pink A. Permanent/ resinous- used for
• Muscle fibers- deeply pink sections that have been
dehydrated and cleared in
xylene.
Mounting- process of placing mountant a. Natural – recommended for thick
and a cover slip over the slide after sections and whole mounts, does
staining not cause shrinkage. (Canada
Purpose: balsam 1.524 extracted by abus
balsamea) disadv (gradual fading
1. Protect the spx from physical of stain)
injury b. Synthetic- recommended for
2. Protect the section from small tissue sections not for
bleaching whole mount because it dries
3. Facilitate ease of handling and quickly and causes shrinkage
storage
4. Prevent damage of sections DPX – 1.532

Characteristics of good mounting Malinol -

1. Should have refractive index Clarite 1.544

close to that of the slide 1.518 Premount XAM 1.520
2. Miscible with xylene
3. Should not fade out tissue Gun’s Neutral Mounting Medium
sections
 B. Aqueous/ Temporary- used 1. Formation of small bubbles, can
when certain substance are to be be remedied by gently pressing
demonstrated, used to mount on the coverslip
water miscible. 2. Sections that are not properly
dehydrated will become cloudy or
COMPOSITION OF AQUEOUS
milky with xylene.
MOUNT
RINGING - Process of margins of the
1. GELATIN, GUM ARABIC OR
GLYCERIN JELLY- these are cover slip
examples of solidifying agent
- Not mandatory step
2. Glycerol- added to mountant to
prevent cracking and drying Purpose of Ringing
3. Sugar – to raise the refractive
index can also act as 1. Prevent escape of fluid
preservative 2. Avoid evaporation of mountant
3. Immobilize coverslip
EXAMPLES OF AQUEOUS 4. Prevent sticking of slides upon
MOUNTANT storage
1. Glycerine Jelly/ Kaiser’s RI: Examples of Ringing
1.470
• Standard moutant if • Kronig cement
dehydration and clearing • Durofix
with xylene cannot be •
made
Routine:
• Stains mounted using the
moutant tend to fade Fixation 10 % formaldehyde
2. Farrant’s/ Gun’s arabic 1.430
Decalcification- nitric acid
• Requires longer time to
harden Dehydration- ethyl alcohol
• Not solidify upon storage
Clearing/ De-alcoholization- xylene
3. Apathy’s 1.52
• For methylene blue Infiltration - paraffin wax
stained nerve prep.
4. Brun’s Embedding
• Recommended for Sectioning- rotary
mounting frozen sections
from water Staining- H&E
5. Karo corn syrup Mounting
6. Levulose/fructose
7. Water- disadv: very low refractive SECTIONING NI DOC LIWANAG AY
index, it evaporates easily; not NAISAMA KO NA SA NOTES SA TAAS
allow examination of tissue NI SIR WOUND
under OIO
STAINING KAY DOC LIWANAG (yung
DIFFICULTIES: iba naisama ko na sa notes ni doc
wounds)
 - Process of applying dyes on the – derived from plants and animals
section to see and study the (Hematoxylin, Cochineal dyes, orcein,
architectural pattern of the saffron)
tissue and physical
characteristics of cells • Hematoxylin- not a stain, active
coloring agent is hematein.
STAINING CATEGORIES: - Formed from the oxidation of
hematoxylin either by exposure
1. Histological – Nuclear/
material to a sunlight or by
cytoplasm or cells and tissues
adding oxidizing agents:
• Tissue components are
➢ Hydrogen peroxide
demonstrated by direct
➢ Mercuric peroxide
interaction with a dye or staining
➢ Sodium iodate
solution
➢ Sodium perborate
• Active tissue component is ➢ Potassium permanganate
colored
• Micro-anatomical stains, - Used in combination with alum, iron,
bacterial stains, Specific tissue chromium & copper salts
stains
1. ALUM HEMATOXYLIN – Uses
2. Histochemical/Histochemistry
alum or iron as mordant
• tissues component studied by
a. Ehrlich’s hematoxylin-
chemical reaction/ demonstrate
potassium alum as mordant,
carbs
sodium iodate
• Perl’s Prussian blue is for hgb b. Harris hematoxylin-
• Periodic acid Schiff is for potassium alum as mordant,
carbohydrates mercuric oxide, for exfoliative
3. Immunohistochemical cytology, sex chromosomes
staining c. Cole’s hematoxylin-
• We used antibodies instead of potassium alum as mordant,
dyes alcoholic iodine
• Combination of histochemical d. Mayer’s hematoxylin- With
and immunologic. citric acid & chloral hydrate
• Detection of phenotypic markers as preservative. Sodium
that are detected by antibodies iodate, for immunochem
(monoclonal, polyclonal) e. Delafield’s- smells like wine
STAINING METHODS FOR FROZEN and gives purple- red shape;
SECTION: Glycerol- stabilize oxidation
f. Gill’s- sodium iodate; stain
1. H&E mucin in goblet cells
2. Thionine g. Carazzi’s- potassium iodate
3. Polychrome methylene blue 2. IRON HEMATOXYLIN- in
4. Alcoholic pinacyanol method general for photomicrography
a. Weigert’s Hematoxylin- For
NATURAL DYES:
Muscle fibers and CT; Ferric
ammonium chloride
 b. Heidenhain’s Hematoxylin- 3. Sudan III – 1st sudan dye to
For nuclei & cytoplasmic introduced in histochem/ good
Inclusion (mitochondria); fat stain for CNS tissues
Ferric ammonium sulfate.
ADDITIONAL NOTES:
3. COPPER HEMATOXYLIN- The
INISCREENSHOT KO NALANG!!!
mordants spermatogenesis,
phosphotungstic acid. • ACID DYE- EOSIN
4. TUNGSTEN HEMATOXYLIN - • BASIC- HEMATOXYLIN
The mordants spermatogenesis, • NEUTRAL/AMPHOTERIC DYE-
phosphotungstic acid. GIEMSA, LEISHMAN,
5. LEAD HEMATOXYLIN – demo of IRISHMAN
granules of endocrine cells of
alimentary tract,
6. MOLYBDENUM
HEMATOXYLIN - demo of
collagen, reticulin and
argentaffin cells
Other natural dyes:
1. Cochineal dyes – extracted
from bug treated with alum
to produce carmine

✓ With picric acid – picocarmine for

neuropathological studies

✓ Aluminum chloride- best carmine

stain for Glycogen

2. Orcein – vegetable dye extracted

from lichens, used for staining
elastic fibers
SYNTHETIC DYES a.k.a Coal tar
dyes/aniline dyes

1. Chromophore – coloring
property; gives or impart color
2. Auxochrome- dyeing property-
retaining the imported color
Lysochrome dyes – w/o auxochrome
dyes (oil soluble dye) – demonstrate fats

1. Sudan Black B- most sensitive

of oil soluble dye
2. Sudan IV - a.k.a Scharlach R;
For neutral lipids, staining them
red