HISTOPATHOLOGIC TECHNIQUES

Disclaimer: HTMLE does not involve much analysis, just memorization. Medtech bioethics and laws are
included in your board exam. JUST READ and memorize

METHODS OF TISSUE PREPARATION

1. Teasing or Dissociation

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a. Selected tissue specimen is IMMERSED in a watch glass containing NSS, carefully
DISSECTED or SEPARATED, and examined under the microscope
b. Examine as stained or unstained
c. — Disadvantage: anatomical relationship is destroyed
2. Squash or Crushing
a. Small pieces of tissue not more than one mm in diameter are placed in a slide and

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forcibly pressed with another slide or with a cover glass.
b. Sandwich the specimen
3. Smearing (for cytological studies)
a. Also used in hematology.
b. Place a drop on the slide and GENTLY spread into the slide using another slide.
4. Streaking
a. Uniform distribution in a zigzag manner with an applicator stick or platinum loop.
b. Material is RAPIDLY AND GENTLY APPLIED.
c. Can be used in mucoid sample
5. Spreading
a. Recommended for mucoid sample, thin samples and bone marrow
b. Pull-Apart: with a single uninterrupted motion (blood smears & thick secretions)
c. Tough/Impression smear: freshly cut tissue is brought in contact & pressed onto the slide
(allows direct transfer of cells; maintains cellular interrelationship/ANATOMY of the cells

SPECIAL PROCESSING TECHNIQUES

1. Quenching
- Rapid freezing of tissue blocks to allow instant cessation of cellular activities
☺ Common Freezing Agent: Liquid Nitrogen
a. Isopentane
b. Pentane
c. Propane
d. Dichlorodifluoromethane
2. Freeze-Drying
- Rapid freezing the tissues at -150 OC
- Desiccation
Disadvantage: Formed tiny ice crystals are removed by sublimation in a vacuum at -40 to -70 OC
- Dry—RT—fix/process/embedded
Ø Molten paraffin
Ø Water soluble waxes
Ø Celloidin
3. Free Substitution
- Similar with freeze-drying
- Instead of dehydration in a vacuum, tissue blocks are:
 § Fixed in Rossman’s fluid or 1% acetone
§ Dehydrated with absolute alcohol at room temperature

Steps in Tissue Processing

1. FIXATION
a. Primary Aim: Preserve the morphological and chemical integrity of the cell in as
life-like manner as possible. (key word: stop, inhibit)
b. Secondary aim: Harden and protect the tissue from the trauma of further handling, so
that it is easier to cut during the gross examination
c. The first and most critical step in Histotechnology.
d. Proper fixation is recommended. Also the amount.
i. Too much of fixative: ma subraan ka gahi
ii. Kulang: dili ma gahi
e. REQUIREMENTS:
i. Tissue size: 2cm2 /10 mm X 4 mm thick;
ii. 1 - 2 mm2 (Electron Microscopy) o
iii. The volume of fixative: 20 times the volume of the specimen; except:
1. Osmium tetroxide: 5 – 10x of the tissue volume;
2. Museum: Not < 50 – 100x
iv. Tissue container: adequate size, wide-mouthed and non-rusting
f. TISSUE ORGAN PREPARATION:
i. Hollow Organs (stomach, intestines) - packed with cotton soaked in fixative or

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completely opened before immersion in fixing solution
ii. Air-filled lungs float in fixative - Organs may be covered in layers of gauze to

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maintain it under the surface.
iii. Eyes - Should NOT be dissected before they are fixed since this may lead to
tissue collapse and wrinkling due to vitreous humor,.Formol alcohol must be

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injected before immersing the organ in fixative.
iv. Brain- Suspended WHOLE in 10% buffered formalin for 2 to 3 weeks to
ensure fixation and hardening prior to sectioning. Human brains should be
washed with Ringer’s Lactate (Intravascular perfusion)
g. Types of Tissue
*Not all tissues can be fixed with formaldehyde. But in practice, we use buffered
formaldehyde (common fixative)
 I. Lipids: Mercuric Chloride or K2Cr2O7
II. Phospholipids: Baker’s formol-calcium
III. Cholesterol: Digitonin
IV. CHO: alcoholic fixatives (eg. Rossman’s fluid)
V. CHON: Neutral buffered formol saline or formaldehyde vapor
VI. Electron Microscopy: 2 fixatives

PLEASE MEMORIZE FOR YOUR BOARD EXAM!

TYPE OF ACCORDING TO ITS NAME OF FIXATIVE DESCRIPTION/ OTHER
FIXATIVE CLASSIFICATION PARTICULARS/
CONSIDERATIONS

SIMPLE FIXATIVE FORMALDEHYDE Gas produced by the

oxidation of methanol;
soluble in water to the
extent of 37-40% wt. in
vol.
PURE STOCK: 40%
(unsatisfactory for routine
fixation)
-FIXATION TIME: 24
hours

GLUTARALDEHYDE -made up of 2
formaldehyde residues,
linked
by 3-C chains
-USES: routine L.M.(light
microscopy) works, E.M.
(electronic Microscopy)
-2.5%: used for small
tissue fragments and
needle biopsies (2-4 hrs)
-4%: recommended for
larger tissues (6-8 hrs
to 1 day)

METALLIC FIXATIVE MERCURIC “MOST COMMON

CHLORIDE METALLIC FIXATIVE”
-5-7% saturated aqueous
solution
 -Fixative of choice for
tissue photography

POTASSIUM -3% aqueous solution

DICHROMATE -does not precipitate
cytoplasmic structures
-preserves lipids
-preserves mitochondria
(pH of 4.5-5.2

CHROMIC ACID -1-2% aqueous solution

- adequately preserves
CHO
-it precipitates all proteins
-“strong oxidizing agent

LEAD FIXATIVE -4% aqueous solution

mucopolysaccharide
-fixes connective tissue
mucin
-It takes up CO2 to form
insoluble lead carbonate
on prolonged standing:
this can be removed by
filtration or adding acetic
acid drop by drop to lower
the pH and dissolve the
residue

PICRIC ACID -normally used in strong

saturated aqueous
solution (1%)
- excellent fixative for
glycogen demonstration
-Yellow stain taken in by
tissues prevents small
fragments from being
overlooked.
-Brilliant staining with
trichrome method
-Highly explosive when
dry, therefore must be
kept moist with distilled
water of saturated
alchohol during storage

GLACIAL ACETIC -normally used in

ACID conjunction with other
fixatives to form a
compound
-same with acetic acid
except sa temperature
-It SOLIDIFIES at 17OC
(Gl. HAc)
Stored in refrigerator
ACETONE -Used at ice cold
temperature ranging from
-5 OC to 4 O
-recommended for study
of water diffusable
enzymes especially
phosphatases and lipases
- Used in fixing brain
tissues for diagnosis of
rabies

ALCOHOL -rapidly denatures and

precipitates proteins (H+
bond is destroyed)
-70%-100%: usual
concentration range
(lesser concentrated
solutionsà produce lysis
of cells)
- ideal for small tissue
fragments
-can be used as
dehydrating agent

METHANOL
- Excellent for
fixing dry and wet
smears, blood
smears and BM
smears

ETHANOL (70-100%)
- SImple fixative
used at
concentrations of
70-100%

OSMIUM -pale yellow powder which

TETROXIDE/OSMIC dissolves water (up to 6%
ACID at 20 OC)à strong
oxidizing solution
-Osmic acid fixed tissues
must be washed in
running water for at least
24 hrs to prevent the
formation of artifacts
-Saturated aqueous
HgCl2 solution (0.5-1
mL/100mL of stock
solution): prevents the
reduction of formed black
deposits

HEAT -involves thermal

coagulation of tissue
 proteins for rapid
diagnosis
-it usually employed for
frozen tissue sections and
preparation of
BACTERIOLOGIC
smears
-prevents microorganism
to infect

COMPOUND MICROANATOMICAL 10% FORMOL -simple fixative

FIXATIVES FIXATIVES SALINE -COMPOSITION:
saturated formaldehyde
(40 grams by wt. vol.)
diluted to 10% NaCl
-RECOMMENDED for
CENTRAL NERVOUS
TISSUES and GENERAL
POST-MORTEM tissues
-Autopsy procedures

10% NEUTRAL -RECOMMENDED for

BUFFERED PRESERVATION AND
FORMALIN STORAGE OF
SURGICAL,
POST-MORTEM, and
RESEARCH
SPECIMENS
-Commonly used in the
laboratory

HEIDENHAIN’S -RECOMMENDED for

SUSA TUMOR BIOPSIES of the
(Sublimatauregemsich SKIN -Excellent cytologic
mercuric chloride fixative
Saure - Trichloroacetic -It permits most staining
acid content-strong procedures to be done:
protein precipitant SILVER IMPREGNATION
(German word) BRILLIANT RESULTS:
sharp nuclear and
cytoplasmic details -It
permits easier sectioning
of large blocks of fibrous
connective tissues
-RBC preservation is poor
NOTE: After using
Heidenhain’s Susa
fixative, the tissue should
be transferred directly to a
high-grade alcohol (96%
alcohol/absolute alcohol)à
to avoid undue swelling of
tissues

FORMOL -FORMOL MERCURIC

 SUBLIMATE CHLORIDE:
(FORMOL-CORROSI recommended for routine
VE) post-mortem tissues

ZENKER’S FLUID -made up of HgCl2 stock

solution to which glacial
acetic acid has been
added just before its use
-mercuric deposits may
be removed by immersing
in ALCOHOLIC IODINE
SOLUTIONS
-RECOMMENDED: liver,
spleen, connective tissue
fibers and nuclei

ZENKER-FORMOL -EXCELLENT FOR

(HELLY’S SOLUTION) MICROANATOMIC
FIXATION: pituitary gland,
bone marrow & blood
containing organs (spleen
and liver) -Brown
pigments are produced
from blood containing
organs (>24hrs of fixation:
RBC lysisà immerse the
tissue in saturated
alcoholic picric acid or
sodium hydroxide
-Nuclear fixation &
staining is better than
Zenker’s fluid -It
preserves cytoplasmic
granules well

BOUIN’S SOLUTION -RECOMMENDED FOR

EMBRYO fixation

BRASIL’S -It is better and less

ALCOHOLIC messy than Bouin’s
PICROFORMOL solution
SOLUTION -Excellent fixative for
glycogen -AUTOMATIC
TISSUE PROCESSING:
Overnight tissue fixation→
3-4 changes of Brasil’s
fixative (1 ½ to 2 hours)
succeeded directly to
absolute alcohol

CYTOLOGICAL FLEMMING’S FLUID -most common

FIXATIVES: NUCLEAR Chrome-Osmium acetic
acid fixative used
-RECOMMENDED for
 NUCLEAR preparation

CARNOY’S FLUID -RECOMMENDED for

fixing CHROMOSOMES,
LYMPH GLANDS,
URGENT BIOPSIES
-“MOST RAPID
FIXATIVE”

BOUIN’S FLUID -can be used for nuclear

fixatives.
-recommended for
embryo

NEWCOMER’S -RECOMMENDED for

FLUID fixing
mucopolysaccharide and
nuclear proteins
-It produces better
reaction in Feulgen’s stain
than Carnoy’s fluid
-Acts as a nuclear and
histochemical fixatives

HEIDENHAIN’S (Same as above. Can be

SUSA used for nuclear fixative)

CYTOLOGICAL FLEMMING’S FLUID -made up only of

FIXATIVES: WITHOUT ACETIC CHROMIC ACID AND
CYTOPLASMIC ACID OSMIC ACID
-RECOMMENDED for
CYTOPLASMIC structure
(mitochondria)

HELLY’S FLUID -EXCELLENT FOR

MICROANATOMIC
FIXATION: pituitary gland,
bone marrow & blood
containing organs (spleen
and liver)
-Brown pigments are
produced from blood
containing organs (>24hrs
of fixation: RBC lysis
immerse the tissue in
saturated alcoholic picric
acid or sodium hydroxide
-Nuclear fixation &
staining is better than
Zenker’s fluid
-It preserves cytoplasmic
granules well
 REGAUD’S FLUID -It hardens tissues better
(MOLLER’S FLUID) and more rapidly than
Orth’s fluid
-RECOMMENDED for the
demonstration of
chromatin, mitochondria,
mitotic figures, golgi
bodies, RBCs and
colloid-containing tissues
-It preserves hemosiderin
less than buffered
formalin
-Glycogen penetration is
poor
-Does not preserve fats

ORTH’S FLUID -RECOMMENDED for

study of EARLY
DEGENERATIVE
processes & TISSUE
NECROSIS

WASHING-OUT: REMOVAL OF EXCESS FIXATIVE

1. Tap Water: removes excess - Chromates (Helly’s, Zenker’s & Flemming’s) - Formalin - Osmic Acid
● Ilhanan nga wash out na ang tissue is clear na ang water.
2. 50 – 70 % alcohol - Picric acid (Bouin’s solution)
3. Alcoholic iodide - Mercuric fixatives

WASHING-OUT: REMOVAL OF PIGMENT AFTER FIXATION

- Fixative that produces stains
• FORMALIN (white ppt.)
o Kardasewitsch’s: 28%NH3 H2O + 70% EA
o Lillie’s: 28%NH3 H2O, acetone + H2O2 → wash in 70% alcohol
O Alkaline Picrate: Picric acid + 95% ethanol

● PICRIC ACID (yellow discoloration)

○ Sat. Li2CO3 in 70 % EA
○ 70% EA then 5% Na thiosulfate
○ Lenoir’s solution: concentrated NH4Ac, 95% ethanol and distilled H2O
● MERCURIC FIXATIVE
○ 0.5% Iodine solution in 70% ethanol (5-10 mins)
○ Langeron’s iodine: I2 crystals, KI, Distilled H2O
● MELANIN
○ KMnO4 (decolorize)
○ Pyrogallic acid (reduce)
○ H2O2 (bleach & final removal)
2. Decalcification (calcium-containing organs)
● Process of calcium, phosphate salt or lime salt removal
 ● Recommended for bone, teeth, teratomas containing bony tissue, calcified pathological
tissues like tuberculosis foci, arteries calcified by atheroma
● Done after fixation and before infiltration
● Ideal time : 24 – 48 hrs or 4 days or MORE
○ Volume of reagent: 20 : 1 tissue volume
○ Concentration of agent used
○ Agitation & Partial/Complete Vacuum : With little accelerating effect
○ Optimum temperature: RT (18 - 30 OC)
● Heat
○ Accelerates demineralization; Promotes destructive action of acids on matrix
○ At 55OC - tissues will undergo complete digestion
○ At 37OC - Impair nuclear staining Van Gieson’s: reduced effectiveness of
Trichrome & PAS
○
DECALCIFYING AGENTS
1. Nitric acid
○ Most common & the fastest
○ Can be used as fixative
○ Inhibits nuclear stains and destroying tissues (concentrated solutions)
○ Carefully watch for endpoint
○ Cleared by 70 - 90 % ethanol
■ Slides are placed in 1% aqueous Lithium carbonate for 1 hour
○ Usually combined with formaldehyde or alcohol
○ Causes spontaneous yellow discoloration (HNO2)
■ Neutralize with 5% NaSO4 → wash for 12 hrs
■ Add 0.1% urea
○ Examples
■ De Castro’s fluid - silver impregnation of nerve fibers
■ 10 % Aqueous Nitric Acid (12 – 24 hrs)
■ Formol-Nitric Acid (1 – 3 days)
■ Perenyi’s fluid with chromic acid: 2 - 7 days
■ Phloroglucin-Nitric acid (12 – 24 hrs)
2. Formic Acid
○ Fixative and a decalcifying agent -
○ Moderate-acting decalcifying agent: better nuclear staining
○ Recommended for routine decalcification of post-mortem research tissues
○ Not suitable for urgent examinations
○ For small pieces of bones & teeth (2 – 7 days) -
○ For large pieces of bones
3. Hydrochloric acid
○ Slower action; with great distortion. Can destroy tissue
○ INFERIOR compared to nitric acidà produces good nuclear staining
○ RECOMMENDED FOR SURFACE DECALCIFICATION OF THE TISSUE
BLOCKS
○ Nuclear staining: 1% with 70% alcohol
○ Cannot be measured by chemical test
4. Von Ebner’s fluid
○ RECOMMENDED for teeth & small pieces of bones -
○ Moderately rapid decalcifying agent -
 ○ Permits relatively good cytologic staining
5. Trichloroacetic acid (TCA)
○ Weak and very slow acting decalcifying agent, not used for dense tissues
○ It permits good nuclear staining
○ It does not require washing-out
6. Sulphurous acid
○ Very weak decalcifying solution -
○ Suitable for minute pieces of bone
7. Chromic acid
○ Acts both a fixative and decalcifying agent -
○ RECOMMENDED FOR MINUTE BONE SPICULES -
○ Nuclear staining with Hematoxylin is inhibited
8. Citric acid-Citrate Buffer Solution (pH 4.5)
○ Permits excellent nuclear and cytoplasmic staining -
○ TOO SLOW for routine purposes -
○ It does not produce cell and tissue distortion
9. Phloroglucin-Nitric Acid
○ MOST RAPID DECALCIFYING AGENT; RECOMMENDED FOR URGENT
WORKS -
○ Yellow color must be neutralized with 5% sodium sulphateà wash with running
tap water (24 hrs) -
○ Complete decalcification cannot be determined by chemical means -
○ Poor nuclear staining
10. Perenyi’s Fluid
○ It decalcifies and softens tissues -
○ Maceration is avoided due to the presence of Chromic acid and Alcohol
○ Relatively slow for dense bone

Chelating Agents for Decalcifying

- Substances which combine with calcium ions and other salts (iron & magnesium deposits) to form
weakly dissociated complexes and facilitate removal of calcium salts

1. EDTA (Ethylene DiamineTetraacetic Acid)

➔ Decalcifying agent & water softener -
➔ 5 – 7% disodium salt @ 7.0 – 7.4
➔ 2 – 4 oz /g of bone -
➔ MOST COMMON CHELATING AGENT -
➔ Commercial Name: Versene -
➔ Slow-acting, but does not interfere with staining, does not distort tissues and enzymes (1
– 3 weeks) -
➔ Requires continuous change (use of transfusion set) - 1 – 3 weeks) for small tissues; 6 –
8 weeks for large ones; or longer
➔ Very slow: NOT RECOMMENDED for urgent and routine purposes
2. Ion Exchange Resin (ammonium form of polysterene resin)
➔ Formic acid and Resin (Ammonium-sulfonated polysterene)
 ◆ o Sequesters liberated Ca++
➔ NOT RECOMMENDED for fluids containing mineral acids such as nitric acid or
hydrochloric acid (HCl)
➔ Ion Exchange Resin Thickness: ½ inch
➔ Volume: 20-30x the volume of the tissue
➔ The tissue may allow to stay in solution for 1-14 days
➔ Measured by Physical/Radiological (X-Ray) methods
◆ When soft na or smooth, sakto na ang decalcifying (physical touch)
◆ Wala nay white sa x-ray, complete na ang process
➔ CANNOT BE MEASURED by chemical means
➔ REACTIVATION OF USED RESIN: immersed with N/10 HCl TWICE →wash
with distilled water THRICE
3. Commercial Decalcifying Reagents
● Best reserved for sections not requiring more than 4 - 8 hours in decalcifying
solutions -
● Avoid over decalcification & use of metal containers
4. Electrophoresis/Electrical Ionization
● Principle: Electrolysis/ Electrical Ionization -
● Process whereby positively charged calcium ions are attracted to a negative
electrode and subsequently removed from the decalcifying solution -
○ Temperature: 30 – 45OC - 90% formic acid & concentrated HCl
○ - Insoluble calcium salts in the specimen are changed to ionizable salts
by the action of the acids -
○ This method is satisfactory for small bone fragments

POST-DECALCIFICATION TREATMENT
● Washing-out
○ Running water: 3 – 8 hrs
● Neutralization: immerse in
○ 2% Li thiumcarbonate or
○ 5 – 10 % Na bicarbonate
○ Frozen Sectioning:
○ thoroughly wash acid-decalcified tissues in water , or store in formol saline with sucrose
or in PO4- buffered saline with sucrose at 4oC
○ EDTA-decalcified: wash and immerse in alcohol

TESTS FOR COMPLETE DECALCIFICATION

1. Physical or Mechanical Test

- Touch/Bending the tissue; Pliability; Resistance to fingernails or needling
- Very vague and inaccurate way of determining if a tissue has been completely decalcified or not
- Alternative way: pricking of the tissue with a fine needle or a probe
- Disadvantage: small calcified foci may not even be detected

2. X-ray or Radiological Method

- Very expensive; MOST IDEAL and MOST RELIABLE METHOD

 - Smallest Calcium deposit can be detected (opaque)
- NOT recommended for Mercuric Chloride fixed tissues
- Cannot be used when radiopaque metallic salts have been used such (HgCl2)

3. Chemical test (Calcium Oxalate Test)

- Simple, Reliable and Convenient method RECOMMENDED for ROUTINE PURPOSES
a. 5ml of the used decalcifying agent
b. Alkalinize w/ NH3 water.
c. Add 0.5 mL saturated aqueous NH4 oxalate (1% Na oxalate) →stand for 15 - 30 mins.

*RESULTS:
- CLEAR fluid = COMPLETE decalcification
- CLOUDINESS = (+) Ca Oxalate = incomplete decalcification

Note: Grossly undercalcified bone: a cloud of Ca(OH)2 upon addition of NH3

- Most laboratories do not rotate their staff to clinical and anatomic. Due to the time processing

☁️ TISSUE SOFTENERS
- Perenyi’s fluid
- 4% Aqueous Phenol (Lendrum’s)
- Molliflex
- 2% HCl
- 1% HCl in 70% alcohol

3. Dehydration
- Removes water from the tissue in preparation for impregnation
- Reagent used: must have high affinity with water
- Reagent: not less than 10 x the tissue volume
- Dehydrating fluids: increasing strengths

Agents for Dehydration

1. ETHANOL
- Fast acting, miscible with water & not poisonous
- RECOMMENDED AND BEST DEHYDRATING AGENT (fast-acting)
- Start from 70% - + 4% phenol to 95% EA: softener for hard tissues
- Not poisonous and not very expensive
2. METHANOL
- TOXIC dehydrating agent - Employed for both blood and tissue films & smear preparations

3. BUTYL ALCOHOL
- For plant and animal tissue microtechniques

4. ISOPROPYL ALCOHOL– substitute to ethanol

TEST FOR COMPLETE DEHYDRATION WITH ALCOHOL

- Layer (1/4 in.) anhydrous CuSO4 crystals at the bottom of container and cover with filter paper
 - Blue discoloration of the crystals indicates full saturation of alcohol with water
5. Acetone
- Rapid-acting, cheap but very volatile and flammable
- Hardens tissues more than ethanol does
- Fixative and dehydrating agent
- Utilized for most urgent biopsies
6. Cellosolve (Ethylene Glycol Monoethyl ether)

- Rapid, but toxic when inhaled, ingested & when in contact with skin
- Combustible at 110-120OF
- Tissues may be stored for months without distortion
- Prefer propylene-based glycol ethers
- Dehydrates rapidly and is not harmful to the tissues

7. Dioxane (Diethylene dioxide)

- Excellent dehydrating and a clearing agent
- Readily miscible with water, alcohol, paraffin wax & xylol
- Tissue may be taken directly into dioxane (except chromate fixatives)
- Not recommended for routine use (expensive & toxic); dangerous
- Dioxane can be recovered by removing the water with CaCl2 or CaO (quicklime)- may create explosive
peroxides
● Graupner’s Method
○ Time schedule of dehydration with Dioxane
a. Pure Dioxane solution: 1 hr
b. Pure Dioxane solution: 1 hr
c. Pure Dioxane solution: 2 hrs
d. Paraffin Wax: 15 minutes
e. Paraffin Wax: 45 minutes
f. Paraffin Wax: 2 hours

● Weiseberger’s Method
○ - (Uses anhydrous CaO): Bottle containing wrapped gauze + Dioxane (removes
chromate fixative) + Anhydrous Calcium Oxide/Quicklime (3-24 hrs)

8. Triethyl PO4
- Soluble in water, alcohol, xylene, ether, benzene, chloroform and acetone
- It is used to dehydrate sections and smears following certain stains and produces minimum
shrinkage

9. THF (Tetrahydrofuran)
- Acts as a dehydrating and a clearing agent
- Miscible w/ alcohol, xylene, benzene, ether, chloroform & acetone
- 6 months: prolonged exposure causes conjunctival irritation
- It causes less shrinkage and easier cutting of sections with fewer artifacts
- With offensive odor & should be used in a well-ventilated room
- It does not dissolve aniline dyes
4. Clearing/ Dealcoholization
- Replacement of the dehydrating fluid with a substance that is miscible with the embedding
medium to be used
- Tissue may become transparent & translucent
- Not all dealcoholizing agents act as clearing agents or vice versa

Applications of Clearing

1. Clearing in Embedding
- Uses a solvent that dealcoholize and act as solvent of paraffin
- Agents: xylene, toluene, dioxane and chloroform
2. Clearing in Mounting
- Solvents w/ high RI → transparent
- Agents must be solvents of the Mounting media
- Agents: xylene, toluene, terpineol, carbol-xylene
3. For the purpose of making the tissues transparent → internal structure
- R.I. of clearing agents is approximately equal to that of the tissues

CLEARING AGENTS
1. Xylol/Xylene
- Most rapid clearing agent (15 - 30 mins/ 30 min – 1 hr: 5 mm thick); Commonly used agent
- Excellent clearing agent (but tends to make tissues excessively hard & brittle (longer than 3 hrs.)
- Can be used for celloidin sections
- It is used for embedding and mounting procedures
- Suitable for urgent biopsies\
- It does not extract aniline dye
- Miscible with Canada Balsam
- Unsuitable for brain & lymph nodes
- Turns milky when dehydration is not complete
- Highly inflammable

2. Benzene
- Rapid agent (15 – 60 mins)→ RECOMMENDED FOR URGENT BIOPSIES
- Doesn’t make tissues hard & brittle but may cause considerable shrinkage
- Highly inflammable; Miscible with absolute alcohol
- Carcinogenic → Aplastic Anemia (bone marrow damage)

3. Toluol/Toluene
- Similar to xylene but does NOT harden tissues that much: SUBSTITUTE for xylene and benzene
- RECOMMENDED CLEARING TIME: 1 – 2 hrs
- Not carcinogenic but emits toxic fumes
- RECOMMENDED for ROUTINE PURPOSES (but it is expensive)
It tends to acidify in a partially filled vessel

4. Chloroform
- Excellent for nervous tissues, lymph nodes and embryos
- Best for large specimens and tough tissues
- Causes little shrinkage & does not harden tissues excessively
- Not flammable - Slow & doesn’t make tissues transparent
 - Not volatile in paraffin oven
- Toxic to the liver on prolonged inhalation
- used for kidnapping

5. Cedarwood oil
- Oil immersion
- It is used to clear both Paraffin and Celloidin (5-6 days) sections
- RECOMMENDED for CNS tissues and Cytological studies (smooth muscles and skin)
- Slow: 2-3 days clearing time
- Remove by xylene or benzene
- Quality is NOT always uniform and good
- Extremely slow clearing agent (NOT RECOMMENDED FOR ROUTINE PURPOSE)
- Turns milky on prolonged storage
- Tissue tends to float (layer absolute alcohol on the surface)
- May form crystals with acetic-alcohol fixed tissues
- Melting Point: 35 OC } Cedarwood Oil has been previously used to clear acetic-
O
- Heated: 200 C } alcohol fixed tissue

6. Aniline Oil
- Recommended for embryos, insects and delicate specimens due to its ability to clear 70% alcohol

7. Clove Oil
- Slow & has the tendency to become adulterated
- This reagent causes minimum shrinkage of tissues

8. Carbon Tetrachloride
- Similar to chloroform
- May be used in clearing tissues for embedding
- It produces considerable hardening of tissues and is dangerous to inhale on prolonged exposure due to
its highly toxic effect

9. Amyl acetate
- For large pieces of tissues & embryonic materials

10. Terpineol (artificial oil of lilac)

- Substitute for cedarwood oil

11. Methyl Benzoate/Salicylate

- Slow
- Double embedding
- Expensive

12. Glycerin, Gum syrup & Brun’s solution

- for tissue to be cleared directly from water
- merely improve the RI

13. Carbo-Xylene
- Used for materials that are difficult to clear
- Should be thoroughly rinsed in xylene

14. Others
- Oil of bergamot
- Phenol in alcohol
- Creosote

15. NEWER CLEARING AGENTS

- Based on limonene: a volatile oil found in citrus peels
- Long chain aliphatic hydrocarbon (Clearite)

5. Impregnation or Infiltration, and Embedding

> Impregnation: Infiltration of the tissues with a medium that will fill all natural cavities, spaces &
interstices of the tissues

To set tissues to firm consistency to allow the cutting

● Volume of medium: 25x the volume of tissue
● MP: 56OC
● Hard tissues require wax with a higher melting point
● Type of Microtome
● Fixed-knife microtomes: hard wax
○ Heavier knives: harder wax

Embedding/Blocking/Casting: Is the process by which the impregnated tissue is placed into a precisely
arranged position in a mold containing a medium which is then allowed to solidify

Materials used to infiltrate, support and enclose tissue specimen.

Same in the infiltrating and embedding
Characteristics of Embedding media
Must be capable of being converted readily from liquid to solid form:
1. Crystallization – (paraffin & carbowax)
2. Evaporation of the solvent – (celloidin)
3. Polymerization – (plastics)

Types of Embedding

1. Paraffin Embedding
- Simplest, most common and best embedding medium
- Shrinks about 10% on cooling
- Melting point for normal routine work: 56 – 58 OC
- Paraffin oven: 2 – 5 OC or higher

Methods of Paraffin Impregnation

1. AUTOMATIC PROCESSING
- Uses automatic tissue processor
- Shortened due to constant agitation
 - Dehydrating & Clearing agents
- once/week changing
- Wax bath: at least 3 OC > MP - Size: 3 by 2 ½ cm in area; Should NOT be more than 4 mm thick

- PRINCIPLE: central rotating spindle that carries a “basket” suspended from the outer end of a
horizontal radial arm (clock-controlled transfer arm)
- A short repetitive, up-and-down motion of the entire head assembly or arm, or a separate motor on the
basket arm, continuously moves the basket in the solution -

A Thermostat is within the walls of the beakers

2. MANUAL PROCESSING
- Requires at least 4 changes at 15 minutes
- The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to
ensure complete embedding or casting of tissue

3. VACUUM EMBEDDING
o Negative atmospheric pressure (400-500 mmHg)
o Heat & vacuum - increases fluid exchange & removal of air bubbles & clearing agents
o 2 - 4 OC above MP of wax

o ADVANTAGE: Effects of heat are prevented

Substitute for Paraffin Wax (mugawas sa board exam ang melting point)
1. Paraplast
- Melting Point (MP): 56 – 57 OC
- More elastic & resilient; does not crumble
- Better ribboning with ease
- Doesn’t require cooling
● Embeddol
○ MP= 56 – 58 OC
○ Less brittle than paraplast
● Bioloid
○ for eyes
● Tissue Mat
● Contains rubber (from paraffin)
2. Ester wax
- MP: 46 – 48 OC
- Soluble in 95% Ethanol & other clearing agent
- Harder than paraffin

3. Water Soluble Waxes

o Polyethylene glycols
§ Carbowax
Does not require dehydration & clearing
- Does not remove neutral fats & lipids
- Suitable for enzyme studies
- Carbowax Processing: 70 % - 90% - 100% (2x) at 56 OC; Blocking at 50 OC
o Precaution:
 § It is highly SOLUBLE in water; hygroscopic
§ Cannot be floated in water
§ Avoid overheating – crumbly blocks

● FLOATING solution:
○ PEARSE solution:
■ Diethyl glycol: 40 parts
■ Formaldehyde: 10 parts
■ Dist. H2O: 50 parts
●
● BLANK and MC CARTHY:
○ 0.02 % gelatin
○ 0.02 % K2CrO4

EMBEDDING
- A previously infiltrated specimen is placed into a precisely arranged position into a mold with
medium which is then allowed to solidify
- In general, the surface of the section to be cut should be placed parallel to the bottom of the
mold
● ORIENTATION
○ Arranging the tissue in the mold
○ Fixing the tissue block on the microtome
○ Arranging the tissue ribbons on the slide

Types of Embedding Molds

1. Leuckhart’s embedding mold

- 2 L-shaped heavy metal or brass arranged on a flat metal surface
- It can be moved to adjust the size of the mold to the size of the specimen
- Recommended for routine use

2. Compound E unit
- With several interlocking plates making several compartments
- Advantage: embedding more specimens at a time

3. Disposable Molds
a. Peel-away (thin plastic embedding molds)
- It may be placed directly in the chuck or block holder
b. Plastic Ice Trays
- Used in ordinary refrigerators
- Recommended for busy laboratories
c. Paper boats
- Advantage: easy and cheap, different sizes, avoid confusion

4. Plastic Embedding Rings & Base Molds

- Base mold is fitted to base ring
- Base ring serves as the block holder

• Tissue –Tek system

- Stainless steel in which the tissue block is embedded
- Plastic mold is place on top
- eg. Mark I & II Tissue-Tek Systems with warm and cold plate

• Celloidin/Collodion Embedding
• Purified pyroxylin nitrocellulose
• Suitable for specimens containing large cavities or hollow spaces which tend to collapse (eyes) & for
larger embryos
• 2% (thin); 4% (moderate); 8% (thick)
• Bell jars: control of evaporation

• DISADVANTAGE: Tissues cannot be cut as thin as they are with paraffin wax.
• ADVANTAGES: Causes much less shrinkage & distortion; slow process (days to weeks)

• LVN (Low Viscosity Nitrocellulose)

- Already wet in 35% alcohol
- Chrome-mordanted tissues may crumble (add plasticizers like Castor oil, Oleum racini)

☔
TYPES
1. WET METHOD

- For bones, teeth large brain sections & whole organs 🦴🧠

- Evaporate solvent (until no fingerprint may be observed)
- Store in 70 – 80 % alcohol

☀️
👀
2. DRY METHOD
- For whole eye section
- No need to be stored in 70% alcohol
- Store in Gilson’s mixture (chloroform & cedarwood oil)

5.Gelatin Embedding 🍮
- For delicate specimens
o Volume : 25x the tissue volume
o For histochemical & enzyme studies
o For frozen sections - Water-soluble - Low MP & Does not overharden - 10% - 20% - 20% (+Phenol)

6. Plastic/Resin Embedding Medium

🐌
1. EPOXY (epoxy plastic, catalyst & accelerator): FOR HARD TISSUES
- Araldite base (bisphenol) - slowest

🐎
- Glycerol base (epon)

🐕
- Cyclohexene Dioxide (spur) – fastest -
Hydrophobic – tissue damage

💪🐽
- Reduce antigenecity – not for immunochemical study
- Sensitization (skin & inhalation)

☠️
- Contains toxic components
o VCD ( vinylcyclohexane dioxide) – carcinogenic

2. POLYSTER - For Electron Microscopy

3. ACRYLIC
- For high resolution Light Microscopy
- Glycol Methacrylate (GMA)
o (hydrophilic): allows wide stain selection
- Methyl Methacrylate (MMA)
o (hardness): for dense tissues
- Benzoyl Peroxide – added as catalyst (for drying)

6. Microtomy
- Process by which a previously processed tissue (tissue block), is trimmed and cut into uniformly thin
slices or sections to facilitate microscopic study

Principle: A spring-balanced teeth (pawl) is brought in contact with, and turns a ratchet feed
wheel connected to a micrometer screw, which is in turn rotated, moving the tissue block at a

😀
predetermined distance towards the knife for cutting sections at uniform thickness
(Just remember your intern days and tong 2nd year pa mo second sem with maam Loms. )

🐘
TYPES OF MICROTOME

🎸
1. Rocking (Cambridge) Microtome
- Simplest, (1881, Paldwell Trefall) (na fall si Paldwell Trefall kay nag rocking siya. )
- For both small & large paraffin embedded blocks
- Ideal for actual practice (60-90 section ribbons)
- Not recommended for cutting serial sections because the sections are cut in a slightly curved plane.
- 10 – 12 u

2. Rotary (Minot) Microtome (1885-86) 🐴

- Most commonly used for routine procedures on paraffin embedded sections
- Up & down VERTICAL movement = perfectly flat plane
- Heavier & more stable
- Models are now available for cutting ultrathin sections and cryostat used
-4–6u

3. Sliding Microtome (Adams, 1789) (na slide c Adam) 🐍🔪

-4–9u
- Standard Sliding
o For celloidin embedded sections
o Most dangerous because of the movable exposed knife
- Base-Sledge
o Consists of 2 movable pillars holding the adjustable knife clamps
o Celloidin sections and used for hard & tough tissue blocks in all forms of media

4. Ultrathin Microtome 🦠
- For Electron microscopy (E.M)
- 0.5 u
- Uses broken plate glass knives

5. Freezing Microtome (Queckett, 1848)

- Undehydrated tissues in frozen state
- For fats and tissue constituents that may be damaged by HEAT
- 10 – 15 u

6. Cryostat/COLD Microtome 🧊
- For fresh tissue microtomy, urgent biopsies, Fluorescent and Antibody staining
- Maintained at -20OC
- 4 u (Freezing Fresh Tissues for 2-3 mins)
- Usual Microtome: Cambridge Rocking
- It provides a means of preparing large, thin, often unwrinkled sections of frozen tissues
(FLUORESCENT ANTIBODY TECHNIQUE OR HISTOCHEMICAL ENZYME STUDIES)
-pero according ni Doc A. dili sya preferred sa mga pathologist kay maot.

NOTE:
● Operating Temperature
○ +10 to -30 OC
● Optimum Working Temperature
○ -18 to -20 OC

-5 to -15 OC -15 to -25 OC - 35 OC

🧠 Brain, lymph nodes, liver,

spleen, kidney, testis, uterine
kidney, testis, uterine
curettings, soft cellular tumor,
kidney, testis, uterine
curettings, soft cellular tumor,
curettings, soft cellular tumor,
thyroid
thyroid
Muscle 💪 , connective tissue,
pancreas, uterus and cervix, skin
thyroid
Muscle, connective tissue,
pancreas, uterus and cervix,

ovary, prostate, tongue 👅

(w/o fat), non- fatty breast tissue,
, gut
skin (w/o fat), non- fatty breast
tissue, ovary, prostate, tongue,
gut
Fatty tissue, skin w/ fatty
subcutis, fatty breast, omental
tissue

SUMMARY for the Table: Body parts in different temperatures

● Brain, lymph nodes, liver, spleen can be processed in -5 to -15 OC only

● Muscle 💪
● kidney, testis, uterine curettings, soft cellular tumor, thyroid : can be processed from -5 to -35oC

👅
, connective tissue, pancreas, uterus and cervix, skin (w/o fat), non- fatty breast tissue,
ovary, prostate, tongue , gut can be processed in -15 to -35

KINDS OF MICROTOME KNIVES 🗡️

MICROTOME KNIVES LENGTH TYPES OF BLOCK MICROTOME

PLANE-WEDGE ✈️ 100 mm Celloidin embedded

sections & for extremely
Sliding

hard tissues;
RECOMMENDED for
FROZEN SECTIONS
 PLANE-CONCAVE ✈️👓 25 mm Paraffin & Celloidin
embedded sections
Rotary
Rocking

BICONCAVE 👓 120 mm Paraffin embedded

sections
CO2 Freezing
Rotary
Rocking

MICROTOME KNIVES INCLINATION/ANGLE

1. Clearance angle:
- between the edge of the knife & the tissue block
- 5 – 15O

2. Wedge angle
- angle of cutting (new knife)
- 15O
3. Bevel angle:
- angle of cutting facet
- 27 – 32O
4. Rake angle:
- edge of the knife at 90O

MICROTOME KNIVES SHARPENING

v Cutting edge – must be of good quality steel
- Too soft: doesn’t maintain the edge
- Too hard: is likely to nick against hard objects
v TESTS:
§ Should cut a paraffin wax block at 2 – 4 um thickness w/o
❖ serrations when examined under the microscope (100X) – final test
❖ *Von Mhol’s criterion: with strong nearby light, the cutting
❖ edge will show a slight reflection as a very narrow continuous straight line

POINT OF HONING STROPPING

DIFFERENCES

Use For the removal of gross section For the removal of

irregularities/burr

Material Smooth stones, Machine hone Shell horse leather

Lubricant Soapy water, Oil (Mineral oil, Castor Vegetable Oil

oil, Clove oil) or Xylene

Direction Heel-to-Toe Toe-to-Heel

Motion Zig zag Zig zag

FIXING & DRYING SECTIONS ON SLIDES

 ❖ Wax oven (56OC - 60OC for 2 hrs)
❖ Incubators (37OC overnight)
❖ Hot plate (45OC - 55OC for 30 - 45 mins., preferably inverted)
❖ Blower-type electric slide dryer (50 - 55OC for 20 – 30 mins)

GENERAL STEPS IN FIXING SECTIONS ONTO SLIDE

1. Floating
- water bath (temp: 100C < MP of wax)
2. Adhesion
- Mayer’ s egg albumin
3. Fishing out
- Transfer of tissue sections/ribbons on the slide (use camel’s hair brush)
4. Orientation
- Correct positioning of the tissue section/ribbon on the slide
5. Deparaffinization
- alcohol lamp or paraffin oven
6. Drying sections
- Wax oven (56OC – 60OC for 2 hrs)
- Incubators (overnight)
- Hot plate (45OC – 55OC for 30 – 45 mins.)
- Alcohol lamp/ Bunsen flame

7. Post-mordanting
- Secondary fixation (post-chroming): aqueous solution of mercuric chloride and aq. picric acid
- Used primarily as mordant & secondary as fixative
- 5 – 10 minutes in ether

7. Adhesives and Mounting media

(Adhesive - put the sample on slide. Mounting - cover slip sa slide)
ADHESIVES
1. Mayer’s Egg Albumin
- Most common, easy and convenient, relatively inexpensive
- Compositions: 50 cc of Egg white, 50 cc of Glycerin
- THYMOL: prevents the growth of mold

2. Dried Albumin
- Compositions: Dried albumin, NaCl, Distilled water, Crystals of Thymol
3. Gelatin
- Compositions: Gelatin, Phenol crystals, Glycerol, Distilled water

4. Starch Paste
- Compositions: Powered starch, Distilled water (10cc cold, 20cc boiling), N/1 HCl, Thymol crystals

5. Plasma
- Readily available from outdated blood stored in blood banks, dispensed into sterile tubes, 0.5 mL
6. Poly-L-lysine
- 0.1% detergent solution
 - 1:10 with distilled water
- Final dilution: 0.01%
- Utilized for Immunohistochemistry

7. APES (3-aminopropyltriethoxysilane)
- Used for cytological studies
- Proteinaceous or bloody material
- Slides dipped with 2% APES in acetone

8. Celloidin sections using egg albumin

- Measured smears
- Transferred: 95% alcohol; Stored: 70% alcohol

MOUNTING SECTIONS
1. Koplin Jar: 5-9 slides
2. Slotted Staining Dishes: 5-19 slides
3. Metal/Glass Stain Racks/Carriers: 10-30 slides

NOTE:
- Albumin fixative: loss of odor and clear color; 2-3 months; >1ounce
- Refractive Index (R.I.) of mountant to that of the glass: 1.518
- Frozen sections: Sudan method
- Metachromatic stain: amyloid
- Common Aqueous media: water-miscible preparation directly from water
o Water: low R.I
o Glycerin: high R.I.; preservative
o Gum Arabic/Farrant medium
o Karo Corn Syrup
§ Glycerin: stabilizer; increased visibility (distilled water for moist sections)
DIFFICULTIES ENCOUNTERED DURING MOUNTING
- Sections that not properly dehydrated: cloudy and milky with xylene; Small bubbles may be formed
- Might be hastened in a hot oven at 50OC for 2 hours

RINGING
- Process of sealing the margin of coverslip
o To prevent the escapage of fluid or semi-fluid mounts
o Prevents the evaporation of mountant
o To immobilize the coverslip
o Prevent the sticking of the slides upon storage
- Kronig Cement: 2 parts of paraffin wax mixed with 4-9 parts of powdered Cophonium Resin
- “Cellulose Adhesive”: Durofix

8. Staining/Dyeing STAINING/DYEING
● Importance:
○ For contrast
○ Better optical differentiation
○ Improve aesthetic value
● Chemical basis or dyestuffs
○ Chromophores: “color-bearers”
○ Auxochrome: “increasers”/ ”electron donors”
STAINING METHODS

1. DIRECT Staining: use of aqueous or alcoholic dye solutions

2. INDIRECT Staining:
Mordant: serves a link/bridge between the tissue and the dye to make staining reaction possible
 Example:
K+ alum – Ehrlich’s hematoxylin; Iron – Weigert’s hematoxylin)
Accentuator: NOT essential to the chemical union of the tissue & dye. It does NOT participate in
the staining reaction but merely accelerates or hasten the speed of the staining reaction by increasing the
staining power and selectivity of the dye.
Examples:
KOH –Loeffler’s Methylene Blue; Phenol – Carbol thionin & Fuschin
Best example: Gram Staining.
3. PROGRESSIVE: tissue elements are stained in a definite sequence, and the staining solution is
applied for specific periods of time or until the desired intensity of coloring of the different tissue elements
is attained (increasing the intensity of the stain)
4. REGRESSIVE Staining: tissue is first overstained to obliterate the cellular details, and the excess stain
is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained
DIFFERENTIATOR/DECOLORIZER: Selective removals of excess stain form the tissue during regressive
staining in order that a specific substance may be stained distinctly from the surrounding tissue
Example: Wash with water or alcohol (differentiator for both acid and basic dyes)
5. METACHROMATICS Staining: the use of specific dyes which differentiate particular substance by
staining them with a color that is different from that of the stain itself. (Orthochromatic – color is similar to
the dye)
6. COUNTERSTAINING: application of a different color or stain to provide contrast and background to the
staining of the structural components to be demonstrated
7. METALLIC IMPREGNATION: sp. Tissue elements are demonstrated, NOT by stain, but by
COLORLESS solutions of metallic salts which are thereby reduced by the tissue, producing an opaque,
usually black deposits on the surface of the tissue or bacteria.; It is NOT absorbed by the tissue but is
held physically on the surface as a precipitate or as a reduction product
8. VITAL Staining: selective staining of LIVING cell constituents, demonstrating cytoplasmic structures by
phagocytosis of the dye particle. Nucleus is a living cell which is resistant to vital stains, and therefore is
not demonstrated
9. INTRAVITAL Staining: injecting dye into any part of the animal body (intravenous, intraperitoneal or
subcutaneous)
Example: India ink
10. SUPRAVITAL Staining: staining of living cells immediately after removal from the living body
Example: Neutral red: best vital dye

LIST OF COMMON HISTOLOGICAL STAINS

● Nice to know ra ang uban, just familiarize with the different stains, ayaw lang kalimot sa
commonly used stains.
● Most common method of staining for microanatomical studies of tissue
○ Hematoxylin and Eosin stain
○ Regressive staining
○ All fixatives can be used except osmic acid because it inhibits hematoxylin
 ○ 10 Hematoxylin: primary stain, basic dye, nuclear stain
○ 20 secondary stain, counterstain, acid dye, cytoplasmic stain
● Results: nuclei: blue to blue black
● Cytoplasm: pink
STAINS USES/IMPORTANCE/CHARACTERISTICS/OTHER
PARTICULARS

Acridine Orange Permits discrimination between dead and living cells (DNA: green
fluorescence; RNA: red fluorescence)

Acridine Red 3B Demonstrates deposits of calcium salts and possible sites of

phosphatase activities

Alcian Blue Stains mucopolysaccharide; more specific for connective tissue

and epithelial mucins

Aldehyde Fuschin Stain (Gomori) Used for differential staining of pancreatic islets of Langerhans

Aniline Blue Counterstain for epithelial cells

Azocarmine Connective tissues

Basic Fuschin Is a plasma stain; deep staining for acid fast organisms

Benzidine Used for staining haemoglobin

Benzidine method For Hemoglobin

Bielschowsky’s Technique Neurons, Axons, Neurofibrils

Bismarck Brown Contrast stain for Gram’s stain, In Acid Fast, Papanicolau method
& Diphtheria organism

Brown and Brenn (B&B) Bacteria, Nocardia, Actinomyces

Cajal’s Gold Sublimate Astrocyte

Carmine Used as a chromatin stain for fresh materials in smear preparations

Celestine Blue Resistant to strong acids; Recommended for routine staining of

fixed sections; ALTERNATIVE to Iron Hematoxylin nuclear stain

Congo Red Best known as an indicator; Utilized as a stain for axis cylinder in
embryos; (4% aqueous solution: elastic tissues, amyloid, myelin)

Cresyl Violet Acetate Helicobacter

Crystal Violet For amyloid, fungi, platelets in blood

Dieterle Legionella pneumophilia

Eosin Most valuable stain; used as a counterstain (Eosin B: blue-deep

red; Eosin Y: yellow-green)

Feulgen’s Most reliable and specific histochemical staining technique for DNA
Giemsa Used for staining blood to differentiate leukocytes

Gmelin’s Test Diagnostic for Bile pigments

Gordon and Sweet’s method Reticular Fibers

Grocott Methamine Silver (GMS) Fungi

Hematoxylin Natural dye derived from the heartwood of a Mexican tree,

Hematoxylin Campechianum RIPENING: Hematoxylin ---[o]---→
Hematin

Iodine Oldest stain; Stains amyloid for microscopic study of starch

granules

Janus Green B For demonstrating mitochondria during intravital staining

Levaditi’s method Spirochetes

Lindquist’s Modified Rhodamine Copper

Lissamine Fast Red Tartrazine Muscle demonstration

Method

Malachite Green Stains Ascaris eggs, RBCs, Bacterial spore (BOTH A

DECOLORIZER AND A COUTERSTAIN)

Mallory’s Phosphotungstic Acid Staining for muscles and bones; Astrocytes

Hematoxylin (PTAH)

Masson Fontana Technique Melanin (Silver Modification): BLACK

Methyl Green Stains chromatin green; It gives false positive reactions with certain
secretions such as mucin

Methyl Green Pyronin DNA: green to blue green; RNA: rose red

Methylene Blue Common basic nuclear stain; valuable for plasma cells

Methylene Violet Metachromatic dye; for leukocytes

Neutral Red Cell granules & vacuole phagocytic cells

Night Blue Is used as a substitute for Carbol Fuschin

Orcein Excellent stain for elastic fiber

Orcein method Hepatitis B Surface antigen

Osmic Acid stain Used for fats

Periodic Acid Schiff (PAS) Histochemical stain used for the demonstration of carbohydrates
(GLYCOGEN)
Perl’s Prussian Blue Hemosiderin (iron-containing pigment of haemoglobin)

Picric acid For the demonstration of connective tissue

Prussian Blue Colored Salt of Ferric ferrocyanide, normally used for the
manufacture of paints

Rhodamine B Stains blood and glandular tissues

Silver nitrate Used in Spirochetes reticulum and fiber stains

Sudan Black Phospholipids

Sudan III Fats (orange)

Sudan IV/Scharlach R Triglycerides and Neutral lipids (deep red)

Toluidine Blue Nuclear stain substitute for thionine for fresh frozen tissue;
Nissl/Tigroid granules and chromophilic bodies

Van Gieson Mixture of picric acid and acid fuschin; for the demonstration of
connective tissue; SIMPLEST method of differential staining of
collagen

Victoria Blue Used for demonstration of neuroglia in frozen sections

Von Kossa Silver Nitrate Calcium: BLACK

Warthin Starry/Levaditi’s Spirochetes

methods

Weigert-Pal Technique Normal Myelin Sheaths

⌛ QUICK SUMMARY FOR MEMORIZING STAINS ⌛

1. Cytoplasmic Stains
a. Red: eosin Y, eosin B, Phloxine B
b. Yellow: Picric acid, orange G, rose Bengal
c. Green: light green, lissamine green
2. Nuclear stains
a. Red: Neutral red, safranin O, carmine, hematoxylin
b. Blue: methylene blue, toluidine blue, celestine blue
3. Natural dyes
a. Hematoxyline: Mexican tree, logwood
b. Cochineal dyes: cochineal bug
c. Orcein: lichen
d. Saffron
4. Stains for carbohydrates:
a. PAS, Best Carmine, Langhan’s iodine
5. Stains for fats and lipid
a. Sudan
b. Oil red O
c. Osmic acid (unstable oxide)
 6. Stains for proteins
a. Alkaline fast green for basic proteins (histones protamins found in the nucle, green)
b. Peracetice acid for cysteine and cysteine (blue-green)
c. Sakuguchi’s for arginine (orange-red)
7. Stains for nucleic acid
a. Fuelgen for DNA (red-purple)
b. Methyl green pyronin (DNA green or blue green; RNA: Red)
c. Acridine Orange (DNA yellow-green, RNA brick to orange red)
d. Acriflavine (DNA fluorescent yellow)
8. Stains for collage
a. Van Gieson
b. Trichrome stain
9. Stain for spirochetes
a. Levaditi
b. Warthin-Starry
c. MOdified Steiner
d. Dieterte (also for Legionella methods)

Immunohistochemical techniques & Cytology IMMUNOHISTOCHEMICAL TECHNIQUES

o Specific and highly selective cellular epitopes/antigens
o Immunohistochemistry: can detect organism in cytologic preparation
§ IgG: fluids, sputum, FNAB
o Epitopes: structural part of the antigen; group of amino acids (globular proteins and sugar-side
chain)
Other Notes to Remember:
CLINICAL APPARATUSES
1. Ayre’s spatula: mucoid for hormonal study
2. Laryngeal cannula: endocervical/endometrial
3. Curette: spoon/curved/blunt: Vagina, cervix, placenta, endometrial
4. Endocervical brush: endocervical
5. Vaginal scrape: hysterectomy
6. Lateral Vaginal scrape: hormonal evaluation
7. 4 quadrant vaginal scrape: vaginal adenosis
8. Vulvular: herpetic lesions or carcinoma
9. Fine needle aspiration (FNA): simple, safe and rapid cytologic technique; replaced the use of tissue
core biopsy in many clinical conditions
For palpable masses: breast, thyroid, soft tissue and lymph nodes
VAGINAL HORMONAL CYTOLOGY (UPPER LATERAL THIRD OF THE VAGINAL WALL)

CRITERIA FOR CYTOLOGIC DIAGNOSIS OF NORMAL PREGNANCY

1. Marked progesterone effect (absence of ferning)
FERNING: mucus, on drying exhibits a “fern”/ “palm-leaf” pattern (ARBORIZATION) due to
formation of salt crystals in high sodium chloride
2. At least 50% intermediate cells in clusters
3. At least some typical pregnancy cells present (average 3-7)
4. Less than 30% superficial cells

😅TMI: Tulog ra ko ani guys pag review namo para boards. Ayaw ka goul pag naka tulog mo. haha
TERMS RELATED TO CYTOPATHOLOGIC CHANGES IN DISEASE
1. Inflammation- the sum of total changes in the living tissues
2. Aplasia- defective development of tissue or organ
3. Agenesia- complete non-appearance of an organ
.4. Hypoplasia- refers to the failure of an organ to reach or achieve its full mature or adult size
5. Atresia- is the failure of an organ to form an opening
6. Atrophy- refers to an acquired decrease in the size of a normally developed or mature tissue or organ
7. Hypertrophy- refers to the increase in size of tissues or organs due to an increase in the size of the
individual cells
8. Hyperplasia- refers to the increase in size of an organ or tissue due to increase in the number of cells
resulting from the growth of new cells
9. Metaplasia- is a reversible change involving the transformation in one type of adult cell to another
10. Dysplasia- is the regressive alteration in adult cells manifested by variation in size, shape, and
orientation
11. Anaplasia (DIFFERENTIATION)- is a marked regressive change in adult cells towards more primitive
or embryonic cell types, usually utilized as a criterion toward malignancy
12. Neoplasia (TUMOR)- is the continuous abnormal proliferation of cells without control, serving no
useful purpose or function, usually accompanied by increase in size and pigmentation, mitosis, number,
metaplastic and anaplastic changes of the cells
13. Parenchyma- refers to the active elements of the tumor/tumor cells
14. Stroma- refers to the connective tissue framework with lymphatic and vascular channels
15. Scirrhous carcinoma- more connective tissue than cells
16. Medullary- more cells than supporting tissues
17. Benign tumors- do not produce death
18. Malignant tumors- will produce death eventually, however, small they may be and wherever they may
be located
19. Degeneration- injury to the cell precedes and results in an accumulation of metabolites
20. Infiltration- overloading of a healthy cell by metabolites causes the injury
21. Necrosis- means “cell death” which is due to disease or injury
22. Necrobiosis- slower process, which refers to the physiologic death of cells, indicating the death of
group of cells
23. Ischemia or Anoxia- loss of blood supply leading to death of cells due to deprivation of their oxygen
and nutrients
24. Pyknosis- indicates the reduction in size and condensation of the nuclear material
25. Karyorrhexis- indicates the segmentation and fragmentation of the nucleus, whereby nuclear
contents are broken up and released into the cytoplasm
26. Karyolysis- means the dissolution of the nucleus where all basophilism is lost and the nucleus
disappears
27. Gangrene- refers to the massive death or necrosis of tissue (bacterial infection and anoxia)
28. Algor Mortis- first demonstrable change observed, characterized by cooling of the body to equalize
that of the surrounding environment, occurring at a definite rate about 7OF (-13.9 CO )
i. per hour, and usually important in establishing the approximate time of death
29. Rigor Mortis-refers to the rigidity or stiffening of the muscles, occurring about 6 to 12 hours of death
30. Livor Mortis- refers to the purplish discoloration or lividity of the skin in the dependent portions of the
body due to stasis and eventually settling down of blood into the dependent vessels, which then usually
dilate because of loss in muscular tone
31. Post-mortem clotting- usually slowly, immediately after death and sometimes complicate the cause of
death
32. Dessication- refers to the drying and wrinkling of the cornea and anterior chamber of the eye due to
the absorption of the aqueous humour
33. Putrefaction- is characterized by the production of foul-smelling gases, due to the invasion of the
tissue by multiplying saprophytic organisms
34. Autolysis- implies the self-digestion of cells by their own ferments, eventually undergone by all tissues
of the body

Bibliography
BOOK

Dr. Jocelyn H. Bruce-Gregorios. (2012). Histopathologic Techniques. (2nd Ed.). Goodwill Trading Co. Inc.:
Makati City, Philippines.

S. Kim Suvarna, Christopher Layton, & John D. Bancroft. (2013). Bancroft’s Theory and Practice of
Histological Techniques. (7th Ed.). Churchchill Livingstone Elsevier Ltd.: Philippines

Source of Handout
Histopathologic Techniques and Medical Technology Laws and Ethics
By: Olibrian P. Mallari, RMT & Ryan Geronimo, RMT

Revised and edited by : Jin Joo M. Son RMT