HISTOTECHNIQUES: preparation, processing and staining of tissue sections of tissue sections for

microscopic study to be interpreted by the pathologist

HISTOPATHOLOGY: study of disease at the tissue level

STORAGE

1. Specimen: 1 month to 1 year

2. Tissue Blocks: 3 to 10 years

3. Slides: Indefinite

4. Records (request and result forms): Permanent

SMEARING

▪ for cytological studies, especially for the diagnosis of cancer

▪ for sections or sediments

▪ performed using a wire loop, applicator stick or another slide

 Pull-Apart: For the preparation of blood and bone marrow smears

FROZEN SECTION

- Prepared using freezing microtome or cryostat

- For rapid diagnosis
- For delicate specimens

Specimen Accessioning/Identification

➢ Performed by the medical technologist

➢ Check label and request form

➢ The specimen is given a label (numeric or alpha-numeric) which allows easy

accessioning/identification

➢ Request form should have a provisional diagnosis and brief clinical details

Tissue Processing

➢ Fixation ➢ Dehydration ➢ Clearing ➢ Infiltration ➢ Embedding ➢ Sectioning (+ Floating, Fishing-

out, Drying) ➢ Staining ➢ Mounting ➢ Labelling
There are two general types of samples handled in the histopathology laboratory. These include autopsy
materials and surgical or biopsy materials.

Autopsy materials are processed to determine the cause of death. Surgical or biopsy materials on the
other hand are processed to provide a diagnosis of the patient’s condition. Surgical specimens may be
collected via fine needle aspiration, core needle, incisional, excisional, punch, shave or curettage
biopsies. As part of quality assurance, the laboratory is required to store specimens for one month to
one year, tissue blocks for three to ten years, slides are to be kept indefinitely and records permanently

FIXATION

✓ first and most critical step in tissue processing

✓ fixing or preserving fresh tissue for examination

✓ should be done immediately to preserve cellular and tissue morphology

Two Purposes of Fixation

1. Preservation: primary purpose

2. Protection: secondary purpose

Tissue/Organ Preparations:

1. Air-filled Lungs
o Cover with several layers of gauze
2. Hollow Organs:
o Dilate with cotton soaked in fixative
o Completely open specimen
3. Brain
o Fix first before sampling
o Suspended by a cord tied under the Circle of Willis
o Intravascular perfusion using Ringer’s lactate
4. Eyes
o Fixed whole
o Inject formol-alcohol
5. Hard Tissues
o Lendrum’s Method: washed out with running water overnight and immersed in 4% aq.
phenol solution for 1-3 days
6. Muscles
o Stretched with sutures on each end ✓ Laid flat in a moist filter paper
TYPES OF FIXATIVES:

I. According to Composition

1. Simple Fixatives: made up of only one component substance

2. Compound Fixatives: made up of two or more fixatives

I. According to Action

1. Microanatomical Fixatives: ➢ permit general microscopic study of tissue structures and normal
intercellular relationship of tissues

2. Cytological Fixatives: ➢ preserve specific cellular components

A. Nuclear Fixatives:

- preserve nuclear structures

- contain glacial HAc

▪ high affinity to nuclear chromatin

▪ destroys mitochondria and Golgi bodies

- pH 4.6 or less
B. Cytoplasmic Fixatives:
- preserve cytoplasmic structures
- do not contain glacial HAc
- pH of more than 4.6

3. Histochemical Fixatives:

- preserve chemical constituents

a. Lipids: mercuric chloride or potassium dichromate

b. Phospholipids: Baker’s formol-calcium

c. Cholesterol: digitonin

d. Carbohydrates: alcoholic fixatives

e. Glycogen: Rossman’s fluid or cold absolute alcohol

f. Proteins: neutral buffered formol saline or formaldehyde vapor

g. Electron microscopy: double fixation

Mixture of Fixatives:

1. Electron Cytochemistry: Karnovsky’s paraformaldehydeglutaraldehyde solution

2. Acrolein: mixture of glutaraldehyde or fomaldehyde

✓ rapid penetration, preserves morphology and enzyme activity at low concentrations

✓ immersion fixation of surgical biopsies

 Fixation is considered as the first and most critical step in tissue processing. There are two
purposes of fixation: (1) preservation and (2) protection of the tissue sample. Preservation is the
primary purpose of fixation and this allows maintaining the morphology and architectural
pattern of tissue constituents in as life-like manner as possible. Protection is the secondary
purpose of fixation and this is achieved by adequately hardening the tissue sample in order to
protect it from the damaging effects of reagents used in the subsequent steps in tissue
processing

ALDEHYDE FIXATIVES:

✓ for routine paraffin sections, electron microscopy, histochemistry and enzyme studies

1. Formaldehyde (Formalin)
o gas produced by the oxidation of methyl alcohol
o buffered at ph 7 to 8 → hypoxia in tissues leads to acidity which favors the formation of
Formalin heme pigments (black, polarizable deposits)
o Pure Stock: 40% formalin
o Dilution: 1:10 (10% solution); 1:20 (5% solution)
o Concentration for fixation: 10% formalin
o Paraformaldehyde: white precipitate due to prolonged storage → may be removed by
filtration or addition of 10% methanol
2. 10% Formol-Saline
o central nervous tissues and general post-mortem tissues for histochemical examination
o ideal with most stains including silver impregnation
o duration of fixation: more than 24 hours (slow fixative)
3. 10% Neutral Buffered Formal
o preservation and storage of surgical, post-mortem and research specimens
o best fixative for frozen sections
o best fixative for iron pigments and elastic fibers
4. Formol-Corrosive
o routine post-mortem tissues
o for lipids, especially neutral fats and phospholipids
o no need for washing-out
 5. Alcoholic Formalin
o used to fix sputum
o for the demonstration of immunoperoxidase activity
6. Glutaraldehyde
o made up of two formaldehyde residues linked by three carbon chains
o used in conjunction with osmium tetroxide
o Fixation time: ½ hour to 2 hours

ALCOHOL FIXATIVES

1. 95 % Ethanol
o Preserves but does not “fix” glycogen granules
2. Methanol
o For dry and wet smears, blood and bone marrow samples
3. Isopropyl alcohol (95%)
o for touch preparations
4. Carnoy’s fluid
o most rapid fixative
o for chromosomes, lymph glands, urgent biopsies and brain tissue for the diagnosis of
rabies
5. Newcomer’s
o demonstration of mucopolysaccharides and nucleoproteins
6. Gendre’s fixative

Decalcification and Acid Decalcifying Agents

DECALCIFICATION:

- process that entails the removal of calcium or lime salts from tissue samples after fixation
- this process is also known as demineralization

Consequences of Performing Decalcification:

- Distortion or damage to tissues

- Affects staining
o Failure of sections to stain properly is compounded by:
(1) overtreatment in acid &
(2) insufficient washing out of the acid
o Basic dyes: hematoxylin is inhibited
 Acid dyes: eosin produces a deep brick red color without differential staining
- Sections Float-off During Staining
o Observed after immersion in acid alcohol
o Collodionize prior to staining
GENERAL CONSIDERATIONS FOR ROUTINE ACID TECHNIQUES

1. Thickness of the Specimen

o Dense/Hard Bone: 2-5 mm thick
o Softer Tissue: 4-6 mm thick
2. Duration
o Ideal: 24-48 hours
o Dense Cortical Bone: 14 days
3. Temperature and Heat
o Required temperature: 18-30 degrees Celsius
o Heat enhances destructive action of acids on matrices
o 37 degrees Celsius: impairs nuclear staining with Van Gieson’s → reduced effectiveness
of Trichrome and PAS
o 55 degrees Celsius: tissues will undergo complete digestion within 24-48 hrs
4. Solution Used
- Concentration of Solutions
o Directly proportional to the rate of decalcification
- Strong Acids
o Affects the antigenicity of cells and tissue components
5. Presence of Additives
- Protect tissues but slows down decalcification
6. Fluid Access
- Tissues are to be suspended in the upper portion of the jar/container
7. Changing of the Solution
- Once or twice a day
8. Volume
- Optimum: 20 times the volume of the tissue
9. Agitation
- Mechanical agitation or moving of tissue in the solution which influences fluid exchange
- Gentle fluid agitation: low speed rotation, rocking, mechanical stirrer, bubbling air into the
solution
- Vigorous agitation: sonication
10. Removal of Decalcifying Solution
- Washing-out or neutralization after decalcification and/or prior to staining
11. Microwave and Electrolytic Methods

DEHYDRATION

- Removal of fixative and intercellular and extracellular water from tissues in preparation for
infiltration
- Increasing strengths of the dehydrating agent is used to prevent distortion of tissue structures
by diffusion currents (flow of molecules)
 FACTORS TO BE CONSIDERED
1. Type of Tissue
✓ DELICATE TISSUES (eg. embryo): start with 30% ethanol up to 70%
✓ NORMAL TISSUES: start with 70% up to 95% or Absolute alcohol
2. VOLUME: 10X the volume of tissue
3. Prolonged Immersion
✓ High Concentrations: tissues become hard and brittle
✓ Low Concentrations: tissues become macerated
4. Temperature
✓ 37 deg Celsius: increases rate of dehydration and used for tissues that require urgent
examination
5. Agitation
✓ Accelerates diffusion of molecules increasing the rate of dehydration
 Anhydrous copper sulfate: ¼ inch at the bottom of the container to facilitate the removal of
water molecules from the dehydrating fluid

ALCOHOL DEHYDRANTS: most common

❖ ETHANOL:

- Clear, colorless, flammable liquid

- Recommended for routine dehydration
- Best dehydrating agent, fast-acting and miscible in water and many organic solvents
- Penetrates tissues easily
- Not poisonous, not very expensive
- Long Immersion in high concentrations should be avoided

❖ ISOPROPYL ALCOHOL

- Substitute for ethanol

❖ METHANOL:

- Also referred to as wood alcohol

- Toxic dehydrating agent (methanol is converted to formaldehyde and can be further converted
to formic acid: both formaldehyde and formic acid are toxic to the body)
- for blood & tissue films and smear preparations

❖ BUTYL ALCOHOL:

- Slow-acting
- For plants & animals
- Also recommended for tissues which do not require rapid processing
- May be used in combination with ethanol
- Used to dehydrate slides after staining
CLEARING

- Removal of dehydrating agent from the tissues and replacing it by a solvent → transparent &
translucent tissue
- Not all dealcoholizing agents act as clearing agents
- Clearing agents only: glycerin, gum syrup and Brun’s solution
- Dealcoholizing agents only: chloroform and carbon tetrachloride

Applications of Clearing

1. Clearing in Embedding:
- Done after dehydration & before infiltration
- Solvent: dealcoholize and act as solvent of paraffin
- Agents: xylene, toluene, dioxane and chloroform
2. Clearing in Mounting
- Done after staining & before mounting
- microscopic preparations transparent (use of solvents with high refractive index)
- Agents must be solvents of the Mounting media: xylene, toluene, terpineol, carbol-xylene
3. For the purpose of making the tissues transparent so that their internal structure is
demonstrable to the naked eye.
- Refractive index of clearing agents: approximately equal to that of the tissues

Characteristics of an Ideal Clearing Agent

- Should be miscible with dehydrating agent and either infiltrating medium or mounting medium
- Should remove alcohol quickly & clear quickly without overhardening the tissues
- Should not dissolve out aniline dyes
- Should not evaporate quickly in the water baths
- Evaporates quickly in paraffin oven
- Could be used in amounts at least 10Xthe volume of tissue

Factors affecting Clearing

- Boiling point
o Agents with low BP are readily replaced by paraffin (Except chloroform)
- Viscosity
o Higher viscosity leads to slow penetration
- Refractive Index
o does not affect the rate but affects the quality of cleared tissue
 Reagent Used
o Special considerations should be noted depending on the clearing agent used
o Some reagents will not clear tissues
 - Xylene/Xylol
o Most rapid (15 - 30 mins/ 30 min – 1 hr)
o Excellent clearing agent but tends to make tissues excessively hard & brittle.
o Turns milky when dehydration is not complete
- Benzene
o Rapid agent (15 – 60 mins)
o Carcinogenic, causes aplastic anemia
- Toluene/Toluol
o Similar to xylene but does NOT harden tissues nearly so much
o Slower than xylene or benzene (1 – 2 hrs)
o Not carcinogenic but emits toxic fumes
- Chloroform
o For nervous tissues, lymph nodes & embryos
o Tissue do not become translucent
o Best for large specimens (up to 1cm thick) and tough tissues
o Toxic to the liver on prolonged inhalation
o Tissues tend to float: remedy → wrap tissues with absorbent cotton gauze
- Cedarwood oil
o Recommended for CNS, smooth muscles & skin
o Slow (2 – 3 days); minimal shrinkage
o For both celloidin and paraffin sections
o Tissue floats – use Absolute alcohol to prevent drying out of tissues
o Must be followed by immersion in xylene or benzene to remove oil from tissues
o Turns milky on prolonged storage

Impregnation & Embedding, and Paraffin Processing IMPREGNATION

- to fill all natural cavities, spaces & interstices of the tissues

- to give tissue samples a firm consistency

EMBEDDING

- impregnated tissue is placed into a precisely arranged position in a mold containing a medium
which is then allowed to solidify

PURPOSES OF INFILTRATION

1. Remove clearing agent

2. Fill cavities, spaces and interstices
3. Render firm consistency to the tissue to facilitate easy cutting and handling
4. Allows storage of processed tissue samples
Infiltration/Embedding media:

- substance used to infiltrate, support and enclose tissue specimen

- should be the same for infiltration and embedding
- most important characteristic: convertible from liquid to solid form
- mechanisms of solidification:
→crystallization
→evaporation of the solvent
→polymerization

PARAFFIN PROCESSING

- Simplest, most common & best embedding medium COSIDERATIONS:

1. Laboratory Temperature
o 20-24 oC (Room Temp): 54 – 58 oC
o 15-18 OC: 50-54 OC
2. Hardness of the Tissue sample
3. Temperature during Infiltration
o Paraffin oven: 2 – 5 oC higher than the MP of the wax
o Beyond 60 oC: deleterious to the tissue
4. Number of changes
o Minimum requirement: at least 2 changes
5. Nature and Size of the Tissue sample
o larger and denser: longer and more frequent changes
6. Clearing Agent Used
o xylene/benzene vs chloroform/cedarwood oil
- Advantages:
o Very thin sections may be obtained with ease
o Permits many staining procedures
- Disadvantages:
o Prolongation – excessive shrinkage & hardening
o Inadequate Infiltration - soft & shrunken - crumbling of tissue blocks - breaking up of
sections

Methods of Paraffin Impregnation:

1. MANUAL PROCESSING
o 4 changes at 15-minute intervals
o 2-5 OC higher than the MP of the wax
2. AUTOMATIC PROCESSING
o 2 – 3 changes with agitation
 o At least 3 OC higher than the MP of the wax

3. VACUUM EMBEDDING
o negative atmospheric pressure (400-500 mmHg)
o Heat & vacuum
o 3 changes
o 2 - 4 OC above MP of wax
o ADV: Effects of heat are prevented
o Applications in the lab: urgent biopsies, dense and fibrous tissues, delicate tissues

✓ Paraffin wax should be pure

- Fresh was should be filtered at 2 oC above its MP

- Wax from trimmings should be filtered with a course filter paper
- Water is removed by heating the wax to 100-105 oC

✓ Paraffin wax may be utilized twice

Paraffin Embedding:

- Melted paraffin: 5-10 oC above the MP of wax

- Cooling
o Ref at -5 OC
o immerse in cold water
o Tissue Tek with cold plate

Tissue Processing: Fixation  Dehydration  Clearing  Infiltration  Embedding/Blocking

After the tissue has been identified, grossly examined and sampled:

1. Wrap the pieces of the cut tissues with gauze and tie up securely with a label or tag, or place in a
tissue cassette with label.
2. Fix the wrapped tissues by immersing them in 10% formalin for 2 - 6 hours.
3. After fixation, wash the wrapped tissues in running water for about 30 minutes to 1 hour.
4. Drain the wrapped tissues and transfer to the succeeding solutions of increasing grades of ethyl
alcohol, changes of xylene and melted paraffin, following a designated time schedule

SECTIONING:

- a previously processed tissue, is trimmed and cut into uniformly thin slices or sections
- this procedure is also referred to as microtomy
3 BASIC PARTS

1. BLOCK HOLDER: where the embedded tissue is held in position

2. KNIFE CARRIER AND KNIFE: perform the actual cutting of tissue sections
3. PAWL, RATCHET FEED WHEEL AND ADJUSTMENT SCREWS: line up tissue block in proper position
with the knife, adjust proper thickness

General Principle:

o 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.

COLD MICROTOME/CRYOSTAT:

- rotary microtome inside a cold chamber

- temperature is maintained between -5 to -30 OC (ave. is -20 OC)
TRIMMING AND CUTTING OF PARAFFIN BLOCKS:

A. Trimming:
o Unmold the paraffin blocks from the paper boat. Trim down the sides of the paraffin
block with scalpel or knife until it fits the block holder of the microtome. Make sure that
all the sides are parallel and even.
B. Cutting of Paraffin:
o Sections Orient the well-trimmed paraffin block on the block holder/chuck. Carefully
adjust the block and make certain that it is completely parallel to the knife edge. Fix the
microtome knife in its proper position, maintaining a correct clearance angle. Make sure
that the upper and lower edges of the paraffin block are parallel to knife edge. Adjust
the thickness scale to thicker cuts.
o Unlock the handwheel and bring the block up to and immediately behind the knife edge.
Shave into the block, taking thin section cuts (approximately 10 – 25 micra) until the
outer layer of the paraffin is removed and the tissue is fully in contact with the knife
edge. Adjust now the thickness scale to about 4 – 6 micra. Start cutting and creating
ribbons by turning the handwheel at a slow and even speed (too rapid sectioning can
cause sections of unequal thickness). As each new section is cut, it duplicates the
previous one. These individual sections adhere to one another edge to edge to form a
ribbon. As sections begin to fold with the knife edge, lift the free end of the ribbon
gently with forceps to draw it towards you, taking care to avoid pulling the ribbon taut
as it will break. When the ribbon is about 6 – 8 inches, detach it from the microtome.
Float on water bath or keep the thin sections cut in small labelled boxes.
 Note: Generally, a hard tissue is cut best with a firm, relatively rapid motion, a soft tissue is best
cut with a slow or gentle motion.
ANGLES DURING SECTIONING:

1. Clearance angle (5-10 degrees)

o between the edge of the knife & the tissue block
2. Wedge angle (15 degrees)
o angle of cutting
3. Bevel angle (27-32 degrees)
o angle of cutting facet

SHARPENING OF MICROTOME KNIVES:

- entails maintaining the cutting edge of the knife

- the cutting edge must be of good quality steel o Too soft: doesn’t maintain the edge o Too hard:
is likely to nick against hard objects
- Tests in order to assess the sharpness of the cutting edge
 Should cut a paraffin wax block at 2 – 4 um thickness w/o serrations when
examined under the microscope (100 X)
 Von Mhol’s criterion
 Will split a hair drawn across it with only their own resistance

GENERAL STEPS IN FIXING SECTIONS ONTO SLIDE

1. Adhesion: performed to prevent washing out of tissue sections during staining

o Adhesive is a substance which can be smeared onto the slides so that sections stick well
to the slides
o Mayer’s egg albumin (egg white + glycerin + thymol)
 Most commonly used (easy to prepare, convenient and relatively inexpensive)
 o Dried Albumin (dried albumin + NaCl + thymol)
 Sections are dried and stored in 70% alcohol until it is ready for staining
o 1% Gelatin (gealtin + glycerol + phenol)
 Added to the waterbath during flotation rather than applying it on slides
o gelatin-formaldehyde mixture (gealtin + formaldehyde)
 slides are coated and allowed to dry at 37 OC for one hour or overnight prior to
use
o poly-L-lysine
 aqueous detergent diluted to 0.01% concentration
 widely used in immunohistochemistry
o APES (3-aminoprophylthriethoxysilane)
 Very useful in cytology, particularly for cytospin preparations of proteinaceous
or bloody material
2. Floating on water bath:
o 45-50 OC or 6-10 OC LOWER than the MP of wax; to straighten and tissue sections

3. Fishing out:
o waterbath should have a temperature that is 10 OC lower than the melting point of the
wax
4. Orientation:
o correct positioning of the tissue section/ribbon on the slide
5. Deparaffinization and Drying Sections
o wax oven (56OC – 60OC for 2 hrs)
o Incubators (overnight)
o Hot plate (45OC – 55OC for 30 – 45 mins.)
o Alcohol lamp/ bunsen flame
o Delicate tissues: 37 OC for at least 24 hours
o Blower-type electric slide dryer (50 – 55 OC for 20 – 30 mins.)
6. Post-mordanting
o Secondary fixation (post-chroming)
o 2.5 – 3% K2CrO4 for 24 hrs
o Used primarily as mordant & secondary as fixative
o 5 – 10 mins. in either
 Aq. Sol’n. of HgCl2
 Aq. Picric acid

Principles & Methods of Staining and H& E Technique PRINCIPLES OF STAINING

- process of applying dyes on the sections to see and study the architectural pattern of the tissue
and physical characteristics of the cells
- tissues and cells display varying affinities for most dyes and stains used during the process
 o Acidic structures—greater affinity for basic dyes
o Basic structures—greater affinity for acidic dyes
 Generally, two contrasting stains are used to facilitate differentiation of cellular structures and
constituents
- Impregnation
o related procedure that makes use of heavy metal salts which are selectively precipitated
on certain cellular and tissue components
o used for silver staining of nervous tissue and demonstration of reticulin
o most commonly used agent: silver nitrate—may also be used as a staining agent

METHODS OF STAINING

1. DIRECT STAINING
o process of giving color to the sections by using aqueous or alcoholic dyes
2. INDIRECT STAINING
o process whereby action of dye is intensified by adding another reagent (MORDANT)
which serves as a link/bridge between tissue and dye making staining reaction possible

- MORDANT: may be applied to tissue before staining or may be included in the staining process,
or may be incorporated as part of the dye solution itself (Eg. potassium alum with hematoxylin
in Ehrlich’s hematoxylin and iron in Weigert’s hematoxylin)
- ACCENTUATOR: not essential to the chemical union of tissue and dye; does not participate in the
staining reaction, but merely accelerates or hastens the speed of staining reaction by increasing
the staining power and selectivity of the dye (Eg: potassium hydroxide in Loeffler’s methylene
blue and phenol in carbol thionine and carbol fuchsin)
3. PROGRESSIVE STAINING
o process whereby 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
o no decolorization or washing after the application of the dye
4. REGRESSIVE STAINING
o tissue is first over stained to obliterate cellular details, and excess stain is removed or
decolorized from unwanted parts of the tissue, until the desired intensity of color is
obtained

H and E Staining Technique:

- Regressive staining due to the decolorization step using acid alcohol

- Major steps: Deparaffinization (Step 1) → Hydration (Step 2) → Nuclear staining (Step 4) →
Differentiation (Step 6) → Bluing (Step 8) → Counterstaining (Step 10) → Dehydration (Step 11)
→ Clearing (Step 12)
Steps in H & E Staining

1. Two changes of xylene

2. Descending grades of alcohol
3. Running water
4. Hematoxylin (Harris’: 5 min/Ehrlich’s: 15-30 min)
5. Running water
6. 1% acid alcohol (1ml conc. HCl + 99ml 80% ethanol) →also used to remove stains on the skin
7. Rinse in tap water
8. Ammonia water/1% lithium carbonate (30 sec)
→ Other Bluing Agents: warm tap H2O: 40-50degC, bicarbonate, potassium/sodium acetate,
ammonia and Scott’s Tap Water Substitute (TWS)
9. Running water
10. 5% aq. eosin (5 min)/alcoholic eosin (30 sec to 1 min)
11. Ascending grade of alcohol
12. Two changes of xylene
**Use paper clips to avoid slide surface contacts

EXPECTED STAINING RESULTS:

- Nuclei: blue to blue black

- Karyosome: dark blue
- Cytoplasm, proteins in edema fluid: pale pink
- RBCs, eosinophilic granules, keratin: bright orange-red
- Basophilic cytoplasm, plasma cells, osteoblast: purplish pink
- Cartilage: pink or light blue to dark blue
- Calcium and calcified bone: purplish blue
- Decalcified bone matrix, collagen, osteoid: pink
- Muscle fibers: deep pink

 Note: Slides taken from the oven should be allowed to cool first before staining, otherwise, the
tissue sections would be detached from the slide

MOUNTING AND LABELLING

MOUNTING:

- after staining, the section is mounted under a coverslip using a suitable medium
- uses a syrupy fluid referred to as a mountant or mounting medium

Purposes of Mounting:
 - protects the specimen from physical injury
- protects the section from bleaching or deterioration due to oxidation
- preserves slides for permanent keeping
- facilitates easy handling and storage

RINGING

- sealing margins of the coverslip

- uses Kronig cement → 2 parts paraffin + 4-9 parts powdered colophonium resin

Characteristics of a Good Mounting Medium

1. to avoid distortion of the image, the refractive index of the mountant should be as near as
possible to that of the glass which is 1.518
2. it should not dry quickly
3. It should not dissolve out or fade tissue sections
4. It should not cause shrinkage and distortion of tissues.
5. It should set hard, thereby producing permanent mounting of sections

TYPES OF MOUNTING MEDIA

A. Aqueous Media: for water-miscible preparations

o Water
 Has a low refractive index (1.33), moderately transparent and evaporates easily
 Good for temporary mounting only
 Tissue slide cannot be examined under OIO
o Glycerin
 Has a high RI (1.46) and lasts for a few minutes
 Provides greater visibility when mixed with water
o Glycerin Jelly (gelatin + glycerol + dist. Water + phenol crystals)
 Also known as Kaiser’s 1880 with a refractive index of 1.47
 Standard mounting medium when dehydration and clearing using xylene are not
performed
- DISADVANTAGES:
o X stain tend to fade
o X does not set in the desired degree of hardness
o X solidifies on storage

o Farrant’s medium (gum Arabic + dist. Water + glycerol + sodium merthiolate)

 Has a refractive index of 1.43

  Does not solidify on storage DISADVANTAGE X takes a longer time to harden
o Apathy’s medium (gum Arabic + cane sugar/sucrose + dist. H2O, thymol)
 Has a higher refractive index of 1.52
 Used as a general purpose aqueous mountant and for methylene blue-stained
nerve preparations
 Sets quite hard
o Brun’s fluid
 Recommended for mounting frozen sections from water
B. Resinous Media
o Canada balsam
 Natural resin extracted from the Canadian tree Abus balsamea
 Has a refractive index of 1.524
 Recommended for whole mounts and thick sections
 Sets hard without granulation
- DISADVANTAGES
o X acidifies and darkens with age and upon exposure to sunlight
o X stains are not usually preserved

o DPX (dibutylphthlate polystyrene xylene)

 neutral colorless solution that dries rapidly
 Has a refractive index of 1.532
 Recommended for small tissue sections
o XAM
 Synthetic resin mixture in xylene with a refractive index of 1.52
 Available in pale, yellow or colorless solutions
 Dries quickly without retraction and preserves stains well
o Clarite
 Has a refractive index of 1.544
 Usually diluted to 60% with xylene

LABELLING:

- Unique identification numbers or codes, patient name or other accessioning information should
be etched or written on each slide.
- Automated instruments that imprint the patient’s information on the glass slide are readily
available
- Chemical resistant pens and pencils are routinely used to label the slide
- Improper labelling is considered a “mortal sin” in histopathology
→theoretically, affects proper diagnosis of at least two patients

ACRONYMS:

PAP SMEAR: PAPANICULAUO TEST

HCT: HEMATOCRIT

PCV: PACKED CELL VOLUME

AIDS: ACQUIRED IMMUNE DEFFICIENCY SYNDROME

ALAT/ALT: ALANINE AMINOTRANSFERASE/ALANINE TRANSAMINASE

ALCL: ANAPLASTIC LARGE CELL LYMPHOMA