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Histopathology Reviewer

General Zoology (Laboratory) (University of the East (Philippines))

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MEDICAL TECHNOLOGY ASSESSMENT PROGRAM

HISTOPATHOLOGY & CYTOLOGIC TECHNIQUES

MT 2022 | MR. EDISON RAMOS (LECTURER)

HISTOPATHOLOGY CYTOPATHOLOGIC CHANGE IN DISEASE

▪ HISTOLOGY – study of normal tissues INFLAMMATION
 Histo = tissue and -logy = study ▪ From the Latin word “inflammare” (to set afire)
▪ HISTOPATHOLOGY – study of abnormal tissues ▪ A positive response meaning that the immune system is
 Histo = tissue, Patho = abnormal and -logy = study working properly
▪ Protective response of the tissue of the body to irritation or
TESTS COMMONLY OFFERED IN THE HISTOPATHOLOGY SECTION injury
▪ It is composed of a series of physiologic and morphologic
HISTOTECHNIQUE CYTOTECHNIQUE
changes in the blood vessels, blood components, and
✓ Biopsy ✓ Cell Cytology
surrounding connective tissues for the purpose of protecting
✓ Autopsy ✓ Cell Block
the body against injury.
✓ Pap’s Smear
✓ Blood Vessels – that are connected to the site of
injury or inflammation
PERSONNEL IN HISTOPATHOLOGY SECTION ✓ Blood components=a – particularly to the liquid
component of the blood
✓ Pathologist ✓ Histotechnician
✓ Histotechnologist ✓ Gross Examiner
Notes: 5 CARDINAL SIGNS OF INFLAMMATION
▪ Pathologist reads the result and the one who signs the paper ▪ Redness
▪ Histopathology section is the only section na hindi ▪ Morphological & physiological change in the
pumipirma ang medtech blood vessel
 Medtechs are assigned sa specimen RUBOR
▪ Due to arteriolar and capillary dilatation with
preparation and processing increased rate of blood flow towards the site
of injury
▪ Swelling
HISTOPATHOLOGIC TECHNIQUES ▪ Morphologic & physiologic change in the
▪ Involves the different procedures that have been adopted for blood vessel and blood components
the preparation of materials and tissues for microscopic TUMOR
▪ Due to increased capillary permeability
investigation (pathologist), whether they are normal or causing extravasation of blood fluid
abnormal ▪ E.g. Blister
▪ Includes examination of smears, preservation and processing ▪ Heat
of tissue sections (medtechs) prior to actual evaluation of ▪ Due to transfer of internal heat to the surface
tissues details (pathologist) CALOR or site of injury, brought about by increased
A well processed tissue can help in the blood content due to the dilation of the blood
confirmation and proper valuation of disease vessel and increase of blood flow
entity leading to a proper mode of treatment ▪ Pain
Pag mali yung sample na na-receive ni medtech, ▪ Mechanism: Extravasated blood components
Importance
kahit tama yung process, mali parin yung occupies the tissue causing the area of the
magiging evaluation ng pathologist DOLOR tissue space to decrease or compress causing
Quality of result ay nakadepende sa medical pressure to the sensory nerve
technologist ▪ Area na inooccupy dapat ng tissue, inooccupy
See to it that the tissues collected have been ng extravasated liquid components
Concern of
properly preserved and adequately prepared for ▪ Diminished Function
Medtech
microscopic study FUNCTIO ▪ Destruction of the functioning units of the
Notes: LAESA tissue
▪ Routine Histopathologic Techniques (FDCIETSSML) ▪ Pag matagal na yung inflammation
▪ Decalcification – special technique; used ONLY when
specimens are calcified such as bines, teeth, tuberculous
lungs, calcified tissues. CLASSIFICATION OF INFLAMMATION
ACCORDING TO
DURATION Character of Exudate
Acute Inflammation Serous, Fibrinous
Subchronic Inlammation Catarrhal, Hemorrhagic
Chronic Inflammation Suppurative/Purulent

ACCORDING TO DURATION
▪ Usually, but not necessarily, of sudden
ACUTE
onset
INFLAMMATION
▪ Protective response
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▪ Non-specific Smaller than normal

RETROGRESSIVE
▪ Vascular and exudative ▪ Did not reach full maturity
CHANGES
 Blood vessels and blood ▪ Matured but undergone abnormality
components PROGRESSIVE
Larger than normal (-megaly)
▪ Predominantly PMNs (neutrophil) CHANGES
▪ When this fails to subside withing several DEGENERATIVE Tissues have abnormalities
weeks → CHRONIC CHANGES Not associated with size
▪ Stimuli:
✓ Infectious ✓ Tissue necrosis
agent ✓ Foreign bodies RETROGRESSIVE CHANGES
✓ Physical ✓ Immune
✓ Chemical response ▪ Incomplete/defective development of a
tissue/organ
▪ Outcomes:
▪ Mos t commonly seen in one of paired
✓ 100% complete resolution
APLASIA structures (kidneys, gonads, adrenals)
✓ Scar
▪ Represented only by a mass of fatty/fibrous
✓ Chronic Inflammation
tissue
SUBCHRONIC ▪ Represents an intergrade between acute
▪ No resemblance to the adult structure
INFLAMMATION and chronic
AGNESIA ▪ Non-appearance of an organ
▪ Persistence of the injuring agent for
▪ Failure of an organ to reach its full mature size
weeks/years HYPOPLASIA
▪ Resemblance to adult structure
▪ Vascular and fibroblastic
▪ Predominantly Mononuclear ATRESIA ▪ Failure of an organ to form an opening
(macrophages, lymphocytes, plasma cells) ▪ Decrease in size of a normally mature
CHRONIC tissue/organ
but PMNs may also be present ATROPHY
INFLAMMATION ▪ Nareach yung full mature size pero nagkaron
▪ Causes:
✓ Persistence of infection ng physiologic changes (pag liit)
✓ Prolonged and continuous What is the difference of atrophy from hypoplasia?
exposure - Resulting from the reduction in cell size or decrease in the
✓ Auto-immunity total number of cells
PHYSIOLOGIC ATROPHY PATHOLOGIC ATROPHY
ACCORDING TO CHARACTER OF EXUDATE ▪ Occurs as a natural ▪ As a consequence of
▪ Extensive outpouring of watery (crustal consequence of disease
clear), low-protein fluid maturation ▪ Vascular, Pressure,
▪ Derived from serum or secretions from the ▪ Atrophy of the thymus Starvation or Hunger,
SEROUS serosal mesothelial cells. (peritoneal, during puberty and/or Endocrine Atrophy
INFLAMMATION pleural, or pericardial cavities) ▪ At about 50 y/o atrophy of
▪ Characteristic of certain forms of the brain and sexual
pulmonary tuberculosis organs
▪ i.e. Blisters
▪ Exudation of large amounts of fibrinogen
FIBRINOUS and the precipitation of masses of fibrin PROGRESSIVE CHANGES
INFLAMMATION ▪ Found in diphtheria, rheumatic pericarditis
Tissue size increase because of size increase of
and in early stage of pneumonia HYPERTROPHY
individual cells
▪ Excessive discharge or build-up of mucus in
Tissue size increases because of increase in
the nose or throat, associated with HYPERPLASIA
the number of cells making up the tissue
inflammation of the mucous membrane
CATARRHAL
(kapag sinisipon)
INFLAMMATION DEGENERATIVE CHANGES
▪ Affects the mucous surfaces
▪ With hypersecretion of the mucosa
Reversible involving transformation in one type
▪ Degenerative changes in the epithelium
of adult cell to another
▪ Admixture of blood and other elements of
A -> B
HEMORRHAGIC the exudate METAPLASIA
One type of Another type
INFLAMMATION ▪ May be found in bacterial infection and
adult cell of adult cell
other infection
▪ Production of large amounts of pus or
Atypical hyperplasia
SUPPURATIVE/ purulent exudate
A -> A1
PURULENT ▪ PUS – thick . creamy fluid composed of One type of Change in
INFLAMMATION large number of living and necrotic PMNs DYSPLASIA
adult cell structural
and necrotic tissue debris
Components

CHANGES IN CELLULAR GROWTH PATTERNS

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Dedifferentiation (maturation) TERATOMAS

 When cells differentiate, they mature ▪ Type of neoplasm
 Dedifferentiation → did not mature ▪ Compound tumors
(immature) → non functional cell ▪ Greek: “monstrous tumors”
ANAPLASIA Usually used as a criterion toward malignancy ▪ Tumor with normal tissue or organ
components that are inappropriate to
Adult cells More primitive surrounding tissues
or embryonic ▪ May contain hair, teeth, bones, and
very rarely eyeballs, torso and hands
Continuous abnormal proliferation of cells
NEOPLASIA without control (no purpose or function)
(TUMOR) Pathologic overgrowth of the tissue
Aggregate of non-functional cells (tumor) TUMORS
DIFFERENTIATION OF TUMORS
NEOPLASIA
BASED ON CAPACITY TO PRODUCE DEATH
▪ Process whereby a neoplasm is formed
BENIGN TUMORS Do not produce death
▪ “Neoplasm” – from the Greek word meaning “new growth”
MALIGNANT TUMORS Will produce death eventually
▪ Study of neoplasia: Oncology
BASED ON HISTOLOGIC CHARACTERISTICS
GROWTH BEHAVIOR OF NEOPLASIA
MEDULLARY More cells than supporting tissue
▪ Advanced type SCIRRHOUS CARCINOMA More connective tissues than cell
TRANSFORMED ▪ Show altered surface antigens
PHENOTYPE ▪ Proliferate to form colonies (clones) GRADING OF TUMORS
▪ Without normal cell orientation
DIFFERENTIATED UNDIFFERENTIATED
TUMORIGENICITY ▪ Will grow into tumors
GRADE I 100-75 0-25
▪ Indefinite replication and can grow as cell
IMMORTALITY GRADE II 75-50 25-50
lines
GRADE III 50-25 50-75
GRADE IV 25-0 75-100
NOMENCLATURE OF NEOPLASTIC CELLS
% Mature cells % Immature cells
BENIGN NEOPLASMS MALIGNANT NEOPLASM
-sarcoma NECROSIS
-oma (mesynchymal/connective tissue)
-carcinoma CAUSES OF NECROSIS
(epithelial tissues) ✓ Ischemia or Anoxia – loss of blood supply
✓ Physical agents – Trauma, Extreme heat/cold, Radiant
MESENCHYMAL/ CONNECTIVE TISSUE TUMOR energy, Electrical energy
BENIGN MALIGNANT ✓ Chemical agents
Fibrous Tissue Fibroma Fibrosarcoma ✓ Biological products (toxics/lason)
Adipose Tissue Lipoma Liposarcoma ✓ Coagulation – most common
Cartilage Chondroma Chondrosarcoma ✓ Liquefaction/Colliquative – pus formation
Bone Osteoma Osteogenic Sarcoma ✓ Fat necrosis
Hematopoietic Lymphoid tissue ✓ Caseous/Caseation
Leukemia ✓ Gangrenous (Dry/Wet)
cells sarcoma
Smooth muscle Leiomyoma Leiomyosarcoma
Striated muscle Rhabdomyoma Rhabdomyosarcoma DEATH
EPITHELIAL TISSUE TUMORS
SOMATIC DEATH CELLULAR DEATH
BENIGN MALIGNANT
▪ Apoptosis – cellular suicide
Stratified Squamous cell Squamous cell (normal; programmed cell
squamous papilloma carcinoma Death of the entire organism
death)
Glands and or the body
Adenoma Adenocarcinoma ▪ Necrosis – pathologic death
Ducts of cells
Renal Renal Tubular
Renal Cell Carcinoma
Epithelium Adenoma
SOMATIC DEATH
Hepatocarcinoma
Liver Cells Liver cell adenoma ▪ Circulatory failure
(Hepatoma) PRIMARY
Neuroectoderm Nevus/Mole Hemangiosarcoma ▪ Respiratory failure
CHANGES/ SIGNS
Testicular ▪ Nervous failure (CNS failure)
Seminoma SECONDARY ▪ Algor Mortis ▪ Desiccation
epithelium
CHANGES ▪ Rigor Mortis ▪ Putrefaction
▪ Livor Mortis ▪ Autolysis
▪ Post-mortem
Clotting

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STAGES OF DEATH ▪ For determination of the internal sex individual in case of

abnormalities in sexes.
PALLOR MORTIS 15 to 20 minutes after death ▪ Assessment of female hormonal status in case of sterility and
Pallor – pale (humihinto yung circulation) endocrine disorders
ALGOR MORTIS 2ºC for 1st hour, 1ºC after
Lumalamig yung bangkay
RIGOR MORTIS 3 to 4 hours, max 12 hours SPECIMENS FOR EXAMINATION
Tumitigas yung bangkay
✓ Peritoneal, Pericardial, ✓ Sputum
LIVOR MORTIS Bluish color
Pleural Fluid ✓ Gastric washings
Yung place ng discoloration is dependent sa
✓ CSF ✓ Urine sediments
pwesto ng deceased
✓ Breast Secretion/ Nipple ✓ Prostatic secretions/fluid
PUTREFACTION Production of putrescine and cadaverine
Discharge ✓ Cervicovaginal (paps) smear
DECOMPOSITION
SKELETONIZATION
FACTORS OF MALIGNANCY
PALLOR MORTIS
1. Hormonal changes
▪ Is a post mortem paleness which happens in those with 2. Trauma (Misused, Overused, Unused)
light/white skin almost instantly because of a lack of capillary 3. Preservatives (common; chemically preserved foods)
circulation throughout the body 4. Radiation (DNA Alteration)
▪ The blood sinks down to the lower parts (due to gravity) of the
body creating livor mortis
SPECIMEN THAT REQUIRES ADHESIVE
ALGOR MORTIS 1. Urinary sediment
▪ Is the reduction in the body temperature following death. 2. Bronchial lavage specimen
▪ This is generally a steady decline until matching ambient 3. Specimen that utilizes proteolytic enzymes during
temperature, although external factors can have a significant processing (eg. Trypsin, Concentrated Sputum and
influence Enzymatic lavage sample from GIT)

RIGOR MORTIS
▪ Is one of the recognizable signs of death, caused by chemical CRITERIA FOR MICROSCOPIC DIAGNOSIS OF CANCER CELLS
changes in the muscles after death, causing the limbs of the Variation is size, shape, form and
corpse to become stiff and difficult to move or manipulate. PLEOMORPHISM
external appearance of cells
▪ In humans, it commences after about three to four hours, Increased in staining affinity above the
reaches maximum stiffness after 12 hours, and gradually HYPERCHROMATISM normal affecting, mainly the nuclear
dissipates from approximately 24 hours after death. structures. (Basophilism)
MULTINUCLEATION Increase in numbers of nuclei per cell
LIVOR MORTIS ATYPICAL MITOTIC Abnormal stages of mitosis or cell
▪ Is a settling of the blood in the lower (dependent) portion of FIGURES division
the body, causing a purplish red discoloration of the skin. Lighter-staining affinity affecting the
When the heart stops functioning and is no longer agitating the VESICULAR cytoplasm affecting the cytoplasm
blood, heavy red blood cells sink through the serum by action STAINING producing a great contrast in staining
of gravity CYTOPLASM affinity with that of the nucleus
(Inversion or reversal of the N:C ratio)

EXFOLIATIVE CYTOLOGY
CRITERIA FOR ABNORMALITIES OF THE CELLS
▪ Branched of science dealing with the study of cells that are (ASIDE FROM CANCER CELLS)
scrapped off or removed off or coming from lining epithelium
and mucosa of different organs of the body ▪ Variation in size, shape or form of the cell
▪ Synonyms: PAPANICOLAOU’S METHOD or PAP’S METHOD CHANGES ▪ Enlargement of cells in swelling
IN THE CELL ▪ Diminution in size of the cells as in shrinking
MEMBRANE ▪ Cloudiness or indistinctness in the cellular
DIVISIONS boundary
CYTOPATHOLOGY CYTOTECHNIQUE ▪ Accumulation of materials or substances within
Study of the methods preparing CHANGES as water, protein, carbohydrate, fat and others
Study of abnormal cells as
the cells for microscopic IN THE ▪ Disintegration or breaking down of the
cancer cells CYTOPLASM cytoplasmic organelles
examinations
▪ Dissolution of the cytoplasmic components
▪ Pyknosis – condensation of the chromatin
PURPOSE, AIMS, & SIGNIFICANCE OF DIAGNOSTIC EXFOLIATIVE materials
CHANGES
CYTOLOGY ▪ Karyorrhexis – breaking down of the nuclear
IN THE
components
▪ For diagnosis or Cancer NUCLEUS
▪ Karyolysis – dissolution of the nuclear
▪ For differentiation between Malignant and Benign Tumors
structures
▪ For differentiation between Tumors from other diseases as .

Infectious, Inflammations, or Degenerations.

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PROCEDURES/METHODS DONE IN BOILING SPECIMENS FOR 4. GASTRIC CONTENTS AND DUODENAL FLUID
EXFOLIATIVE CYTOLOGY
▪ Specimens obtained by the intubation technique using rubber
1. SCRAPPINGS tubing of specimen diameter and length known as nasogastric
tube
▪ Specimens come from lining epithelium and mucosa or
✓ Levine Tube – designed for the nostrils
secretions of the female reproductive tract as various vagina,
✓ Rhefuss Tube – designed for the mouth
cervix and endometrium of the uterus.
Instruments Used for the Aspiration of Secretion Gastric Content Duodenal Fluid
1. Rubber Bulb with Watery, Acidic and plenty of Greenish-Yellow, Alkaline
For aspiration of fluid in cancer
Glass Pipette Food Debris Fluid, Mucoid
For obtaining mucoid specimens for
2. Aryes Spatula
hormonal studies
Spoon, curved or blunt types –
commonly used for much biopsies to 5. CEREBROSPINAL FLUID
3. Curette obtain pieces of the tissues from the ▪ Obtained by lumbar
vaginal and cervix or placental tissues puncture or lumbar tap
for the endometrium down in the
intervertebral spaces
between the second and
third lumbar vertebrae
4. Vaginal Speculum or between the third
and fourth lumbar
vertebrae
▪ The fluid is a secretion of the choroidal plexus of the brain
circulating within the ventricles and central canal of the spinal
Diagnostic Curettage (DC) Minor procedure for diagnosis cord
Dilatation and Curettage Major operation for treatment Tube Section Temperature
1 Chemistry, Serology Freezing
2 Microbiology Room Temp
3 Hematology Ref Temp

6. SEROUS FLUID
PLEURAL ▪ Fluids are obtained by thoracentesis
CAVITY ▪ Done commonly on the left lateral thoracic
wall
PERICARDIAL ▪ Fluids are obtained by pericardiocentesis
CAVITY ▪ Done commonly on the left sterna border
2. PROSTATIC SECRETION PERITONEAL ▪ Fluids are obtained by paracentesis
CAVITY abdominis
▪ Done by massage through an
▪ Done on the midline of the abdomen below
intra-rectal route using the
the navel (at the linea alba)
middle and index finger
aseptically.
▪ Three (3) samples are
collected (urine before
massage, prostatic secretion
by massage, urine after
massage)

3. BRONCHIAL SECRETION OR SPUTUM

▪ “Deep cough” specimen is the best study

▪ Three consecutive morning sputum sample
▪ SPUTUM collected in a wide-mouth container containing
SACCOMANO FLUID (50% ETOH and 2% carbowax)

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7. BONE MARROW SUBSTANCE EXCISION ▪ Process of cutting the tissues under

(Solid tissue) examination and removing either partially,
▪ Specimen from the bone marrow cavities (red bone marrow completely, or by pieces for laboratory
or myeloid tissue) microscopic examination and diagnosis.
▪ Obtained by puncture using a bone marrow borer at the ▪ This is commonly done for masses in any
sternum or iliac crest portion of the body suspected to be tumors.
ASIPIRATION ▪ Type of biopsy commonly done for fluid
8. URINE (Liquid specimen only.
tissue) ▪ This method utilizes an aspiration in the form
▪ Voided urine of male is usually sufficient for cytological of needle and syringe or a suction tube or
evaluation pump to obtain fluid specimens directly or
▪ But for female patients, catheterized specimen is indirectly from the source.
recommended (less contamination) ▪ The specimens are commonly utilized for
smear preparation especially for Pap’s smear
and can also be utilized for cell block
preparation.

ASPIRATION INCISION
Liquids that are normally Fluids that are caused or
produced by the body (e.g. brought about by some
urine) abnormalities (exudates)

AUTOPSY
▪ Also known as:
✓ post-mortem examination
NIPPLE DISCHARGE ✓ necropsy (particularly as to non-human bodies)
▪ Spontaneous nipple discharge is ✓ autopsia cadaverum
collected with a cotton swab ✓ obduction
▪ Procedure: ▪ The term “autopsy” derives from the Ancient Greek autopsia
1. Gently strip the sub-areolar meaning “to see for oneself”
area and nipple with the ▪ Process of taking pieces of tissue (representative out
thumb and forefinger sections) from a dead person (Cadaver) for the purpose of
2. Place the slide upon the examination or investigation, in order to determine the
nipple and draw it quickly across the nipple. If 1 drop is cause of death or extent of injury leading to the death of the
obtained, get another slide and do the pull-apart person.
technique.
3. Immediately immersed the slide into a bottle of 95% PURPOSE OF AUTOPSY
isopropyl ROH or use spray fixative.
1 To determine or ascertain the causer of death of a person
4. If the secretion is very little in amount, the smear should
2 To determine or know the final diagnosis
be restricted to a small area only to prevent drying.
To investigate the cause of death or extent of injury leading
5. Label the secretions obtained indicating from where 3
to the death of the person
they’re collected. (left or right breast)
To preserve the tissue of the dead body for further
4 examination, investigation, and future study as in research or
scientific study
PRINCIPLES OF AUTOPSY & BIOPSY
BIOPSY TYPES OF AUTOPSY
▪ Biopsy is of Greek origin, coming from the words bio, ACCORDING TO ITS PURPOSE
meaning life, and opsia, meaning to see
Routine Hospital Autopsy
▪ Biopsy is the process of taking pieces of tissue from a living
▪ Done in private hospitals for the cause of death of the
patient for the purpose of microscopic examination and
A person especially if the cause cannot be determined
diagnosis
clinically or the cause of death is problematic to the
▪ This is commonly used in histopathology for laboratory
clinician.
interpretation of tumors, especially in differentiation
Medico-Legal Autopsy
between benign and malignant tumors
▪ Done at NBI (National Bureau of Investigation) or other
▪ The main purpose of biopsy is attained if the tissue under B
government institution for the purpose of persecution
examination turns out to be cancer (CA) or malignant.
(criminal case).
ACCORDING TO ITS COMPLETENESS OF THE PROCEDURE OR
TYPES OF BIOPSIES TECHNIQUE
INCISION ▪ Process of opening and cutting the tissue Partial
(Liquid under examination in order to obtain ▪ Autopsy request involved only the examination of a
A
tissue) specimen and also to a tube for drainage of region or regions of the body as head only, thorax only
fluid secretion or exudates or abdomen only
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Complete EXAMINATION OF TISSUES

▪ Autopsy request involved the examination of the whole
B
body from head to foot for complete diagnosis and METHODS OF TISSUE EXAMINATION
investigation 1 Fresh Tissue Examination
ACCORING TO THE MANNER OF INCISION OR OPENING THE 2 Preserved/Fixed Tissue Examination
CADAVER
Y – Shaped Incision FRESH TISSUE EXAMINATION
▪ Cadaver is open from both shoulder regions down to the 1 Teasing or Dissociation
A xiphoid area, and then incised down to the pubis. This 2 Squash Preparation (Crushing)
commonly done in adult and female cadaver. 3 Smear Preparation
▪ Sometimes “U” shaped daw 4 Frozen Section
Straight Cut Incision
▪ Cadaver is open from the middle of the body from the 1. TEASING OR DISSOCIATION
B
suprasternal notch down to the pubis. This is commonly
done in children and infants. ▪ A selected tissue specimen is immersed in a watch glass
containing NSS, carefully dissected or separated and
examined
FORENSIC AUTOPSY ▪ NSS – isotonic; preserves integrity of the cells
▪ Used to determine the cause and manner of death. ▪ Unstained preparation
▪ Forensic science involves the application of the sciences to  Bright field microscope – reduced light
answer questions of interest to the legal system  Phase contrast – recommended for unstained
▪ Deaths are classified under one of five manners:
a. Natural d. Suicide 2. SQUASH PREPARATION
b. Accident e. Undetermined
▪ Small pieces of tissues are placed in a microscopic slide and
c. Homicide
forcibly compressed with another slide or coverslip
▪ Undetermined category may include deaths in absentia, such
▪ Can directly be observed in the microscope, stained or
as deaths at sea and missing persons declared dead in a court
unstained
of law; in others, such deaths are classified under "Other".
 For stained – di na kailangan pag-hiwalayin yung
▪ Medical examiners also attempt to determine the time of
slide para di madisturb yung preparation using a
death, the exact cause of death, and what, if anything,
pipette, slowly drop the preferred stain sa gilid ng
preceded the death, such as a struggle.
slde. Capillary attraction principle
▪ A forensic autopsy may include obtaining biological specimens
from the deceased for toxicological testing, including stomach
contents 3. SMEAR PREPARATION
▪ Smears should be from fresh material (specifically liquid
samples)
PRE-REQUISITE OF AUTPOSY PROCEDURE
 Though may specific na smear preparation na
Written consent or permission from the nearest of kin or ginagamit for solid sample → Touch/Impression
relative. smear
Deceased (w/fam) → Wife → Anak → Magulang → ▪ See requisition form (patient’s ID: name, age; date and type
1
Kapatid → Kamaganak of specimen requested
Deceased (w/o fam) → Magulang → Kapatid → Family ▪ Label the slide
member
Type of autopsy procedure or technique must be specified Methods of Smear Preparation
2
according to purpose and completeness. Streaking
Common apparatus or instruments needed during autopsy ▪ Used for preparing mucoid secretions
procedures as: vaginal secretions, sputum and gastric
✓ Bone marrow borer 1 content)
✓ Kwaksaw or electric saw for bones ▪ Use a spatula, dissecting needle or
✓ Forceps – different sizes and types applicator stick and streak in a zigzag fashion
✓ Sterile syringes and needles ▪ Directly into the slide
✓ Specimen bottles with fixation Spreading
3 ✓ Surgical needles and sutures ▪ Used for thick mucoid secretions (smears of fresh sputum
✓ Pails – container for organs or specimens 2
[AFB] and bronchial aspirates)
✓ Knives ✓ Spatula Weighing ▪ Usually done in blood specimen
✓ Scissors scale Pull - Apart
✓ Culture medium ✓ Sterile cotton ▪ For serous fluids,
✓ Clean dry slide ✓ Surgical gloves concentrated sputum, and
✓ Alcohol lamp ✓ Surgical mask enzymatic lavage form the
(secondary) 3 GIT, smears of urinary
4 Pathologist on duty – performer of the procedure sediment, vaginal pool and
breast secretions
▪ Done to maximize the amount of the collected sample
(e.g. sample from nipple discharge)

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Touch or Impression Smear DECALCIFICATION

▪ Sample is a solid mass tissue ▪ Special technique, not all specimen will undergo decalcification
▪ PREPARATION: for preparation ▪ Done after fixation and before dehydration
4 of direct impression from the ▪ Used ONLY when specimens are calcified such as bones, teeth,
cut surface of tissue like the tuberculous lungs, calcified tissues
lymph nodes and other ▪ Tuberculous lungs – accumulation if calcium sa lungs kaya
surgical or autopsy secretion tumitigas at hindi nag fufunction

ROUTINE HISTOPATHOLOGIC TECHNIQUES

4. FROZEN SECTION
1 Fixation 6 Trimming
▪ Utilized when rapid diagnosis of tissue is required (STAT or 2 Dehydration 7 Sectioning
intraoperative) 3 Clearing 8 Staining
▪ Recommended for lipids and nervous tissue 4 Impregnation 9 Mounting
▪ Thickness: 10-15 micra 5 Embedding 10 Labelling
▪ Temperature: -10 to -20°C
“FDCIETSSML”
INDICATION LIMITATION
▪ Rapid diagnosis (guide for ▪ Limited section sampling FIXATION
intra-operative patient ▪ Ice crystal or freezing artifact
management) ▪ Inferior quality compared to ▪ This is the process by which the constituents of cells and
▪ To optimally process paraffin sections tissues are fixed in a physical and chemical (carbohydrate,
tissues for special studies  Routinely done, but protein, fats) state so that they will withstand subsequent
for diagnosis, treatment, or time consuming treatment with various reagents with minimum loss of
research ▪ Lack of special studies (time architecture. This is achieved by exposing the tissue to
▪ To confirm that lesional constraint) chemical compounds called FIXATIVES/PRESERVATIVES
tissue is present for ✓ Special stains, ▪ First and most critical step
diagnosis on permanent immunohistochemistry, ▪ The quality of the section in the slide is as good as the quality
sections (sample adequacy) culture of the fixed tissue specimen
▪ Lack of consultation for ▪ Primary purpose: preserve the morphological and chemical
difficult cases integrity of the cell
▪ Secondary purpose: harden and protect the tissue, thus
Consider These During RFS easier to cut
1 Relevant clinical information ▪ It prevents DEGENERATION, DECOMPOSITION,
2 Type of tissue or location of biopsy PUTREFACTION, DISTORTION of tissue after removal from
To determine beforehand what information the surgeon body
3
required from the FS and how the information will be used
4 Optimal turn-around time is ≤ 15 mins (maximum) FIXATIVES
5 Coordination between laboratory and OR personnel EFFECTS OF FIXATIVES
6 Check cryostat, temperature should be -17ºC
No fixative is used (di na dadaan sa routine, deretso 1 Hardens soft and friable tissues
7 2 Makes cells resistant to damage and distortion
freezing na the cutting in the microtome [freezing])
Protection of laboratory personnel (possible source of 3 Inhibit bacterial decomposition
8 4 Increase optical differentiation of cells
infection)
9 Selection of the part of tissue for FS Acts as mordant or accentuator thereby facilitating staining
5
process
Freezing Agents 6 Reduce the risk of infection
Liquid nitrogen – most common and most rapid and is
1 CHARACTERISTICS OF A GOOD FIXATIVE
readily available
2 Isopentane 1 Must be cheap
3 Carbon dioxide gas 2 Must be stable
4 Aerosol spray 3 Must be safe to handle
4 Must kill the cell quickly
5 Must inhibit bacterial decomposition and autolysis
HISTOPATHOLOGIC TECHNIQUES Must produce minimum shrinkage of tissue (should be
6
isotonic)
EXAMINATION OF FIXED (PRESERVED TISSUES
Must permit rapid and even penetration of tissue (can also
HISTOPATHOLOGIC TECHNIQUES/STEPS 7
preserve the innermost part of the tissue)
1 Fixation 7 Blocking
8 Must be isotonic
2 Decalcification 8 Trimming
9 Must harden tissue
3 Dehydration 9 Sectioning
Must make cellular components insoluble to hypotonic
4 Clearing 10 Staining 10
solution
5 Impregnation 11 Mounting
11 Must permit subsequent application of many stains
6 Embedding 12 Labelling

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IMPORTANT FACTORS TO BE CONSIDERED IN FIXATION CYTOLOGICAL FIXATIVE

Size of tissue to be processed NUCLER CYTOPLASMIC
1 pH ≤ 4.6 >4.6
▪ Ideal size: 3mm
Glacial Acetic YES NO
Amount of fixatives
Acid (High affinity to nuclear (because it destroys
2 ▪ 10 – 20 x volume of specimen (except Osmium chromatin) mitochondria and Golgi bodies)
tetroxide – used in lesser amounts very expensive) Examples ▪ Bouin’s Fluid ▪ Flemming’s Fluid w/o
Duration of fixation ▪ Flemming’s Fluid acetic acid
3 ▪ Duration of fixation varies depending on the fixatives ▪ Newcomer’s Fluid ▪ Helly’s Fluid
used – hindi pareparehas sis pati precautions ▪ Carnoy’s Fluid ▪ Formalin w/ “post
4 Precautions ▪ Heidenhain’ s Susa chroming”
Hydrogen concentration ▪ Regaud’s /Moller’ s
5 ▪ Orth’s Fluid
▪ pH of 6.0 to 8.0
Temperature
6 ▪ Routine: Room Temp
▪ Electron Microscopy & Histochemistry: 0- 4°C
Osmolality
7
▪ Isotonic solution

FACTORS TO BE CONSIDERED IN CHOOSING THE RIGHT FIXATIVE

1 Need for immediate evaluation
Type of specimen to be processed
2 ▪ Walang all purpose fixative it depends sa tissue
specimen (e.g. formalin)
3 Tissue structure to be studied
4 Type of section to be made
Staining technique to be applied
▪ Fixative should be compatible to the stain that’s I. ALDEHYDE FIXATIVE
5 going to be used ▪ Satisfactory for routine paraffin sections
▪ There are specific fixatives that are immiscible to  Formaldehyde – most satisfactory for paraffin
stains sections
▪ For electron microscopy – Glutaraldehyde
▪ For histochemical and enzyme studies
TYPES OF FIXATIVE
ACCORDING TO ITS COMPOSITION FORMALDEHYDE (FORMALIN)
SIMPLE COMPOUND ▪ Most common, widely used
▪ Gas produced by oxidation of methanol
One component substance Made up of 2 or more fixative
▪ 10% recommended, buffered to 7.0
ACCORDING TO ITS ACTION  Remember: 6.0-8.0 accepted hydrogen ion
MICROANATOMICAL concentration
A
▪ Permit general microscopic study  Maximum concentration of formalin = 37-40%
CYTOLOGICAL ▪ Types:
▪ Preserves particular microscopic elements of the cell a. 10% formol saline
b. 10% neutral buffered formalin
Nuclear Cytoplasmic c. Formol corrosive (formol sublimate)
✓ Contain glacial acetic ✓ Does not contain d. Formol calcium
acid (affinity to nuclear glacial acetic acid e. Unbuffered zinc formalin
chromatin) ✓ pH 4.6 and above f. Alcoholic formalin
B
✓ pH 4.6 or less
✓ Temperature usually PRECAUTIONS FOR FORMALDEHYDE
20-22°C
1 Paraformaldehyde formation
✓ Ethanol, Methanol,
2 Well ventilated room
Carnoy’s – common for
3 Not neutralized if concentrated – explosion
nuclei acid
Buffered or neutralized by adding magnesium
4
HISTOCHEMICHAL carbonate/CaCO3 – wide mouth bottle
C
▪ Preserves chemical constituents of cells and tissue 5 Bleaching prevented by changing formalin
REMEMBER !!! Prolonged storage may induce precipitation (white
6
▪ Lipid – frozen section precipitate) – filter or add 10% methanol
▪ Carbohydrates – alcoholic fixative
▪ Protein – formaldehyde or neutral buffered formol-saline

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ADVANTAGE DISADVANTAGE ▪ Disadvantage of Picric Acid – it will leave a yellowish stain

Cheap, readily available, easy ▪ More common – Kardasewitch’s Method
to prepare, stable, compatible Irritating fumes, prolonged
w/ stains, penetrates tissues fixation may bleach tissues GLUTARALDEHYDE
well, preserves fat, mucin, ▪ Made up of 2 formaldehyde residues linked by 3 carbon chains
glycogen, for tissue ▪ For LM, EM
photography
needle biopsies
2.5% Glutaraldehyde
10% FORMOL SALINE 2 to 4 hours at RT
▪ Made up of formaldehyde dilute to 10% NaCl larger tissues
4% Glutaraldehyde
▪ Recommended for CNS tissues and post mortem tissues for 6 to 24 hours at RT
histochemical exam
Advantage Disadvantage ADVANTAGE DISADVANTAGE
Penetrates and fixes tissues More stable effect, less tissue More expensive, slow
well, minimum shrinkage & Slow (>24 h) shrinkage, less irritating penetration
distortion, does not
Than formaldehyde
overharden tissues

10% NEUTRAL BUFFERED FORMALIN ALDEHYDE FIXATIVE FIXATION TIME

▪ Formalin buffered with Na dihydrogen PO4 , Disodium H PO4 Formaldehyde 24 hours
▪ For preservation and storage of surgical, post mortem and 24 hours – 35ºC
10% Formol-Saline
research specimens 48 hours – 20-25ºC
Advantage Disadvantage 10% Neutral Buffered Formalin 4-24 hours
Best fixative for Fe pigments, Longer to prepare – time Formol-Corrosive/
3-24 hours
elastic fibers consuming, inert towards lipids FormolSublimate
2.5% Glutaraldehyde 2-4 hours – room temp
FORMOL CORROSIVE (FORMOL SUBLIMATE) 4% Glutaraldehyde 6-24 hours
▪ Formol mercuric chloride Formal Calcium 12-24 hours
▪ Recommended for routine post-mortem tissues Unbuffered Zinc Formalin 4-8 hours
Advantage Disadvantage
Minimum shrinkage and Slow
hardening, No need for wash Forms mercuric chloride II. METALLIC FIXATIVES
out from fixative to ROH deposits
▪ More commonly used
▪ Varying indications
FORMAL CALCIUM
▪ HCHO + CaCl
MERCURIC CHLORIDE
▪ Recommended for the preservation of lipids especially
phospholipids ▪ Most common metallic fixative; 5-7 %
▪ Recommended for renal tissue, fibrin, connective tissues and
UNBUFFERED ZINC FORMALIN muscles
▪ HCHO + Zinc Sulphate ▪ Produces black precipitates of mercury – remove by 0.5%
▪ Alternatives to mercuric chloride formulations iodine solution in 70% ethanol then decolorize iodine using
▪ Improved results with IHC absolute alcohol
ADVANTAGE DISADVANTAGE
ALCOHOLIC FORMALIN (GENDRE’S) Hardens outer layers only,
▪ Coagulates mucus For tissue photography,
black granular deposits formed
▪ Can be used to fix sputum recommended for renal
(removed by adding iodine),
tissues, fibrin, CT, muscles
corrosive to metals
REMOVAL OF FORMALIN PIGMENT
1 Bring down to water TYPE DESCRIPTION
Kardasewitch Place in mixture of 70% ETOH and 28% ▪ HgCl2 + Glacial Hac
2
’s Method Ammonia water for 5mins-3hrs ▪ Recommended for liver, spleen, CT fibers,
3 Wash with water nuclei
ZENKER’S
▪ Poor penetration
1 Bring down to water FLUID
▪ Wash thoroughly in running H20
Place in mixture of Acetone, H2O2 , 28%
Lilie’s 2 ▪ Recommended for trichrome staining
Ammonia water for 1-5 mins
Method ▪ With glacial acetic acid added before use
3 Wash with 70% ETOH
▪ Excellent micro anatomic fixative for
4 Wash with water ZENKER-
pituitary, BM, spleen, liver;
FORMOL/
1 Bring down to water ▪ Brown pigment produced if fixed for more
HELLY’S
Picric Acid Place in saturated alcoholic picric acid for than 24 hours due to lysis of RBC– remove by
2 SOLUTION
Method 5mins-2hrs saturated picric acid or NaOH
3 Wash in running water for 10-15mins

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▪ HgCl2 , NaCl, TCA, HCHO BRASIL’S

▪ For skin biopsies; place in high grade ROH ALCOHOLIC ▪ Good for glycogen; better
HEIDENHAIN’ 24 hours
▪ Excellent cytological fixative PICROFORMO & less messy than Bouin
S SUSA
▪ Black precipitate produced – immerse in L (W/TCA)
alcoholic iodine ▪ For GIT specimens and
SCHAUDINN’S endocrine tissues;
HOLLANDE’S
FLUID/SUBLI ▪ HgCl2 + ETOH produces less lysis than 4-18 hours
FIXATIVE
MATED ▪ For making smears of loose cells on slides Bouin; Has decalcifying
ALCOHOL properties
▪ HgCl2 + anhydrous NaAc + HCHO
B-5 FIXATIVE
▪ For hematopoietic and lymphoid tissue
IV. ALCOHOLIC FIXATIVE
▪ Acts as both fixative and dehydrating agent
CHROMATE FIXATIVE
 Dehydrating agent - ETHANOL
1-2% CHROMIC ▪ Preserves CHO
▪ Rapidly denatures and precipitates proteins
ACID
POTASSIUM ▪ K2Cr2O7
TYPE DESCRIPTION FIXATION TIME
DICHROMATE ▪ Preserves lipids, mitochondria
▪ Slow, for BM/blood
▪ 3% K2Cr2O7 + HCHO METHANOL 6-24 hours
REGAUD’S/ smears
▪ For chromatin, mitochondria, Golgi, RBC,
MOLLER’S ETHANOL ▪ Strong reducing agent 18-24 hours
colloid, mitotic figures; slow
FLUID ▪ Absolute ROH, CHCl3,
▪ Not for fats CARNOY’ S
▪ 2.5% K2Cr2O7 + HCHO gHAC 1-3 hours
FLUID
▪ For Rickettsia, bacteria, myelin ▪ Most rapid; RBC hemolysis
ORTH’S FLUID ALCOHOLIC
▪ Recommended for early degenerative ▪ ETOH, HCHO, glacial acetic
process and tissue necrosis FORMALIN/
acid 4-18 hours
GENDRE’S
▪ Sputum
FIXATIVE
LEAD FIXATIVES
▪ Isopropyl ROH, propionic
▪ For acid MPS
NEWCOMER’ acid, petroleum ether,
▪ Fixes connective tissue mucin 12-18 hours
S FLUID acetone, dioxane
▪ Forms insoluble lead carbonate – remove by filtering or
▪ For MPS
adding HAc
▪ ETOH + gHAC
CLARKE’S
▪ For frozen section and 3-4 hours
SOLUTION
smear
METALLIC FIXATIVE FIXATION TIME
Zenker’s Fluid 12-24 hours
Zenker-Formol/ Helly’s
12-24 hours V. OSMIUM TETROXIDE (OSMIC ACID) FIXATIVES
Solution
Heidenhain’s Susa 3-12 hours ▪ Fixes fats, for EM
Schaudinn’s Fixative 30 minutes-24 hours ▪ Expensive, poor penetration, reduced w/ sunlight → black
B-5 Fixative 4-8 hours deposit; dark amber bottle
Chromic Acid 24-48 hours ▪ Acid vapor → conjunctivitis, osmic oxide in cornea → blindness
Potassium Dichromate 24-48 hours ▪ Inhibits hematoxylin - Extremely volatile
Regaud’s/ Moller’s Fluid 12-48 hours  Hematoxylin – most common stain in histopath
Orth’s Fluid 36-72 hours
TYPE DESCRIPTION FIXATION TIME
▪ Aq. Chromic Acid, Aq.
III. PICRIC ACID FIXATIVE FLEMMING’S
Osmium tetroxide, Glacial 24-48 hours
SOLUTION
▪ Normally used in strong aqueous solution (1%) Hac – nuclear fixative
▪ Yellow in color FLEMMING’S
 Intense yellow color → golden yellow SOLUTION
24-48 hours
 Remove color by dipping 70% ETOH followed by 5% WITHOUT
▪ Cytoplasmic fixative
sodium thiosulfate & running water ACETIC ACID
▪ Highly explosive when dry CHAMPY’S
24-48 hours
▪ Excellent for glycogen demonstration FLUID

TYPE DESCRIPTION FIXATION TIME

▪ For embyros, glycogen,
BOUIN’ S does not need washing
(PICRIC, out; poor penetration, not
6-24 hours
HCHO, good for kidneys,
GLACIAL) mitochondria, hemolyzes
RBC

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VI. GLACIAL ACETIC ACID DECALCIFICATION

▪ Solidifies at 17ºC → Glacial ▪ Process of removing Calcium or Lime
▪ Glacial acetic acid alone is not a good fixative, it must be Salts from tissues
combined with another fixative ▪ Performed on bones, teeth, calcified
▪ For nucleoproteins, chromosomes tissues (e.g. tuberculous lungs,
▪ Contraindicated in cytoplasmic fixatives → destroys arteriosclerotic vessels)
mitochondria & golgi ▪ Prevents poor cutting of hard
tissues/knife damage because calcified
VII. TRICHLOROACETIC ACID (TCA) sample are harder than normal tissue samples
▪ Incorporated into compound fixatives ▪ If tissue size is very large – use saw (fine-fret)
▪ Weak decalcifying agent ▪ Decalcifying agent should be changed regularly
▪ Poor penetration ▪ “GRATING” sensation during cutting = place block in 10 %
HCl for 1 hour
▪ Rapid decalcification – produces effect on nuclear staining –
VIII. ACETONE
(failure of nuclear chromatin to take up hematoxylin)
▪ Use at ice cold temp (-5ºC to 4ºC) ▪ Not a routine step, because not all specimen will go though
▪ Fixes brain – for rabies decalcification
▪ For enzyme studies
▪ Dissolves fat, evaporates rapidly, preserves glycogen poorly DECALCIFYING AGENT
HNO3 , HCl, Formic, TCA, Sulfurous,
ACIDS Chromic, Citric
OTHER TYPES OF FIXATIONS Most common
1 Heat fixation CHELATING AGENTS EDTA (Versene)
2 Microwave fixation (Ammonium form of polystrene resin) –
ION EXCHANGE
Secondary fixation 1 – 14 days – spread on bottom of
3 RESIN
▪ Process of placing a fixed tissue in a second fixative container
Post-Chromatization (Electrophoresis) – attraction of Ca to
ELECTRICAL
4 ▪ Form of secondary fixation which utilizes 2.5% to 3% IONIZATION
negative electrode
potassium dichromate as mordant Method of separation
Washing Out – done if the sample is left in a fixative for a
longer period of time suggested; removal of excess fixative
5 ▪ Tap Water – for chromates, formalin, osmic acid I. ACIDS
▪ 50-70% Alcohol – for picric acid ▪ Most common
▪ Alcoholic Iodine – for mercuric fixatives ▪ Stable
▪ Easily available
FACTORS THAT AFFECT FIXATION ▪ Cheap
▪ Examples: Nitric, Hydrochloric, Formic, TCA, Sulfurous,
RETARDED BY ENHANCED BY Chromic, Citric Acids
Size & Thickness
Mucus Size & Thinness
Blood Agitation NITRIC ACID
Fatty tissues Moderate heat (37-56 ºC) ▪ Most common
Cold temperature ▪ Fastest
▪ Inhibits nuclear stain (nitric acid alone)– prevented by
combining with formaldehyde or alcohol
METHODS RESORTED TO IF CHEMICAL FIXATION IS TO BE ▪ Not compatible for nuclear stains
AVOIDED
[

FREEZE-DRYING NITRIC ACID BASED USE FIXATION TIME

▪ Preserving tissue by rapid freezing (quenching) and 10% Aqueous Nitric Urgent biopsy,
12-24 hours
1 removing water (dessication) by a physical process Acid Needle Biopsy
from the still frozen tissue block without the use of Formol–Nitric Acid Nuclear staining 1-3 days
any chemical fixative Nuclear and
FREEZE SUBSTITUTION Perenyi’s Fluid Cytoplasmic 2-7 days
▪ The frozen tissue is fixed in Rossman’s fluid or staining
2 Osmium tetroxide in 1% acetone for 1-6 days at Most Rapid
Phloroglucin–Nitric
temperature of -60° c to -70° c and dehydrated in Poor nuclear 12-24 hours
Acid
70% absolute alcohol staining
3 FRESH FROZEN TISSUE SECTIONING

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NITRIC ACID BASED DECALCIFYING AGENTS MISCELLANEOUS ACIDS

▪ Diluted nitric acid ▪ TCA + 10% Formol Saline
▪ 10mL Conc. Nitric acid + 100 mL dH2O TRICHLOROACETIC ▪ 4-8 days – very slow acting
▪ Rapid, with minimal tissue distortion (if ACID (TCA) ▪ Permits good nuclear staining
10% AQUEOUS
prolonged) ▪ Weak decalcifying agent
NITRIC ACID
▪ Recommended for urgent biopsy, needle ▪ Very weak decalcifying agent
SULFUROUS ACID
biopsy ▪ Suitable only for minute pieces of bone
▪ Yellow color imparted ▪ Chromic acid + Osmium tetroxide +
▪ Conc. Nitric Acid + 40% Formalin + dH2O glacial HAc
▪ Rapid acting ▪ Fixative and decalcifying agent
▪ Good nuclear staining ▪ Nuclear staining with hematoxylin is
 Presence of formalin prevents the CHROMICACID inhibited
FORMOL–NITRIC inhibition of nuclear staining (FLEMMING’S  However, hematoxylin is most
ACID ▪ Less tissue destruction than 10% Aqueous FLUID) important stain in histopathology
nitric acid ▪ Forms precipitate at the bottom
▪ Use fume hood  Can be inhibited by frequently
▪ Lessen yellow tissue discoloration by 5% changing of solution
sodium sulfate or 0.1 % urea ▪ Carcinogenic, corrosive to skin
▪ 10% Nitric acid + 0.5% Chromic acid + ▪ 7% Citric Acid + 7.4% Ammonium Citrate
absolute ETOH + 1% Zinc sulfate + Chloroform
▪ Decalcifies and softens CITRIC ACID– (preservative)
PERENYI’S FLUID ▪ Good nuclear and cytoplasmic staining CITRATE BUFFER ▪ 6 days – slow
▪ Maceration avoided by chromic/ethyl ▪ Permits good nuclear and cytoplasmic
▪ Disadvantages: slow, difficult to assess staining
complete decalcification by chemical means
▪ Conc. Nitric + Phloroglucin → dense white
fumes are formed, add 10% Nitric acid after II. CHELATING AGENTS
disappearance ▪ Chelating agents are mostly alternatives
PHLOROGLUCIN– ▪ Most rapid ▪ Substances which combine with Calcium ions and other salts
NITRIC ACID ▪ Poor nuclear staining (Fe, Mg)
▪ EDTA (Versene, Sequetrene) – most common chelating agent,
when decalcification is complete, acid must be will not bind Ca at pH below 3.0
removed by 3 changes of 70 to 90% ethanol
▪ Permits excellent staining
▪ Very slow – 1-3 weeks
HYDROCHLORIC ACID ▪ Slight tissue hardening produced
▪ Inferior compared to Nitric Acid as decalcifying agent ▪ EDTA inactivates alkaline phosphatase – add magnesium
▪ Slower action, greater tissue distortion chloride
▪ Good nuclear staining
▪ Recommended for surface decalcification
HLC BASED DECALCIFYING AGENTS III. ION EXCHANGE RESINS
▪ NaCl, HCl, H20 ▪ Ammonia form of Polystrene Resin
VON EBNER’S ▪ Good cytologic staining ▪ Hastens decalcification by removing Ca ions
FLUID ▪ Recommended for teeth and small pieces of from Formic acid-containing decalcifying
bones solutions
▪ Artifacts produced, usually caused by CO2
FORMIC ACID bubbles
▪ Better nuclear staining with less tissue distortion ▪ Slow – 1-14 days
▪ Safer to handle than nitric acid and HCL ▪ Degree of calcification cannot be measured
▪ Recommended for postmortem research tissues upon the by chemical means
addition of sodium citrate
FORMIC ACID BASED DECALCIFYING AGENTS
▪ NaCl, HCl, H20 IV. ELECTROPHORESIS (ELECTIRAL IONIZATION)
▪ Good cytologic staining ▪ Process whereby positively
10% FORMIC
▪ Recommended for teeth and small pieces of charged Ca ions are
ACID
bones attracted to a negative
▪ Fixation Time: 2-7 days electrode and subsequently
▪ 45% Formic Acid + 20% Na Citrate removed from the
▪ Slow, not recommended for routine decalcifying agent
FORMIC ACID-
purposes ▪ Satisfactory for small bone
SODIUM
▪ Permits better nuclear staining than Nitric fragments
CITRATE
Acid ▪ Uses electricity
SOLUTION
▪ Requires neutralization with 5% Na sulfate ▪ Solutions used for Electrolytic Decalcification: Formic Acid
▪ Fixation Time: 3-14 days 88%, Conc. Hydrochloric Acid, distilled water

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TEST FOR COMPLETENESS OF DECALCIFICATION ▪ Hindi basta basta lulubos sa alcohol, (kailangan several changes
▪ Physical/Mechanical (routine) of alcohol in different concentration usually 3 changes
a. bending of bones; if bones are bendable, increasing concentration)
decalcification has taken place ▪ 10:1 ratio of dehydrating agent and tissue
b. pricking the bones with needle
▪ X–ray/Radiological (standard, very expensive)
▪ Chemical (Ammonium Oxalate)
 BLUE litmus paper + 5mL discarded decalcifying agent
→ RED due to acidity
 Add NH3 drop by drop to neutralize (litmus change to
BLUE )
 If CLOUDY → still with calcium; if CLEAR: add
ammonium oxalate, 30 mins → CLOUDY if incomplete
▪ In fixation/decalcification process, agents are diluted with
WATER, and water is removed during dehydration using
TISSUE SOFTENERS ALCOHOL
1 Perenyi’s Fluid– 12-24 hours ▪ The tissue is then packed with ALCOHOL after dehydration, and
2 4% Aqueous phenol – 1-3 days then alcohol will be removed by the clearing agent which is
3 Molliflex (swollen & soapy appearance) XYLENE (most common)
4 2 % HCl ▪ After clearing the tissue is now packed with XYLENE and will
5 1 % HCl in 70 % alcohol then undergo impregnation and will be put into PARAFFIN
 Paraffin is solid and liquid at 2 different temperature
POST DECALCIFICATION  Liquid at high temp, and solid at cool temp
▪ Remove acid by saturated lithium carbonate solution or 5-10%  Liquid form is used in impregnation
aqueous NaHCO3 for several hours
▪ Use running tap water
▪ If EDTA is used – use 70% alcohol QUALITIES OF AN IDEAL DEHYDRATION SOLUTION
1 Rapid acting w/ no tissue shrinkage or distortion
RATE OF DECALCIFICATION 2 Should not evaporate fast
▪ More concentrated acid solutions – more rapid but more 3 Can dehydrate even fatty tissue
harmful to tissue 4 Non toxic
▪ Heat hastens decalcification, but increases damaging effect of 5 Not fire hazard
acids to tissues 6 Should not harden tissue excessively
▪ Mechanical agitation, sonication
▪ Ideal time: 24-48 hours
DEHYDRATING AGENTS
1 Alcohol (most common)
TISSUE PROCESSING 2 Acetone
3 Dioxane
▪ Designed to remove all extractable water from the tissue, 4 Cellosolve
replacing it with a support medium that provides sufficient
5 Triethyl phosphate
rigidity to enable sectioning of the tissue without damage or
6 Tetrahydrofuran (THF)
distortion.
▪ Preparing the tissue for actual cutting/sectioning
▪ Includes:
✓ Dehydration I. ALCOHOL
✓ Clearing Progressively increasing
✓ Infiltration Prolonged storage in 70% Directly places in high grade
✓ Embedding (tissue must be firm enough to cut) alcohol alcohol
Shrinkage & hardening of
Macerates tissue
tissues
DEHYDRATION
▪ Removal of intercellular and extracellular water from the TYPES
tissue ETHYL Best, fast, mixes with water and penetrates easily,
▪ Make sure that the solution used in dehydration process is not poisonous, cheap, very much available
miscible to the prior set and miscible to the next step METHYL Toxic, for blood films
BUTYL/ Slow, for Plant and Animal microtechnique
BUTANOL Not used in histopathologic techniques

 Oil based in not used in the first few steps because it is

very much immiscible with several solutions II. ACETONE
▪ Increasing concentration of alcohol (alcohol – miscible to
✓ Cheap
water)
✓ Routine – starts with 70% ethyl alcohol ✓ Rapid acting
✓ Embryonic tissues – starts with 30% of ethyl alcohol ✓ Volatile
✓ Tissue shrinkage
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III. DIOXANE Clearing Agent Description

▪ Most common and readily available,
✓ Excellent dehydrating & clearing agent
colorless
✓ Less tissue shrinkage ▪ Used as cleaning agent for microscope
✓ Tissues can be left here for a long time before but can destroy the lenses
 Ribbons poorly XYLENE ▪ Cheap
 Expensive ▪ Milky if incomplete dehydration
 Dangerous – vapor - cumulative ▪ Hardens/Shrinks tissue – not for nervous
system and lymph nodes
GRAUPNER’S METHOD WEISEBERGER’S METHOD ▪ Hard/brittle tissues if left >3 hours
Dioxane, Paraffin Wax Gauze, Ca Oxide ▪ Not carcinogenic: but will emit fumes that
are toxic upon prolonged exposure
▪ Rapid acting
IV. CELLOSOLVE TOLUENE ▪ Tissues do not become excessively hard and
✓ Ethylene Glycol Monoethyl Ether brittle even if left here for 24 hours
▪ Slower than xylene & benzene
✓ Dehydrates rapidly
▪ Expensive
✓ Storage without producing hardening or distortion
▪ Rapid acting
▪ Volatilizes rapidly in paraffin oven
V. TRIETHYL PHOSPHATE BENZENE ▪ Tissue shrinkage if left for a long time
▪ Carcinogenic; may damage bone marrow
✓ Minimum shrinkage resulting to aplastic anemia
✓ Minimum distortion and hardening of tissue ▪ For tough tissues – decalcified, nervous,
✓ Used to dehydrate sections and smears following certain lymph nodes, embyros
stains ▪ Minimum shrinkage and hardening
CHLOROFORM ▪ For large specimens
▪ Toxic to liver (prolonged inhalation)
VI. TETRAHYDROFURAN (THF) ▪ Does not make tissues transparent
✓ Dehydrates and clears ▪ Tissues tend to float
✓ Easier cutting w/ fewer artifact ▪ Very penetrating
Non toxic ▪ Extremely slow
✓ ▪ For CNS, smooth muscles, skin, cytology
6 months exposure – conjunctival irritation
CEDARWOOD ▪ Milky on prolonged storage (filter)
✓ Offensive odor (use well ventilated room)
OIL ▪ May produce crystals – heat to 200 C
▪ Very expensive
ADDITIVE TO DEHYDRATING AGENTS ▪ Maybe used for paraffin and celloidin
4 % phenol added to softens hard tissues sections
95 % ethanol ▪ For clearing embryos, insects and delicate
Hard tissues Immerse in Glycerol/alcohol ▪ specimens due to its ability to clear 70%
ANILINE OIL
mixture or in Molliflex ▪ alcohol without excessive tissue shrinkage
and hardening
▪ Causes minimum shrinkage of tissues
CLEARING/DEALCOHOLIZATION ▪ Wax impregnation here – slow / difficult
CLOVE OIL
▪ Tissues become brittle
▪ Removal of dehydrating solution, making the tissue ▪ Expensive
components receptive to infiltrating medium CARBON ▪ Properties and disadvantages are like
▪ After staining – transparent tissues TETRACHLORIDE chloroform but cheaper
▪ Improve refractive index of the tissue (CCL4 ) ▪ Hepatotoxic
TETRA- ▪ Dehydrating and clearing agent
CLEARING AGENTS HYDROFURAN ▪ Non toxic but with offensive odor and
1. Xylene 7. Clove oil (THF) should be used in a well-ventilated room
2. Toluene 8. Carbon tetrachloride METHYL
3. Benzene 9. Methyl benzoate BENZOATE/ Slow acting – used for double embedding
4. Chloroform 10. Methyl salicylate METHYL
5. Cedarwood oil 11. THF SALICYLATE
6. Aniline oil

CHARACTERISTICS OF GOOD CLEARING AGENTS

✓ Miscible with alcohol and paraffin &/or mounting medium
✓ Should not produce excessive tissue shrinkage and
hardening
✓ Should not evaporate quickly in a water bath
✓ Should make tissue transparent

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IMPREGNTION / INFILTRATION NITROCELLULOSE METHOD

▪ Permeating the tissue with a support medium ▪ LVN – low viscosity nitrocellulose
▪ Clearing medium is completely removed from the tissue and ▪ A form of celloidin but more expensive
replaced by a medium that will completely fill all the tissue ▪ Forms harder tissue block; makes thinner sections
cavities ▪ When dry, striking or dropping the container will cause
explosion.
TYPES OF TISSUE IMPREGNATION/EMBEDDING
A. Manual – 4 changes of wax, 15 mins. Interval
B. Automatic – Autotechnicon, Elliot Bench GELATIN IMPREGNATION
Paraffin
type
Wax ▪ Rarely used – when dehydration is avoided; for histochemical
C. Vacuum – fastest, negative atmospheric
and enzyme studies
pressure, gives the fastest results
▪ Does not require dehydration & clearing
Celloidin A. Wet – bones, teeth, large brain section
▪ Embedding medium for delicate specimen and frozen
(Collodion) B. Dry – whole eye sections
sections
Gelatin ▪ Low melting point

Plastic
EMBEDDING
▪ Casting /Blocking
PARAFFIN WAX IMPREGNATION ▪ Place tissue in a precisely arranged position in a mold with a
✓ Simplest, most common, best medium which is then allowed to solidify (orientation)
✓ Meting point: 54-58 ºC at 20-24 ºC ▪ Orientation – process by which tissue is arranged in precise
✓ Ease in cutting positions in the mold during embedding, on the microtome
✓ Permanent paraffin blocks before cutting, and on the slide before staining
✓ Good staining result
✓ Not recommended for fatty tissues BLOCKING OUT MOLDS
✓ Overheated paraffin → brittle specimen ✓ Leuckhart’s Embedding Mold
✓ Inadequate impregnation → clearing agent retained ✓ Compound Embedding Unit
✓ Plastic Embedding Rings & Base Mold
SUBSTITUTE FOR PARAFFIN WAX  Tissue Tek – machine w/ warm plate
▪ More elastic and resilient (56-57 ºC) ✓ Disposable Embedding Molds
 Peel Away
Types  Plastic Ice Trays
PARAPLAST Embeddol Less brittle (56-57 ºC)  Paper boats
Bioloid For eyes  TIMS tissue processing and embedding system
Tissue Mat With rubber
ESTER ▪ Low melting point (46-48ºC) DOUBLE EMBEDDING
▪ Harder than paraffin; water insoluble, can be
used for impregnation without prior clearing ✓ 2% Celloidin for 3 days and subsequent paraffin
CARBOWAX ▪ Water soluble (no dehydration or clearing) ✓ For large blocks of dense firm tissues – brain
▪ For enzyme histochemistry ✓ Obsolete – due to paraffin waxes with different types of
▪ Hygroscopic resins

PLASTIC (RESIN) EMBEDDING

CELLOIDIN ✓ Epoxy, polyester, acrylic
✓ For hard tissues, renal and bone marrow biopsies
✓Permits cutting of thicker tissue section
✓ For ultrathin sections requiring minimal distortion
✓Crumbling of tissues avoided
✓ With VCD – vinylcyclohexane dioxide-carcinogenic
✓Very slow
✓Thin sections difficult to cut
✓Serial sections difficult to prepare
✓Photo micrographs difficult to obtain TRIMMING
✓Very volatile ▪ Process of removing excess wax from the block to expose the
Suitable for specimens with large hallow cavities which tissue surface in preparation for actual cutting
✓
tend to collapse ▪ Sides, top and bottom of tissue block are trimmed
PARAFFIN BLOCKS CELLOIDIN BLOCKS
WET CELLOIDIN METHODS DRY CELLOIDIN METHOD Require chilling in ice cold Do not require chilling or
Recommended for bones, Preferred for whole eye water or refrigerator refrigeration
teeth, large brain sections and section
whole organs
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MICROTOME ANGLES
Microtome Inventor Description Purpose Angle formed between the cutting edge,
BEVEL ANGLE
Serial sections of normally 27°to 32°
Paldwell
large paraffin Angle formed by the sides of the wedge
ROCKING Trefall Simplest WEDGE ANGLE
embedded sections, knives, normally 14° to 15°
1881
10-12u Angle formed between the cutting facet
CLEARANCE
George presenting to the block and the surface of the
Most Celloidin embedded ANGLE
SLIDING Adams block, normally 5° to 15°
Dangerous sections
1789
ROTARY Charles
Sedgwick Most Paraffin embedded HONING
Minot common sections
▪ Heel to toe
1885
▪ Sharpening
FREEZING John
For Frozen Unembedded frozen
Quekett
Section section, 4u TYPES OF HONING
1881
ULTRATHIN Marfred COARSE HONING Gross nicks
Sections for EM, HONING PROPER Even edge acquired
von
Ardenne
0.5u HONE –
Belgium Yellow Best result
CARBORUNDUM
Arkansas More polishing
– 8” x 3”
Fine Much coarser
ROCKING MICROTOME ROTARY MICROSCOPE Carborundum For badly nicked
Belgium Black
or Belgium Blue
Green

COMMON LUBRICANT USED FOR HONING

1 Mineral oil
SLIDING MICROTOME ULTRATHIN MICROTOME 2 Clove oil
3 Xylene
4 Liquid paraffin
5 Soapy water

STROPPING
▪ Toe to heel
▪ Polishing
▪ Burrs formed during honing is
MICROTOME KNIVES removed
Used for trimming and actual section-cutting ▪ Cutting edge of knife is polished
PLANE Celloidin ▪ One side is flat, the other is / sharpened
CONCAVE embedded, concave ▪ Paddlestrop –made of horse leather
sliding ▪ Less concave – celloidin ▪ 40-120 double stokes
embedded tissue using sliding ▪ Treated with vegetable oil – Castor Oil
microtome ▪ Should never be treated with Mineral oil
▪ More concave – paraffin ▪ Should not be used 24-48 hours after treatment
embedded tissue using rotary ▪ Wax should not come in contact with the strop
and rocking microtome
BICONCAVE Paraffin ▪ Both sides concave
embedded, ▪ For paraffin embedded
rotary sections using rotary
microtome microtome
PLANE Frozen ▪ Both sides straight
WEDGE sections or ▪ For frozen sections, extremely
hard hard and tough specimen in
specimens in paraffin blocks using base-
paraffin sledge microtome
blocks with
base
sledge/sliding
DIAMOND For ultrathin
EDGE sections
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FAULTS REASON REMEDY

Prolonged fixation
Prolonged dehydration
Tissue may be softened by soaking in a small
Prolonged clearing
Brittle or hard tissues dish or bowl containing water with detergent,
Prolonged paraffin infiltration
phenol or Molliflex
Overheated paraffin infiltration
Drying out of tissue before actual fixation
Clearing agent turns milky as soon as tissue Water is not completely removes due to Repeat dehydration with absolute alcohol then
is placed in it incomplete dehydration clear again
Block is trimmed down nearest to the tissue.
Remaining wax is melted in embedding oven
On trimming, tissue smells of clearing Clearing agent not completely removes due to
and paraffin impregnation is repeated,
agent insufficient impregnation
changing the paraffin at least once before
blocking
Repeat clearing; if object has already been
Tissue is opaque, section cutting is difficult
Insufficient clearing embedded, prolong clearing up to 12 hours,
due to presence of alcohol
then re-embed
Tissue shrinks away from wax when Insufficient dehydration therefore incomplete
Repeat the whole process
trimmed clearing and impregnation
Tissue block is soft when block is trimmed Incomplete fixation Repeat the whole process
Air holes found on tissue during trimming Incomplete impregnation Repeat impregnation
Contaminated wax
On trimming, wax appears crystalline Re-embed in freshly filtered wax
Block not cooled rapidly enough
Paraffin block, after cooling is moist and
Insufficient paraffin impregnation Repeat paraffin impregnation, then re-embed
crumbles
Surface and edges of the block is not parallel to
Re-trim the block
the knife
Horizontal surface of the block is not parallel to
Re-adjust and re-orient the block
the block
Section fails to form ribbons Coat horizontal edges of the block with wax of
Paraffin wax is too hard
lower melting point
Knife is tilted too much Reduce the tilt
Sections are too thick Readjust the thickness of the loop
Knife is dull Hone and strop
Knife is blunt Sharpen the knife
Sections roll up on cutting Tilt of knife is too great Reduce the tilt
Knife edge is dirty Clean the knife edge
Blunt of dull spot on the knife producing an Adjust the knife so that knife edge will present
irregular knife edge a uniformly sharp edge to the block or sharpen
Ribbon is curved, crooked or uneven Edged of the block are not parallel but round
Re-trim the block
instead of straight or wedge shape
Knife is not parallel to the block Readjust the knife and block
Paraffin is impure
Knife is blunt or dull Re-sharpen the knife
Paraffin block is warm and soft Cool the block on ice water until firm
Sections are compressed, wrinkled, or Knife edge is coated with paraffin Clean the knife edge
jammed Sections are too thin Readjust thickness of section
Microtome set screw is loose Tighten the screw
Tilt of knife is too vertical Reduce the tilt
Sections are squashed (width of each Bevel of knife is lost due to incorrect Re-sharpen, using a knife back or automatic
section is less than that of block) sharpening knife sharpener
Bubble or dirt formed in the embedding
Re-embed in freshly filtered wax if necessary
medium
A hole is formed in the section
Once embedded in paraffin wax, decalcification
Hard spot on tissue due to calcium
is impractical’ use a base sledge
Tilt of knife is too great or bevel is not cleared,
hence object is compressed against the knife Reduce tilt
Sections of unequal thickness are produced edge
Clamp set screw knife or block holder is loose Tighten the screw
Blocks are too large Cut blocks into smaller fragments
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Blocks are too hard Soften the block s in detergent or alcohol

Tilt of knife is too small, paraffin block is
Resistance is felt on the lower part of the
therefore compressed against the base of the Increase the tilt
section during cutting
knife towards the end of the stroke
Horizontal or parallel lines or furrows Treat with phenol during processing or
Knife edge vibrates due to hardness of tissue
across the section (chatters) are seen, collodionize
forming thin and thick zones Tilt of knife is too great Reduce the tilt
Knife is blunt Sharpen knife
Knife is not clamped properly Adjust the knife
Section cut is sometimes thin, sometimes Tilt of knife is too great Reduce the tilt
thick Knife or block holder is loose Tighten adjusting and locking screws
Knife tilt is too small that block is compressed
Increase the tilt
by bevel and section is not cut
Tilt of knife is too slanted of too big Readjust the angulation of the knife
Knife makes a hard metallic scraping or
Take a fresh block treated with phenol during
ringing sound on backstroke when section Tissue is too hard
processing
is cut
Knife blade is too thin Change the knife
Frozen tissue crumbles and comes off the
Freezing is not adequate Refreeze the tissue block
block holder when cut
Frozen tissue chips into fragments when
Tissue is frozen too hard Warm the tissue with fingers
cut

SECTIONING DRIED ALBUMIN

FISHING OUT Dried Albumin 5g

▪ After cutting, tissue sections are floated out on a water bath NaCl 5g
▪ When the sections are flattened out, slide is immersed in Distilled water 100 mL
water bath and tissue is fished out Thymol
▪ Adhesives are used to promote adhesion of sections
1% GELATIN
CHARACTERISTICS OF A RIBBON Mix with water in float out bath
✓ Thin Gelatin 1g
✓ Transparent Distilled water 100 mL
✓ Without irregularities Glycerol 15 mL
✓ Not wrinkled Phenol crystal 2g
✓ Uniform thickness
✓ Can easily be separated from each other STARCH PASTE
✓ Continuous
Powdered starch 1g
METHODS TO FLATTEN RIBBON Distilled water (30 mL) 10 ml cold
✓ 50% Alcohol 20 ml boiling
✓ Flout out bath - 45-50°C Thymol
✓ Distilled water in hot plate
DEPARAFFINIZATION
ADHESIVES Paraffin surrounding the tissue section is removed to prevent
1 Mayer’s Egg Albumin over staining of tissue
2 Dried Albumin Methods
3 1% Gelatin 1 Alcohol lamp
4 Gelatin-Formaldehyde 2 Xylene bath
5 Starch Paste 3 Hot oven (55-60 c)
6 Plasma (0.5mL)
7 Poly-L-Lysine - IHC
8 APES (3-aminopropylthriethoxysilane) - cytology STAINING
PURPOSE
1 Better microscopic visualization
MAYER’S EGG ALBUMIN
2 Contrast (background)
Egg white 50 mL 3 Differentiation of tissue and cell components
Glycerin 50 mL To prevent drying
Thymol To prevent growth of molds TYPES OF STAINING
HISTOLOGICAL ▪ Tissue constituents are demonstrated
STAINING in section by direct interaction with dye

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▪ Also called as micro-anatomical ALUMINUM HEMATOXYLIN

staining Components Use
HISTOCHEMICAL ▪ Various constituents of the tissue are Hematoxylin, Absolute
EHRLICH’S
STAINING studied through chemical reactions ETOH, Alum, Glycerin, Regressive staining
HEMATOXYLIN
IMMUNO- Dist H2O, GAc
▪ Combination of immunologic and
HISTOCHEMICAL Widely used for
histochemical techniques
STAINING Hematoxylin, Absolute routine nuclear
HARRIS
ETOH, Alum, Dist H2O, staining in Exfoliative
HEMATOXYLIN
TYPES OF STAIN Mercuric Chloride, GAc Cytology and sex
ACCORDING TO SOURCE chromosomes
NATURAL DYES SYNTHETIC (ARTIFICIAL) DYES Hematoxylin, 1%
Hematoxylin Aniline COLE’S Iodine in 95% ETOH, Recommended for
Cochineal Dyes Coal Tar Dyes HEMATOXYLIN Sat Aq Ammonium routine purposes
Orcein Alum, Dist H2O
Saffron Hematoxylin, NaIO3 ,
ACCORSING TO EFFECT MAYER’S Alum, Citric Acid, Recommended for
Acts on tissue but first treated with HEMATOXYLIN Chloral Hydrate, Dist routine purposes
ADJECTIVE H2O
another substance
Acts immediately, directly on the Others: Lillie’s Hematoxylin, Delafield Hematoxylin
SUBSTANCE
object
AMPHOTERIC Acid and basic group IRON HEMATOXYLIN
Components Use
Hematoxylin, Hematoxylin,
WEIGERT’S
SYNTHETIC DYES (PROPERTIES) Absolute ETOH, Absolute ETOH,
HEMATOXYLIN
CHROMOPHORE The COLORING property FeCl, HCl, Dist H2O FeCl, HCl, Dist H2O
AUXOCHROME The DYEING property Hematoxylin,95% Hematoxylin,95%
HEIDENHAIN’S ETOH, Fe ETOH, Fe
HEMATOXYLIN Ammonium Sulfate, Ammonium
NATURAL DYES Dist H2O Sulfate, Dist H2O
DYES SOURCE PHOSPHOTUNGSTIC Demonstration of
Hematoxylin,
Logwood ACID structures in
Hematoxylin Phosphotungstic
(Hematoxylin campechianum) HEMATOXYLIN paraffin, celloidin,
Acid, Dist H2O
Female Cochineal bugs (PTAH) frozen sections
Cochineal Dyes & Derivatives
(Coccus cacti)
Lichen ALUM (BLUE SALT LAKES)
Orcein Component Use
(Roccella tinctoria
Saffron EHRLICH’S Na iodate) Regressive staining
Saffron For routine nuclear staining,
(Crocus sativus)
Mercuric cytology, sex chromosome; large
HARRIS’
chloride beaker used – explosion (O2
I. HEMATOXYLIN liberation)
▪ Staining solution most commonly used for routine histologic COLE’S Celestine blue
studies MAYER’ S More vigorous than Ehrlich’ s
▪ Active coloring agent is HEMATIN
 Formed by the oxidation of Hematoxylin in a process FE (BLUE BLACK SALT LAKES)
called “RIPENING” (natural and artificial) Component
▪ Ripening/oxidation of hematoxylin is done by: WEIGERT’ S Ferric Ammonium Chloride
 Natural Ripening - Exposing to sunlight and air (slow) HEIDENHAIN’S
Ferric Am. SO4 (Fe alum)
 Artificial Ripening - Adding strong agents: hydrogen SOLUTION
peroxide, mercuric oxide, potassium permanganate,
sodium perborate, sodium iodate Chromium
▪ Ripened Hematoxylin – rarely used due to low affinity to Copper
tissue
HEMATOXYLIN RIPENING AGENTS
TYPE OF HEMATOXYLIN ACTIVE MORDANT Hematoxylin Ripening Agent
ALUMINUM HEMATOXYLIN
Aluminum/ Potassium Weigert’s
Aluminum Sulfate Ferric Chloride
Hematoxylin
IRON HEMATOXYLIN
Iron Alum/ Ferric Ammonium Heidenhain’s
Sulfate Ferric Ammonium Sulfate
Hematoxylin
Ehrlich’s Hematoxylin
Sodium Iodate
Mayer’s Hematoxylin
Harris Hematoxylin Mercuric Oxide
Cole’s Hematoxylin Alcoholic Iodine Solution

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II. COCHINEAL DYES METHYL GREEN ▪ Chromatin

BISMARCK
▪ From female cochineal bug ▪ Contrast, diphtheria
BROWN
 (Coccus cacti) + alum → carmine
▪ Powerful chromatin and nuclear stain PRUSSIAN BLUE ▪ Use to manufacture paint
▪ With PICRIC ACID – picrocarmine – neuropath ORCEIN ▪ Elastic fibers
▪ With ALCL3 (BEST’S CARMINE) – glycogen ▪ Taenzer unna; dermatologic studies –
fibers
PICRIC ACID ▪ Contrast stain to acid fuchsin –
demonstration of CT
III. ORCEIN
▪ Counterstain to crystal violet, tissue
▪ Vegetable dye from Lichens fixative, decalcifier
▪ Colorless and if treated with ammonia and exposed to air CARMINE + AlCl3 ▪ Glycogen- Best Carmine and Fucin
produces blue or violet colors (Mucicarmine)
▪ For elastic fibers MAYER’S
▪ Litmus – used as indicator CARMALUM ▪ Basic dye and stains acidic structures
SOLUTION
IODINE ▪ Oldest, starch, amyloid, cellulose, starch,
IV. EOSIN carotenes, glycogen
✓ Gram’s iodine
▪ Counterstain after hematoxylin and before methylene blue
✓ Lugol’s iodine – glycogen, amyloid,
2 SHADES corpora amylacea
Bluish (Eosin B) Deeper red color ALCIAN BLUE ▪ Phthalocyanin dye similar to chlorophyll
Yellowish Most commonly used; in both aqueous ▪ Stains acid mps – more specific for ct and
(Eosin Y) and alcoholic solutions (yellow epithelial mucin
fluorescence) NEUTRAL RED ▪ Basic dye – cell granules
CONGO RED ▪ Indicator – stain for axis cylinders in
embryos
5% Aqueous Eosin Y ▪ Krajan’s method – elastic tissues,
Eosin Y 5g amyloid, myelin
Distilled H2O 100mL JANUS GREEN B ▪ Mitochondria (intravital)
Thymol VICTORIA BLUE ▪ Neuroglia (FS)
NIGHT BLUE ▪ Carbol Fuchsin – Acid fast
Eosin (Alcoholic Solution) ACRIDINE RED 3B ▪ Calcium deposits
Eosin Y 1g ACRIDINE ▪ Green fluorescence for DNA and red if
Distilled H2O 20mL ORANGE RNA
95% Alcohol 80mL RHODAMINE B ▪ W/ osmic acid-blood/glands
GAc(optional) 0.5mL -to give a deeper red stain BENZIDINE ▪ Hemoglobin
OSMIUM ▪ Fixative
TETROXIDE ▪ Stains fat
OTHER STAINS SILVER NITRATE ▪ Stains spirochetes, reticulum and other
METHYLENE BLUE ▪ Basic nuclear stain with eosin fiber stain
▪ For plasma cells; sputum, bacterial stain, Silver Nitrate solution 10mL
vital staining of ns, indicator Distilled H2O 90mL
METHYLENE AMMONIA ▪ Used to blue stains
▪ Metachromatic dye WATER Strong Ammonium Hydroxide 2mL
VIOLET
TOLUIDINE BLUE ▪ Nuclear stain Tap Water 98mL
▪ Nissl granules, chromophilic bodies
CRYSTAL VIOLET ▪ If w/ methyl violet & dexterin
▪ Gentian violet OIL SOLUBLE DYES - LYSOCHROMES
ANILINE BLUE ▪ Cytoplasmic counterstain ▪ No auxochrome
BASIC FUCHSIN ▪ Acid fast, mitochondria, smooth muscles ▪ Gives color to lipids because they are more soluble in lipid
▪ Carbol fuchsin medium of the tissues, than in their medium of 70 % alcohol
✓ Coleman’s feulgen reagent ▪ Sudan Black, Sudan IV (Scharlach R), Sudan III
✓ Schiff’s reagent
✓ Mallory’s fuchsin
✓ Aldehyde fuchsin (gomori’s stain)
GOLD SUBLIMATE
VAN GIESON’S ▪ Picric acid, acid fuchsin
▪ Masson stain – for ct ▪ Used for Metallic impregnation – made up of gold chloride
GIEMSA ▪ Blood (brown) and mercuric chloride
CELESTINE BLUE ▪ Good nuclear stain with Alum Mercuric Chloride 0.4g
hematoxylin Distilled H2O 60mL
MALACHITE ▪ Ascaris ova, RBC, spore, decolorizer, 1% Gold Chloride 10mL
GREEN counterstain

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CHIEF SOLVENTS USED FOR STAINS Picric Acid Lissamine Green

1 Water – always used unless otherwise stated Methyl Violet Bismarck Brown
2 Alcohol – ETOH, MTOH; should be Acetone-free Crystal Violet Methylene Blue
Metachromatic
3 Aniline water Cresyl Blue Thionine
Stains
4 Phenol Safranin Toluidine Blue
Basic Fuchsin Azure A, B, C
METHODS OF STAINING
STAINING OF PARAFFIN SECTIONS
▪ Sections are stained with simple of
DIRECT STAINING HEMATOXYLIN & EOSIN STAINING PROCEDURE
aqueous solution of the dye.
▪ Requires mordant and accentuator for REAGENT PURPOSE
INDIRECT STAINING
staining Xylene (2 changes) Deparaffinization
PROGRESSIVE ▪ Tissues are stained in a definite Descending Grades of ROH Hydration of Alcohol
STAINING sequence Hematoxylin Primary Stain
REGRESSIVE ▪ Tissues are first overstained & excess 1% Acid Alcohol Differentiation
STAINING stain is removed or decolorized Ammonia Water Blueing Agent
▪ Stain imparts a different color to the Eosin Counterstain
METACHROMATIC tissue Ascending Grades of ROH Dehydration of Alcohol
STAINING ▪ E.g. Toluidine blue – imparts red- Xylene (2 changes) Clearing
purple to Mast cells
▪ Application of a different stain to MATERIALS FOR STAINING
provide contrast and background to
COUNTERSTAINING
the staining of the structural
components to be demonstrated
▪ Demonstrate the general relationship
of tissues with general differentiation
MICROANATOMICAL
of nucleus and cytoplasm without
STAINING Slide Dish with Metal of Glass
necessarily emphasizing the inclusion Coplin Jar
Staining Rack/ Carrier Staining Rack/ Carrier
bodies
▪ Specific tissue elements are
demonstrated by colorless solutions
METALLIC
of metallic salts which are reduced by
IMPREGNATION
tissues producing an opaque black
deposit on the surface of the tissue Slotted Staining Dish Watch Glass Section Lifter
▪ Stain that can be applied on living
cells without killing them.
▪ Examples:
✓ Neutral Red (the best vital
dye)
VITAL STAINING ✓ Janus Green (mitochondria)
✓ Trypan blue Pipette Screw Cap Vial Staining Rack
✓ Nile blue
✓ Thionine
✓ Toluidine blue
RESTAINING
▪ Non-toxic dye injected into the body
Intravital 1 Xylene – for 24 hours
to selectively stain certain cells or
staining 2 Coverslip is removed using dissecting needle
tissues.
3 Xylene – 30 minutes
Supravital ▪ Stain living cells immediately after
staining removal from the living body 4 0.5% Potassium Permanganate – 5-10 minutes
5 Rinse with tap water
6 5% Oxalic Acid – 5 minutes
Intravital Stains India Ink Carmine 7 Rinse with tap water
Lithium 8 Allow to dry and restain
Supravital Neutral Red Nile Blue
Stains Janus Green Thionine
Trypan Blue Toluidine Blue
Nuclear Stains Neutral Red Methylene blue
Safranin O Toluidine Blue
Carmine Celestine Blue
Hematoxylin
Cytoplasmic Eosin Y Orange G
Stains Eosin B Rose Bengal
Phloxine B Light Green SF

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STAINS AND STAINING SOLUTION

STAINS OF CARBOHYDRATES
STAIN USE RESULTS
PAS (+) → Red or Magenta
Periodic Acid Schiff (PAS)
Nuclei → Blue
Method of choice for glycogen
Nuclei → Blue-black
PAS with Diastase Glycogen → Red
demonstration
Control → Only the nuclei is stained
Nuclei → Blue or grayish blue (Ehlich’s
hematoxylin as counterstain)
Best Carmine For glycogen demonstration
Glycogen → Bright red granules
Mucin, Fibrin → Weak red
Glycogen → Mahogany brown
Langhan’s Iodine Oldest stain (obsolete)
Tissue constituents → Yellow
Fresh Frozen Azure A Glycosaminoglycans → Red-purple
Staining for glycosaminoglycans
Metachromatic Tissue background → Blue
Glycosaminoglycans → Red-purple
Metachromatic Toluidine Blue
Tissue background → Blue
Acid mucin → Blue
Alcian Blue Technique
Nuclei → Red
Combined Alcian Blue-PAS
Acid mucin → Blue
For acid and neutral mucin Nuclei → Pale blue
technique
Nuclei mucin → Magenta
Mucin → Red
Mucarmine Nuclei → Blue
Background → Unstained
Acid mucin → Dark blue
Hale’s Dialyzed Iron Technique
Nuclei → Red
Acid mucopolysaccharide → Black
Fluorescent Acridine Orange Fungi → Greenish red fluorescent
Background → Reddish orange fluorescent

STAINS OF FAT
STAIN USE RESULTS
Lipids → Blue black
Sudan Black B Most sensitive
Nuclei → Red
Lipids (triglycerides) → Red
Sudan IV (Scharlach R)
Nuclei → Blue/black
Fats → Brilliant red
Oil Red O in Dextrin
Nuclei → Blue
Fats → Black
Osmic Acid
Nuclei → Yellow orange

STAINS FOR PROTEIN, ENZYMES AND NUCLEIC ACID

STAIN USE RESULTS
Alkaline Fast-Green Histones and Protamine → Green
Alkaline phosphatase activity → Brownish black
Gomori Calcium
Nuclei → Green
DNA → Red-purple
Feulgen For nuclear DNA
Cytoplasm → Green
DNA → Green or blue green
Methyl Green Pyronin For RNA and DNA RNA → Rose red
Plasma cell cytoplasm → Purple

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STAINS OF CONNECTIVE TISSUE

STAIN USE RESULTS
Gomori’s Silver
For reticulin Reticulin fibers → Black
Impregnation Stain
Collagen (fibrous connective → Pink, deep red
tissue)
Van Geison’s Stain
Nuclei → Brownish black
For collagen Muscle, Cytoplasm, → Yellow
Collagen and mucus → Blue
Masson’s Trichrome Stain Muscle, RBC, Keratin → Red
Nuclei → Blue-black
Mallory’s Aniline Blue, Azocarmine, Krajian’s Aniline Blue

FOR ELASTIC FIBERS FOR TISSUE PIGMENTS AND DEPOSITS

STAIN RESULTS ▪ Perl’s Prussian Blue
Elastic fiber – dark blue, blue HEMOSIDERIN ▪ Gomori’s Prussian Blue
Weigert’s Elastic Tissue Stain ▪ Turnbull’s Blue Reaction
black
Elastic fibers – black HEMOGLOBIN ▪ Benzidine-Nitroprusside Stain
Nuclei – gray to black BILE PIGMENTS ▪ Modified Fouchet’s Technique
Verhoeff’s ▪ Gmelin’s Technique
Collagen – red AND
Cytoplasm – yellow HEMATOIDIN ▪ Stein’s Iodine
Taenzer-Unna Orcein ▪ Gomori’s Aldehyde Fucshin Technique
LIPOFUCSHIN
Gomori’s Aldehyde-Fuschin ▪ Mallory’s Fuschin Stain
Gomori’s Aldehyde-Fuschin ▪ Masson Fontana Technique – also for
MELANIN
Krajian’s Argentaffin granules
▪ Von Kossa’s Silver
CALCIUM
▪ Nitrate Method
MORE MORE STAINS COPPER ▪ Lindquist Modified Rhodanine Technique
FIBRIN ▪ Mallory’s PTAH
AMYLOID ▪ Gram’s iodine
▪ Congo Red STAINS FOR MICROORGANISMS
▪ Methy violet-Crystal violet FOR BACTERIA
MUSCLE & ▪ Modified Gomori’s Trichrome Stain Grams method
BONES Result: Brown and Brenn Nocardia and Actinomyces
✓ Muscle fiber – red Ziehl Neelsen Mycobacterium
✓ Collagen – green Wade-Fite Technique M. leprae , Nocardia
✓ Nuclei – blue to black Auramine – Rhodamine Mycobacteria
▪ Mallory’s PTAH Toluidine Blue and Cresyl
▪ Heidenhain’s Iron Hematoxylin Violet Acetate
H. pylori
▪ Lissamine Fast Red – tartrazine method for
Dieterle Method L. pneumophilia
muscles and bones
Levaditi’s
BONES ▪ Schmorl’s Picro-Thionin Method
Modified Steiner and Steiner Spirochetes
BONE MARROE ▪ Rapid Toluidine-Eosin Stain
Warthin - Starry
AND BLOOD ▪ Romanowsky
ELEMENTS ▪ Wright’s stain
VIRUS Lendrum’s Phloxine-Tartrazine Method – viral
▪ Giemsa stain
inclusion
▪ Peroxidase Reaction – for myeloid cells
Orcein Method – HBsAg
FUNGI Grocott Methamine Silver
CENTRAL NERVOUS SYSTEM PROTOZOA Giemsa
Bielschowsky’s
For neurons, axons and neurofibrils
Technique
Bodian’s Stain Nerve fibers and nerve endings MOUNTING
Siever-Munger ▪ After staining and before examination, sections should be
Neural tissues
Technique mounted with a suitable medium under cover slips .
Cresyl Fast Violet Nissl bodies ▪ To facilitate ease of handling and storage and to prevent
Weigert-Pal Technique Kluver and Barrera damage to the section.
MYELIN SHEATH
Luxol Fast Blue Stain Weil’s Method ▪ The liquid used for mounting slides is known as “Mountant”.
Cajal’s Gold Sublimate Modified PTAH
ASTROCYTES
Modified Holzer’s Method

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QUALITIES OF AN IDEAL MOUNTANT AQUEOUS MOUNTING MEDIUM

The refractive index should be near as possible to that of ▪ It is used in cases where the stain would be decolorized or
1
the glass – 1.518 removed by alcohol and xylene
2 It should not dry quickly ▪ Requires the addition of a bacteriostatic agent such as phenol
3 It should not dissolve out or fade tissue sections to prevent the growth of molds
4 It should not cause shrinkage and distortion of tissues ▪ Once the medium has hardened, a permanent seal can be
It should set hard, thereby producing permanent mounting obtained by ringing the cover glass with clear nail polish
5
of sections ▪ Very useful for mounting sections for
fluorescent microscopy
AN IDEAL MOUNTANT Gum Arabic 50g
1 It should not cause stains to “bleed” Sucrose 50g
APATHY’S
2 It should not crack or appear granular on setting Distilled water 50mL
MEDIUM
3 It should be non-sticky Thymol
4 It should not shrink back from the edges of the cover glass ▪ Remains fluid in storage and hardens by
5 It must be free flowing evaporation
6 It must be free from bubbles ▪ Should be stored in well-stoppered bottle
7 It should be resistant to contamination ▪ Remains fluid in storage therefore, it is more
8 Once set, the mountant should remain stable convenient than glycerin jelly
Gum Arabic 50g
Glycerin 50mL
TYPES OF MOUNTING MEDIA FARRANT’S
Distilled water 50mL
AQUEOUS MOUNTING MEDIA MEDIUM
Sodium Merthiolate 0.025g
▪ Cannot be used for permanent mount ▪ Its disadvantage lies in the fact that it takes
▪ Temporary: last for a week to a month longer time to set. Store in a well stoppered
▪ Used for mounting sections from distilled water bottle
▪ Recommended as a temporary mountant and
FRUCTOSE
AQUEOUS MOUTANT REFRACTIVE INDEX for special techniques
(LEVULOSE)
Water 1.455 Fructose 75g
SYRUP
Apathy’s Medium 1.52 Distilled water 25Ml
Farrant’s Medium (Gum Arabic) 1.43 ▪ An excellent routine mountant for fat stains
Fructose (Levulose) Syrup 1.47 Gelatin 10g
Glycerin Jelly 1.47 Distilled water 60mL
Highman’s Medium 1.52 Glycerin 70mL
Phenol 0.25g
GLYCERIN
RESINOUS MOUNTING MEDIA ▪ The gelatin causes this medium to solidify and it
JELLY
must be melted before use by placing in oven
▪ Semi-permanent
or water bath at 60ºC prior to use.
▪ Used for preparations dehydrated and cleared in xylene or
▪ Do not shake or stir the medium during melting
toluene
as this will produce bubbles that are difficult to
RESINOUS MOUTANT REFRACTIVE INDEX remove
Canada Balsam 1.524 ▪ This medium is recommended for use with
Coverbond 1.530 metachromatic dyes especially methyl violet
DPX (distyrene plasticizer and xylene) 1.532 Gum Arabic 20g
Gurr’s Neutral Mounting Media Sucrose 20g
1.510 HIGHMAN’S
Histoclad 1.540 Potassium acetate 20g
MEDIUM
Permount 1.526 Sodium merthiolate 10mL
Pro-Texx 1.495 Distilled water 40mL
▪ Remains fluid in storage and hardens by
Technicon Resin 1.620
evaporation. Store in a well-stoppered bottle.
UVinert 1.517
XAM 1.520
Clarite 1.544
RESINOUS MOUNTING MEDIA
Eukitt 1.510
▪ Resinous mountants are either natural or synthetic resins
Entellan 1.500
dissolved in solvents such as benzene, toluene or xylene.
▪ A very expensive natural resin
It is important to note also that the clearing agent used must be ▪ Prepared by collecting the resin exuded by
compatible with the mounting medium, or the sections must be Abies balsamea (Balsam fir) and dilute it in the
thoroughly dried prior to mounting solvent e.g., Xylene
CANADA ▪ This may be purchased as a ready made
BALSAM solution of 60% resin by weight in xylene or in
the dry form  If it is obtained in the dry form,
it is made up as a 55 – 70% solution in xylene.
▪ A few chips of marble in the bottom of the
bottle can be seen to help reduce acidity
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▪ It is HSR (Harleco Synthetic Resin) dissolved as COVER GLASSES

a 60% solution by weight in toluene
COVERBOND ▪ Cover glasses are either square or rectangular
▪ It may be purchased as a dry resin and may be
▪ Glass coverslips are used to protect specimens from physical
dissolved in toluene or xylene
damage
▪ DPX is a polysterene resin dissolved as a 20%
▪ They come in different sizes and
solution in xylene
widths
▪ Because this mountant tends to shrink on
DPX ▪ They are either 22mm or 24mm
hardening, it should be added in excess.
wide
Overflow is easily removed with a scalpel after
▪ Comes in four different lengths:
drying is complete
22mm, 30mm, 40mm or 50mm
GURR’S ▪ This is a mixture of coumarone and other ▪ Manufactured to a specified thickness to complement the
NEUTRAL resins as 76% solution in cineol optical specifications of microscope objectives lenses
MOUNTING ▪ This mountant is a rather viscous solution (recommended thickness is indicated on the barrel of the lens
MEDIUM and is normally 0.17 mm)
▪ Clay Adam’s Histoclad is a 60% solution of a ▪ Various thicknesses are available (with some variation between
HISTOCLAD
synthetic resin in toluene manufacturers) and are designated
▪ Fisher’s Permount is a 60% solution of
PERMOUNT
naphthalene polymer in toluene
▪ Pro-Texx from Lerner Laboratories is a
THICKNESS OF COVERGLASS
mounting medium of neutral pH and with an
PRO-TEXX
antioxidant additive to preserve stain quality No. 1 0.13 - 0.17 mm
▪ It is soluble to both toluene and xylene No. 1½ 0.16 - 0.19 mm
▪ This mountant from the Technicon No. 2 0.19 - 0.25 mm
Corporation is a 60% solution of
TECHNICON couramoneindene polymer in benzene and
RESIN xylene in equal parts GLASS SLIDES
▪ It has a tendency to form bubbles on
hardening due to high volatility of benzene.
▪ A patented mounting medium that is non
UVINERT
fluorescent from Gurr
▪ Eukitt is a very fast drying general-purpose
resin-based mounting medium.
▪ It will solidify within 20 minutes.
▪ The specimens must be free of water and
EUKITT
placed first in alcohol and then in xylene prior
to mounting
▪ Eukitt can also be diluted by xylene to adjust it
viscosity
▪ Entellan is a new mounting medium for
coverslippers and is specially suited for
ENTELLAN
commercial automated mounting instruments
that operate with coverslippers
▪ Nail polish is used to seal the sides of the
coverslip when using aqueous mounting media SLIDE SIZE USE
▪ It can also be used directly as a mounting 75 x 25 mm
medium. Ordinary (3x1 inches) Routine microscopy
CLEAR NAIL 1 mm thickness
The specimens must first be dehydrated in
POLISH 75 x 25 mm Routine microscopy
alcohol and can then be directly mounted
(without xylene) in nail polish. Frosted (3x1 inches) which requires
It tends to shrink a lot when making very thick 1 mm thickness labelling; Cytology
mounts. 75 x 50 mm
Macro
(3x2 inches) Geological; Serology
Slides
1 mm thickness
USUAL CAUSE OF MESSY SLIDES 46 x 27 mm
Petrography; For thin
1 Too much mountant Micro Slides (2x1 inches)
sections; Serology
Mountant too thick and has not been spread to the edges 1mm thickness
2 of the cover glass (if the mountant os too thick it can be 76 x 26 mm
For examining living
diluted with either toluene or xylene) 1.5 thickness
Hanging microorganisms such
Concavity:
drop as bacteria and yeast
0.5 mm depth
in hanging drop
18 mm diameter

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GLASS SLIDE SPECIFICATION RINGING

EDGES DESCRIPTION
▪ Fine but sharp edges; for ▪ Process the margins of the coverslip to prevent escapage of
CUT fluid or semi-fluid mounts and evaporation of mountant, to
routine microscopy use
▪ Rough and grainy edges immobilize the coverslip, and to prevent sticking of the slides
▪ Manufactured using a wet grinding process; upon storage
GROUND
▪ Reduces the danger of cuts ▪ The name comes from the way it was
and infections to laboratory personnel originally done, i.e. with circular coverslips and a
turntable.
▪ Not perpendicular to the
BEVELLED
faces of the piece; for easier handling
RINGING MEDIA

TECHNIQUE FOR MOUNTING SECTIONS 1 Varnish

2 Nail polish
Select the appropriately sized coverslip and place on a
1 3 Kronig cement
white paper sheet
4 Durofix
2 Place a drop of mountant in the center of the coverslip
Invert the slide (section face down) over the coverslip and
3 with one end resting on the paper sheet; gradually lower
the other end until the mountant touches the section
Mountant will spread quickly over the section, between
4
slide and coverslip
5 The slide, with coverslip attached, is then turned upright
Any trapped air is gently squeezed out whilst aligning the
6
coverslip
The mountant is allowed to set (time required will depend
7
upon the particular agent used)
If the result is inadequate, slides are returned to the solvent
8 (appropriate to the mountant) to have the coverslip
removed and the process repeated

OTHER TECHNIQUES FOR MOUNTING

Place slide on a level surface,
1 and apply a drop of mountant
using the dispenser rod
Hold the cover slip at a 45°
If you have problems, you'll be needing a FIXATIVE if you're not sure
angle to the surface of the
of what to do, what you need is an EMBEDDING MEDIUM if you're
slide, and allow the bottom
lost, you have to do LABELING if you're sad, you do STAINING to give
edge to touch the drop of
2 more colors to your life and if you're happy, you add
mountant. When the drop has
PRESERVATIVE... That's the histopathologic technique of life! :)
spread along the edge of the
slip, let go of the slip and allow
the mountant to spread slowly

REMOVAL OF EXCESS MOUNTANT

Excess mounting medium may be removed with
WHEN WET
a tissue
Excess mounting medium may be removed with
WHEN DRY
a razor blade
Mountant will dry sufficiently to be read in 30
minutes

LABELLING
▪ After staining and mounting apply a
paper label with:
Name of tissue
Date
Laboratory number
Staining method

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