Histopathologic & Cytologic Techniques

SCHOOL OF NATURAL SCIENCES
Histopathologic and Cytologic

HSTCYT1
Techniques
A Self-regulated Learning Module
A Self-regulated Learning Module 1

SELF-REGULATED LEARNING
MODULE IN HISTOPATHOLOGIC
AND CYTOLOGIC TECHNIQUES
Kathleenjoy Rosalia Katleya Almondia-Gili

Erlinda P. Sanchez
HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES 1ST SEM AY 2021
TABLE OF CONTENTS
CONTENT PAGE
Introduction 2
Study Schedule 6
Lesson 1: Introduciton to Tissue Processing 8
Lesson 2: Fixation 22
Lesson 3: Decalcification 39
Lesson 4: Dehydration 52
Lesson 5: Clearing 58
Lesson 6: Infiltration and Embedding 64
Lesson 7: Sectioning and Adhesion of 82
Tissue Sections
Lesson 8: Staining 98
Lesson 9: Mounting and Labelling 116
Lesson 10: Cell Blocking 124
Lesson 11: Automated and Rapid Tissue 130
Processing
Lesson 12: Immunohistochemistry 141
Lesson 13: Exfoliative Cytology 149
Answers to Self-Assessment Activities 160
Endorsed by:
Teresa N. Villanueva, RMT, MACT

Dean, School of Natural Sciences
CHANCE FAVORS THOSE WHO ARE PREPARED. 1

INTRODUCTION
Course Code: HSTCYT1
Course Title: Histopathologic and Cytologic Techniques
Course Description:
HSTCYT1 is a professional subject with a 2-unit-lecture and 1-unit laboratory class. The
course deals with the of histologic and cytologic techniques essential in the production of tissue
and cytologic slides and smears for the diagnosis of diseases including special procedures and
other related techniques.
Requirements of the Course:

As per Article IX. Section 8 of the 2010 ed UB Student Handbook, the lowest passing
score for board programs is 70% which is equivalent to 75. As such, students should incur at
least 70% out of the total number of points in order to pass the course per grading period.
Therefore, students are enjoined to perform well in the different forms of assessments and
activities to be given during the entirety of this course.
This learning module contains the different topics in histopathologic and cytologic
techniques which are presented comprehensively for your perusal. Each lesson includes
enrichment activities which are to be accomplished individually or in collaboration with your
assigned groupmates. You may submit your individual or group outputs via several ways:
(1)online through our Google classroom if you have a strong internet connectivity, (2)through
Facebook messenger or SMS if you have a weak connectivity, and if you have no connectivity at
all--(3)through courier services if you reside outside Baguio City, or (4) submit it personally at
the security office of the University of Baguio if you reside within Baguio City. In addition to
these, each lesson also includes activities for self-assessment to help you to review and prepare
for the summative assessments and for you to keep track of your progress in understanding the
different topics. Self-assessment activities will not be graded, however, you are required to
compile them as part of your journal which will be passed and evaluated every grading period.
Please take note that summative assessments will be given separately in the lecture and
laboratory class periods. All enrichment activities will be part of the coverage of the summative
assessments in the laboratory. The coverage of the summative assessments in the lecture will
be determined and communicated by your instructor. Periodical examinations will also be given
as part of individual student assessment.
Please follow the following format in accomplishing your reports and case analysis
outputs. For offline learners, you are required to accomplish all tasks individually regardless if it
is an Individual Task or a Group Task. This is to avoid any misunderstandings which may be
brought about by the limited ways by which you may communicate with your other classmates.
Individual and Group Reports/Case analysis:
1. References: Always indicate the reference(s) used for each question, immediately after the
corresponding answer.
a. Book or E-book: Title; Author; Edition; Page Number

b. Internet sources: complete website address (Uniform or Universal Resource

Locator/URL; Date Retrieved).
c. Written reports without references shall NOT be considered.
2. Individual reports shall be accomplished using the spaces provided in this module. Group
reports shall be written or encoded on a short bond paper. Always encode or rewrite
questions first before the corresponding answers.
3. Use short bond paper with margins (1.0 inch left; 0.5 inch to top, right and bottom) for all
drawings.
The following are the rubrics which will be utilized by your instructor in grading your
individual and group outputs.
Rubrics for Individual Outputs:
Criteria 4 3 2 1
Completeness
Output contains Some labels were
-Completeness of all Some labels were Complete non
all necessary data left out and there
required illustrations left out but group drawing of a
required by the was no group
-Proper labelling result is present particular item
activity. result
(4pts)
Content was
Organization
correct, no errors More than 5
-Sequencing (for 1-3 errors were 4-5 errors were
were noted (e.g. errors were
procedural steps) noted noted
correct color of noted
(4pts)
samples / tubes)
• 1 point is given if all Technical aspects required by instructor is met
Technical Aspects • 1 point is given if there is organized arrangement of drawings submitted
(2pts) • No point (0) is given if any error in technical aspect required by instructor
is seen
TOTAL
NB: Total Score may vary depending on the number of required drawings/illustrations or as
deemed applicable by your instructor.
Rubrics for Individual/Group Research:
Criteria 1 2 3 4
Some data were Some data were Output contains all
Complete non
left out and left out or answers necessary data required
Completeness answering of a
there was no were complete but by the activity (ex. Label).
(4pts) particular
discussion of discussion was not The answer was complete
question
answer comprehensive and comprehensive
Output was Answers were Answers were Content was correct,
copied summarized summarized and concise yet important
verbatim; haphazardly; derived from details were seen;
Content
Answers were some answers reliable sources but Answers were derived
(4pts)
copied from were copied a few errors in the from reliable sources;
unreliable from unreliable content of the there is evidence of
sources sources output were seen personal input

Technical • 1 point is given if all Technical aspects required by staff is met

Aspects • 1 point is given if there is organized arrangement of drawings submitted
(2pts) • No point (0) is given if any error in technical aspect required by staff is seen
TOTAL
NB: Total Score may vary depending on the number of questions for research to be answered or
as deemed applicable by your instructor.
In the event when face-to-face classes will be allowed, a major requirement for this
course would be for you to perform manual tissue processing with your assigned groupmates.
At the end of this activity, you will be required to individually submit one paraffin block and one
H and E stained tissue slide together with the following evaluation forms containing the rubrics
which will be utilized to grade your outputs.
EVALUATION FORM
NAME: _____________________________________Class Schedule: ____________________
PARAFFIN (TISSUE/CELL) BLOCK: Proper preparation of paraffin block.
Type of specimen: ___________________________________
CRITERIA 5 4 3 2 1
Tissue block is Tissue block

Tissue block is properly Tissue block is
INCOMPLETELY is NOT
labelled with: properly labelled
--- labelled with --- labelled; or
▪ Student’s complete with the student’s
student’s name with
name; and complete name and
only or with detached
▪ Type of specimen type of specimen.
specimen type only. label.
▪ Tissue is properly
ORIENTED into the
paraffin block (parallel
Tissue Tissue
& centered).
block block
▪ Tissue block is well- Tissue block
meets Tissue block meets meets
trimmed/ have EVEN Tissue block meets does not
three (3) two (2) out of four only one
sides. all four (4) criteria. meet all four
out of (4) criteria. (1) of the
▪ Label is NOT (4) criteria.
four (4) four (4)
protruding from any
criteria. criteria.
of the five (5) sides.
▪ Tissue block FITS into
block holder.
Tissue Tissue Tissue block
▪ Tissue block must Tissue block has NO Tissue block has
block has block has has crack/s
have NO crack/s NOR crack/s NOR air crack/s or air
crack/s crack/s or or air
air space/s in all of its space/s in all of its space/s in two (2)
or air air space/s in
sides. sides. side.
space/s space/s more than

in one on three three (3)

(1) side. (3) sides. sides.
▪ Tissue must:
➢ be with FIRM Tissue Tissue
consistency; block block
Tissue block
➢ NOT smell like meets Tissue block meets meets
Tissue block meets does not
fixative; and three (3) two (2) out of four only one
all four (4) criteria. meet all four
➢ NOT smell like out of (4) criteria. (1) of the
(4) criteria.
clearing agent. four (4) four (4)
▪ Paraffin must not be criteria. criteria.
soft.
Tentative score X 2
Total Score:
HISTOPATHOLOGY SLIDE (H & E - Stained)

Type of specimen: ___________________________________
CRITERIA 3 2 1
Slide is properly
Labelling: Slide is INCOMPLETELY
labelled with the Slide is NOT in
Slide is properly labelled with: labelled with student’s
student’s complete any way
▪ Student’s complete name; and name only or with
name and type of labelled.
▪ Type of specimen specimen type only.
specimen.
Orientation:
▪ Minimum of 2 IDENTICAL tissue Slide does NOT
Slide meets both Slide meets only one
sections are mounted on the slide. meet both
criteria. (1) of the criteria.
▪ Tissue sections are properly criteria.
ORIENTED on the slide (parallel).
Staining:
▪ Cell’s nucleus appears blue/violet Slide does NOT
Slide meets both Slide meets only one
against pink cytoplasm. meet both
criteria. (1) of the criteria.
▪ Slide is thoroughly cleaned of excess criteria.
stain.
Mounting:
Slide does NOT
▪ Coverglass is fixed firm on the slide. Slide meets both Slide meets only one
meet both
▪ No bubbles are superimposed on the criteria. (1) of the criteria.
criteria.
specimen
Tentative score X 2
TOTAL SCORE:

STUDY SCHEDULE
ACTIVITIES
WEEK TOPIC
Lecture Laboratory
Lesson 1: Introduction to *Online Discussion *Activity 1: Glasswares,
Histotechniques *Summative Assessment Equipment and Materials in
1 Histopathology
-Individual Task
*Summative Assessment
Lesson 2: Fixation *Online Discussion *Activity 2a: Fixation
2 *Summative Assessment -Group Task
Lesson 3: Decalcification *Online Discussion *Activity 2a: Decalcification
Lesson 4: Dehydration *Online Discussion *Activity 2a: Dehydration
Lesson 5: Clearing *Online Discussion *Activity 2a: Clearing
FIRST GRADING EXAMINATION
6
Coverage: Lessons 1 to 5
Lesson 6: Infiltration and *Online Discussion *Video Demonstration
Embedding Part I *Summative Assessment *Activity 2a: Manual Tissue
Processing
7
-Individual Task
-Group Task
Lesson 6: Infiltration and *Online Discussion *Video Demonstration
Embedding Part II *Summative Assessment *Activity 2b: Manual Tissue
Processing—Embedding
8
-Individual Task
-Group Task
Lesson 7: Sectioning and *Online Discussion *Video Demonstrations
Adhesion of Sections *Summative Assessment *Activity 3: Trimming and
Cutting of Paraffin Blocks
*Activity 4: Sharpening of
9
Microtome Knives
*Activity 5: Adhesion and
Drying to Tissue Seions onto
the Slide

-Individual Task
-Group Task
Lesson 8: Staining Part I *Online Discussion *Video Demonstration
& II *Summative Assessment *Activity 6: Hematoxyling
and Eosin Staining
10
-Individual Task
-Group Task
Lesson 8: Staining Part III *Online Discussion
11
& IV *Summative Assessment
Lesson 9: Mounting and *Online Discussion *Video Demonstration
Labelling *Summative Assessment *Activity 7: Coverslipping
12 -Individual Task
-Group Task
MIDTERM GRADING EXAMINATION
13
Lesson 10: Cell Blocking *Online Discussion *Video Demonstration
*Summative Assessment *Activity 8: Cell Block
Technique
14
-Individual Task
-Group Task
Lesson 11: Automated *Online Discussion *Activity 9: Automation and
and Rapid Tissue *Summative Assessment Rapid Tissue Processing in
15 Processing HIstopathology
-Individual Task
Lesson 12: *Online Discussion
16
Immunohistochemistry *Summative Assessment
Lesson 13: Exfoliative *Online Discussion *Video Demonstration
Cytology *Summative Assessment *Activity 10: Papanicolaou
Staining Technique
17
-Individual Task
-Group Task
FINAL GRADING EXAMINATION
18

LESSON 1: INTRODUCTION TO TISSUE PROCESSING

Desired Learning Outcomes
At the end of the lesson, you are expected to:
1. know the different methods of tissues examination and select the appropriate method
to be performed depending on the specific tissue sample;
2. discriminate the different specimens handled in the histopathology laboratory;
3. discuss the advantages and disadvantages of fresh and preserved tissue examination;
4. identify and explain the relevance of the initial steps prior to tissue processing; and
5. identify the different equipment, glasswares and materials used in the histopathology
laboratory as well as the corresponding use of each.
Tapping Prior Knowledge

Try to answer the following activity by remembering what you have learned in
your previous subjects. This is a self-assessment activity and will not be graded
but should be compiled as part of your journal to be checked every grading
period. Refer to Appendix A for the answers.
Across Down
3. dye used in fresh tissue preparation & 1. process that makes use of aromatic oils
examination 2. reagent used to preserve tissues
8. cutting embedded tissues into very thin slices 4. reagent for dehydration
9. process of replacing aromatic oil with paraffin 5. performed to provide optical differentiation
10. also known as blocking or casting 6. process of removing water from the tissue sample
7. first step in tissue processing

Lesson Proper
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
SPECIMENS
1. Autopsy Materials: examined to determine the cause of death
2. Surgical Materials: otherwise referred to as surgical or biopsy materials; examined to provide
a diagnosis
A. FINE NEEDLE ASPIRATION: removal of cells from the area of abnormality
➢ considered as the simplest and least invasive method of collecting biopsy
specimens
➢ method of collection for fluid-containing tumors
B. CORE NEEDLE BIOSY: removal of cells and small amount of surrounding tissue
C. INCISIONAL BIOPSY: removal of cells with more surrounding tissue
D. EXCISIONAL BIOPSY: removal of the entire area in question
➢ Ensure complete removal of the lesion
➢ Confirm that the diagnosis is correct
E. PUNCH BIOPSY: removal of 3 to 4 mm cylindrical core of tissue samples
➢ small: 2mm; large: 4mm
➢ lesion should be at the center
F. SHAVE BIOPSY: removal of small fragments of tissue from a surface
G. CURETTINGS: removal of tissue or growths from body cavities
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
FACTORS TO BE CONSIDERED IN CHOOSING A METHOD

1. Structural and chemical components to be studied
2. Nature and amount of sample to be evaluated
3. The need to provide an immediate diagnosis
METHODS OF PREPARATION AND EXAMINATION

A. Diagnostic Laboratories
1. FRESH TISSUE EXAMINATION
➢ No fixative required
➢ Examined using a Brightfield or Phase-Contrast microscope
➢ Stained with supravital or differential dyes

ADVANTAGES
✓ Observation of physiologic processes or protoplasmic activities (motion, mitosis,
phagocytosis and pinocytosis)
✓ Relatively simple and easy to perform
DISADVANTAGES
✓ Limited use
✓ Liable to develop changes observed after death (putrefaction and autolysis)
TEASING ✓ Dissection or separation of tissue components in NSS or Ringer’s solution

✓ Examined as stained or unstained
✓ Anatomical relationship is destroyed
SQUASH ✓ Tissue (<1mm) is sandwiched between two slides
✓ Stain is applied on one side of the slide and allowed to spread via capillary
action
SMEARING
▪ for cytological studies, especially for the diagnosis of cancer
▪ for sections or sediments
▪ performed using a wire loop, applicator stick or another slide
➢ Streaking ✓ Uniform distribution in a direct or zigzag manner
➢ Spreading ✓ Thick or mucoid specimens
✓ Teasing on a slide
✓ Maintains intercellular relationship
➢ Pull-Apart ✓ For the preparation of blood and bone marrow smears
➢ Touch ✓ One side of a slide is allowed to touch a surface of the sample
Preparation ✓ Intercellular relationship is maintained
FROZEN SECTION ✓ Prepared using freezing microtome or cryostat
✓ For rapid diagnosis
✓ For delicate specimens
2. PRESERVED TISSUE EXAMINATION

Initial Steps in Tissue Processing
A. 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
B. Gross Examination and Sampling
➢ Performed by the pathologist
➢ Describing the sample macroscopically
➢ Weight and dimensions of the sample are determined

C. Tissue Processing
➢ Fixation
➢ Dehydration
➢ Clearing
➢ Infiltration
➢ Embedding
➢ Sectioning (+ Floating, Fishing-out, Drying)
➢ Staining
➢ Mounting
➢ Labelling
B. Research Laboratories
1. MICROINCINERATION
✓ Used to locate the presence and position of mineral elements in the tissue
✓ Two duplicate sections of alcohol-fixed tissues
2. AUTORADIOGRAPHY
✓ Injection of radioactive isotopes into organs
Fix→Section→Mount + Photographic Emulsion (Ag Halide)→Stain
✓ Determines the relationship and location of the isotopes and cells to be studied
✓ Provides qualitative and quantitative information

Enrichment Activity
ACTIVITY 1: GLASSWARES, EQUIPMENT and MATERIALS IN HISTOPATHOLOGY
OBJECTIVES
At the end of this activity, you are expected to:
1. acquaint oneself with the apparatuses and equipment used in the study of
histopathologic and cytologic techniques; and
2. know the function(s) and parts of these apparatuses and equipment.
Individual Task
Compile images/draw and indicate the use/s of the following glasswares, materials, and equipment
used in the histopathology laboratory. Label them accordingly.
Glasswares:
Erlenmeyer flask Graduated cylinder
Alcohol lamp Beaker
Coplin jar Storage jars

Test tubes Glass slides (Plain & with frosted end),

Coverslip
Instruments:
Compound Microscope

Clinical Centrifuge Water Bath
Incubator Paraffin Oven

Hot plate Fret saw Surgical blade with

handle
Staining rack Test tube rack

Test tube holder Tissue tong Timer
Other Materials
Applicator stick Camel’s hair brush Gauze cloth

Diamond tipped pen and Scissor Kitchen knife

Leaded pencil
Labeling tape Ruler Chopping board

Slide box
Summing It Up
Histotechniques deals with the preparation, processing and staining of tissue
sections for microscopic study to be interpreted by the pathologist. This requires
medical laboratory scientists to have the skills and knowledge on the methods used
to prepare and process tissue samples for examination.
There are two general types of samples handled in the histopathology laboratory. These
include autopsy materials and surgical or biopsy materials. The purpose of processing these materials
also differs. 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.
Samples submitted to the laboratory are processed using any of the different methods of
tissue preparation and examination. There are three factors which are to be considered in choosing
the method to be performed: (1) structural and chemical components of the cells or tissues, (2)
nature and amount of the tissue sample to be evaluated, and (3) if there is a need to provide an
immediate diagnosis. In diagnostic laboratories, the methods which may be performed include fresh

tissue, preserved tissue and frozen section preparations. In research laboratories, the methods which
may be performed include microincineration and autoradiography.
Fresh tissue preparation and examination permits the observation of physiologic or
protoplasmic processes which include motion, mitosis, phagocytosis and pinocytosis. Since this
method does not involve the use of any fixative, immediate changes brought about by cell death and
tissue degradation are likely to occur. Specifically, the tissue sample undergoes autolysis and
putrefaction, thus, a fresh tissue preparation is not permanent and can only be utilized for a limited
period of time. Techniques under fresh tissue preparation and examination include teasing, crushing,
smear preparation (streaking, spreading, pull-apart and impression smear), and frozen section
preparation.
Preserved tissue preparation and examination involves a series of sequential steps which
have to be performed with utmost care. Prior to the performance of this method, specimen
accessioning and gross examination have to be performed. The routine steps for preserved tissue
preparation and examination are fixation, dehydration, clearing, infiltration, embedding, trimming,
sectioning, staining, mounting and labelling. Decalcification is an additional step performed after
fixation and before dehydration in order to remove mineral deposits from tissue samples. Tissue
slides required for examination by the pathologist have to be of diagnostic quality.
Microincineration is a method that is performed to locate the presence and position of
mineral elements in the tissue. This involves the preparation of two duplicate sections of alcohol-
fixed tissues. One section is stained and the other one is incinerated which will produce an ash
pattern known as spodogram.
Autoradiography involved the injection of radioactive isotopes into organs or tissues. After
which, the organ or tissue is fixed and sections are produced. The sections are then mounted on
slides containing a photographic emulsion (silver halide) and stained. This method is utilized to
determine the relationship and location of the isotopes and cells to be studied.

Assessing Oneself
Accomplish the following activities in order to assess your knowledge and
comprehension regarding the lesson. Once you start answering the activities, do not
go back to the Lesson Proper notes as this would defeat the purpose of this portion
of the module. These activities will not be recorded but you are required to compile
them as part of your journal to be checked every grading period.
Level I: Easy Breezy

Answer the following crossword puzzle.
Across Down
2. method to be performed to remove a uterine 1. equipment used to prepare frozen sections
lesion 3. location of lesion in punch biopsies
5. recommended storage duration for 4. personnel who performs gross examination and
histopathology results sampling
7. method that gives qualitative and quantitative 6. purpose of processing and examining surgical
information materials
9. stain for fresh tissue processing 8. change that occurs after cells are removed from
the body
10. fixative used for microincineration

Level II: Modified True/False

Read and understand the statement and evaluate whether it is true or false. If the statement is true,
write/type “true” on the space provided before the number. If the statement is false, underline the
word/s that make/s the statement incorrect. After which, change or remove the underlined word/s
to make the statement correct. Write/Type the corrected statement after the given statement.
__________ 1. Test tubes are used to view chemical reactions and prepare cell cultures is
histopathology.
__________ 2. Spreading is the recommended technique for the preparation of mucoid samples.
__________ 3. Teasing allows the intercellular relationship of tissue components to be observed.
__________ 4. Tissue blocks should be stored for 5-10 years.
__________ 5. Beakers with fixatives are utilized to hold tissue slides in an upright position during
staining.
__________ 6. Shave biopsy is performed to collect lesions on the inner surface of hollow organs.
__________ 7. Floating and fishing-out are important steps performed prior to mounting with the
use of a paraffin oven.
__________ 8. Smear preparation involves impression prep, streaking, spreading and dissociation.
__________ 9. Leaded pencils are used to label plain glass slides containing tissue sections or smears.
__________ 10. Specimen accessioning performed by the medical technologist involves the
macroscopic evaluation of the submitted sample.
Check the key answers provided in Appendix A of this module. Assess your
performance before the scheduled summative assessments.
Summative Assessment
A. Get ready for summative assessments in the lecture and laboratory to be scheduled by your
instructor.
B. Summative assessments will be given by the instructor via several modalities depending on
your connectivity status.
✓ If you have a strong internet connectivity, you may take the quiz via the online platform
Quizziz.
✓ If you have a weak internet connectivity, you may take the quiz via Facebook
messenger.
✓ If you have no internet connectivity, you may take the quiz via SMS/text message.
References
1. Allen, D., & Cameron, R.I. (2004). Histopathologic specimens: clinical, pathological and
laboratory aspects. USA: Springer
2. Armed Forces Institute of Pathology Laboratory. Methods in histotechnology. Washington
DC: American Registry of Pathology
3. Bruce-Gregorios, J. (2012). Histopathologic techniques (2nd ed.). Philippines: Goodwill
Trading Co., Inc.
4. Raphael, S. (1983). Lynch’s medical laboratory technology (4th ed.). Philadelphia: W.B.
Saunders Co.

LESSON 2: FIXATION
1. explain the importance of tissue fixation as well as the mechanisms involved in this process;
2. apply the different guidelines and precautions observed during tissue fixation;
3. identify the different factors that affect fixation, relate the how these factors affect fixation
and formulate steps or procedures in modifying these factors in order to carry out fixation
efficiently and optimally;
4. assess and recommend what fixative should be used for a given sample and explain why such
fixative is the most appropriate one; and
5. identify the hazards associated with the use of each of the different fixatives and know how
to avoid or mitigate such hazards.

Try to answer the following activity by remembering what you have learned in your
previous subjects. This is a self-assessment activity and will not be graded but should
be compiled as part of your journal to be checked every grading period. Refer to
Appendix A for the answers.
Across Down
5. shape of nucleic acids 1. storage form of carbohydrates in humans
6. examples are stomach, uterus and gallbladder 2. protein fiber that provides tensile strength in
8. portion of the brain that contains the Circle of hard tissues
Willis 3. organelle that contains proteolytic enzymes
9. microscope used in studying ultrastructures 4. colloid-containing gland responsible in
10. soluble in organic solvents and serves as the producing calcitonin
second source of energy 7. biomolecule known as the body's building block

Lesson Proper
Part I: Overview of Fixation
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
Fixative agents
✓ capable of forming cross-links between proteins
➢ stabilizes tissue components making them insoluble to lysosomal enzymes
Types of Fixative:
1. Additive: becomes part of the cross-link itself
2. Non-Additive: facilitates the removal of water in order for cross-links to form
✓ capable of inactivating lysosomes
RETARDED BY:
• Increase in size and thickness • Presence of blood
• Presence of mucus • Decrease in temperature
• Presence of fats
ENHANCED BY:
✓ Decrease in size and thickness
✓ Presence of agitation
✓ Presence of heat
FACTORS TO BE CONSIDERED
✓ pH: 6 to 8 ✓ Osmolality
✓ Temperature: ▪ Light microscopy: slightly
▪ Routine Manual: Room Temp (20 to 22oC) hypertonic (400-450 mOsm)
▪ Routine Automated: 40 oC ▪ Electron Microscopy: more or
▪ Electron Microscopy: 0 to 4oC less isotonic (340 mOsm)
▪ Formalin at 60 oC: very urgent biopsies ✓ Concentration
▪ Formalin at 100 oC: diagnosis of ▪ Formaldehyde: 10%
tuberculosis ▪ Glutaraldehyde: 3%
▪ DNA: 65 oC ✓ Volume
▪ RNA: 45 oC ▪ Routine: 10 to 25 times the
✓ Size and Thickness volume of the specimen
▪ Light Microscopy: 2cm2 by 0.4cm thick ▪ Museum: 50 to 100 times the
▪ Electron Microscopy: 1 to 2 mm2 volume of the specimen
▪ Lung Edema: 1 to 2 cm thick

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

1. Cheap & economical
2. Stable and safe to handle
3. Fast acting, permits rapid an even penetration
4. Inhibits bacterial decomposition & autolysis
5. Must harden tissues
6. At least isotonic
7. Must render tissues insensitive to subsequent processing
8. Must be compatible with many staining procedures
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 paraformaldehyde-
glutaraldehyde solution
2. Acrolein: mixture of glutaraldehyde or fomaldehyde
✓ rapid penetration, preserves morphology and enzyme
activity at low concentrations
✓ immersion fixation of surgical biopsies
CYTOLOGIC
MICROANATOMICAL HISTOCHEMICAL
NUCLEAR CYTOPLASMIC
• 10% Formol saline • Heidenhain’s SuSa • Helly’s • 10% Formol saline
• 10% Neutral buffered • Newcomer’s • Orth’s • Absolute ethanol
formalin • Bouin’s • Regaud’s/Muller’s • Newcomer’s
• Heidenhain’s SuSa • Flemming’s • Flemming’s • Acetone
• Formol sublimate/corrosive • Carnoy’s • Formalin with
• Zenker’s solution post-chroming
• Zenker-formol/Helly’s
solution
• Bouin’s solution
• Brasil’s solution

Summing It Up
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.
The rate at which fixation takes place is an important consideration in this step.
Generally, the rate of fixation should be high in order to adequately fix even the internal
portion of tissue samples. The rate of fixation is retarded by an increase in the size and
thickness of the tissue sample, presence of mucus, fats and/or blood, and a decrease in
temperature. In contrast, the rate of fixation is enhanced by a decrease in the size and
thickness of the tissue sample, presence of agitation and heat.
Factors that affect fixation include the temperature, size and thickness of the tissues
sample, duration of fixation, pH, osmolality, concentration and volume of the fixative. In
addition to these factors, specific organs require additional considerations and preparations in
order for these to be fixed properly and adequately. These organs include air-filled lungs,
hollow organs, brain samples, eyes, hard tissues and muscles.
The fixatives used in histopathology may be classified based on different criteria.
Fixatives may be classified according to composition. Simple fixatives are those that contain
only one active component, while compound fixatives are those that contain two or more
active components. Another basis of classification is according to action. Microanatomical
fixatives are those that preserves all components and thus, permits the general study of
structures and normal intercellular relationship. Cytological fixatives are those that allows a
certain tissue component to be fixed. This type includes nuclear and cytoplasmic fixatives.
Nuclear fixatives are those that fix the nucleus and its associated structures. These fixatives
contain glacial acetic acid which has a high affinity to nuclear chromatin but tends to destroy
organelles such as mitochondria and Golgi bodies. On the contrary, cytoplasmic fixatives are
those that fix the cytoplasm as well as the organelles. These fixatives do not contain glacial
acetic acid. Histochemical fixatives are those that preserve the biochemical components of
tissues. Biochemical components of tissues include lipids, proteins and carbohydrates. Specific
solutions may be utilized depending on the solubility characteristics of the biochemical
component to be fixed.

Assessing Oneself
comprehension regarding the lesson. Once you start answering the activities,
do not go back to the Lesson Proper notes as this would defeat the purpose of
this portion of the module. These activities will not be recorded but you are
required to compile them as part of your journal to be checked every grading period.

Across Down
6. diagnosis of this requires the use of 1. reagent that differentiates nuclear from
formalin heated at 100 deg C cytoplasmic fixatives
9. mixture of glutaraldehyde or formaldehyde 2. chemical component that is fixed using
10. organelle destroyed by nuclear fixatives Baker's formol-calcium
3. solution that can act as a microanatomical
and nuclear fixative
4. reagent used in Lendrum's method
5. secondary purpose of fixation
7. fixatives with only one active component
8. effect of the presence of blood in the tissue
sample to the rate of fixation


Read and understand the statement and evaluate whether it is true or false. If the statement
is true, write/type “true” on the space provided before the number. If the statement is false,
underline the word/s that make/s the statement incorrect. After which, change or remove the
underlined word/s to make the statement correct. Write/Type the corrected statement after
the given statement.
__________ 1. Routine fixation using a tissue processor requires a temperature of 20 to 22 oC.
__________ 2. For electron microscopy, fructose is added to glutaraldehyde to achieve a high
vehicle osmolality.
__________ 3. The minimum requirement for the osmolarity of the fixative is that it should be
slightly hypertonic.
__________ 4. Sampling brain tissues should be performed prior to fixation to prevent it from
floating out of the fixative.
__________ 5. Maximum effectiveness of the fixative is observed when the volume is 20x the
volume of the tissue.
Part II: The Different Fixatives

ALDEHYDE FIXATIVES:
✓ for routine paraffin sections, electron microscopy, histochemistry and
enzyme studies
1. Formaldehyde (Formalin)
▪ gas produced by the oxidation of methyl alcohol
▪ buffered at ph 7 to 8
→ hypoxia in tissues leads to acidity which favors the formation of
Formalin heme pigments (black, polarizable deposits)
▪ Pure Stock: 40% formalin
▪ Dilution: 1:10 (10% solution); 1:20 (5% solution)
▪ Concentration for fixation: 10% formalin
▪ Paraformaldehyde: white precipitate due to prolonged storage
→ may be removed by filtration or addition of 10% methanol
2. 10% Formol-Saline
▪ central nervous tissues and general post-mortem tissues for histochemical
examination
▪ ideal with most stains including silver impregnation
▪ duration of fixation: more than 24 hours (slow fixative)
3. 10% Neutral Buffered Formalin
▪ preservation and storage of surgical, post-mortem and research specimens

▪ best fixative for frozen sections

▪ best fixative for iron pigments and elastic fibers
4. Formol-Corrosive
▪ routine post-mortem tissues
▪ for lipids, especially neutral fats and phospholipids
▪ no need for washing-out
5. Alcoholic Formalin
▪ used to fix sputum
▪ for the demonstration of immunoperoxidase activity
6. Glutaraldehyde
▪ made up of two formaldehyde residues linked by three carbon chains
▪ used in conjunction with osmium tetroxide
▪ Fixation time: ½ hour to 2 hours
METALLIC FIXATIVES
1. Mercuric Chloride
✓ most common metallic fixative
✓ tissue photography
✓ tissues contain black precipitates of mercury (except Susa)
A. Zenker’s Fluid
✓ fixing small pieces of liver, spleen, connective tissues fibers and nuclei
De-zenkerization: removal of mercuric deposits in tissues
B. Zenker-formol
✓ pituitary gland, bone marrow and blood containing organs (spleen and
liver)
C. Heidenhain’s Susa Solution
✓ for tumor biopsies
D. B-5 Fixative
✓ for bone marrow biopsies
2. Chromate Fixatives
A. Chromic Acid
✓ preserves carbohydrates
B. Potassium Dichromate
✓ preserves lipids and mitochondria
C. Regaud’s Fluid
✓ demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies,
RBC and colloid-containing tissues
D. Orth’s Fluid
✓ study of early degenerative processes and tissue necrosis
✓ demonstrates Rickettsia and other bacteria
3. Lead Fixatives
✓ demonstration of acid mucopolysaccharides

PICRIC ACID FIXATIVES:

❖ Picrates are soluble in water, therefore, tissues should not be washed in running tap
water (use 70% alcohol instead)
1. Bouin’s Solution
✓ fixation of embryos and pituitary biopsies
2. Brasil’s Alcoholic Picroformol Fixative
✓ fixative for glycogen
GLACIAL ACETIC ACID

✓ fixation of nucleoproteins
ALCOHOL FIXATIVES
1. 95 % Ethanol
▪ Preserves but does not “fix” glycogen granules
2. Methanol
▪ For dry and wet smears, blood and bone marrow samples
3. Isopropyl alcohol (95%)
▪ for touch preparations
4. Carnoy’s fluid
▪ most rapid fixative
▪ for chromosomes, lymph glands, urgent biopsies and brain tissue for the
diagnosis of rabies
5. Newcomer’s
▪ demonstration of mucopolysaccharides and nucleoproteins
6. Gendre’s fixative
OSMIUM TETROXIDE
1. Flemming’s (chrome-osmium HAc)
▪ Permanently fixes fats and recommended for fixing nuclear structures
2. Flemming’s w/o acetic acid
▪ For cytoplasmic structures
TRICHLOROACETIC ACID
✓ may also be used as a weak decalcifying agent
ACETONE
✓ use at cold temperature (-5 to 4oC)
✓ fixation of brain tissues for rabies diagnosis

Summing It Up
ALDEHYDE FIXATIVES
Formaldehyde Routinely used fixative
Glutaraldehyde Used for electron microscopy
10% Formol-Saline For enzymes, nucleoproteins, fats and mucins
10% Neutral Buffered Formalin Best for frozen sections, iron pigments and elastic fibers
Formol Corrosive/Sublimate For lipids, especially neutral fats and phospholipids
Gendre’s For immunoperoxidase activity, glycogen, microincineration & sputum
METALLIC FIXATIVES
Mercuric Chloride-Based
Zenker’s For small pieces of liver, spleen, connective tissue fibers & nuclei
Zenker-Formol For pituitary, bone marrow and blood-containing organs
Heidenhain’s SuSa For tumor biopsies of the skin
B-5 For bone marrow biopsies
Chromate-Based
Chromic Acid For carbohydrates
Potassium dichromate For lipids and mitochondria
Regaud’s For chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and
colloid-containing tissues
Orth’s For early degenerative processes, tissue necrosis, Rickettsiae and
other bacteria
Lead-Based
4% Basic Lead acetate For acid mucopolysaccharides
PICRIC ACID FIXATIVES
Bouin’s For embryos and pituitary biopsies
Brasil’s Alcoholic Picroformol For glycogen
ALCOHOL FIXATIVE
95% Ethanol For glycogen
100% Methanol For dry and wet smears, blood and bone marrow samples
95% Isopropyl alcohol For touch preparations
Carnoy’s For chromosomes, lymph glands, urgent biopsies and brain fixation for
rabies diagnosis
Newcomer’s For mucopolysaccharides and nuclear proteins
Gendre’s
OSMIUM TETROXIDE FIXATIVES
Flemming’s with HAc For fats and nuclear structures
Flemming’s without HAc For cytoplasmic structures
OTHERS
Glacial Acetic acid For nuclear structures
Trichloroacetic acid For dense fibrous tissues
Acetone For brain fixation for rabies diagnosis and water-diffusible enzymes

Assessing Oneself
Accomplish the following activities in order to assess your knowledge and comprehension
regarding the lesson. Once you start answering the activities, do not go back to
the Lesson Proper notes as this would defeat the purpose of this portion of
the module. These activities will not be recorded but you are required to
compile them as part of your journal to be checked every grading period.

Across Down
2. fixative that penetrates the worst 1. removal of black mercuric deposits using
6. for colloid-containing tissue samples alcoholic iodine
7. aka osmium tetroxide 3. used to fix dry and wet smears
8. white precipitate of formaldehyde 4. formaldehyde-containing solution that does
not require washing
11. su in Haidenhein's
5. alcoholic formalin

12. physical removal of paraformaldehyde 9. non-alcohol-based fixative used for rabies

13. buffer used in formaldehyde solution diagnosis
14. for the fixation of Rickettsiae 10. group of fixative that dissolves fats and
lipids
15. most rapid fixative, used for rabies
diagnosis
Level II: Master Column

Match the following fixatives (Column A) to their respective chemical group (Column B) and
characteristics (Column C).
A B C ANSWERS
1. B-5 A. Aldehyde fixatives a. oxidation of methyl alcohol
2. Acetone B. Mercuric fixatives b. embryos and pituitary
biopsies
3. Bouin’s Solution C. Chromate fixatives c. colloid-containing organs
4. Heidenhain’s SuSa D. Lead fixatives d. bone marrow biopsies
5. Formaldehyde E. Picric acid fixatives e. study of early degenerative
processes and tissue necrosis
6. Gendre’s fixative F. Alcohol fixatives f. touch preparations
7. Orth’s fluid G. None of the above g. water diffusible enzymes
8. 95% isopropyl h. blood-containing organs
alcohol
9. Regaud’s Fluid i. tumor biopsies of the skin
10. Helly’s Solution j. fixes and dehydrates tissues
Part III: Post-fixation Procedures and Physical Fixation

POST MORDANTING/SECONDARY FIXATION
✓ placing a fixed tissue in a second fixative
✓ Post-chromatization
➢ 2.5-3% potassium dichromate as the secondary fixative
➢ acts as a mordant for better staining effects
WASHING-OUT: REMOVAL OF EXCESS FIXATIVE

1. Tap Water: removes excess
✓ Chromates (Helly’s, Zenker’s & Flemming’s)
✓ Formalin
✓ Osmic Acid

2. 50 – 70 % Alcohol
✓ Picric acid (Bouin’s solution)
3. Alcoholic iodide
✓ Mercuric fixatives
WASHING-OUT: REMOVAL OF PIGMENT AFTER FIXATION

1. FORMALIN
➢ (white ppt): filtration; + 10% methanol
➢ brown/black: Alkaline Picrate (Picric acid + 95% EA)
: 1% KOH in 80% alcohol
➢ Kardasewitsch’s: 28%NH3 H2O + 70% EA
➢ Lillie’s: 28%NH3 H2O, acetone + H2O2 (wash in 70% Alcohol)
2. PICRIC ACID
➢ Sat. Li2CO3 in 70 % EA
➢ 70% EA then 5% Na thiosulfate
➢ Lenoir’s solution: conc. NH4Ac, 95% EA & dist. H2O
3. MERCURIC FIXATIVE
➢ 0.5% Iodine solution in 70% ethanol (5-10 mins
➢ Decolorizer: 5% Na thiosulfate
➢ Langeron’s iodine: I2 crystals, KI, Dist H2O
4. MELANIN
➢ KMnO4 (decolorize)
➢ Pyrogallic acid (reduce)
➢ H2O2 (bleach & final removal)
5. Others
➢ Carnoy’s – 100% alcohol
Fixation for Electron Microscopy

FIXATIVES: preserve cellular structure & require short fixation time
➢ ALDEHYDE FIXATION
1. Karnovsky’s paraformaldehyde - glutaraldehyde
2. Acrolein – glutaradehyde / formaldehyde
POST FIXATION: Buffered Osmium Tetroxide
➢ OSMIC ACID FIXATION
1. Palade’s fixative
2. Zetterqvist Osmium fixative
3. Millonig’s Osmium tetroxide
Enzyme Histochemistry
1. 4% formaldehyde or formol-saline
2. Frozen sections: acetone or formaldehyde

Fixatives for Smears

1. Schaudinn’s
➢ Mercuric Chloride
➢ Absolute Ethanol
➢ Acetic acid
2. Ether-Alcohol
3. Methanol
Other Methods of Fixation

1. Heat
2. Microwave Fixation – vacuum, heat & agitation (10 – 15 mm) + formaldehyde fixatives
SPECIAL TECHNIQUES
✓ Chemical fixation is avoided
✓ Recommended for histochemical studies
Quenching
➢ Rapid freezing of tissue blocks to allow instant cessation of cellular activities
Freezing Agents
1. Liquid Nitrogen
2. Isopentane
3. Pentane
4. Propane
5. Dichlorodifluoromethane
FREEZE-DRYING
➢ Rapid freezing: tissue plunged in isopentane or propane-isopentane
(-160 to -180 OC)
➢ Desiccation: removal of tiny ice crystals by sublimation in a vacuum
(-30 to -40 OC)
FREEZE-SUBSTITUTION
➢ Similar with freeze-drying
➢ Instead of dehydration in a vacuum, tissue blocks are:
✓ Fixed in Rossman’s fluid or 1% acetone
✓ Dehydrated with abs. alcohol
Enrichment Activity
Group Task: Let’s Collaborate
ACTIVITY 2a: FIXATION
Work with your assigned groupmates and answer the following questions for
research. Make sure to understand the concepts being asked in each question.
1. What are the different methods of fixation? Discuss each method.

2. What are the general precautions in handling specimens for tissue

processing?
3. Explain the compatibility and incompatibility of fixatives and stains.
4. Discuss how to remedy improperly fixed tissue blocks.
Summing It Up
Post-fixation procedures are performed in order to enhance the initial
fixation process. One post-fixation procedure is referred to as secondary
fixation. Depending on the specific procedure performed, secondary fixation
can improve the demonstration of certain substances (double fixation) or
improve the staining characteristics of tissue constituents (post-mordanting).
Another post-fixation procedure is the washing of the fixed tissue sample. There are
two purposes of washing-out after fixation. One is to remove excess fixative and another is to
remove precipitates and pigments from the tissue sample. Washing solutions to remove
excess fixatives include tap water, 50 to 70% alcohol and alcoholic iodine. Examples of
solutions used to remove precipitates and pigments include 10% methanol, alkaline picrate,
Lenoir’s solution, Langeron’s iodine, potassium permanganate, pyrogallic acid, hydrogen
peroxide, and absolute alcohol.
In addition to the different chemical fixatives, there are also physical methods
particularly the use of high temperatures, which may be performed to carry-out the fixation
of tissue samples. Heat fixation is a simple fixation method which is often times performed to
easily fix smears. Microwave fixation is another special physical method that may also be
performed in the laboratory. It is often times employed in conjunction with the use of
formaldehyde-based fixatives.
There are also special techniques which may be performed in the laboratory when
there is a need to avoid chemical fixation such as in the case of histochemical studies. These
special techniques include freeze-drying and freeze substitution. Both of these techniques are
based on the concept of rapid freezing or quenching. Tissue constituents are fixed using very
low temperatures. This environmental condition needed to perform quenching is achieved
using freezing agents such as carbon dioxide and liquid nitrogen. Freeze-drying involves the
desiccation of water molecules from the tissue sample in a vacuum. Freeze-substitution on
the other hand involves the replacement of water molecules by solutions such as Rossman’s
fluid or osmic acid.

Assessing Oneself
this portion of the module. This activity will not be recorded but you are
required to compile it as part of your journal to be checked every grading period.

Across Down
2. purpose of post-chromatization is the 1. secondary fixation using potassium
enhancement of this dichromate
4. used to remove formaldehyde 3. used to remove picric acid fixatives
5. pigment that is reduced by pyrogallic acid 6. alcohol present in Schaudinn's solution
8. rapid freezing 7. Lenoir's solution is used to remove this type
9. fixation procedure for electron microscopy of fixative
10. physical fixation that makes use of
formaldehyde fixatives

Check the key answers provided in Appendix A of this module. Assess your performance
before the scheduled summative assessments.
A. Get ready for summative assessments in the lecture and laboratory to be scheduled by
your instructor.
B. Summative assessments will be given by the instructor via several modalities depending
on your connectivity status.
✓ If you have a strong internet connectivity, you may take the quiz via the online
platform Quizziz.
messenger.
✓ If you have no internet connectivity, you may take the quiz via SMS/text
message.
References
1. Allen, D., & Cameron, R.I. (2004). Histopathologic specimens: clinical, pathological
and laboratory aspects. USA: Springer
2. Armed Forces Institute of Pathology Laboratory. Methods in histotechnology.
Washington DC: American Registry of Pathology
3. Bancroft, Layton, Suvarna. (2012) Bancroft’s theory and practice of histological
techniques. (7th ed.). Churchill Livingstone: Elsevier
4. Bruce-Gregorios, J. (1974). Histopathologic techniques. (1st ed.). Philippines:
Goodwill
Trading Co., Inc.
5. Bruce-Gregorios, J. (2012). Histopathologic techniques. (2nd ed.). Philippines:
Goodwill Trading Co., Inc.
6. Bruce-Gregorios, J. (2016). Histopathologic techniques. (3rd ed.). Philippines:
7. Finkbeiner, W.E. et.al. (2009). Autopsy pathology: a manual and atlas (2nd ed.). USA:
Elsevier
8. Raphael, S. (1983). Lynch’s medical laboratory technology. (4th ed.). Philadelphia:
W.B. Saunders Co.

LESSON 3: DECALCIFICATION
1. explain the basic concepts, principles that govern the decalcification process as well as
the different factors that affect it;
2. identify the most appropriate reagent or method to be used when decalcifying a given
specimen;
3. discern the various plausible causes of an improperly decalcified sample;
4. explain the different tests to determine complete decalcification and to utilize the
result of the test as a guide in performing the decalcificaiton process; and
5. identify the toxicities involvd with the use of decalcifying solutions and how to prevent
or mitigate the exposure to such toxicities.

Across Down
2. commercial name of the sodium salt of this 1. examples of this are inorganic acids such as nitric and
anticoagulant is versene sulfuric acid
5. bone is what type of tissue? 3. negatively-charged electrode
9. type of bone tissue found in the ends of long 4. process by which anticoagulants sequesters Ca2+
bones 6. type of bone tissue found in flat bones
10. tissue component made up of substances and 7. positively-charged electrode
fibers
8. examples are organic acids such as formic and citric acids

Lesson Proper
Part I: Overview of 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 NOT Performing Decalcification

✓ Poor cutting of hard tissues
✓ Damage to the knife edge during sectioning
✓ Bone dust and other cellular debris obscures microanatomic details
Consequences of Performing Decalcification

✓ Distortion or damage to tissues
✓ Affects staining
➢ Failure of sections to stain properly is compounded by: (1) overtreatment in
acid & (2) insufficient washing out of the acid
➢ Basic dyes: hematoxylin is inhibited
➢ Acid dyes: eosin produces a deep brick red color without differential staining
✓ Sections Float-off During Staining
➢ Observed after immersion in acid alcohol
➢ Collodionize prior to staining
SAMPLES THAT REQUIRE DECALCIFICATION

1. Bones and other calcified samples (tuberculous organs, atherosclerotic vessels,
teratomas)
➢ cut into small pieces using fret-saw, trimmed with a hand razor and fixed with
10% neutral buffered formalin
2. Teeth
➢ partial or complete decalcification is required before cutting samples
3. Microcalcified samples
➢ detected during sectioning or examination
➢ REMEDY IF DETECTED DURING SECTIONING: surface decalcification using a
pad of cotton/gauze soaked with 10% HCl for 1 hour
➢ APPEARANCE UNDER THE MICROSCOPE: dark purple granular masses with
lighter purple halos
➢ commonly found in malignancy
GENERAL CONSIDERATIONS FOR ROUTINE ACID TECHNIQUES

1. Thickness of the Specimen
➢ Dense/Hard Bone: 2-5 mm thick
➢ Softer Tissue: 4-6 mm thick

2. Duration
➢ Ideal: 24-48 hours
➢ Dense Cortical Bone: 14 days
3. Temperature and Heat
✓ Required temperature: 18-30 degrees Celsius
✓ Heat enhances destructive action of acids on matrices
✓ 37 degrees Celsius: impairs nuclear staining with Van Gieson’s
→ reduced effectiveness of Trichrome and PAS
✓ 55 degrees Celsius: tissues will undergo complete digestion within 24-48 hrs
4. Solution Used
❖ Concentration of Solutions
➢ Directly proportional to the rate of decalcification
❖ Strong Acids
➢ 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
DECALCIFYING AGENTS: acids or chelating agents

Characteristics of a Good Decalcifying Agent:
1. Remove calcium salts completely.
2. Does not produce considerable destruction of cells and tissue components.
3. Does not adversely affect the staining capacity of the cell
DECALCIFYING METHODS: use of acids, use of chelating agents, ion exchange resins and
electrical ionization
USE OF ACID SOLUTIONS:

❖ Injurious to the organic ground substance of tissues

NITRIC ACID
✓ most common and fastest
✓ 5 to 10% is the recommended concentration when used as a simple solution
✓ rapid decalcifying agent: may inhibit nuclear stains and damage tissues
✓ formaldehyde or alcohol and chromic acid may be added as additives
✓ Washing of tissue: acid removed by 3 changes of 70-90% ethanol
✓ Washing slide: brought to water and placed in 1% aq. lithium carbonate for 1
hour→wash for 15 min
✓ causes spontaneous yellow discoloration
➢ impairs staining reaction of the tissue
➢ IF PRESENT IN TISSUES: neutralize with 5% NaSO4 → wash in running tap
water (at least 12 hours)
➢ IF PRESENT IN SOLUTION: add 0.1% urea to pure conc. nitric acid
NITRIC ACID FORMULATIONS

1. De Castro’s Fluid: silver impregnation of nerve fibers
✓ Composition: chloral hydrate, distilled water, alcohol, nitric acid
2. 10% Aqueous Nitric Acid
3. Formol-Nitric Acid: nitric acid, 40% formaldehyde
4. Perenyi’s Fluid: nitric acid, 0.5% chromic acid, absolute alcohol
❖ DISADVANTAGES:
o slow-acting
o complete decalcification cannot be determined by chemical testing
→ precipitate forms upon addition of ammonia even in the
absence of calcium ion
→ re-dissolve by adding glacial HAc drop by drop
→ add 0.5ml saturated aqueous ammonium oxalate
→ reappearance of white precipitate after 30 minutes
→ presence of calcium ions
→decalcification is not yet complete
5.Phloroglucin-Nitric Acid: conc. nitric acid, phloroglucin, 10% nitric acid

✓ most rapid decalcifying agent
✓ recommended for urgent work
❖ WASHING OF TISSUES:
o 3 changes of 70 to 90% ethanol
❖ WASHING OF SECTIONS:
o bring slides to water
o place in 1% aqueous lithium carbonate for 1 hour
o wash for 15 minutes
FORMIC ACID
✓ fixative and decalcifying agent
✓ for small and large pieces of bones, and teeth

✓ gentler on tissues
✓ post-mortem research tissues
✓ concentrated reagent: 90%
➢ Aqueous Formic Acid: formic acid, formalin
➢ Formic Acid-Sodium Citrate
❖ WASHING: neutralize with 5% sodium sulfate
HYDROCHLORIC ACID
✓ slower action, with greater distortion
✓ provides good nuclear staining
✓ surface decalcification: 1% HCl with 70% alcohol
✓ cannot be measured by chemical testing
1. Von Ebner’s Fluid: 36% saturated aqueous NaCl, concentrated HCl
✓ for teeth and small pieces of bones
OTHER ACIDS
1. Trichloroacetic Acid (TCA)
✓ for minute samples
✓ provides good nuclear staining
✓ does not require washing-out
✓ very slow and weak decalcifying acid
2. Flemming’s Fluid
✓ also for minute samples
✓ fixative and decalcifying agent
3. Citric Acid-Citrate Buffer
✓ provides excellent nuclear and cytoplasmic staining
✓ does not produce distortion
4. Sulfurous Acid
✓ very weak decalcifying agent, thus it is recommended for very minute samples
only
STAINS USED AFTER ACID DECALCIFICATION

H and E
Masson’s Hematoxylin-Phloxine-Safran
Giemsa Stain

Summing It Up
Decalcification, also known as demineralization, is the process that involves the
removal of mineral deposits (commonly calcium salts) from tissue samples.
Improperly performing decalcification or if it is not performed at all presents
consequences that will affect the subsequent steps in tissue processing.
Notable effects include (1) poor cutting of tissue sections, (2) possible damage to the
knife edge and (3) the presence of bone dust and other cellular debris which might
obscure microanatomic details. Similarly, performing decalcification presents
unavoidable consequences that will likewise affect the subsequent steps in tissue
processing. Effects of performing decalcification includes (1) failure of tissue sections
to stain properly and (2) washing-out of sections during the staining process.
Bones, tuberculous organs, atherosclerotic vessels, teratomas, teeth and
microcalcified samples have to be decalcified. These samples contain high amounts
of mineral deposits that have to be removed before proceeding to the next steps in
tissue processing.
In order to properly perform decalcification, several factors have to be
considered. These factors include the thickness of the specimen, duration of
decalcification, temperature, the solution to be used, presence of additives, fluid
access, changing of the solution, volume of the decalcifying agent, presence of
agitation, removal of decalcifying solution and the use of microwave and electrolytic
methods.
There are four decalcifying methods which may be performed: (1) the use of
decalcifying agents, (2) the use of chelating agents, (3) electrophoresis, and (4) ion-
exchange resin. Each method involves the use of a decalcifying agent which could
either be an acid or a chelating agent.
NITRIC ACID DECALCIFYING AGENTS

5 to 10% Nitric acid Most commonly used decalcifying agent
De Castro’s fluid For silver impregnation of nerve fibers
Formol-Nitric acid Nitric acid + 40% formaldehyde
Perenyi’s fluid Slow-acting decalcifying agent
Phloroglucin-Nitric acid Most rapid decalcifying agent
FORMIC ACID For small and large pieces of bones and teeth
HYDROCHLORIC ACID DECALCIFYING AGENTS
1% HCl with 70% alcohol For surface decalcification
10% HCl For surface decalcification
Von Ebner’s fluid For teeth and small pieces of bones
OTHER ACIDS
Trichloroacetic acid For minute samples
Flemming’s fluid For minute samples
Citric acid-Citrate buffer Excellent nuclear and cytoplasmic staining
Sulfurous acid For very minute samples

Assessing Oneself

Across Down
3. sample that requires partial or complete 1. material used to cut calcified samples
decalcification prior to sampling 2. recommended stain for acid decalcification
5. acid for very minute samples 4. HCl-containing fluid
8. presence of these in decalcifying solutions 6. reagent used to remove nitrous acid from
retards the process tissues

11. step primarily affected if decalcification is 7. slow-acting nitric acid formulation

performed 9. formulations of this acid are used for
13. calcium deposits associated with surface decalcification
malignancy 10. used to remove nitric acid formulations
14. step primarily affected if decalcification is from tissues
not performed 12. decalcifying solutions that damage tissue
15. acid that does not require washing out antigens

__________ 1. Decalcification maybe performed after dehydration and before staining.
__________ 2. Microcalcifications are commonly found in extra-pulmonary tuberculosis and
appear as dark red masses without differentiation during sectioning.
__________ 3. Removal of the decalcifying solution may be performed after decalcification
and
prior to sectioning to completely remove calcium salts.
__________ 4. Failure of decalcified samples to stain properly is dependent upon how long
the
tissue remains in contact with acid-decalcifying solutions.
__________ 5. Weak acids such as hydrochloric acid may be used for immunohistochemistry.
Part II: Other Decalcifying Methods and Post-Decalcification Procedures

USE OF CHELATING AGENTS
✓ EDTA:
✓ excellent bone decalcifying agent for immunohistochemistry and
enzyme studies
✓ acts as both decalcifying agent and water softener
✓ does not interfere with staining
✓ does not distort tissues and enzymes
✓ available in 2 formulations
❖ 5-10% EDTA Disodium
❖ EDTA Tetrasodium
o pH adjusted to 7.4 using concentrated HAc

✓ DURATION:
➢ 1 to 3 weeks: small specimens
➢ 6 to 8 weeks: dense cortical bone
✓ pH: very important factor
➢ pH 3: inhibits calcium binding
➢ pH 8: optimum binding
➢ pH 7.0 to 7.4: allows binding and does not destroy tissue components
ION EXCHANGE RESIN

✓ uses ammonium-sulfonated polysterene + formic acid
✓ volume of the acid solution: 20-30 times the volume of the sample
✓ Resin and Formic Acid (RAF)
➢ Cellular detail is well preserved
➢ Decalcification is faster
➢ Daily changing of solution is eliminated
❖ 10 % and 20 % RAF
➢ cancellous bone 2-3 mm thick (2-3 hours)
➢ 5-6 mm thick (4-8 hours)
❖ 40 % RAF: large pieces of dense bone (24 hrs)→trimmed to 3 mm thick→40 % RAF
(24 hrs)
❖ REACTIVATION OF USED RESIN:
→ 2 washings with N/10 HCl
→ final washing with distilled water
✓ end-point of decalcification is measured using physical or X-ray methods
ELECTROPHORESIS
✓ positively charged calcium ions are attracted to a negative electrode
✓ uses heat and electrolytic reaction
✓ dependent on electricity for the removal of calcium
✓ temperature: 30 to 45 degrees Celsius
✓ uses 90%/88% formic acid and concentrated HCl as the acid solution
TESTS FOR COMPLETE DECALCIFICATION

1. PHYSICAL TEST
✓ done by touching or bending to determine consistency or pricking with fine
needle or probe
✓ vague and not a reliable method
2. RADIOLOGIC TEST
✓ calcium and mineral salts produce opaque areas
✓ very expensive, most ideal method, most sensitive and most reliable method
✓ DO NOT use for mercuric chloride fixed tissues
✓ EQUIPMENT: Faxitron and Kodak X-OMAT X-ray film

3. CHEMICAL TEST
✓ Simple, reliable and convenient method for routine purposes
❖ PROCEDURE:
➢ Aliquot 5 ml of used reagent.
➢ Alkalinize with ammonia water
→ (+) precipitate = (+) for calcium = INCOMPLETE
DECALCIFICATION
→ if clear, proceed to the next step
➢ Add 0.5 ml ammonium oxalate or 1% sodium oxalate
➢ Stand for 15 to 30 minutes
→ (+) cloudiness or precipitate = (+) for calcium =
INCOMPLETE DECALCIFICATION
POST-DECALCIFICATION TREATMENT
1. WASHING-OUT
❖ Water Rinsing
➢ 30 minutes for small samples
➢ 1 to 4 hours for larger samples
➢ quick rinsing and blotting for small needle biopsies
2. NEUTRALIZATION
❖ 2% Lithium Carbonate
❖ 5 to 10% Aqueous Sodium Bicarbonate
3. FROZEN SECTIONING
❖ thorough washing in water
❖ storage in any of the following:
➢ formol saline with 15% sucrose
➢ phosphate-buffered saline (PBS) with 15 to 20% sucrose at 4 degrees
Celsius
4. EDTA
❖ wash in water NEVER IN ALCOHOL
❖ storage in formol-saline or PBS overnight
TISSUE SOFTENERS
1. Perenyi’s Fluid
✓ surface blocks submerged for 1 to 2 hours
✓ tissues immersed for 12 to 24 hours
2. 4% Aqueous Phenol
3. Molliflex
✓ may cause swelling or make tissues soapy
4. 2% HCl
5. 1% HCl in 70% alcohol

Enrichment Activity
ACTIVITY 2a: DECALCIFICATION
1. What are the precautionary measures and guidelines observed in
decalcifying tissues?
2. What are the different ways of assessing the degree or extent of
decalcification? Make a schematic diagram of the steps performed in
each.
3. Discuss tissue softening emphasizing on its importance in relation to
decalcification.
Summing It Up
In addition to the use of acids, other methods of decalcification include
the use of chelating agents, electrophoresis and ion exchange resin. The most
commonly used chelating agent in histopathology is ethylenediaminetetraacetic
acid (EDTA) particularly the sodium salt (commercially known as versene). EDTA
is considered as an excellent bone decalcifying agent for immunohistochemistry. Another
method is ion exchange resin. This method involves the use of ammonium-sulfonated
polysterene and formic acid. Lastly, electrophoresis may also be performed to remove mineral
deposits from tissues. This process makes use of electrodes, formic and hydrochloric acids for
decalcification.
In performing decalcification, it is important to evaluate whether the process is
already complete. This is to avoid undue contact between the tissue sample and the
decalcifying acid solution. Take note that acid solutions are particularly injurious to the tissues
matrices and prolonged contact may lead to the distortion and breakdown of tissue
components. There are three ways by which extent of decalcification may be evaluated: (1)
physical test, (2) radiologic test, and (3) chemical test.
Once decalcification is complete, removal of the decalcifying solution is done via
washing-out using water or alcohol. This may also be performed via neutralization using
alkaline solutions like lithium carbonate or sodium bicarbonate. Unduly hard tissue samples
may require tissue softening in order to prevent any damage to the microtome during
sectioning. Tissues softening is performed using Perenyi’s fluid, 4% phenol, 2% HCl, 1% HCl in
70% alcohol or Molliflex.

Assessing Oneself

Across Down
1. acid used for resin reactivation
2. frequency of changing EDTA solution during
3. appearance of calcifications in the X-ray
the final stage of decalcification
film
5. EDTA that requires pH adjustment
4. involves touching or bending of decalcified
7. requires storage in PBS with sucrose samples
8. indicates presence of calcium ions in the 6. weak acid used in electrophoresis
chemical test
10. used to remove excess EDTA from tissues
9. tissues appear swollen and soapy in this
solution
11. enzyme inhibited by EDTA


__________ 1. The mechanical test performed to assess the extent of decalcification yields white
precipitates of either calcium hydroxide or calcium oxalate indicating that the
process is complete.
__________ 2. Hydrochloric acid can be used in both ion exchange resin and electrophoresis
for the removal of calcium deposits.
__________ 3. In the radiologic method of testing complete decalcification, waterproof
polysterene sheet is overlaid on the X-ray film and 40kV is applied for 2
minutes.
__________ 4. In the calcium oxalate test, 5 ml of unused reagents is acidified before adding
sodium ammonia.
your instructor.
platform Quizziz.
messenger.
message.
References
W.B. Saunders Co.

LESSON 4: DEHYDRATION
1. have a clear understanding of the different concepts and factors that affect the
dehydration process;
2. discriminate the uses of the different reagents-how and when to use these reagents;
&
3. identify the toxicities attributed to the use of dehydrating agents and how to prevent
or mitigate the exposure to such toxicities.

Across Down
5. eye inflammation 1. carboxylic acid formed from methanol
7. means no water 2. solvent for organic molecules
8. solvent for water 3. in between cells
9. 100% alcohol 4. aka soluble
10. simplest tertiary alcohol 6. effect of a higher concentration gradient on
the rate of diffusion of molecules

Lesson Proper
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
ACETONE
✓ cheap, rapid and used for most urgent biopsies
✓ duration: 30 minutes to 2 hours
✓ removes lipids from tissues
✓ penetration is poor and causes brittleness
CELLOSOLVE (Ethylene Glycol Monoethyl Ether)

✓ Rapid and does not cause any harmful effect on tissues
✓ 4 baths: 30 min → 30-60 min → 60 min → 90 min
✓ Toxic to the reproductive, fetal, urinary and blood systems
✓ Combustible at 110-120 OF
✓ Propylene-based glycol ethers may be used in place of EGME
DIOXANE (Diethylene dioxide)

✓ Excellent dehydrating and clearing agent
✓ Fixatives → Dioxane→paraffin
**dehydration and clearing may be done at the same time using dioxane
✓ Sections ribbon poorly
✓ Expensive and extremely dangerous
✓ Vapor tends to accumulate in the body and is highly toxic
✓ Creates explosive peroxides
✓ Removal of water
➢ Graupner’s Method: uses several changes of pure dioxane
➢ Weiseberger’s Method: uses pure dioxane and anhydrous calcium
oxide or quicklime to facilitate removal of water
THF (Tetrahydrofuran)
✓ May be used as dehydrating and clearing agent
✓ Can dissolve fats in tissues
✓ Vapors cause nausea, dizziness, headache and anesthesia
✓ Skin and eye irritant
✓ Offensive odor; may cause conjunctivitis during prolonged exposure
TRIETHYL PO4
✓ May be used to dehydrate sections and smears after staining

ADDITIVES TO DEHYDRANTS
✓ 4% phenol: added to 95% alcohol to soften tissues
✓ Glycerol-alcohol mixture and Molliflex: used to soften hard tissues
Enrichment Activity
ACTIVITY 2a: DEHYDRATION
1. What are the properties of a good dehydrant?
2. What are the precautionary measures, guidelines and conditions
required for dehydration?
Summing It Up
Dehydration involves the removal of the fixative, intercellular end
extracellular water from tissues in preparation for infiltration. Since water is not
miscible in paraffin, it is essential to remove and replace water molecules
present in tissues with a solvent that is compatible with paraffin.
Dehydration is carried out by first immersing the tissue sample in increasing strengths
of the dehydrating agents-starting with lower concentrations and moving to higher
concentrations. This is to facilitate the smooth diffusion of molecules, thereby preventing the
distortion of tissue components.
The dehydration process is affected by several factors including the type of tissue
sample, volume of the dehydrating agent, duration of the process, temperature and presence
of agitation.
The most commonly used dehydrating agents are alcohols. Ethanol is considered to be
the best dehydrating agent for routine processing. Substitutes for ethanol are isopropyl
alcohol and butyl alcohol. Butanol can also be used for the dehydration of plant and animal
tissues. Since it is a slow-acting dehydrant, it causes less shrinkage and hardening which are
common disadvantages associated with other dehydrating agents. Methanol is another
alcohol which can be used for dehydration. However, its use in the laboratory is limited to
blood and tissue smears only. This is due to its toxic nature since it is metabolized inside the
body to harmful substances, particularly formaldehyde and formic acid.
Non-alcohol-based dehydrants are also available. These include acetone, cellosolve,
dioxane, tetrahydrofuran and triethyl phosphate. Proper precautionary measures should be
strictly observed when using these dehydrants due to their various hazardous nature. Acetone
is an extremely volatile and flammable substance. Cellosolve is toxic to the human body by
inhalation, skin contact and ingestion, Dioxane is highly toxic and extremely dangerous due to
the possible formation of explosive peroxides. Tetrahydrofuran causes nausea, dizziness,
headache and anesthesia. It is also considered as a skin and eye irritant.
Since dehydration can cause hardening of tissue samples, tissue softening is often
times performed as a remedy to this. Additives to dehydrants that may be used to soften
tissues include 4% phenol, glycerol-alcohol mixture and Molliflex. These additives are
incorporated in the last concentration of alcohol used for dehydration.

Assessing Oneself

Across Down
3. toxic alcohol dehydrant 1. method that uses pure dioxane and
6. other name of cellosolve anhydrous calcium oxide
8. dehydrant with an offensive odor 2. type of tissue that is first immersed in 30%
alcohol
9. non-alcohol-based dehydrant that removes
lipids 4. expensive and may form explosive peroxides
10. effect of prolonged immersion of tissues in 5. best dehydrating agent for routine
low concentrations of dehydrants processing
7. purpose of additives to dehydrants


__________ 1. Additives are incorporated in the lowest concentration of acetone to prevent tissue
maceration.
__________ 2. Tert-butyl alcohol is a slow-acting dehydrant thus causing less tissue shrinkage
and hardening.
__________ 3. Acetone is a rapid-acting dehydrant that penetrates tissue samples well.
__________ 4. Tetrahydrofuran can be used as a fixative and dehydrating agent but it causes
dissolution of glycogen.
__________ 5. Graupner’s method makes use of cellosolve which can act as a decalcifying and
dehydrating agent.
your instructor.
platform Quizziz.
messenger.
message.
References
Elsevier
W.B. Saunders Co.

LESSON 5: CLEARING
1. explain the various concepts and effects of factors that affect clearing;
2. identify the most appropriate clearing agent when processing a given specimens;
3. discern the different plausible causes and identify steps to prevent and remedy
improperly cleared tissue samples; and
4. identify toxicities and hazards attributed to the use of clearing agents and how to
prevent or mitigate the exposure to such toxicities.

Across Down
1. otherwise referred to as fluidity 2. substances that cause malignancy
3. benign overgrowth usually composed of 5. combustible
muscle tissue and fibers 7. cyclic organic compounds
4. benzene ring with two methyl groups
6. methyl benzene
8. CHCl4
9. CCl4
10. C6H6

Lesson Proper
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
▪ Agents with low BP are readily replaced by paraffin (Except chloroform)
✓ Viscosity
▪ Higher viscosity leads to slow penetration
✓ Refractive Index
▪ does not affect the rate but affects the quality of cleared tissue
**Reagent Used
▪ Special considerations should be noted depending on the clearing agent used
▪ Some reagents will not clear tissues

Xylene/Xylol
✓ Most rapid (15 - 30 mins/ 30 min – 1 hr)
✓ Excellent clearing agent but tends to make tissues excessively hard & brittle.
✓ Turns milky when dehydration is not complete
Benzene
✓ Rapid agent (15 – 60 mins)
✓ Carcinogenic, causes aplastic anemia
Toluene/Toluol
✓ Similar to xylene but does NOT harden tissues nearly so much
✓ Slower than xylene or benzene (1 – 2 hrs)
✓ Not carcinogenic but emits toxic fumes
Chloroform
✓ For nervous tissues, lymph nodes & embryos
✓ Tissue do not become translucent
✓ Best for large specimens (up to 1cm thick) and tough tissues
✓ Toxic to the liver on prolonged inhalation
✓ Tissues tend to float: remedy → wrap tissues with absorbent cotton gauze
Cedarwood oil
✓ Recommended for CNS, smooth muscles & skin
✓ Slow (2 – 3 days); minimal shrinkage
✓ For both celloidin and paraffin sections
✓ Tissue floats – use Absolute alcohol to prevent drying out of tissues
✓ Must be followed by immersion in xylene or benzene to remove oil from tissues
✓ Turns milky on prolonged storage
OTHER CLEARING AGENTS

Aniline oil ✓ Clears 70% alcohol
✓ Recommended for embryos, insects and delicate specimens
Clove Oil ✓ Slow; may be adulterated; removes aniline dyes
CCl4 ✓ Similar to chloroform
Dioxane ✓ Both dehydrating and clearing
Amyl acetate ✓ For large pieces of tissues & embryonic materials
Terpineol ✓ Substitute for cedarwood oil
Methyl ✓ Used in double embedding.
Benzoate/Salicylate
Glycerin, Gum ✓ when the tissue is to be cleared directly from water (no de-
syrup & Brun’s alcoholization, it merely improves the RI)
solution

Carbo - Xylene ✓ For materials that are difficult to clear (eg. Thick mucinous
Papanicolaou smears)
✓ Should be thoroughly rinsed in xylene prior to mounting
Others
✓ Oil of bergamot
✓ Phenol in alcohol
✓ Creosote
Newer Clearing agents
✓ Based on limonene: a volatile oil found in citrus peels
✓ Clearite - Long chain aliphatic HC
Enrichment Activity
ACTIVITY 2a: CLEARING
1. What are the precautionary measures and guidelines observed in
clearing tissues?
2. Enumerate and describe clearing agents that may partially act as
dehydrants.
Summing It Up
Clearing, also referred to as dealcoholization, involves the removal of
the dehydrating agent and replacing it with a solvent that is miscible in paraffin.
Another purpose of clearing is to render transparency to the tissue sample.
Take note that not all clearing agents are dealcoholizing agents and vice versa.
Some reagents used for this procedure are only able to remove alcohol but do not make
tissue samples transparent. Examples of these are chloroform and carbon tetrachloride. On
the other hand, some reagents are only capable of making tissues transparent but do not
remove alcohol. Examples of these are glycerin, gum syrup and Brun’s solution.
There are three applications of clearing: (1) clearing in embedding, (2) clearing in
mounting, and (3) to render transparency to the tissue sample. Factors that affect clearing
include boiling point, viscosity and refractive index of the reagent. The reagent that is used for
clearing will affect the subsequent process which is infiltration. The following table shows the
different commonly used reagents for this procedure:
Xylene Most commonly used and most rapid clearing agent
Benzene Rapid-acting and clears tissues without causing hardness or brittleness
Causes aplastic anemia
Toluene Used as a substitute for xylene and benzene
CHCl3 and CCl4 Versatile reagents as they can be used for different types of tissues
Used for delicate as well as tough tissue samples
Cedarwood oil Recommended for CNS, smooth muscle and skin tissues

Assessing Oneself

Across Down
4. organ affected by chloroform toxicity 1. BP of chloroform compared to xylene
7. factor that does not affect clearing rate 2. anemia caused by benzene
8. turns milky if dehydration is incomplete 3. clearing agent that is prone to adulteration
10. clearing in _______; reagent should be 5. also known as artificial oil of lilac
miscible in the stain 6. turns milky on prolonged storage
9. aromatic oil used for insects

your instructor.
platform Quizziz.
messenger.
message.
References
4. Bruce-Gregorios, J. (1974). Histopathologic techniques. (1st ed.). Philippines:
Elsevier
W.B. Saunders Co.

LESSON 6: INFILTRATION AND EMBEDDING

1. explain the imporatnce of tissue impregnation and embedding;
2. identify the various media and the respective use/s of each; and
3. discuss the importance advantages and disadvantages of each type of mold.

Across Down
2. water-fearing 1. substance used to soften hard tissues
7. temperature at which a solid substance 3. direction of the plane of cut in transverse
liquefies section
8. plane of cutting is at an angle 4. plane of cut is parallel to the longest
9. histological section in which the lumen & dimension of the organ
layers of hollow organs are observed 5. equipment used to float sections
10. water-loving 6. substance that hastens a reaction

Lesson Proper
Part I: Overview of 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
✓ 20-24 OC (Room Temp): 54 – 58 OC
✓ 15-18 OC: 50-54 OC
2. Hardness of the Tissue sample
3. Temperature during Infiltration
✓ Paraffin oven: 2 – 5 OC higher than the MP of the wax
✓ Beyond 60 OC: deleterious to the tissue
4. Number of changes
✓ Minimum requirement: at least 2 changes
5. Nature and Size of the Tissue sample
✓ larger and denser: longer and more frequent changes
6. Clearing Agent Used
✓ xylene/benzene vs chloroform/cedarwood oil

Advantages:
✓ Very thin sections may be obtained with ease
✓ 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
❖ 4 changes at 15-minute intervals
❖ 2-5 OC higher than the MP of the wax
2. AUTOMATIC PROCESSING
❖ 2 – 3 changes with agitation
❖ At least 3 OC higher than the MP of the wax
3. VACUUM EMBEDDING
❖ negative atmospheric pressure (400-500 mmHg)
❖ Heat & vacuum
❖ 3 changes
❖ 2 - 4 OC above MP of wax
❖ ADV: Effects of heat are prevented
❖ 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
➢ Ref at -5 OC
➢ immerse in cold water
➢ Tissue Tek with cold plate

Substitutes for Paraffin Wax

1. Paraplast
➢ MP: 56-57 OC
➢ More elastic & resilient
➢ Doesn’t require cooling
❖ Embeddol
➢ MP= 56 – 58 OC
➢ Less brittle than paraplast
❖ Bioloid
➢ Semisynthetic wax that is used in impregnating and embedding eye samples
❖ Tissue Mat
➢ Product of paraffin that contains rubber
2. Ester wax
➢ MP: 46 – 48 OC
➢ Lower melting point but harder than paraffin
➢ Soluble in 95% EA & other clearing agents
➢ PROCESSING: cellosolve/xylene → equal parts clearing agent + ester wax for 3-
6 hours → 3-4 changes of pure ester wax → embedding → sectioning using
heavy duty microtome
3. Water Soluble Waxes

➢ Polyethylene glycols
➢ MP: 38-42 OC or 45-56 OC
❖ Carbowax
➢ Does not require dehydration & clearing
Fixation → wash-out → carbowax processing
✓ Reduces the processing time
➢ Does not remove neutral fats & lipids
➢ For histochemical and enzyme studies
❖ Carbowax Processing
➢ 70 % (30 min) → 90% (45 min) → 100% (2 changes for 1 hr each) at 56 OC
➢ Blocking at 50 OC →rapidly cooled at ref temp
➢ Applications: histochemical and enzyme studies; for the demonstration of
neutral fats and lipids
➢ DISADVANTAGE: hygroscopic reagent
➢ REMEDY:
→ Add soap or 10% polyethylene glycol 900 in waterbath
→ Use floating solutions: Pearse solution or
Blank and McCarthy solution

Enrichment Activity
ACTIVITY 2a: MANUAL TISSUE PROCESSING
OBJECTIVES:
1. identify the various steps in tissue processing;
2. exercise systematic and time-controlled sequence of the procedure; and
3. understand the principle involved in each step of the procedure.
The histologic technician is concerned with the preparation, processing and staining of
tissue sections from surgical or autopsy material for microscopic study and interpretation by
the pathologist. Conscientious handling of tissue and care in producing the best possible slides
can substantially affect and improve the accuracy of the diagnosis. Thus, quality
histopathology depends on quality histotechnology.
A video demonstration showing the different procedures will be presented by your
instructor. Make sure to listen attentively and take note of the important concepts and
principles which will be applied in performing the different steps in preserved tissue
preparation.
Part I: Gross Examination and Sampling (Done by the Pathologist)

1. The specimen is identified and grossly examined.
2. The tissue is placed on a chopping board/dissecting pan and cut with a sharp scalpel to a
size of about 2 by 4 mm2.
Part II: 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.
Note: The specimens are usually obtained from the anatomic pathology department of
hospitals. Be sure to obtain information on the source and type of the specimens. The
specimens have been fixed or preserved in 10% formalin, thus, the procedure on fixation
then may be omitted and processing shall start with dehydration after an overnight
washing in running water.

SCHEDULE OF ACTIVITIES
PROCEDURE & REAGENT TIME REQUIRED PROCESSING SCHEDULE

1. FIXATION
10% formalin 2 – 6 hours _____________
Wash in running tap water 30 mins - 1 hr _____________
2. DEHYDRATION
70% Ethyl Alcohol (2 changes) 1 hr in each reagent _____________
80% Ethyl Alcohol 1 hour _____________
95% Ethyl Alcohol (2 changes) 1 hr in each reagent _____________
3. CLEARING
Xylene ( 2 changes) 30 min each reagent _____________
4. INFILTRATION (Inside paraffin oven)

Melted paraffin 1 1 – 1 ½ hours _____________
Notes:
▪ The time of processing may vary depending on the type and size of tissue.
▪ The temperature setting of the paraffin oven should be at least 5 – 10O higher than the
melting point of the medium being used.
Individual Task
Copy and paste images from reliable sources or draw the following:
1. Preparation of tissues for processing (step no. 1 of Part II procedure)
2. Procedural steps in tissue processing (fixation, washing, dehydration & clearing – indicate
reagents)
3. Infiltration (in a paraffin oven)
Preparation of tissues for processing

Procedural steps in tissue processing
Infiltration


1. What are the different ways in performing paraffin infiltration? Make a
schematic diagram showing the procedural steps performed in each.
2. How many times may a paraffin wax be used for infiltration? Justify your
answer.
Summing It Up
Impregnation involves the process of completely removing the clearing
agent from the tissue and replacing it with a medium to completely fill the
tissue cavities. An impregnated tissue sample will then be embedded using the
same medium used in infiltration. Embedding is a process wherein an
impregnated tissue is placed precisely in a mold with a medium. Both of these procedures
allow easier handling and sectioning of the tissue sample.
There are four general types of media used in histopathology. These are paraffin,
celloidin, gelatin and plastic. An embedding medium must be capable of being converted
readily from liquid to its solid form via any of the following mechanisms: (1) crystallization, (2)
evaporation of the solvent, or (3) polymerization.
Paraffin processing is considered as the simplest, most common and best method for
routine purposes. Factors that affect this type of processing include the nature and size of the
tissue sample, clearing agent used, temperature and the number of changes required. Paraffin
impregnation may be performed via manual, automatic or vacuum techniques. Manual
technique is performed at a temperature 2-5 OC higher than the melting point of the wax with
4 changes. Automatic technique is carried out at a temperature of at least 3 OC higher than
the melting point of the wax, 2-3 changes with constant agitation. Vacuum technique requires
a temperature that is 2-4 OC higher than the melting point of the wax with 3 changes at a
negative pressure.
Paraffin embedding requires the use of a melted paraffin with a temperature of 5 to
O
10 C above the melting point of the wax. After which, paraffin blocks are cooled via (1)
refrigeration at -5 OC, immersion in cold water or with the use of a cold plate.
Substitute media for paraffin may also be utilized. Examples are paraplast, embeddol,
bioloid, tissue mat, ester wax or water-soluble waxes. Ester wax has a lower melting point
than paraffin but it is harder, thus, it requires the use of heavy-duty microtomes during
sectioning. Ester wax is also soluble in 95% ethanol and as such, the clearing procedure may
be omitted. Water-soluble waxes, the most common of which is carbowax, have the
advantage of being miscible in water. Since it is already miscible in water, the dehydration and
clearing procedures may be omitted. The disadvantage of water-soluble waxes is their
hygroscopic nature. Tissues embedded in these media should never come in contact with
water or even ice and this becomes a problem during flotation of sections. In order to remedy
this, soap or 10% polyethylene glycol 900 may be added to the waterbath or floating solutions
such as Pearse or Blank & McCarthy solutions may be utilized.

Assessing Oneself

Across Down
4. mechanism of solidification of 1. microtome used for ester wax blocks
carbowax 2. paraffin wax substitute that contains rubber
7. characteristic of water-soluble waxes 3. paraffin substitute used for eye samples
9. mechanism of solidification of plastics 5. fastest paraffin impregnation technique
10. floating solution for ester wax 6. mold that contains a cold plate
sections
8. dehydration and clearing are omitted with the use
of this medium

Level II: True/False – Right Minus Wrong

Read and understand the statements and evaluate whether they are true or false. Write/Type
true on the space before the number if the statement is correct, otherwise, write/type false if
the statement is incorrect. Do not guess your answer since wrong answer will be deducted
from your score. If you are not sure of your answer, you may leave the item blank in order to
avoid incurring any deduction.
__________ 1. Three changes of carbowax is needed for routine processing. One each in 70%, 90%
and 100% concentrations.
__________ 2. Tissue mat is a product of paraffin. It has the same property as paraplast.
__________ 3. Paraplast has a melting pint of 56-57 oC. Serial sections are obtained easily.
__________ 4. Pearse solution and Blank & McCarthy are used for floating ester wax sections.
Both are added to the waterbath to straighten sections.
__________ 5. If the laboratory temperature is 20 oC, a wax with a melting point of 55 oC may
be used. If manual processing is performed, the temperature should not
exceed 60 oC.
Part II: Other Media and Embedding Molds

CELLOIDIN PROCESSING
✓ Purified pyroxylin nitrocellulose
✓ Suitable for specimens containing large cavities or hollow spaces which
tend to collapse (eyes) & for larger embryos
✓ Available in thin (2%), medium (4%) and thick (8%) solutions
✓ DIS: Tissues cannot be cut as thin as they are with paraffin wax
✓ ADV: Causes much less shrinkage & distortion; slow process
➢ With rubbery consistency – w/o distortion
➢ Does not require heat
❖ Low Viscosity Nitrocellulose
➢ Soluble in equal concentrations of ether and alcohol
ADVANTAGES:
✓ Can be used in higher concentrations
✓ Produces harder tissue blocks
DISADVANTAGES:
o Cracking of tissue sections and chrome-mordanted tissues may crumble
→ REMEDY: add plasticizers like Castor oil, Oleum ricini
o More explosive than celloidin
o Cannot be stored

WET METHOD
❖ for bones, teeth large brain sections & whole organs
❖ Fixation → Dehydration → Equal parts ether and alcohol (12-24 hours) → Thin
celloidin: 2-4 % (5-7 days) → Medium celloidin: 4-6 % (5-7 days) → Thick celloidin: 8-
12 % (3-5 days) → Embedding: fresh thick celloidin in a jar or desiccator (fingerprint no
longer leaves a mark on block surface) → Storage: 70-80% alcohol
DRY METHOD
❖ Same with wet method EXCEPT for the following steps
✓ Gilson’s mixture (chloroform + cedarwood oil) is added to the fresh thick
celloidin during embedding
✓ Storage in alcohol is contraindicated
PLASTICS
❖ Provided superior results for light microscopic studies especially of hard tissues and
samples for high resolution microscopy
TYPES OF PLASTIC MEDIA

1. EPOXY (epoxy plastic, catalyst & accelerator)
➢ Araldite base (bisphenol) - slowest
➢ Glycerol base (epon)
➢ Cyclohexene Dioxide (spurr) - fastest
Disadvantages:
➢ Reduce antigenicity
➢ Sensitization on skin contact and inhalation
➢ Contains toxic components (eg. Vinylcyclohexane dioxide)
2. POLYESTER
3. ACRYLIC
➢ For high resolution LM
➢ Glycol Methacrylate (GMA): valued for its hydrophilic nature
➢ Methyl Methacrylate (MMA): valued for its hardness
➢ Benzoyl Peroxide – added as catalyst
→ acts as an active site (polymerization of acrylics)
GELATIN PROCESSING
✓ Histochemical and enzyme studies
✓ Delicate specimens
✓ Frozen sections
✓ Water-soluble
✓ Low MP and does not overharden
❖ Fixation → washing → 10 % (24 hours) → 20 % (12 hours) → 20% until
impregnation and embedding are complete → 10 % formalin (12-24 hours)
**All gelatin reagents contain 1 % phenol

ORIENTATION
✓ Arranging the tissue in the mold: embedding
✓ Fixing the tissue block on the microtome: prior to sectioning
✓ Arranging the tissue ribbons on the slide: flotation
Types of Embedding Molds

1. Leuckhart’s embedding mold
✓ 2 L-shaped heavy metal arranged flat on a metal surface
2. Compound E unit
✓ with several interlocking plates making several compartments
3. Disposable Molds
a. Peel-away (thin plastic Embedding molds)
b. Plastic Ice Trays
c. Paper boats
✓ ADV: cheap, diff sizes, avoids confusion
4. Plastic Embedding Rings & Base Molds
Enrichment Activity
ACTIVITY 2b: MANUAL TISSUE PROCESSING – EMBEDDING
OBJECTIVES:
At the end of this activity, you are expected to
1. know the different kinds of embedding media; and
2. acquaint yourself with the different kinds of molds used in embedding.

principles which will be applied in performing the different steps in preserved tissue
preparation.
Following complete infiltration, the following are the procedural steps in paraffin
embedding:
1. Remove the pieces of tissues from the gauze/tissue cassette.
2. Pour freshly melted paraffin into the molder (paper boats).
Note: Depth of the paraffin should be about twice the size of the tissue
3. Orient (observe correct plane of sectioning) a piece of the processed tissue into
the melted paraffin using warm forceps of applicator stick.
4. Place the label on top of the melted paraffin and transfer the filled paper boat
in a shallow pan of cold water or in a chilled enamel tray.
5. After the paraffin has hardened into block, keep paraffin block inside the
refrigerator overnight.
❖ Guidelines on Orientation: Orientation should be such that the resistance the tissue
offers the knife proceeds from the lesser amount toward the greater amount as the block
is sectioned. This presents the harder tissue from compressing the softer tissues above

and produces much smoother sections. There must be an adequate margin (2mm
minimum) of embedding medium surrounding all sides of the tissue.
1. Tubular structures such as vas deferens, veins and fallopian tubes must be embedded
so that the knife cuts across/perpendicular to the long axis of the tube. Placement
should be as vertical as possible.
2. Tissues with a epithelial surface such as skin, intestines, gallbladder, urinary bladder
and uterus must be positioned so that the epithelial surface is at the top of the block
so that it will be cut last.
3. Multiple soft tissue fragments and lymph nodes should be place side by side with
space between them.
4. Multiple small rectangular tissues should be oriented with their long axis nearly
parallel to the knife edge to minimize pressure distortion and wrinkling.
5. Small bisected cysts have a dome shape. The cyst should be embedded with the cut
surface down so that the knife cuts through all layers of the cyst wall.
Note: Paraffin blocks may be stored for an indefinite period of time
Individual Task
1. Procedural steps in making a paper boat
2. Procedural steps in embedding
3. Different kinds of embedding molds
4. Proper orientation of tissues (tissue types 1 – 5)
Steps in making a paper boat

Types of Embedding molds
Embedding procedure

PROPER ORIENTATION OF TISSUES

Tubular structures Tissues with an epithelial Small bisected cysts
surface
Multiple soft tissue fragments and lymph Multiple small rectangular tissues
nodes

1. Aside from paraffin and its substitutes, what are the other media used for
infiltration and embedding? Make a schematic diagram showing the
procedural steps performed in each.
2. Give the working principle and briefly describe three automated
machines that can be used in infiltration and embedding.

Summing It Up
Other media which may be used for infiltration and embedding includes
celloidin, plastic and gelatin. Celloidin is a purified form of pyroxylin
nitrocellulose. It is available in three concentrations: (1) thin (2%), (2) medium
(4%) and thick (8%). Celloidin is utilized for specimens with large cavities and
hollow spaces which tend to collapse. It is also used for larger embryos, hard and dense tissue
specimens. Low-viscosity nitrocellulose is a type of celloidin that is preferred over ordinary
celloidin. The two types of celloidin processing are the wet and dry methods. The wet method
requires storage of celloidin blocks in 70-80% alcohol and is utilized for bones, teeth, large
brain sections and whole organs. On the other hand, the dry method does not require storage
in alcohol but incorporates Gilson’s mixture during the embedding procedure. The dry
method if used for whole eye samples.
Plastics provide superior results for light microscopic studies especially of hard tissues.
In addition to this, plastics are the ones which are used for electron microscopy. There are
three types of plastic media: (1) epoxy, (2) polyester and (3) acrylic. Polyester plastics were
once used for electron microscopic but have already been replaced by epoxy plastics (araldite,
glycerol-based and cyclohexene dioxide). Acrylic plastics (glycol methacrylate and methyl
methacrylate) on the other hand are used for high resolution light microscopy.
Gelatin medium is utilized for delicate specimens, frozen sections, histochemical and
enzymes studies. It has the advantage of being water-soluble, thus, dehydration and clearing
procedures may be omitted. However, due to the advent of better media, gelatin is rarely
used nowadays in tissue infiltration and embedding.
Orientation is a very important step in embedding. This involves properly arranging the
tissue sample in the mold in order to make suitable tissue sections. Depending on the
orientation of the tissue sample, a medical laboratory scientist may be able to produce cross,
longitudinal or oblique sections from the tissue block.
The embedding procedure requires the use of a mold in order to form tissue blocks.
Depending on the laboratory, several embedding molds are available for use. These include
Leuckhart’s embedding mold, compound embedding unit, peel-away mold, plastic ice trays,
paper boats, tissue tek system, plastic embedding rings and base molds.

Assessing Oneself

Across Down
2. acts as a catalyst in acrylic polymerization 1. acrylic that is hydrophilic
6. ideal medium for undecalcified bone 3. jar utilized in celloidin embedding
samples 4. slowest epoxy plastic
8. fastest epoxy plastic 5. mixture composed of CHCl3 & cedarwood oil
9. plasticizer added to LVN 7. reagent that prevents the growth of molds
10. hazardous solvent used in celloidin in gelatin medium


__________ 1. Methyl methacrylate is an epoxy plastic that is compatible with a wide range of
staining techniques because of its hydrophobic nature.
__________ 2. Epoxy plastics have to be stored in amber bottles to prevent premature
polymerization.
__________ 3. During tissue infiltration, the surface of the section to be cut should be
perpendicular to the bottom of the mold.
__________ 4. Celloidin is suitable for hard and dense tissue samples and it is also
recommended for processing neurological tissues.
__________ 5. Lower viscosity of acrylic plastics allows staining to be faster and at a longer
period of time.
your instructor.
platform Quizziz.
messenger.
message.
References
W.B. Saunders Co.

LESSON 7: SECTIONING AND ADHESION OF TISSUE SECTIONS

1. identify the different types of microtome, explain the working principle and use/s of
each;
2. describe the microtome knives and identify the use/s of each;
3. enumerate ways on proper handling and maintenance of microtomes and microtome
knives;
4. identify appropriate measures to prevent and resolve problems encountered during
sectioning; and
5. explain the procedures performed to ensure proper adhesion of tissue sections on
slides.
Lesson Proper
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:
✓ 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.
TYPES OF MICROTOME
Microtome Thickness Inventor Characteristics
ROCKING 10-12 um Paldwell *simplest microtome
Trefall *for small and large paraffin-embedded blocks
*not for serial sections
ROTARY 4-6 um Minot *most common microtome
*for paraffin embedded blocks
SLIDING 4-9 um Adams *most dangerous
STANDARD SLIDING: celloidin
BASE-SLEDGE: hard and tough tissue blocks in all forms
of media

ULTRATHIN 0.5 um *for electron microscopy

*small specimen fixed in osmium tetroxide and
embedded in plastic
FREEZING 10-15 um Queckett *undehydrated tissues in frozen state
*fats and heat sensitive tissue constituents
COLD MICROTOME/CRYOSTAT
✓ rotary microtome inside a cold chamber
✓ temperature is maintained between -5 to -30 OC (ave. is -20 OC)
Enrichment Activity
ACTIVITY 3: TRIMMING AND CUTTING OF PARAFFIN BLOCKS
OBJECTIVES:
1. acquire the skill in cutting paraffin sections using the rotary microtome
and know the difficulties encountered during the cutting process, their
causes and possible remedies; and
2. develop the skills in flattening cut paraffin sections, “fishing-out” of
ribbons, and mounting of sections onto the slide.
principles which will be applied in performing the different steps in trimming blocks and
sectioning.
A. Trimming
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 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.
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.
Individual Task
1. Cutting thin sections with the rotary microtome (show ribbon formation)
2. Relationship of tissue block with the microtome knife showing all the angles (bevel,
wedge, clearance and rake angles; indicate angle degrees)
3. Other Microtomes: Cambridge, Sliding and Freezing
Cutting (ribbon formation)

Relationship of tissue block with the microtome knife
Bevel angle:
____________________
Wedge angle:
____________________
Clearance angle:
____________________
Rake angle:
____________________
Cambridge microtome

Sliding microtome
Freezing microtome

1. In a tabular form, identify the problems encountered during sectioning,
their causes and possible remedies.
2. What do serial sections mean?

KINDS MICROTOME KNIVES

Length Use
Light Plane-Concave 25 mm Plane side → celloidin
Microscopy Concave → paraffin
Plane-Wedge 100 mm Frozen sections
Extremely hard and tough specimens in paraffin
Biconcave 120 mm Paraffin section
Electron Diamond-edge Resin blocks
Microscopy Broken Glass Trimming
Knives Semi-thin sectioning
Others Disposable Sharp cutting edge capable of producing 2-4 um
blades sections
Safety razor 10 um and above
blades Partially calcified tissues, paraffin and frozen
sections
ANGLES DURING SECTIONING

1. Clearance angle (5-10 degrees)
✓ between the edge of the knife & the tissue block
2. Wedge angle (15 degrees)
✓ angle of cutting
3. Bevel angle (27-32 degrees)
✓ 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
HONING STROPPING
Purpose Removal of gross nicks and blemishes Removal of burrs, for final polishing and
sharpening of the cutting edge
Material Smooth stones Horse leather
Belgium yellow: best result
Arkansas: more polishing effect
Fine carborundum: coarsest

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

oil, Clove oil) , Xylene, or liquid
paraffin
Direction Heel-to-Toe, edge first Toe-to-Heel, edge last
No. of 20-30 strokes in each direction 40-120 double strokes
strokes 10-20 strokes each surface (Minot and
Plane-wedge knives)
OTHER HONING PROCEDURES

❑ Plate Glass Honing
➢ Diamantine: used for final polishing
❑ Factory Grinding
➢ After repeated sharpening
➢ Widened Bevel angle (> 35O)
❑ Automatic hones
PRECAUTIONS IN MICROTOMY
✓ Cutting edge must be sharp & smooth
✓ Cutting edge must be thinner than the section to be cut
✓ Knife and its corresponding knife back should not be interchanged
✓ Honing and stropping
PROPER CARE OF THE MICROTOME KNIFE

The microtome knife is an expensive piece of equipment and great care must be taken of it.
The following points must be observed.
1. Store the knife in its box when not in use.
2. Never lay a knife flat n the bench.
3. Never allow an edge to become badly “nicked”. It is advisable to have a second knife
for cutting bone and hard tissues.
4. The edge of the knife should be “touched up” daily before use.
5. Always use a back, when required, for sharpening. This is not necessary with bi-
concave knives.
6. The knife should be cleaned with xylene or toluene before and after use. Never allow it
to become rusty.
7. Never lend or borrow a knife.

Enrichment Activity
ACTIVITY 4: SHARPENING OF MICROTOME KNIVES
(HONING and STROPPING)
OBJECTIVES:
1. gain adequate knowledge and skills in proper sharpening techniques; and
2. gain adequate knowledge in the proper care for the microtome knife.
The degree of sharpness is proportional to the fineness of the abrasive used in

sharpening. The final sharpening materials used must have a grain size smaller than the
permissible serrations remaining in the edge. Sharpening may be carried out either by hand or
machine.
principles which will be applied in performing the proper sharpening of microtome knives.
HONING:
The hone is first wiped clean with soft cloth (moistened with xylene to remove loose
particles of stones and metals). The knife is fitted with its own back and laid obliquely on
the stone, edge forward. Gentle, even pressure is made on the knife with the thumbs or
forefingers, and the knife is drawn obliquely forward on the stone in an easy, steady
motion, “heel” (handle end) first, so that when the other end of the stone is reached, the
“toe” is on the stone.
The knife is then rotated on its back, so that the other cutting facet rests on the
stone, and the knife is drawn toward the operator, again from heel to toe direction. Such
sequence forms a double stoke, and about 20 to 30 such strokes on the medium-grain
stone and an equal number on the fine-grain stone until all the nicks in the knife edge
have been eradicated. The knife is then wiped clean with a rag moistened in xylene.
The edge may then be inspected under the microscope whether it is ready for
stropping. Occasional small nicks in the knife-edge may be left, as they will gradually be
honed out, and the knife can be set in the microtome so that the nicks can be avoided.
Large nicks demand regrinding of the edge, and this is best done at the factory.
Precautions:
▪ Excessive honing and excessive pressure may turn the edge or make the edge round
▪ Too little pressure on the blade results to inadequate honing
▪ Jerky motion leads to nicking
STROPPING:
Theoretically, a perfectly honed knife does not require stropping, however, in
practice, some stropping is still required to remove “burrs” formed during honing. Strops
should be of best quality shell-horse leather made from the rump or thick part of the

horsehide. The leather or strop surface must be of good size (3 to 4 by 18 inches) and
should be mounted on a solid wooden block.
To strop, the knife is first fitted with its appropriate knife-back, then laid
obliquely on the strop and pushed backward and drawn forward (toe to heel), edge last
(the opposite of honing). About 40 to 120 double strokes are usually required. Finally,
when stropping is completed, the knife edge is oiled or greased to prevent rusting. The
knife is then placed in its suspension box and stored.
Precautions:
▪ Excessive stropping will turn the edge and may “burn” it (may produce localized dark
areas).
▪ The knife-edge must be wiped clean after each series of stropping, and before changing
from coarse to fine strop, so as not to carry over any particles.
Individual Task
1. Proper honing and stropping techniques
2. The different kinds of microtome knives.
(Indicate the knife description, type of tissue block and type of microtome the knife is
used)
Honing (forward and backward strokes) Stropping (forward and backward strokes)

Microtome knife (Description) Tissue block & Microtome

Used on:
Tissue block/s:
______________________
______________________
______________________
Microtome:
Plane-wedged knife: ______________________
__________________________________________________ ______________________
__________________________________________________ ______________________
Used on:
Tissue block/s:
______________________
______________________
______________________
Microtome:
Plane-concave knife: ______________________
__________________________________________________ ______________________
__________________________________________________ ______________________
Used on:
Tissue block/s:
______________________
______________________
Microtome:
Biconcave knife: ______________________
__________________________________________________ ______________________
__________________________________________________ ______________________
__________ _____
Disposable blades

GENERAL STEPS IN FIXING SECTIONS ONTO SLIDE

1. Adhesion: performed to prevent washing out of tissue sections during staining
❖ Adhesive is a substance which can be smeared onto the slides so that sections stick
well to the slides
• Mayer’s egg albumin (egg white + glycerin + thymol)
➢ Most commonly used (easy to prepare, convenient and relatively
inexpensive)
• Dried Albumin (dried albumin + NaCl + thymol)
➢ Sections are dried and stored in 70% alcohol until it is ready for staining
• 1% Gelatin (gealtin + glycerol + phenol)
➢ Added to the waterbath during flotation rather than applying it on slides
• gelatin-formaldehyde mixture (gealtin + formaldehyde)
➢ slides are coated and allowed to dry at 37 OC for one hour or overnight
prior to use
• poly-L-lysine
➢ aqueous detergent diluted to 0.01% concentration
➢ widely used in immunohistochemistry
• APES (3-aminoprophylthriethoxysilane)
➢ Very useful in cytology, particularly for cytospin preparations of
proteinaceous or bloody material
2. Floating on water bath: 45-50 OC or 6-10 OC LOWER than the MP of wax; to straighten and
tissue sections
3. Fishing out: waterbath should have a temperature that is 10 OC lower than the melting
point of the wax
4. Orientation: correct positioning of the tissue section/ribbon on the slide
5. Deparaffinization and Drying Sections
✓ wax oven (56OC – 60OC for 2 hrs)
✓ Incubators (overnight)
✓ Hot plate (45OC – 55OC for 30 – 45 mins.)
✓ Alcohol lamp/ bunsen flame
✓ Delicate tissues: 37 OC for at least 24 hours
✓ Blower-type electric slide dryer (50 – 55 OC for 20 – 30 mins.)
6. Post-mordanting
✓ Secondary fixation (post-chroming)
✓ 2.5 – 3% K2CrO4 for 24 hrs
✓ Used primarily as mordant & secondary as fixative
✓ 5 – 10 mins. in either
➢ Aq. Sol’n. of HgCl2
➢ Aq. Picric acid

Enrichment Activity
ACTIVITY 5: ADHESION AND DRYING OF TISSUE SECTIONS ONTO THE SLIDE
OBJECTIVES:
1. develop the skills of stretching tissue sections and mounting them onto
the glass slide; and
2. gain knowledge on the different adhesives used in mounting tissue
sections.
principles which will be applied in performing the different steps in adhesion and drying of
tissue sections on slides.
PROCEDURE:
1. Dip a clean glass slide into 95% ethyl alcohol and xylene. Wipe dry.
2. Thinly smear Mayer’s egg albumin on one side of the slide.
3. Carefully spread a selected strip of tissue ribbon onto the slide, arranging it to ensure
adhesion
4. Add a drop of water on one edge of the tissue ribbon. By capillary action, the water
will spread throughout and underneath the tissue ribbon.
5. Place the slide on the warming plate or stretching table set at about 43 – 45 OC.
6. Carefully tease out the paraffin surrounding the tissue by pointed sticks and wipe off
with gauze pad (Do this carefully and cautiously so as not to damage the tissue
section)
- or -
1. Float a strip of paraffin ribbon on the flotation bath. Visually select the best sections
and carefully tease these away from the other sections with a teasing stick or heated
scalpel.
2. Hold the selected ribbon and fish-out by slipping the slide directly under the ribbon.
3. Lift the slide from the bath, and let it stand upright for a few seconds to allow the
water to drain off.
4. Blot the end of the slide and transfer to warming plate.
Individual Task
1. Fishing-out of the paraffin ribbons from the water bath.
2. Transferring the paraffin sections (from the microtome) onto a slide.
3. Proper flattening and stretching of the paraffin ribbons/sections,
a. on a flotation bath
b. on a slide

Fishing-out of the paraffin ribbons from the flotation bath
Transferring of the paraffin sections (from the microtome) into a slide
Flattening and stretching of the paraffin ribbons/section

on a flotation bath on a slide

Work with your assigned groupmates and answer the following questions
for research. Make sure to understand the concepts being asked in each
question.
1. What is the effect of too much application of Mayer’s egg albumin onto
the slide?
2. How do you clear prepared slides prior to staining?

Summing It Up
Sectioning, also known as microtomy, involves cutting of uniformly
thin slices or sections from a previously processed tissue block. Sectioning is
performed using a microtome which is an instrument capable of cutting
sections at a predetermined thickness by sliding the block into a cutting tool
(steel knife or blade).
There are several types of microtome which may be used in sectioning. The type of
microtome to be utilized is dependent upon the type of tissue sample, the medium used and
the type of microscope which will be used for examination. The rocking microtome is
considered as the simplest type. It is used produce sections that are 10-12um thick from
small and large paraffin embedded blocks. The rotary microtome is the most commonly
used both in routine and research laboratories. It is the type of microtome that is
incorporated in cryostats. It is used to produce sections that are 4-6um thick from paraffin
blocks. Sliding microtomes are heavy-duty microtomes often times used for hard tissues and
blocks. There are two types of sliding microtomes. The standard sliding microtome is used
for celloidin blocks, while the base-sledge sliding microtome is used for hard and tough
tissues blocks. Both types are capable of producing sections that are 4-9um thick. Ultrathin
microtomes are used for electron microscopy and is capable of producing sections that are
0.5um thick. The freezing microtome is used to produce frozen sections that are 10-15um
thick. It makes use of carbon dioxide as the freezing agent.
The type of microtome knife to be used during sectioning depends upon the type of
microscopic examination of be performed. Plane-concave, plane-wedge and biconcave
knives are used for light microscopy. Diamond-edge and glass knives are used for electron
microscopy. In addition to these knives, disposable and safety razor blades may also be used
in cutting tissue sections.
It is essential that regular sharpening is performed on the microtomes knives in order
to maintain its cutting edge. This involves honing and stropping the knives using hones and
horse leather respectively.
After microtomy, adhesion, flotation, orientation, deparaffinization and drying of
sections are performed in preparation for staining

Assessing Oneself
comprehension regarding the lesson. Once you start answering the
activities, do not go back to the Lesson Proper notes as this would defeat the
purpose of this portion of the module. These activities will not be recorded
but you are required to compile them as part of your journal to be checked every grading
period.

Across Down
2. heel-to-toe, edge first 1. microtome invented by Cambridge
6. freezing agent used in freezing microtome 3. knife that measures 120mm
9. substance that prevents the growth of 4. inventor of the rotary microtome
molds in Mayer's egg albumin 5. microtome referred to as the cold knife
11. angle of cutting 7. used for final polishing of glass knives
13. substance that prevents the growth of 8. removes burrs from knives for final
molds in 1%gelatin polishing
14. adhesive for immunohistochemistry 10. type of block sectioned by the plane side
15. angle between the edge of the knife and of a plane-concave knife
the tissue block 12. adhesive that is useful for cytospin
preparations


Match the following microtomes to the thickness of the sections produced (Column A), the
inventor (Column B), the type of sections produced (Column C) and the characteristic of
each (Column D).
MICROTOME A B C D ANSWERS
1. Cambridge A. 0.5 u A. Minot A. paraffin sections A. neurological
structures
2. Freezing B. 4-9 u B. Adams B. celloidin sections B. most
common type
3. Ultrathin C. 10-15 u C. Paldwell C. frozen sections C. most
Trefall dangerous
4. Standard D. 4-6 u D. Queckett D. None of the D. small tissues
Sliding above embedded in
plastic
5. Rotary E. 3-4 u E. None of E. simplest type
the above
F. 10-12 u
your instructor.
B. Summative assessments will be given by the instructor via several modalities
depending on your connectivity status.
platform Quizziz.
messenger.
message.
References
W.B. Saunders Co.

LESSON 8: STAINING
1. discuss and differentiate the various principles and methods of staining;
2. enumerate in the correct sequence the different reagents used in Hematoxylin &
Eosin staining technique and explain the purpose of each;
3. identify the appropriate special stain used to demonstrate a given tissue component
or organism as well as the respective color exhibited; and
4. explain the procedures performed in re-staining old sections and broken tissue slides.

Try to answer the following activity by remembering what you have learned
in your previous subjects. This is a self-assessment activity and will not be
graded but should be compiled as part of your journal to be checked every
grading period. Refer to Appendix A for the answers.
Across Down
1. storage form of carbohydrates 2. iodine solution used to determine the
3. tests that utilizes Milton's reagent presence of starch
4. storage form of iron in macrophages 5. substance that bridges the tissue & the dye
7. storage form of lipids 6. type of fiber demonstrated by Taenzer-Unna
orcein
9. most abundant connective tissue fiber
8. deposits demonstrated by Von Kossa
10. structural protein present in chromosomes

Lesson Proper
Part I: Principles & Methods of Staining and H& E Technique
PRINCIPLES OF STAINING
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
➢ Acidic structures—greater affinity for basic dyes
➢ Basic structures—greater affinity for acidic dyes
**Generally, two contrasting stains are used to facilitate differentiation of cellular structures
and constituents
Impregnation
✓ related procedure that makes use of heavy metal salts which are selectively
precipitated on certain cellular and tissue components
✓ used for silver staining of nervous tissue and demonstration of reticulin
✓ most commonly used agent: silver nitrate—may also be used as a staining agent
THREE MAJOR TYPES

1. HISTOLOGICAL STAINING
✓ tissue constituents are demonstrated in sections by direct interaction with a
dye or staining solution
✓ producing coloration of the active tissue component
✓ employed to demonstrate the general relationship of tissues and cells with
differentiation of nucleus and cytoplasm
✓ Examples: microanatomic stains, bacterial stains and specific tissue stains
(muscles, CT, and neurologic stains)
2. HISTOCHEMICAL STAINING/HISTOCHEMISTRY
✓ tissue constituents are studied through chemical reactions that will permit
microscopic localization of a specific tissue substance
✓ Examples: Perl’s Prussian blue reaction for hemoglobin, Periodic Acid Schiff
staining for carbohydrates
✓ Enzyme histochemistry: active reagent serves as the substrate upon which the
enzymes act
➢ final opacity of coloration produced from the substrate rather than the
tissue
3. IMMUNOHISTOCHEMICAL STAINING
✓ combination of immunologic and histochemical techniques that allow
phenotypic markers to be detected and demonstrated

✓ makes use of different labels: monoclonal/polyclonal, fluorescent-labeled,

enzyme-labeled antibodies
METHODS OF STAINING
1. DIRECT STAINING
✓ process of giving color to the sections by using aqueous or alcoholic dyes
2. INDIRECT STAINING
✓ 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
✓ 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
✓ no decolorization or washing after the application of the dye
4. REGRESSIVE STAINING
✓ 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
DIFFERENTIATION/DECOLORIZATION: selective removal of excess stain from the tissue so
that a specific substance may be stained distinctly from surrounding tissues
5. METACHROMATIC STAINING
✓ use of dyes which differentiate particular substances by staining them with a
color that is different from that of the stain itself (metachromasia)
✓ basic dyes belonging to the thiazine and triphenylmethane groups
Examples: methyl violet or crystal violet
Cresyl blue for reticulocytes
Safranin
Bismarck brown

**ORTHOCHROMATIC STAINING→stain tissues in color shades that are similar to the

color of the dye itself
6. COUNTERSTAINING
✓ application of a different color or stain to provide contrast and background to
the staining of structural components to be demonstrated
Cytoplasmic stains:
Red→ eosin Y, Eosin B, Phloxine B
Yellow→ piciric acid, orange G, Rose Bengal
Green→ light green SF, Lissamine green
Nuclear stains:
Red→ neutral red, safranin 0, carmine, hematoxylin
Blue→ methylene blue, toluidine blue, Celestine blue
7. METALLIC IMPREGNATION
✓ process where specific tissue elements are demonstrated, not by stains, but by
colorless solutions of metallic salts which are thereby reduced by the tissue,
producing an opaque, usually black deposit on the surface of the tissue
✓ agent is not absorbed by the tissue, but is held physically on the surface as a
precipitate or as a reduction product
✓ Examples: ammoniacal silver →reduced by argentaffin cells (melanin and
intestinal glands)
Gold chloride
8. VITAL STAINING
✓ selective staining of living cell constituents, demonstrating cytoplasmic
structures by phagocytosis of the dye particle
**nucleus is resistant to staining
***if nuclear structures are demonstrated→death of cell already
✓ Examples: trypan blue→reticuloendothelial cells; Janus green→mitochondria
INTRAVITAL STAINING
✓ done by injecting dye into any part of the body (intravenous, intraperitoneal or
subcutaneous), producing specific color
✓ Examples: lithium, carmine and India ink
SUPRAVITAL STAINING
✓ stain living cells immediately after removal from the living body
✓ Examples: neutral red, Janus green, trypan blue, etc.

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

Enrichment Activity
ACTIVITY 6: HEMATOXYLIN AND EOSIN STAINING
OBJECTIVES:
1. properly perform the technique of routine Hematoxylin and Eosin
staining;
2. study the chemical basis of stains; and
3. understand fully the principles and purposes of staining.
A video demonstration showing the procedure will be presented by your

instructor. Make sure to listen attentively and take note of the important concepts
and principles which will be applied in performing the different steps in H and E
staining.
PROCEDURE:
REAGENT TIME SCHEDULE
Xylene 2 minutes
Xylene 2 minutes
Absolute alcohol 1 minute
Absolute alcohol 1 minute
95% Alcohol 1 minute
Running water then Rinse for 1 minute
Distilled Water Rinse briefly
Harris Hematoxylin 4 to 8 minutes
Tap Water 4 dips
Acid Alcohol 4 dips
Tap Water 4 dips
Ammonia Water 4 dips (or until blue)
Distilled Water 2 minutes
Eosin 2 minutes
Xylene 2 minutes
Xylene 2 minutes
Drain on filter paper and mount
Note: Slides taken from the oven should be allowed to cool first before staining,
otherwise, the tissue sections would be detached from the slide.

Individual Task
1. H & E procedure (Label the chronological order of reagents and stains)
2. H&E-stained tissue section - Show the macroscopic and Microscopic details (with
distinct nuclei, cytoplasm and basement membrane)
H & E procedure
Stained Slide Microscopic view


question.
1. Discuss the following terms in relation to staining:
a. Fluorescent dyes
b. Argyrophilic
c. Stain oxidizer
2. Describe the quality control in H and E staining.
Summing It Up
Staining allows the study of architectural pattern of the tissue. It
provides optical differentiation and makes microscopic examination easier.
The general rule in staining is that acidic structures have a greater affinity for
basic dyes. On the other hand, basic structures have a greater affinity for
acidic dyes. Impregnation is another technique that is utilized to demonstrate tissue
components. This involves the precipitation of heavy metals on specific tissue components.
There are three types of staining performed in the laboratory—histological,
histochemical and immunohistochemical. Histological staining involves a direct interaction
between the dye and the tissue. Histochemical staining involves a reaction to take place in
order that results to the coloration of tissue components. Immunohistochemical staining
involves the use of labels and antibodies that detect the presence of specific phenotypic
markers on tissue components.
Methods of staining include direct, indirect, progressive, regressive, metachromatic,
counterstaining, metallic impregnation and vital staining techniques.
Hematoxylin and Eosin staining technique is the recommended staining technique for
histological sections. It incorporates two methods of staining—regressive and
counterstaining. The major steps performed in H and E staining include: (1)
deparaffinization, (2) hydration, (3) nuclear staining, (4) differentiation, (5) bluing, (6)
counterstaining, (7) dehydration and (8) clearing.

Assessing Oneself
period.

Across Down
5. color of proteins after H and E staining 1. Perl's Prussian blue technique is an
6. method that involves administration of the example of this type of staining
dye into the body 2. involves overstaining followed by
7. functions as the decolorizer in H and E decolorization of sections

8. silver staining of nervous tissue is an 3. method that involves staining after the
example of this removal of tissue from the body
10. purpose of ammonia water in H and E 4. method in which the color produced is
11. hastens the speed of staining different from the color of the dye
13. reagent that differentiates direct from 9. hematoxylin that requires 15-30 min of
indirect staining immersion
15. staining that involves antigen and 12. color produced by the cytoplasmic stain
antibody reaction rose Bengal
14. color produced by the nuclear stain
hematoxylin
underline the word/s that make/s the statement incorrect. After which, change or remove
the underlined word/s to make the statement correct. Write/Type the corrected statement
after the given statement.
__________ 1. A specific staining technique may involve more than one staining principle.
__________ 2. In H and E staining, hydration performed after the bluing step makes use of
ascending grades of acetone.
__________ 3. Regressive staining is less preferred than progressive staining due to the use
of a mordant.
__________ 4. Microanatomic stains are capable of directly interacting with tissue
constituents, thus, they are examples of histological staining.
__________ 5. Nuclei should have a blue to blue back color and the cytoplasm should have a
pale pink color after H and E staining.
Part II STAINS AND STAINING SOLUTIONS

NATURAL DYES
HEMATOXYLIN
✓ extracted from the core/heartwood of a Mexican tree (Hematoxylin
campechianum)
✓ Ripening/Oxidizing: exposure to air & sunlight or to strong oxidizing agents
Aluminum Ehrlich’s Sodium iodate Tissues subjected to acid
Hematoxylin decalcification and acidic tissues
Harris’ Mercuric chloride Routine nuclear staining,
exfoliative cytology and staining
sex chromosomes
Cole’s Alcoholic iodine Routine purposes, esp in
sequence to Celestine blue
Mayer’s Sodium iodate Cytoplasmic glycogen

Iron Weigert’s Ferric ammonium Muscle fibers and connective

Hematoxylin chloride tissues
Heidenhain’s Ferric ammonium Nuclear and cytoplasmic
sulfate inclusions (chromatin,
chromosomes, nucleoli,
centrosomes, mitochondria)
Voluntary muscle striations and
myelin
Phosphotungstic 1% aq. phosphotungstic acid Nuclei, fibrin, muscle striations,
Acid myofibrils, fibroglia
Hematoxylin Collagen, bone, cartilage
Paraffin, celloidin and frozen
sections
Copper For demonstration of spermatogenesis
Hematoxylin
COCHINEAL DYES
✓ Extracted from a female cochineal bug (Coccus cacti)
✓ Treated with alum to produce carmine
Carmine + Picric acid → Picrocarmine: neuropathological studies
Carmine + aluminium chloride → Best carmine: glycogen
ORCEIN
✓ Vegetable dye from lichens treated with ammonia and exposed to air
✓ For staining elastic fibers: dark brown
SYNTHETIC DYES
✓ Coal tar dyes or aniline dyes
✓ Chromophores: produces color
✓ Auxochromes: promotes color retention
✓ DYE: Chromophore + auxochrome
ACID DYES BASIC DYES NEUTRAL DYES
Picric acid Methylene blue Romanowsky
Giemsa
Irishman
OIL SOLUBLE DYES (Lysochromes)
Sudan Black B Most sensitive
Stains phospholipids and neutr1al lipids (BLACK)
Sudan IV Recommended for staining neutral lipids (RED)
(Scharlach R)
Sudan III First to be introduced
Fat stain for CNS tissues (ORANGE)
Other Non-Lysochrome Lipid Stains
✓ Oil Red O: for neutral fats and lipofuchsin (brilliant red)
✓ Osmic Acid: stains lipids black

Assessing Oneself
purpose of this portion of the module. This activity will not be recorded but
you are required to compile it as part of your journal to be checked every grading period.

Across Down
7. synthetic dyes 1. process demonstrated by copper
8. AlCl3 + carmine hematoxylin
9. promotes color retention in synthetic 2. compound produced after oxidizing
dyes hematoxylin
10. oxidizing hematoxylin 3. oil soluble dyes
4. most sensitive oil soluble dye
5. oil soluble dye that colors neutral lipids red
6. color of lipids after osmic acid staining

COMMON STAINING SOLUTIONS

Eosin Most valuable for differential staining of CT & cytoplasm
Acid Fuchsin-Picric Acid Connective tissues
Acridine Orange Discrimination between dead and living cells
DNA: green fluorescence
RNA: red fluorescence
Acridine Red 3B Calcium salts and sites of phosphatase activities
Alcian Blue Acid mucopolysaccharides, more specific for connective tissue and
epithelial mucin
Aniline Blue Conterstaining of epithelial sections
Basic fuschin Deep staining of acid-fast organisms, mitochondria
With picric acid: differentiation of smooth muscles
In Feulgen’s and Schiff’s reagent: detection of aldehydes
In Van Gieson’s: connective tissues, mucin and elastic tissues
Benzidine Hemoglobin
Bismarck Brown Contrast stain for Gram’s, acid fast and Pap’s method
Used for staining diphtheria
Carmine Chromatin stain for fresh materials (smear prep)
With aluminum chloride → glycogen
Celestine blue Routine staining of fixed sections
Congo Red Axis cylinders in embryos
In Krajians method: elastic tissues, amyloid and myelin
Crystal violet Amyloid and platelets
Giemsa Leukocyte differentiation
Gold sublimate Metallic impregnation
Iodine Oldest stain
GRAM’S: microorganisms and fibrin
LUGOL’s: glycogen, amyloid and corpora amylacea
Janus Green B Mitochondria
Malachite Green Ascaris eggs, erythrocytes and bacterial spore
Methyl green Chromatin
Methylene blue Plasma cells
Cytologic examinations of fresh sputum for malignant cells
Methylene violet Nuclei of leukocytes
Neutral red Observing cell granules and vacuoles in phagocytic cells
Night blue Carbol fuchsin substitute in acid-fast staining
Orcein Elastic fibers (Taenzer Unna Orcein Method)
Dermatological studies to demonstrate finest and most delicate skin
fibers
Osmium tetroxide Stains fat
Prussian blue Demonstration of circulatory system by injection
Rhodamine B Fix and stain blood and glandular tissues
Silver nitrate Spirochetes, reticulum and other fiber stains
Toluidine blue Substitute for thionine in fresh frozen sections
Recommended for Nissl granules or chromophilic bodies
Victoria blue Neuroglia in frozen sections

Assessing Oneself
period.

Across Down
4. used for metallic impregnation 1. used to differentiate dead from living cells
6. oldest stain 2. used to demonstrate the finest and most
7. method that makes use of Congo red to delicate skin fibers
stain elastic fibers 3. used to identify Ascaris eggs
8. demonstration of granules in phagocytes 5. demonstration of plasma cells
9. color of DNA in acridine orange
10. used to demonstrate hemoglobin

Part IV: SPECIAL STAINS
GLYCOGEN STAINS
1. Periodic Acid Schiff (PAS) Magenta Red
**PAS with Diastase Control Stain of choice for glycogen (red)
2. Best Carmine Selective and specific for glycogen (bright red)
3. Langhan’s Iodine Mahogany Brown
PROTEINS
1. Alkaline-fast Green Histones and protamines (green)
2. Peracetic acid-Alcian blue Cystine and cysteine (blue-green)
3. Sakaguchi’s Test (Milton’s Arginine (orange-red)
Reagent)
NUCLEIC ACIDS
1. Feulgen Technique Most reliable and specific stain for DNA
DNA: Red-purple
2. Methyl Green-Pyronin DNA: Green or Blue-green
RNA: Rose-red
3. Fluorescein Most widely-used fluorochrome
Apple green
4. Rhodamine Orange-red
5. Acridine Orange DNA: yellow-green
RNA: brick to orange-red
6. Acriflavine DNA: yellow
RETICULIN FIBERS
1. Gomori’s Silver Impregnation Black
COLLAGEN
1. Van Gieson’s Pink or Deep red
2. Masson’s Trichrome Blue
3. Mallory’s Aniline Blue Blue
ELASTIC FIBERS
1. Weigert’s Elastic Tissue Dark-blue or Blue-black
2. Taenzer-Unna Orcein Dark brown
3. Verhoeff’s Black
4. Krajian’s Bright red
HEMOSIDERIN
1. Perl’s Prussian Blue Deep blue

MELANIN AND ARGENTAFFIN GRANULES

1. Masson Fontana Black
CALCIUM DEPOSITS
1. Von Kossa’s Silver Nitrate Black
SPIROCHETES
1. Levaditi’s Black
2. Warthin-Starry Method Black
MYCOBACTERIA AND NOCARDIA
1. Wade-Fite Technique Red
HBsAg
1. Orcein Method Brown-black
RE-STAINING OLD SECTIONS:

1. xylene (24 hours)/gentle heating
2. remove coverslip
3. xylene (30min)
4. bring down to water
5. 0.5 potassium permanganate
6. 5% oxalic acid
7. running tap water
8. re-stain
BROKEN SLIDES:
✓ mount onto another clean xylene-moist slide with Clarite or Permount
-----or-----
✓ transfer to another slide if sections are still intact

Assessing Oneself

Across Down
5. color of glycogen PAS staining 1. protein demonstrated by Sakaguchi's test
7. color of collagen fibers after Van Gieson 2. color of fluorescence in fluorescein staining
staining 3. stains spirochetes black
8. color of hemosiderin in Perl's Prussian blue 4. color of histones and protamines after
10. stains argentaffin granules black alkaline-fast green staining
6. most specific stain for DNA
9. fiber stained black by Verhoeff's

Check the key answers provided at the back portion of this module. Assess
your performance before the scheduled summative assessments.
your instructor.
platform Quizziz.
messenger.
message.
References
4. Bruce-Gregorios, J. (1974). Histopathologic techniques (1st ed.). Philippines:
5. Bruce-Gregorios, J. (2012). Histopathologic techniques (2nd ed.). Philippines:
6. Bruce-Gregorios, J. (2016). Histopathologic techniques (3rd ed.). Philippines:
7. Finkbeiner, W.E. etal.AUTOPSY PATHOLOGY A MANUAL AND ATLAS, 2nd edition,
2009
8. Raphael, S. (1983). Lynch’s medical laboratory technology (4th ed.). Philadelphia:
W.B. Saunders Co.

LESSON 9: MOUNTING AND LABELLING

1. explain the importance of mounting;
2. identify the different types of mounting media and the respective use/s of each;
3. discuss the proper technique of labeling; and
4. explain the precautions observed in labeling tissue samples and slides.

in your previous subjects and lessons. This is a self-assessment activity and
will not be graded but should be compiled as part of your journal to be
checked every grading period. Refer to Appendix A for the answers.
Across Down
5. piece of glass that holds the sample during 1. used to clear frozen sections
microscopic examination 2. used to label frosted-end glass slides with
6. used to label plain glass slides containing tissue sections
tissue sections 3. solvent miscible in mountants for
8. thin sheet of glass that holds the sample in permanent slides
place 4. water-containing substance
7. prevents mold formation in gelatin

Lesson Proper
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
❖ 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
❖ Glycerin
✓ Has a high RI (1.46) and lasts for a few minutes
✓ Provides greater visibility when mixed with water
❖ 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
X stain tend to fade
X does not set in the desired degree of hardness
X solidifies on storage

❖ 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
❖ 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
❖ Brun’s fluid
✓ Recommended for mounting frozen sections from water
B. Resinous Media
❖ Canada balsam
✓ Natural resin extracted from the Canadian tree Abus balsamea
✓ Recommended for whole mounts and thick sections
✓ Sets hard without granulation
DISADVANTAGES
X acidifies and darkens with age and upon exposure to sunlight
X stains are not usually preserved
❖ DPX (dibutylphthlate polystyrene xylene)
✓ neutral colorless solution that dries rapidly
✓ Recommended for small tissue sections
❖ 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
❖ Clarite
✓ 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

Enrichment Activity
ACTIVITY 7: COVERSLIPPING
OBJECTIVES:
1. discuss the proper mounting of stained sections; and
2. properly identify, label and store mounted slides.
A video demonstration showing the procedure will be presented by your instructor.

Make sure to listen attentively and take note of the important concepts and principles which
will be applied in performing the different steps in mounting.
PROCEDURES:
Method A
1. Place small round drop of the mounting medium at the center of the lower extreme
edge of a clean coverslip.
2. Bring the slide up to the edge of the coverslip and invert the side so that the
mounting reagent just touches it.
3. Gently complete the inversion.
Method B
1. Place the slide flat on a clean paper. Place a round drop of mounting medium over
the tissue section.
2. “Roll” coverslip onto slide to expel trapped air by lowering it on one side first with
the other side following.
PROCEDURAL NOTES:
1. Work on a clean, flat surface, such as a lint-free, white, folded towel or paper.
2. The mounting medium should be of good spreading consistency not too thick, nor
too thin. It should drop readily off the rod. If the media is too thin the solvent will
evaporate, leaving air spaces. If it is too thick it will not spread evenly.
3. The size of the drop of mounting media must be sufficient to occupy the entire space
between the coverslip and slide and will vary slightly with the size of the coverslip
used. Experience is necessary to obtain complete film without excess.
4. Following application of the coverslip, excess mounting media must be cleaned from
the slide. The coverslip will not move as much if a few minutes drying time has
elapsed. Clean coverslip edges if necessary, with a camel’s hairbrush moistened with
xylene and gently wiped edges. A light touch is necessary so as not to disturb the
coverslip.
5. Do not allow tissue to dry out at any time during coverslipping or it may later become
opaque.
6. Excess xylene, will mix with the mountant and form bubbles on the slide.
7. Too little mounting medium may cause improper setting of the coverslip.

8. Bubbles formed on the section could be teased out by gently pressing on the
coverslips with a mounting needle.
9. If bubbles cannot be easily removed, place the slide back into the xylene until the
coverslip drops off, rinse, and remount.
Individual Task
1. Procedural steps in Method A
2. Procedural steps in Method B
Procedural steps - Method A
Procedural steps - Method b


question.
1. In a tabular form, enumerate difficulties which may be encountered
during mounting. Give the cause and remedy for each.
Summing It Up
Mounting, otherwise referred to as coverslipping, is performed after
staining. This step is important in order to protect the tissue section from
damage especially during storage. A mounting medium/mountant is applied
between the section on a slide and the coverslip. This will set the section
firmly and prevent the movement of the coverslip.
The following table shows the different mounting media used in coverslipping with
their corresponding refractive indices and uses.
MOUNTANT RI USE/S
AQUEOUS
Water 1.33 For temporary mounting
Glycerin 1.46 For moist sections
Glycerin jelly 1.47 Standard mountant when dehydration and clearing with
xylene are not performed
Farrant’s medium 1.43 For temporary mounting
Apathy’s medium 1.52 For general purpose mounting and methylene blue-stained
nerve preparations
Brun’s fluid For frozen sections
RESINOUS
Canada Balsam 1.524 For whole mounts and thick sections
DPX 1.532 For small tissue sections
XAM 1.52 For permanent mounting
Clarite 1.544 For permanent mounting
Proper labelling of tissue slides is very important in histopathology. Regardless of

whether an automated or manual labelling system is used, adequate policies and procedures
have to be in place to ensure positive identification of processed tissue slides. Labelling may
be accomplishes routinely using chemical resistant pens and pencils. Nowadays,
electronically generated labels are also being used.

Assessing Oneself

Across Down
3. sealing margins of the coverslip 1. type of section mounted using Brun's fluid
4. mounting medium diluted with xylene 2. mortal sin in histopathology
5. recommended for small tissue sections 6. RI is 1.52 and preserves stains well
9. recommended for whole mounts 7. mounting medium that uses pure cane sugar
10. Kaiser's 1880 8. mountant that does not allow OIO
examination

your instructor.
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References
2. Bruce-Gregorios, J. (1974). Histopathologic techniques (1st ed.). Philippines:
W.B. Saunders Co.

LESSON 10: CELL BLOCKING

1. enumerate and describe the specimens suitable for cell blocking;
2. discuss the appropriate method of fixation for specimens used in cell blocking; and
3. explain the various procedures and techniques in cell blocking.

in your previous subjects and lessons. This is a self-assessment activity and
will not be graded but should be compiled as part of your journal to be
checked every grading period. Refer to Appendix A for the answers.
Across Down
2. involves distortion and possible breakdown 1. process of bringing slides to water
of protein structure 3. solid portion formed at the bottom after
5. type of tissue that undergoes exfoliation centrifugation
7. routine embedding medium 4. fluid formed after centrifugation
8. fluid found in the peritoneal cavity 6. fluid surrounding the lungs

Lesson Proper
CELL BLOCKING
✓ involves the preparation of cells by histopathologic procedure
✓ used for diagnosis when cancer is suspected or to provide evidence
of metastatic disease
✓ performed in conjunction with Papanicolaou smear preparation
Materials suitable for the Cell Block procedure:

➢ Sputum containing cells in thick mucus.
➢ Effusions in which cells are growing as a state of cell culture.
➢ Washings containing cell clusters, which are mechanically exfoliated.
**fluids should be collected in an anticoagulant (potassium oxalate, heparin or sodium
citrate-citric acid) when possible
→ 2 ml anticoagulant for each 100 ml of fluid
**samples should be processed in the fresh state promptly
Gross Examination
▪ Describing the general characteristics of the sample and volume received
→ descriptive terms: blood-tinged, clear, yellow, amber, frothy, mucinous
General Processing of Samples

1. Agitate the fluid slightly and pour into test tubes.
2. Centrifuge at medium speed for 15 to 30 min.
3. Pour off supernatant.
4. Prepare two smears for the Papanicolaou technique from the sediment.
5. Pour a small amount of fixative directly into the test tube containing the sediment to
coagulate it and let it stand for 15 to 30 minutes.
6. Wrap the recovered sediment using lens paper or gauze.
7. Immerse in the fixative for 6 to 24 hours depending on the size of the coagulated
sample.
8. Perform routine procedure for dehydration, clearing, infiltration, embedding,
sectioning and staining.

Enrichment Activity
ACTIVITY 8: CELL BLOCK TECHNIQUE
OBJECTIVES:
1. learn the techniques on cell blocking; and
2. know how to handle specimens for cell blocking.

Make sure to listen attentively and take note of the important concepts and principles which
will be applied in performing the different steps in cell blocking.
PROCEDURE:
A. Preparing and processing a sputum
1. Fix sputum and sediments with Gendre’s fluid, Bouin’s fluid or Picric Acid Alcohol
solution made by dissolving Picric Acid 13 g in 70% ethyl alcohol.
2. Enwrap the sputum with gauze and place it in a small wire basket to be dehydrated. In
case of a fluid specimen, the sediment should be dehydrated in each step within
centrifuge tubes throughout the process to embedding as follows: Centrifugation at
1000 r.p.m. for 5 minutes – fixation of the buffy coat in a centrifuge tube containing the
fixative for about 10 minutes – 80% alcohol – absolute alcohol.
3. Embedding in paraffin (same embedding method as in manual tissue processing)
Fixation
1. Place cell block in Zenker’s-formalin solution that has been neutralized by adding 2
grams of lithium magnesium carbonate to 500 ml of the Zenker’s formalin solution.
2. Leave cell block there for 2 hours.
3. Wash in running water 1 to 24 hours, and then prepare for embedding.
Staining
1. Deparaffinize and bring slides to water.
2. Stain with hematoxylin.
3. Wash in running water 5 to 10 minutes, then wash in distilled water 5 minutes.
4. Stain with eosin-azure overnight.
5. Differentiate in 95% ethyl alcohol 2 to 3 hours: The overall microscopic appearance
should be blue with slight green tinge.
6. Dehydrate, clear, and mount.
Individual Task
1. Preparation of specimen for cell block processing
2. A cell block

Preparation of specimen for cell block processing
Cell block (show surface/face of the block)

1. Enumerate at least 5 specific specimens suitable for cell blocking.
Describe and discuss the purpose of performing cell block technique of
each enumerated specimen.
Summing It Up
Cell blocking is a technique involves processing of body fluids that
contains an adequate amount of cellular elements. Samples which are
processed using this technique include effusions, sputum and washings from
cavities or hollow organs. The use of an anticoagulant is recommended when
collecting samples. The samples will then be grossly examines taking note of its
appearance and volume. After which, samples are centrifuged for 10 to 15 minutes
to allow the cells to settle at the bottom (sediment) of the tube. The sediment
formed will then be processed following the routine steps in performing tissue
preparation from fixation up to staining.

Assessing Oneself

Across Down
4. role of 95% ethanol in staining cell block 1. instrument used to separate cells in cell block
sections samples
6. first step to be performed when staining 2. anticoagulant used for cell block samples
cell block sections 3. fixative used for sputum
8. duration of immersing sections of sputum 5. alcohol present in picric acid alcohol
cell block in eosin-azure
7. accumulation of fluid in body cavities or
10. smear preparation technique performed spaces
along with cell blocking
9. volume of anticoagulant per 100 ml of fluid
sample

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References
2009
W.B. Saunders Co.

LESSON 11: AUTOMATED AND RAPID TISSUE PROCESSING

1. describe the different components of automated tissue processors and identify the
function/s of each;
2. discuss the working principle of automated tissue processors; and
3. explain the advantages and disadvantages of automated tissue processing.

your previous subjects and lessons. This is a self-assessment activity and will
not be graded but should be compiled as part of your journal to be checked
every grading period. Refer to Appendix A for the answers.
Across Down
1. other name for cryostat 2. presence of this leads to consistent results in
7. freezing agent in freezing microtome automatic processors
9. step following clearing 3. type of pressure used in vacuum processing
10. also known as freezing microtome 4. step following fixation
5. layer of fatty tissue found under the dermis
6. microtome incorporated in cryostats
8. effect of heat on the diffusion of molecules

Lesson Proper
Part I
AUTOMATED TISSUE PROCESSING
✓ makes use of an automatic tissue processing machine that fixes,
dehydrates, clears and infiltrates tissue
✓ decreases time (approximately 16 hours) and labor needed during tissue
processing → more rapid diagnosis with less technicality
Components of Tissue Processors

1. Electrical Time Clock: controls the time intervals in the immersion and transfer of
tissue samples from one reagent to another
2. Circular Superstructure: contains the basket carrier, receptacle basket and receptacles
3. Transfer Arm: moves the tissues from one processing reagent to another
4. Receptacle Basket: holds the specimen
5. Circular Deck: holds the reagent beakers and paraffin wax baths
6. Beakers: contain the reagents
7. Wax Bath: contains the paraffin
Care of the Tissue Processors

✓ Change reagent solutions depending on the number and sizes of tissue samples being
processed in the laboratory
CHANGING OF REAGENTS
PARAFFIN: presence of odor of clearing agent
**In general, when there are 3 to 4 beakers containing the same solution, only the last
requires a fresh change. Remove the first beaker and discard the solution. Move the others up
in place, decanting the reagent from one to the other beaker to replace evaporated liquids
and make the last change a fresh one.
DEHYDRATING AGENT: 100% → AFTER TWO COMPLETE LOADS
LOWER DEHYDRATING AND CLEARING AGENTS: at least once a week
✓ Presence of odor, contamination or cloudiness in a reagent solution indicates that
changing is required
✓ Wax baths should be filled to the appropriate level
✓ Accumulated wax in beakers or on any surface of the machine must be removed and
any spillage should be wiped
✓ Receptacles and baskets are cleaned of residual paraffin by immersion in xylene
✓ Clean all nylon parts with detergents (do not use acid)
✓ wax bath thermostats should be set at least 3oC above the melting point of the was
✓ check the timing when loading the machine
RAPID TISSUE PROCESSING

❖ Vacuum Tissue Processor
▪ tissues are placed inside a retort chamber

▪ reagents and melted paraffin are moved sequentially into and out of the retort
chamber using vacuum and pressure
▪ each step is customized by adding time, temperature and/or vacuum/pressure
▪ employ alarm systems and diagnostic programs for trouble-shooting
instrumentation malfunction
ADVANTAGES:
✓ vacuum and heat can be applied at any stage
✓ customized schedules for tissue processing produced
✓ fluid spillage contained
✓ fumes eliminated
❖ Microwave Ovens
▪ Shortens processing time (hours → minutes)
▪ Diffusion of the solutions into the tissue is stimulated by increasing the
specimen’s internal heat
▪ Reagents: ethanol, isopropanol or proprietary alcohol mixtures, and paraffin
ADVANTAGES
✓ Eliminates toxic fumes and the use of carcinogens
✓ Provides uncompromised morphology and antigenicity of specimens
✓ Increased efficiency
✓ Environmentally friendly reagents
✓ Greater profitability
DISADVANTAGES
X manual manipulation of solutions
X cost of laboratory-grade microwaves
X proper use of microwave requires calibration and monitoring
❖ Continuous Input Rapid Tissue Processor
▪ Enclosed processor that uses microwave technology, vacuum infiltration and
“molecular-friendly” proprietary reagents
▪ Tissue cassettes are moved through stations with the following reagents:
1. Acetone
2. Isopropanol
3. Polyethylene glycol
4. Mineral oil
5. Paraffin
▪ Microwaves and agitation accelerate the diffusion of solvents in the tissue
ADVANTAGES
✓ Acceptance of tissues into the system at time intervals
✓ Reagents used are environmentally safe
✓ Morphology and quality of the specimens is consistent with that of
traditional tissue processing
DISADVANTAGES
X added cost of the processor
X grossing of the tissue samples requires standardized specimen dissection

Assessing Oneself

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3. moves the tissues from one reagent to 1. glassware used to hold reagents in automatic
another processors
5. clearing agent in continuous input rapid 2. fixative in continuous input rapid tissue
tissue processor processors
9. component that holds the reagent beakers 4. type of reagents used in continuous input rapid
and paraffin wax baths tissue processors
10. controls time intervals in tissue processing 6. frequency of changing dehydrants & clearants
7. type of fixative generally used in microwave
processing
8. procedure that needs to be standardized in
continuous input rapid tissue processors

Part II
FROZEN SECTIONS
❖ Preparation of Frozen Sections
Advantages
▪ Rapid production of sections for intraoperative diagnosis
▪ Diagnostic and research enzyme and non-enzyme histochemistry
▪ Immunofluorescent and Immunohistochemistry methods
▪ Specialized silver stains in neuropathology
Disadvantages
X no serial sections
X structural details tend to be distorted during cutting and handling
X staining is rarely as satisfactory
X freezing artifact may be produced by inappropriate technique
Specimens
✓ Fresh, unfixed tissue
✓ Previously fixed tissue
o 10% buffered formalin
o 10% formol calcium (4oC) for histochemistry and lipids
o Alcohol
o Mercuric chloride fixative
o Potassium dichromate
A. COLD KNIFE PROCEDURE

✓ Required thickness of the tissue: 3 to 5 mm
✓ Freezing agent used: CO2
✓ Temperature requirements
▪ Knife: -40 to -60 oC
▪ Tissue: -5 to -10 oC
▪ Environment: 0 to -10 oC
✓ Thickness of sections produced: 10u (dew line)
B. CRYOSTAT PROCEDURE
✓ Components
▪ An anti-fogging air-circulating system
▪ A drain for defrosting and sterilizing
▪ A shelf-metal block carriers

▪ Rotary microtome mounted at a 45 o angle

▪ Knife, mostly 120 mm wedge knife equipped with antiroll
devices
✓ Temperature requirements
▪ Operating Temperature: 10 to -30 oC
▪ Optimum Working Temperature: -18 to -20 oC
▪ -35 oC: for fatty tissue, skin with fatty subcutis, fatty breast,
omental tissue
▪ -5 oC to -15 oC: for brain, lymph nodes, liver, spleen, kidney,
testis, uterine curettings, soft cellular tumor, and thyroid
▪ -15 to -25 oC: for muscle, connective, pancreas, uterus, cervix,
non-fatty skin, non-fatty breast tissue, ovary, prostate, tongue
and gut
✓ Freezing methods
▪ Liquid nitrogen
➢ Allow tissue to equilibrate
➢ For histochemistry and intraoperative procedures
▪ Isopentane
▪ CO2
▪ Aerosol spray
➢ Fluorinated hydrocarbons
✓ Mounting methods
▪ Media
➢ Water
➢ 20 to 30% bovine albumin
➢ Von Apathy’s gum syrup
➢ Synthetic water-soluble glycols and resins
▪ Procedures
➢ Fresh unfixed sections: wave for a few seconds, or fix
immediately in formol-alcohol (15 + 85 ml)
➢ Formalinized/Fixed tissues: use albumin or Zwemer’s
chrome-glycerin jelly
➢ Place mounted section in a covered coplin jar containing
40% formaldehyde for 1 to 5 minutes.
✓ Operation and maintenance
▪ Leave ON at all times
▪ Special low-temperature oil should be applied on the microtome

Enrichment Activity
ACTIVITY 9: AUTOMATION and RAPID TISSUE PROCESSING IN HISTOPATHOLOGY
OBJECTIVES:
1. learn and understand the principle involved in the automated tissue
processing; and
2. familiarize oneself with the working principle and the working
mechanism/s of each machine.
PROCEDURE:
Examine the charts or illustrations of the automated histopathologic equipment. Identify and
locate the functional parts of the equipment used.
Individual Task
Copy and paste images from reliable sources or draw the following equipment. Indicate the
working principle of each automated equipment
1. Automatic tissue processor
2. Cryostat
3. Microwave processor
Automatic tissue processor (Indicate description and working principle)

Cryostat (Indicate description and working principle)
Microwave processor ((Indicate description and working principle)

Summing It Up
The use of instrumentation in tissue processing is becoming common
nowadays. With the advent of new machines and techniques, automation is
becoming more common especially in healthcare institutions that handle and
process a large number of tissue samples. This is due to the fact that
automation in histopathology decreases the time and labor needed in processing, thereby
increasing efficiency and improving the turnaround time.
Several tissue processors are currently available for use. The carousel-type processor
and the self-contained fluid exchange systems were the first automated tissue processors
used in the histopathology laboratory. Another type of tissue processor is the enclosed, self-
contained vacuum tissue processor which later became the mainstay in most laboratories.
Also, specially designed microwave ovens for tissue processing are now common. This
equipment shortens the processing time from hours to minutes, thereby further improving
the turnaround time. Recent advances in technology have led to the development of an
enclosed continuous input rapid tissue processor. This equipment makes use of microwave
and vacuum that significantly shortens the processing time.
Another type of rapid tissue processing is the preparation of frozen sections. This
technique is often times employed when the sample cannot be processed using the
conventional methods of tissue processing or in the presence of heat. There are two
procedures which may be performed to produce frozen sections: (1) cold knife procedure and
(2) cryostat procedure. Both procedures are based on the principle of quenching that involves
rapid freezing of the tissue sample.
Cold Knife Cryostat
Microtome Freezing microtome Rotary microtome
Freezing agent CO2 Liquid nitrogen
Thickness of sections 10 um 4-6 um

Assessing Oneself

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4. silver stained frozen sections are used in 1. temperature requirement for this is -40 to -
_____ 60 deg C
7. freezing agent in cryostat for intraoperative 2. temperature requirement for this tissue is -
procedures 35 deg C
8. temperature requirement for this is 0 to -10 3. fixative for lipids
deg C 5. type of specimen for frozen section
10. reagent in which mounted frozen sections preparation
are placed 6. type of tissue sample that uses albumin as
the mountant
9. thickness of frozen section in the cold knife
procedure

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References
2009
W.B. Saunders Co.

LESSON 12: IMMUNOHISTOCHEMISTRY

1. discuss the concepts of immunohisto/cytochemistry in the histopathology laboratory;
2. explain the significance of immunohisto/cytochemistry; and
3. apply the various techniques of immunohisto/cytochemistry.

Across Down
4. cartilage cell 1. type of epithelium that lines the urinary
5. substance produced by the immune system bladder
in the presence of an antigen 2. neuronal process that receives signals
7. phenotypic determinants found on cells, 3. pigment-producing integument cell
tissue components and microorganisms 6. other name for antibody
9. epithelial malignant tumor 8. cells that produce antibodies
10. support cells in nervous tissue

Lesson Proper
IMMUNOHISTOCHEMISTRY
✓ routinely used for the identification of specific or highly selective cellular
epitopes or antigens in frozen or paraffin-embedded tissues
IMMUNOCYTOCHEMISTRY
✓ detect organism in cytologic preparations (fluids, sputum samples & fine
needle aspirates)
**PRINCIPLE: antigen-antibody interactions
APPLICATIONS OF IMMUNOHISTOCHEMISTRY
✓ disease diagnosis
✓ drug development
✓ biological research
ANTIBODIES
1. Polyclonal Antibodies
▪ Antibody is produced by several clones of plasma cells
▪ Uses laboratory animals (rabbit, goat, pig, sheep, horse, guinea pig)
▪ Lab animal is immunized with a purified specific immunogen having the antigen
of interest
▪ Lab animal responds by producing humoral antibody against the antigen
▪ Immunoglobulin-rich serum is collected from the lab animal
2. Monoclonal Antibodies
▪ Antibody is produced by a single clone of plasma cells
▪ Used hybridoma and cloning techniques
▪ Uses mice as the laboratory animal
SAMPLE PREPARATION
1. Proteolytic Enzyme Digestion
▪ Break down formalin cross-links to unmask and allow certain antigenic sites to
be exposed
▪ Useful for heavy chain immunoglobulins, complement and specific antigens
▪ Commonly used enzymes are trypsin (0.1% trypsin in 0.1% CaCl) and protease
(0.05 to 0.1% protease)
➢ pH of both are adjusted to 7.8 with NaOH and preheated to 37 oC
2. Microwave Antigen Retrieval
▪ Boiling to formalin-fixed deparaffinized sections
➢ 0.01 M citrate buffer (pH 6.0)
➢ EDTA (pH 8.0)
➢ Tris EDTA (pH 8 or 10.0)
▪ Optimal exposure to heat: 10 to 60 minutes
▪ Most satisfactory duration: 20 minutes
3. Pressure Cooker Antigen Retrieval
▪ Less time consuming
▪ More consistent antigen recovery
▪

ANTIGENS
EPITHELIAL TUMOR MARKERS
KERATIN: highly sensitive marker for epithelial cells
✓ Epithelial tumors (carcinoma)
✓ Non-epithelial tumors (mesotheliomas and non-seminomatous germ cell tumors)
▪ CK7 (cytokeratin 7): lung, breast, uterus and ovaries carcinomas
▪ CK20 (cytokeratin 20): colon and stomach carcinomas
▪ Positive for CK7 and CK20: bladder transitional cell carcinomas and mucinous
ovarian tumors
EMA (epithelial membrane antigen)
✓ High molecular weight protein that aids in determining the site of the tumor
✓ Positive in adenocarcinomas of the breast, lung and kidneys
✓ Nonreactive in hepatocellular, adrenal or embryonal carcinomas
✓ Negative in non-epithelial tumors (sarcomas, lymphomas, melanomas) and other
tumors (meningiomas, mesotheliomas, anaplastic large cell lymphomas and plasma
cell tumors)
CEA (carcinoembryonic antigen)
✓ Oncofetal antigen present in GIT, pancreas, lung, breast, ovary, uterus and cervix
carcinomas
✓ Differentiates adenocarcinoma from mesothelioma
✓ Nonreactive in prostate, thyroid and renal carcinomas
TTF-1 (thyroid transcription factor-1)
✓ Distinguishes lung adenocarcinoma from mesothelioma
✓ Positive in thyroid, lung and neuroendocrine tumors (medullary thyroid carcinomas,
carcinoid tumors and small cell tumors of the lung)
PSA (prostate specific antigen)
✓ Extremely useful in the diagnosis of prostatic adenocarcinoma
✓ Positive in certain pancreatic and salivary gland tumors
INTERMEDIATE FILAMENT MARKERS
Actin
✓ Contractile intermediate filament protein
✓ Sensitive marker for muscle differentiation
✓ Identify tumors derived from smooth, skeletal and cardiac muscle
Vimentin
✓ 57 kD intermediate filament in normal mesenchymal cells and their neoplastic
counterparts
✓ Always positive in melanomas and Schwanommas
Desmin
✓ 53 kD intermediate filament in smooth and striated muscles
✓ Highly specific for myogenic tumors including leiomyoma (smooth muscle) and
rhabdomyosarcoma (skeletal muscle)
✓ Demonstrate myogenic component of mixed tumors (carcinosarcomas or malignant
mixed mesodermal tumors)

GFAP (glial fibrillary acidic protein)

✓ 51 kD intermediate filament protein expressed by CNS glial cells, particularly astrocytes
✓ Used to confirm astrocytoma diagnosis
✓ Also present in other CNS tumors (ependymomas, oligodendrogliomas
medulloblastomas)
NF (neurofilament)
✓ Expressed by cells of neural origin, particularly neurons, neuronal processes, peripheral
nerves, sympathetic ganglia, adrenal medulla and neuroendocrine cells
✓ Positive in tumors that show neuronal or neuroendocrine differentiation
(neuroblastomas, ganglioneuromas, neuromas, chemodectomas,
pheochromocytomas)
S-100 protein
✓ Low molecular weight calcium-binding protein expressed by CNS glial cells, Schwann
cells, melanocytes, histiocytes, chondrocytes, skeletal and cardiac muscle,
myoepithelial cells and some epithelial cells of breast, salivary and sweat gland
epithelium
NEUROENDOCRINE MARKERS
NSE (neuron-specific enolase)
✓ Isoenzyme marker that provides a strong evidence of neural or neuroendocrine
differentiation
Chromogranin
✓ Found in neural secretory granules of endocrine tissues
✓ Marker for neuroendocrine differentiation
✓ (+) chromogranin, (+) keratin: neuroendocrine carcinoma
✓ (+) chromogranin, (-) keratin: paraganglioma
Synaptophysin
✓ 38 kD transmembrane protein associated with presynaptic vesicles of neurons
✓ Identified in normal neurons and neuroendocrine cells
GERM CELL TUMOR MARKERS
HCG (human chorionic gonadotropin)
✓ Synthesized by placental syncytiotrophoblasts
✓ Marker for choriocarcinoma
AFP (alpha fetoprotein)
✓ Synthesized by normal hepatocytes
✓ Marker for endodermal sinus tumors showing yolk sac differentiation
✓ Positive in embryonal carcinomas, teratomas and hepatocellular carcinomas
PLAP (placenta-like alkaline phosphatase)
✓ Produced by placental syncytiotrophoblasts in late pregnancy
✓ Marker for germ cell tumors, particularly germinomas
✓ Positive in most embryonal carcinomas, choriocarcinomas, endodermal sinus tumors
and majority of seminomas

MESENCHYMAL TUMOR MARKERS

Myogenic tumors
(+) actin, desmin and/or myo-D1, myoglobin, myogenin
Fibrohistiocytic tumors
Malignant: (+) CD68 or FAM 56 with alpha-1-antitrypsin and alpha-1-antichymotrypsin
Undifferenitated: (+) vimentin
Vascular tumors
(+) Factor VII-related antigen, CD31 and Ulex europaeus 1
Melanomas
(+) S100 protein, melanosome (HMB-45), Melan-A (MART-1)
Lymphomas
(+) Leukocyte common antigen (LCA)
T cells: CD3, CD4, CD5
B cells: CD19, CD20, CD23
Reed-Sternberg cells (CD15, CD30, Ig LC and HC)
Cell Proliferation Markers: increased expression is associated with greater aggressiveness
and higher likelihood of recurrence of metastasis
✓ Ki-67 (MIB-1)
✓ PCNA (proliferating cell nuclear antigen)
LABELLING
✓ Enzyme labels
✓ Colloidal metal labels
✓ Fluorescent labels
✓ Radiolabels
TECHNIQUES
✓ Traditional direct technique
▪ Primary antibody is conjugated directly to the label
▪ Direct interaction between the labeled antibody and antigen in the histological
or cytological preparation
✓ Two-step indirect technique
▪ Unconjugated primary antibody binds to the antigen
▪ Labelled-secondary antibody directed against the primary antibody is then
applied
✓ Three-step indirect technique
▪ Another labelled-antibody is used directed against the labelled-secondary
antibody

Summing It Up
The recent introduction of prognostic and predictive markers in
immunologic techniques has made a tremendous impact on patient treatment
and management. This has been possible by the gradual development of
immunohistochemical and immunocytochemical methodologies over the past
years. These techniques allow the identification of specific and highly selective epitopes in
formalin-fixed paraffin-processed tissues with an antibody and appropriate labelling system.
Immunohistochemistry in particular is used for disease diagnosis, drug development
and biological research. It involves the use of polyclonal or monoclonal antibodies which are
allowed to react with antigens present in cells and tissue components. This is in conjunction
with the use of enzyme labels, colloidal metal labels, fluorescent labels or radiolabels.
There two steps employed in immunohistochemistry: (1) sample preparation and (2)
labelling techniques. Sample preparation involves the unmasking of antigens which might
have been hidden as a consequence of formalin fixation and paraffin processing. This will
allow antigens to be exposed to the antibodies used as reagents and permit antigen-antibody
interaction. The techniques used in sample preparation include proteolytic enzyme digestion,
microwave antigen retrieval and pressure cooker antigen retrieval.
The presence of antigens, particularly in abnormal cells, provides evidence for the
diagnosis of malignancies. Depending on the antigen detected, a specific malignancy is
correlated to it. An example is the detection of cytokeratin 7 and 20 (CK 7, CK20) in bladder
transitional cell carcinomas. Prostate specific antigen (PSA) is extremely useful in the
diagnosis of prostatic adenocarcinoma. Detection of alpha fetoprotein on the other hand, is
useful in the diagnosis or embryonal carcinomas and teratomas. Many other highly specific
and selective antigens detected via immunohistochemistry are correlated with different
malignancies (please refer to the table in the Lesson Proper).
Labelling in immunohistochemistry are classified into three general techniques:
(1)traditional direct techniques, (2)indirect two-step technique and (3)indirect three-step
technique. The different types of technique provide varying degrees of sensitivity which is
determined by their ability to amplify signals that depends on the number of antigen-antibody
complexes formed.

Assessing Oneself

Across Down
1. (+)chromogranin, (-)keratin 2. positive in teratomas
3. indicative of neuronal or neuroendocrine 4. CNS glial cell that expresses GFAP
differentiation 5. type of antigen retrieval that provides more
7. skeletal muscle malignancy,(+) desmin consistent antigen recovery
8. used in microwave antigen retrieval with a 6. type of antibody derived from several plasma
pH of 8 or 10 cell lineages
9. cells that produce hCG 10. expresses CD15 and CD30

12. highly sensitive marker for epithelial cells 11. always positive in Schwanommas
14. (+)actin, desmin, myoglobin 13. technique in producing monoclonal
15. enzyme for proteolytic digestion antibodies
14. laboratory animal used in monoclonal
antibody production
your instructor.
platform Quizziz.
messenger.
message.
References
2009
W.B. Saunders Co.

LESSON 13: EXFOLIATIVE CYTOLOGY

1. explain the significance of cytologic examination;
2. identify the specimens processed in exfoliative cytology;
3. discuss the significance of fixation and adhesion in exfoliative cytology;
4. enumerate in the correct sequence the different reagents used in Hematoxylin &
Eosin staining technique and explain the purpose of each; and
5. correlate results in exfoliative cytology with various physiologic and pathologic
conditions.

your previous subjects and lessons. This is a self-assessment activity and will
not be graded but should be compiled as part of your journal to be checked
every grading period. Refer to Appendix A for the answers.
Across Down
3. staining characteristic of the nucleus 1. staining characteristic of the cytoplasm
5. otherwise referred to as cancer 2. anatomical term for the cheeks
7. type of tissue that undergoes desquamation 4. recommended stain in cytology
8. nucleic acid stain 6. hormone secreted by the corpus luteum
10. hormone primarily produced by the ovaries 9. connects the vaginal canal to the uterus

Lesson Proper
EXFOLIATIVE CYTOLOGY
✓ branch of medicine which deals w/ the study of cells that are exfoliated
or scraped off from the lining epithelium & mucosa of different organs
✓ deals with cells that have been desquamated from epithelial surfaces.
DIVISIONS
1. CYTOPATHOLOGY – study of abnormal cells (e.g. CA cells)
2. CYTOTECHNIQUE – study of the different methods of preparing the cells for
microscopic examination
DIAGNOSTIC EXFOLIATIVE CYTOLOGY

1. For diagnosis of Cancer
2. For differentiation between malignant & benign tumors
3. Differentiate tumors from other diseases (e.g. infections, inflammations or
degenerations)
4. Assessment of hormonal status of an individual (determine fertility conditions among
males & females)
5. Determination of the “true sex” of an individual
EQUIPMENT FOR COLLECTION
Glass pipet and rubber bulb Vaginal aspiration
Ayre’s spatula Swab smear
Laryngeal cannula attached to a 10 cc Endocervical or endometrial
syringe aspiration
SPECIMEN COLLECTION FOR CONVENTIONAL PAP SMEAR
Endocervical brush Samples of endocervical canal
Vaginal scrape Hysterectomy patients
Lateral vaginal scrape Hormonal evaluation
Four quadrant vaginal scrape Localization of vaginal adenosis
Vulvar scrape Detection of herpetic lesions or
carcinoma
Transformation Zone (T-zone): extremely important for detection of dysplasias and
carcinomas of the cervix
ADHESION
Specimens that require an adhesive:
✓ Urinary sediment
✓ Bronchial lavage specimen
✓ Specimen that utilize proteolytic enzymes during processing
✓ Adhesives
➢ Pooled human serum or plasma
➢ Celloidin ether alcohol
➢ Leuconostoc culture

FIXATION
✓ Smears should be placed into the fixative immediately after preparation
✓ Common Fixatives:
➢ Equal parts of 95% ethyl alcohol and ether
➢ 95% ethyl alcohol
➢ Isopropyl alcohol with ethyl alcohol
➢ Acetone with glycol
➢ Carnoy’s fluid
➢ Delaunoy’s fluid
Papanicolaou staining
1. Harris Hematoxylin – nuclear stain / basophilic stain
2. OG 6 (Orange – Green 6) – made up of 0.5 – 1.0% solution of OG in 95% ethyl
alcohol & PTA (phosphotungstic acid)
✓ Counterstain for cytoplasm of mature superficial cells
3. EA (eosin azure) – 36 or EA – 50 - made up of light green SF, Bismarck brown, eosin
Y, PTA, lithium carbonate
✓ Counterstain for cytoplasm of immature cells (parabasal and intermediate
cells)
Advantages of Pap’s stain
1. Transparent blue stain of the cytoplasm; allows overlapped cells to be identified
2. Excellent nuclear details
3. Color range is predictable & of great value in identification & classification of cells
4. Valuable in comparing cellular appearances in smears
CRITERIA for MICROSCOPIC Dx of CANCER

1. POLARITY OF CELLS
2. HYPERCHROMATISM – increase in staining affinity above the normal, affecting mainly
the nuclear structures
3. ATYPICAL MITOTIC FIGURES – abnormal stages
4. Reversal of the NUCLEO - CYTOPLASMIC Ratio
CHANGES IN MALIGNANT CELLS
1. CHANGES IN THE INTERCELLULAR STRUCTURAL PATTERN
a. Increase in size
b. Irregular shape
c. Irregular pattern
d. Anisocytosis and Anisokaryosis observed in clusters
e. Excessive grouping and crowding of cells
f. Indistinctness cell membrane
2. CHANGES IN THE CYTOPLASM
a. Acidophilia or marked orangeophilia
b. Excessive cytoplasmic inclusions
c. Abnormal vacuolation

3. CHANGES IN THE NUCLEUS

a. Larger and irregular nucleus
b. More deeply pigmented
c. Multinucleation
d. Increase in number and size of nucleoli
e. Increased distribution and irregular size of chromatin materials
f. Markedly thickened nuclear membrane
g. Necrotic or degenerative changes
REPORTING OF CYTOLOGIC SMEAR
CLASS I Absence of atypical or abnormal cells
CLASS II Atypical cytologic picture but no evidence of malignancy
CLASS III Cytologic picture suggestive but not conclusive of malignancy
CLASS IV Cytologic picture strongly suggestive of malignancy
CLASS V Cytologic picture conclusive of malignancy
SEX DETERMINATION
 Specimen: scrapings from the buccal & vaginal mucosa
 True sex is determined based on the presence of Barr bodies (inner aspect of
nuclear membrane)
 Important in the field of genetics
VAGINAL HORMONAL CYTOLOGY

✓ Vaginal hormonal cytology may be performed regularly without undue risk.
CELLS IN VAGINAL SMEARS
1. Mature superficial cells
➢ Dark pyknotic nuclei
➢ True acidophilia (characteristic of superficial vaginal cells under Estrogen
influence)
2. Intermediate cells
➢ medium-sized polyhedral or elongated cells with basophilic cytoplasm showing
vacuoles
➢ Navicular cells – boat-shaped w/ tendency to fold or curl on edges
• combined estrogen-progesterone effect
• associated with the latter half of menstrual cycle, pregnancy and
menopause
➢ Pregnancy cells – round, oval or boat-shaped cells with translucent basophilic
cytoplasm (greatest at the center due to glycogen)
• double-walled boundary appearance
3. Parabasal cells – fried egg appearance
➢ Strongly basophilic cytoplasm & a large vesicular nucleus
➢ Seen two weeks of age up to puberty, after childbirth, abortions and after
menopause

4. Endometrial cells – slightly cylindrical w/ less basophilic cytoplasm; found during & 1-4
days after menstruation
5. Basal cells – small, round to slightly oval cells w/ relatively large nuclei
➢ Found before puberty & after menopause
FERNING
✓ presence of a “palm leaf” pattern (arborization) on drying of the vaginal or cervical
secretions due to formation of salt crystals under the influence of estrogen
Criteria for cytologic diagnosis of normal pregnancy
1. Marked progesterone effect
2. At least 50% of intermediate cells in clusters
3. Some typical pregnancy cells present
4. Less than 30% superficial cells
5. Doderlein-filled “dirty” background
Other methods
1. Acridine Orange – binds with nucleic acids
➢ Under fluorescence microscope:
RNA – brick to orange red
DNA – green & yellow
 Increased basophilia (RNA) – signifies growth
 Increased acidophilia (DNA) – signifies malignancy
2. Phase – Contrast Microscopy – 2nd best choice after Pap’s staining
➢ Used for hormonal evaluation of gynecologic specimen & for CA detection
3. Interference Microscopy (IM) – determines dry weight of individual cells or cellular
constituents
➢ CA cell nucleus & cytoplasmic dry weight is LESS than that of normal cells
Enrichment Activity
ACTIVITY 10: PAPANICOLAOU STAINING TECHNIQUE
OBJECTIVES:
1. properly perform the Papanicolaou staining technique; and
2. gain adequate knowledge of the different sources of cytologic materials
prepared in Pap’s, their manner of collection and processing.
Papanicolaou method is popular for the cytological examination of smears of the

female reproductive tract. This is partly due to the variegated staining which is helpful in
hormonal studies and in the detection of Candida and Trichomonas infection, and partly
due to the fact that the type of picture produced results in less eye-fatigue.

Make sure to listen attentively and take note of the important concepts and principles
which will be applied in performing the different steps in Pap’s staining.

PROCEDURE:
Smears must not be allowed to dry as this distorts the morphology and alters the
staining reactions of the cells. As soon as they have been made (unless otherwise
specified), and while still moist, smears are placed in the fixative.
STAINING PROCEDURE:
Reagent Time

70% Alcohol 2 minutes
Distilled Water Rinse
Harris Hematoxylin 3 minutes
Tap Water Rinse
1% Acid Alcohol 3 dips
Ammonia Water Until blue
70% Ethyl Alcohol 2 minutes
95% Ethyl Alcohol 30 seconds
OG 6 1 minute
EA 36 1 minute and 30 seconds
95% Ethyl Alcohol 1 minute
95% Ethyl Alcohol 1 minute
Xylene 2 minutes
Xylene 2 minutes
Results:
Nuclei - blue
Superficial (cornified cells) - pink
Intermediate (non-cornified) cells) - green
Candida (Monilia) - red
Trichomonads - grey-green
Parabasal cell cytoplasm - deep green
Red Blood Cells - orange

Individual Task
Copy and paste images from reliable sources or draw the following equipment.
1. Cells seen in cervico-vaginal smears
2. Lactobacilli on vaginal smear
Mature superficial cells Intermediate cells
Parabasal cells Navicular cells
Pregnancy cells Endometrial cells

Endocervical glandular cells Lactobacilli

1. How soon after preparing the smear should it be placed in the fixative?
Explain your answer.
2. How are fluids with low cellular content and large in volume processed?
3. How are smears best prepared for mailing or transporting to a laboratory?
Summing It Up
Exfoliative cytology deals with the microscopic study of cells that have
been desquamated from epithelial surfaces. Exfoliated cells may be found in
smears that have spontaneously been shed or physically removed from epithelial
surfaces and mucous membranes. Spontaneous exfoliation is observed in normal
cells due to constant growth and replacement with new cells and this is more readily observed
in malignant tumor cells.
The method of preparing and evaluating exfoliated cells developed by Papanicolaou has
become an important diagnostic adjunct in daily use in today’s pathology laboratory. The
primary purpose of the examination of a Papanicolaou smear is for the cytologic diagnosis of
cancer. However, this technique is also used to determine chromosomal sex, the effects of
hormones and the presence as well as the type of infection.
Cytology materials may be collected from various regions like vaginal, endocervical,
endometrial, gastric, bronchial, and body cavities. Other samples also include urine, sputum
and prostatic fluid. Once the sample arrives in the laboratory, it is then processed using the
following procedures: (1) smearing, (2) adhesion, (3) fixation and (4) staining. The prepared
smears will then be examined under the microscope for proper evaluation.
Other methods performed in cytology in addition to the preparation of Pap’s smear
include acridine orange technique, phase-contrast microscopy and interference microscopy.

Assessing Oneself

Across Down
5. cells stained by EA-36 or EA-50 1. structure observed in chromosomal sex
7. basis in comparing normal from determination
malignant cells in interference 2. specimen collection for the detection of
microscopy herpetic lesions

11. most commonly used fixative for 3. color of DNA in acridine orange
cytologic smears 4. indicated by increased basophilia
13. palm-leaf pattern 6. equipment for swab smear
14. increased in staining affinity 8. other term for acidophilia
15. important region for the detection of 9. appearance of mature superficial cell
cervical cancer nuclei
10. increase in the level of this hormone
indicates normal pregnancy
12. vaginal scrape is utilized for these
patients

Match the cervico-vaginal cells (Column A) to their corresponding correlations (Column B) and
microscopic appearance (Column C). Choices may be used more than once.
A B C ANSWERS
1. Mature A. Abortions A. fried egg appearance
Superficial Cells
2. Navicular Cells B. pregnancy B. boat-shaped w/ tendency to fold
or curl on edges
3. Pregnancy Cells C. 1-4 days after C. small, round to slightly oval cells
menstruation w/ relatively large nuclei
4. Parabasal Cells D. menopause D. True acidophilia
and pregnancy
5. Basal Cells E. before puberty E. slightly cylindrical w/ less
and after basophilic cytoplasm
menopause
6. Endometrial F. None of the F. round, oval or boat-shaped cells
Cells above with translucent basophilic
cytoplasm

your instructor.
platform Quizziz.
messenger.
✓ If you have no internet connectivity, you may take the quiz via SMS/text message.
References
2. Bruce-Gregorios, J. (1974). Histopathologic techniques (1st ed.). Philippines: Goodwill
Trading Co., Inc.
3. Bruce-Gregorios, J. (2012). Histopathologic techniques (2nd ed.). Philippines: Goodwill
Trading Co., Inc.
2009
5. Raphael, S. (1983). Lynch’s medical laboratory technology (4th ed.). Philadelphia: W.B.
Saunders Co.

APPENDIX A
ANSWERS TO SELF-ASSESSMENT ACTIVITIES
Lesson 1: Tapping Prior Knowledge
1. clearing 6. dehydration
2. fixative 7. fixation
3. supravital 8. sectioning
4. alcohol 9. infiltration
5. staining 10. embedding
Lesson 1: Assessing One’s Self
1. cryostat 6. diagnosis
2. curetting 7. autoradiography
3. center 8. autolysis
4. pathologist 9. differential
5. permanent 10. alcohol
1. Test tubes are used to prepare samples for cell blocking.
2. True
3. Spreading/Touch preparation allows the examiner to view the intercellular relationship
of tissue components.
4. Tissue blocks should be stored for 3-10 years.
5. Coplin jars with stains are utilized to hold tissue slides in an upright position during
staining.
6. Curetting is performed to collect lesions on the inner surface of hollow organs.
7. Floating and fishing-out are important steps performed prior to staining with the use of
a water bath.
8. Smear preparation involves impression prep, streaking, spreading and pull-apart.
9. Leaded pencils are used to label frosted-end glass slides containing tissue sections or
smears.
10. Grossing performed by the pathologist involves the macroscopic evaluation of the
submitted sample.

1. glycogen 6. hollow organs
2. collagen 7. protein
3. lysosome 8. base
4. thyroid 9. electron
5. helix 10. lipids
Lesson 2 Part I: Assessing Oneself
1. glacial HAc 3. Bouin’s
2. phospholipid 4. phenol

5. protection 8. decrease
6. tuberculosis 9. acrolein
7. simple 10. mitochondria
1. Routine fixation using a tissue processor requires a temperature of 40 oC.
2. For electron microscopy, sucrose is added to glutaraldehyde to achieve a low vehicle
osmolality.
3. True
4. Sampling brain tissues should be performed after fixation to ensure that the tissue
constituents remain intact.
5. True
Part II: Assessing Oneself
1. de-zenkerization 9. acetone
2. glutaraldehye 10. alcohol
3. methanol 11. sublimat
4. formol-corrosive 12. filtration
5. Gendre’s 13. phosphate
6. Regaud’s 14. Orth’s
7. osmic acid 15. Carnoy’s
8. paraformaldehyde
1. B-5: B, D 6. Gendre’s fixative: A/F, J
2. Acetone: G, G 7. Orth’s fluid: C, E
3. Bouin’s solution: E, B 8. 95% Isopropyl alcohol: F, F
4. Heidenhain’s SuSa: B, I 9. Regaud’s fluid: C, C
5. Formaldehyde: A, A 10. Helly’s solution: C, H
Part III: Assessing Oneself
Level I Easy Breezy
1. post-chromatization 6. ethanol
2. staining 7. picric acid
3. alcohol 8. quenching
4. water 9. double fixation
5. melanin 10. microwave
Lesson 3 Tapping Prior Knowledge

1. strong acid 6. spongy
2. EDTA 7. anode
3. cathode 8. weak acid
4. chelation 9. compact
5. connective 10. ground matrix

Part I: Assessing Oneself

1. fret saw 9. hydrochloric
2. Giemsa 10. ethanol
3. teeth 11. staining
4. Von Ebner’s 12. strong acids
5. sulfurous 13. microcalcifications
6. urea 14. sectioning
7. Perenyi’s 15. trichloroacetic
8. additives
1. True
2. Microcalcifications are commonly found in malignancies and appear as dark purple
granular masses with lighter purple halos during microscopic examination.
3. Removal of decalcifying solution may be performed after decalcification and prior to
staining to completely remove acids from tissues.
4. True
5. Weak acids such as formic acid may be used for immunohistochemistry.
1. hydrochloric 7. frozen section
2. everyday 8. cloudiness
3. opaque 9. Molliflex
4. physical test 10. water
5. tetrasodium 11. alkaline phosphatase
6. formic acid
1. The chemical test performed to assess the extent of decalcification yields white
precipitates of either calcium hydroxide or calcium oxalate indicating that the process if
incomplete.
2. Formic acid can be used in both ion exchange resin and electrophoresis for the removal
of calcium deposits.
3. In the radiologic method of testing complete decalcification, waterproof polyethylene
sheet is overlaid on the X-ray film and 30kV is applied for 1 minute.
4. In the calcium oxalate test, 5 ml of used reagent is alkalinized before adding ammonium
oxalate or sodium oxalate.

1. formic acid 6. increased
2. nonpolar 7. anhydrous
3. intercellular 8. polar
4. miscible 9. absolute
5. conjunctivitis 10. tert-butanol

Lesson 4: Assessing Oneself

1. Weiseberger’s 6. EGME
2. delicate 7. softening
3. methanol 8. tetrahydrofuran
4. dioxane 9. acetone
5. ethanol 10. maceration
1. Additives are incorporated in the highest concentration of alcohol to promote tissue
softening.
2. True
3. Acetone is a rapid-acting dehydrant but penetrates tissues poorly.
4. Tetrahydrofuran can be used as a dehydrating and clearing agent but is causes
dissolution of lipids.
5. Graupner’s method makes use of dioxane which can act as a dehydrating and clearing
agent.
1. viscosity 6. toluene
2. carcinogenic 7. aromatic
3. fibroid 8. chloroform
4. xylene 9. carbon tetrachloride
5. flammable 10. benzene
1. lower 6. cedarwood oil
2. aplastic 7. refractive index
3. clove oil 8. xylene
4. liver 9. aniline
5. terpineol 10. mounting
1. phenol 6. catalyst
2. hydrophobic 7. melting point
3. perpendicular 8. oblique
4. longitudinal 9. cross section
5. waterbath 10. hydrophilic
Part I: Assessing Oneself
1. heavy-duty 6. tissue tek
2. tissue mat 7. hygroscopic
3. bioloid 8. carbowax
4. crystallization 9. polymerization
5. vacuum 10. Pearse

Level II: True/False

1. False
2. False
3. True
4. False
5. True
1. glycol methacrylate 6. methyl methacrylate
2. benzoyl peroxide 7. phenol
3. desiccator 8. spur
4. bisphenol A 9. castor oil
5. Gilson’s 10. ether
1. glycol methacrylate is an acrylic plastic that is compatible with a wide range of staining
techniques because of its hydrophilic nature.
2. True
3. During tissue orientation, the surface of the section to be cute should be parallel to the
bottom of the mold.
4. True
5. Lower viscosity of epoxy plastics allows infiltration to be faster and at a shorter period of
time.

1. rocking 9. thymol
2. honing 10. celloidin
3. biconcave 11. wedge
4. Minot 12. APES
5. freezing 13. phenol
6. carbon dioxide 14. poly-L-lysine
7. diamantine 15. clearance
8. stropping
1. Cambridge: F, C, A, E
2. Freezing: C, D, C, A
3. Ultrathin: A, E, D, D
4. Sliding: B, B, B, C
5. Rotary: D, A, A, B


1. glycogen 6. elastic
2. Lugol’s 7. triglyceride
3. Sakaguchi 8. calcium
4. hemosiderin 9. collagen
5. mordant 10. histone

1. histochemical 9. Ehrlich’s
2. regressive 10. bluing
3. supravital 11. accentuator
4. metachromatic 12. yellow
5. pale pink 13. mordant
6. intravital 14. red
7. acid alcohol 15. immunohistochemical
8. impregnation
1. True
2. In H and E staining, hydration performed after the deparaffinization step makes use of
descending grades of alcohol.
3. Regressive staining is more preferred than progressive staining due to the use of a
differentiator/decolorizer.
4. True
5. True
Lesson 8 Part II: Assessing Oneself

Part II Level I: Easy Breezy
1. spermatogenesis 6. black
2. hematein 7. coal tar dyes
3. lysochromes 8. best carmine
4. Sudan black B 9. auxochrome
5. Scharlach R 10. ripening
Lesson 8 Part III: Assessing Oneself
1. acridine orange 6. iodine
2. orcein 7. Kraijan’s
3. malachite green 8. neutral red
4. gold sublimate 9. green
5. methylene blue 10. benzidine

Lesson 8 Part IV: Assessing Oneself

1. arginine 5. magenta red
2. apple green 6. Feulgen
3. Levaditi’s 7. deep red
4. green 8. deep blue

1. Brun’s fluid 5. slide
2. leaded pencil 6. diamond-tipped pen
3. xylene 7. phenol
4. aqueous 8. coverslip

1. frozen 6. XAM
2. improper labelling 7. Apathy’s
3. ringing 8. water
4. Clarite 9. Canada balsam
5. DPX 10. glycerin jelly

1. hydration 5. epithelium
2. denaturation 6. pleural
3. sediment 7. paraffin
4. supernatant 8. ascitic

1. centrifuge 6. deparaffinization
2. heparin 7. effusion
3. Gendre’s 8. overnight
4. decolorizer 9. two ml
5. ethanol 10. Papanicolaou

1. cold chamber 6. rotary
2. agitation 7. carbon dioxide
3. negative 8. increase
4. dehydration 9. infiltration
5. subcutaneous 10. cold knife


1. beaker 6. once a week
2. acetone 7. alcohol
3. transfer arm 8. grossing
4. molecular friendly 9. circular deck
5. mineral oil 10. time clock
1. knife 6. formalinized
2. fatty breast 7. liquid nitrogen
3. formol calcium 8. environment
4. neuropathology 9. ten u
5. unfixed 10. formaldehdye

1. transitional 6. immunoglobulin
2. dendrite 7. antigen
3. melanocyte 8. plasma
4. chondrocyte 9. carcinoma
5. antibody 10. glial cells
Lesson 12 Assessing Oneself
1. paraganglioma 9. syncytiotrophoblast
2. alpha fetoprotein 10. Reed Sternberg
3. neurofilament 11. vimentin
4. astrocyte 12. keratin
5. pressure cooker 13. hybridoma
6. polyclonal 14. myogenic tumor
7. rhabdomyosarcoma 15. protease
8. tris EDTA
Lesson 13 Tapping Prior Knowledge

1. acidophilic 6. progesterone
2. buccal 7. epithelium
3. basophilic 8. acridine orange
4. Papanicolaou 9. cervix
5. malignancy 10. estrogen

Lesson 13 Assessing Oneself

Level I Easy Breezy
1. Barr bodies 9. pyknotic
2. vulvar scrape 10. progesterone
3. yellow 11. ethanol
4. growth 12. hysterectomy
5. immature 13. arborization
6. Ayre’s spatula 14. hyperchromatism
7. dry weight 15. T zone
8. orangeophilia
Level II Master Column
1. E, D
2. D, B
3. B, D
4. A, A
5. E, C
6. C, E

A Self-regulated Learning Module 1

Histopathologic &amp; Cytologic Techniques

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SCHOOL OF NATURAL SCIENCES

Histopathologic and Cytologic

A Self-regulated Learning Module

A Self-regulated Learning Module 1

Kathleenjoy Rosalia Katleya Almondia-Gili

Teresa N. Villanueva, RMT, MACT

CHANCE FAVORS THOSE WHO ARE PREPARED. 1

Requirements of the Course:

CHANCE FAVORS THOSE WHO ARE PREPARED. 2

b. Internet sources: complete website address (Uniform or Universal Resource

CHANCE FAVORS THOSE WHO ARE PREPARED. 3

Technical • 1 point is given if all Technical aspects required by staff is met

Tissue block is Tissue block

CHANCE FAVORS THOSE WHO ARE PREPARED. 4

in one on three three (3)

HISTOPATHOLOGY SLIDE (H & E - Stained)

CHANCE FAVORS THOSE WHO ARE PREPARED. 5

CHANCE FAVORS THOSE WHO ARE PREPARED. 6

CHANCE FAVORS THOSE WHO ARE PREPARED. 7

LESSON 1: INTRODUCTION TO TISSUE PROCESSING

Tapping Prior Knowledge

CHANCE FAVORS THOSE WHO ARE PREPARED. 8

FACTORS TO BE CONSIDERED IN CHOOSING A METHOD

METHODS OF PREPARATION AND EXAMINATION

CHANCE FAVORS THOSE WHO ARE PREPARED. 9

TEASING ✓ Dissection or separation of tissue components in NSS or Ringer’s solution

2. PRESERVED TISSUE EXAMINATION

CHANCE FAVORS THOSE WHO ARE PREPARED. 10

Fix→Section→Mount + Photographic Emulsion (Ag Halide)→Stain

CHANCE FAVORS THOSE WHO ARE PREPARED. 11

Alcohol lamp Beaker

Coplin jar Storage jars

CHANCE FAVORS THOSE WHO ARE PREPARED. 12

Test tubes Glass slides (Plain & with frosted end),

CHANCE FAVORS THOSE WHO ARE PREPARED. 13

Clinical Centrifuge Water Bath

Incubator Paraffin Oven

CHANCE FAVORS THOSE WHO ARE PREPARED. 14

Hot plate Fret saw Surgical blade with

Staining rack Test tube rack

CHANCE FAVORS THOSE WHO ARE PREPARED. 15

Test tube holder Tissue tong Timer

Applicator stick Camel’s hair brush Gauze cloth

CHANCE FAVORS THOSE WHO ARE PREPARED. 16

Diamond tipped pen and Scissor Kitchen knife

Labeling tape Ruler Chopping board

CHANCE FAVORS THOSE WHO ARE PREPARED. 17

CHANCE FAVORS THOSE WHO ARE PREPARED. 18

CHANCE FAVORS THOSE WHO ARE PREPARED. 19

Level I: Easy Breezy

CHANCE FAVORS THOSE WHO ARE PREPARED. 20

Level II: Modified True/False

CHANCE FAVORS THOSE WHO ARE PREPARED. 21

Tapping Prior Knowledge

CHANCE FAVORS THOSE WHO ARE PREPARED. 22

Two Purposes of Fixation

CHANCE FAVORS THOSE WHO ARE PREPARED. 23

CHARACTERISTICS OF A GOOD FIXATIVE

CHANCE FAVORS THOSE WHO ARE PREPARED. 24

✓ contain glacial HAc

CHANCE FAVORS THOSE WHO ARE PREPARED. 25

CHANCE FAVORS THOSE WHO ARE PREPARED. 26

Level I: Easy Breezy

CHANCE FAVORS THOSE WHO ARE PREPARED. 27

Histopathologic & Cytologic Techniques

Histopathologic & Cytologic Techniques