Estelles Hct Lab Exercises

SAFETY IN HISTOPATHOLOGIC LABORATORY
Exposure to blood, body fluids and chemicals are the greatest risks associated with
histopathologic laboratory. Occupational Safety and Health Administration issued the rule for
universal precaution. This states that all human blood, body fluids and tissues must be treated
as if they were infectious. Blood borne pathogens when present in human blood can cause
disease. These include Hepatitis B virus, Hepatitis C virus and Human Immunodeficiency Virus
(HIV). These risks can be minimized by adopting universal precautions as well as standard
laboratory practices. The following are good laboratory practices that should be enforced in
histopathologic laboratory.
1. Handwashing.
Hands must be washed:
 Whenever there is visible contamination with blood and body fluids
 Before and after work
 After gloves are removed and between glove changes
 Before leaving the laboratory
2. Eating, drinking, smoking and applying cosmetics are prohibited inside laboratory work
area
3. Sharps should be disposes in a puncture-resistant container. This should be leak proof.
4. Gloves must be worn when handling biologic specimens.
5. Areas or equipment used by personnel who are not gloved should not be touched with
contaminated gloves.
6. Any accident must be reported immediately to instructor so that appropriate prophylactic

precautions may be taken.
7. Mixing of chemicals must be done inside the fume hood.
8. Properly dispose all contaminated supplies in the appropriate container.
9. Disinfect and clean biohazard spills immediately.
10. Personal protective equipment should always be worn inside the laboratory.
Name: MENCHAVEZ, ESTELLE A. Date: June 29, 2021
EXERCISE 1
SUPPLIES & EQUIPMENT IN HISTOPATHOLOGY LABORATORY

In order to study tissues and cells under the microscope, they must undergo
various steps in processing. As students in histopathology and cytology, it is essential to
familiarize yourselves with the different materials and equipment that is being used in this
section of the laboratory. Skills is as important as how you will care and maintain this
equipment to come up with good histology and cytology slides to be examined by the
pathologist that will aid in the correct diagnosis of patients.
1. OBJECTIVES
At the end of the laboratory exercise, the students should be able to:
1.1 Identify the different materials and equipment used to process tissue and body fluids
for histopathologic and cytologic examinations.
1.2 Explain the different uses and maintenance measures of these materials and
equipment to ensure their usability and functionality.
2. MATERIALS
2.1 Laboratory manual
2.2 Ball pen
3. PROCEDURE
3.1 Identify the material or equipment presented in the picture.
3.2 Give its uses, purpose, or application in histopathologic or cytologic techniques.
3.3 Give 2 – 5 sentences on how to maintain the material and equipment in good
condition.
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4. RESULT/OBSERVATION
4.1 Label and provide required information provide.
Name: Rotary Microtome
Uses/Application: It is used in obtaining sections from

paraffin-embedded samples or for section-cutting step in
tissue processing.
Maintenance: The equipment is cleaned daily from

paraffin debris. Further, this equipment requires a yearly
preventive maintenance.
Name: Cryostat or Cryostat Machine
Uses/Application: It is used for cutting frozen samples and

its reduced temperature increases the sample’s hardness.
Maintenance: Microtomy debris must be removed from

the cryostat chamber. The knife and exposed surfaces
must be cleaned with 70% alcohol. Lastly, the equipment
must be placed outside the ref chamber to avoid heat-
expansion and cold-contraction effects.
Name: Tissue Embedding Center
Uses/Application: It is used in moderate to heavy

workloads in preparing wax tissue blocks and minimizes
wax accumulation.
Maintenance: Maintain in a clean condition and use only

minimal quantities of solvent in absorbent cloth and use
plastic or wooden implements when scraping wax off.
Remember to switch off before removing plug and unplug
while cleaning.
2
Name: Automatic Tissue Processor
Uses/Application: It is used to prepare tissue samples for

sectioning and microscopic examination in the laboratory.
Maintenance: For the maintenance of the equipment,

change the reagent when usage limit is reached before
running the next process to obtain well-processed blocks.
Before exchanging fuses, the main switch must be
switched off and instrument is unplugged.
Name: Microscope
Uses/Application: Microscope is used to magnify small

objects and observe a specimen at a cellular level.
Maintenance: Lenses should be kept away from anything

abrasive especially when cleaning. In cleaning, use
moistened lens paper with lens cleaning solution and
clean in circular motion.
Name: Microtome Knife or Blade Holder
Uses/Application: This is used to accommodate

disposable blades for paraffin and cryostat sectioning to
allow histologist maximum flexibility.
Maintenance: This equipment is less susceptible to nicks

and normal wear and tear. Further, this is maintenance-
free and durable.
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Name: Tissue/Embedding Cassettes
Uses/Application: These tissue/embedding cassettes hold

and identify tissue samples in processing, embedding, and
sectioning procedures.
Maintenance: Such equipment is soaked in xylene to

remove the dehydrating agent. It should be noted to not
overload so as to avoid compressed tissues which will
then cannot be adequately dehydrated.
Name: Glass Slides and Cover Slips
Uses/Application: These are used for microscope

specimen observation. It is where the specimen is
mounted.
Maintenance: These should be clean and fingerprint-free

and thus, held by their edges. In cleaning the slides, use
70% ethanol and afterwards, dried with a lint-free tissue
or lens tissue. Remember, these are non-reusable
materials.
Name: Teasing Needle
Uses/Application: This is used to straighten tissue section,

remove tissue folds, and readjust samples.
Maintenance: Teasing needle must be cleaned after each

use. The stainless steel needle and durable handle must
be sanitized well to prepare it for next or future usage.
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Name: Tissue Floatation Water Bath or Floating Out Bath
Uses/Application: It is used to assist with the handling of

samples in the laboratory, allowing manipulation and
location onto slides, and to flatten or spread tissue
section.
Maintenance: The equipment must be stored in a clean

and ventilated room (less than 80% humidity). Keep it
away from flammable chemicals and avoid high heat
areas and prolonged LCD contact.
Name: Glass Staining Dish
Uses/Application: It is used to hold microscope slides

during staining procedures.
Maintenance: It should be cleaned properly for

maintenance. Also, follow suitable number of slides to be
used when handling the equipment/material.
Name: Coplin Jar
Uses/Application: Coplin Jar is used to stain specimens in

lesser quantities and for cytologic studies.
Maintenance: To maintain its functionality, filter out

solution before staining. Remember to clean the
laboratory material properly after each use.
Name: Electric Paraffin Pitcher or Electric Paraffin Wax

Dispenser
Uses/Application: This equipment is used for wax melting

and embedding.
Maintenance: Ensure that there is no debris left in the

equipment. Moreover, filter out wax and check
temperature constantly.
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Name: Electric Paraffin Bath
Uses/Application: This equipment is used for immersing

processed tissues.
Maintenance: The accuracy of temperature is vital since it

might greatly affect the processing of immersed sample.
Therefore, temperature should be checked from time to
time.
Name: Electric Paraffin Knife
Uses/Application: Electric Paraffin Knife is used to pick up

the wax, as well as ensure that tissues won’t adhere to it.
Maintenance: Ensure that there is no leftover wax from

previous use. The equipment must be cleaned well prior
to next use/test.
5. STUDY QUESTIONS
5.1 What are the data that should be included in the equipment maintenance log in
Histopathology section?
The data included in equipment maintenance log in histopathology section are the
following; name, manufacturer, model number, serial number, date of inspection,
validation or performance evaluation, significant action to remedy deficiencies, and
the daily temperature recordings for all temperature-controlled equipment.
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5.2How would you label reagent and stain containers in histopathology section?
By placing the name of all the ingredients, manufacturer’s name and address if
commercially purchased or name of person making the reagent, the date
purchased or made, expiration date (if known), and the hazard warnings and
safety procedures.
5.3 How do you decontaminate materials and equipment in histopathology section?
The following are the ways on how to decontaminate materials and equipment in
histopathology section:
a) Use Lysol solution in decontaminating.
b) Water baths should be washed, rinsed and dried daily.
c) Cutting boards are covered with disinfectant and left overnight.
d) Auto-Technicon processors should also be washed/dried once weekly.
e) For tissue cassettes, wax should be removed and placed in detergent bath,
wash, clean, scald and towel dry.
f) Sinks should also be cleaned with Lysol daily.
g) Wet tissues should be stored in formalin in leak-proof container and

dispose by incineration after 4 weeks.
h) Door knobs, cabinet handles, light plates, etc. should be disinfected with
gauze containing disinfectant solution.
i) Waste should be disposed appropriately in properly identified containers

such as sharps box.
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Student’s Name MENCHAVEZ, ESTELLE A. Group& Sec: 3 – G Date: June 29, 2021
Exercise No. 1: SUPPLIES & EQUIPMENT USED IN HISTOPATHOLOGY LABORATORY
CRITERIA BEGINNING DEVELOPING ACCOMPLISHED EXEMPLARY SCORE

(5) (10) (15) (20)
Ability to Answer Able to answer Able to answer Able to answer Able to answer
Questions 25% of the 50% of the 75% of the item 100% of the
Able to answer all items required. items required. required. item required.
the required items
in the activity.
Content/Knowledge Presents 25% Presents 50% Presents 75% Presents 100%
Presented of the of the of the of the
Presents knowledge knowledge knowledge knowledge
scientific/practical required. required. required. required.
facts based on
knowledge of the
subject.
Finding Relevant 25% of the 50% of the 75% of the 100% of the
Information answers were answers were answers were answers were
Presents facts and supported by supported by supported by supported by
relevant facts & relevant facts & relevant facts & relevant facts & relevant
information to information. information. information. information.
support the
answers.
Sentence Fluency & 25% of the 50% of the 75% of the 100% of the
Organization of sentences were sentences were sentences were sentences were
Thoughts clear & clear & clear & clear &
Sentences are understandable. understandable. understandable. understandable.
very clear & Lacks Few ideas Most of the All ideas
understandable. spontaneity of showed ideas showed showed
ideas. spontaneity. spontaneity. spontaneity.
Ideas were
presented in order
and spontaneous.
Punctuality Did not able to Submitted on Submitted on Submitted on or
Submitted the submit on the the closing date the closing and before the due
output on the due date & but exceed 1 a day after the and closing
specified date & closing date. day on the due due date. dates.
time. date.
TOTAL SCORE
Preceptor’s Signature: __________________
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Name: MENCHAVEZ, ESTELLE A. Date: JULY 01, 2021
EXERCISE 2
ACCESSIONING & CUTTING OF SPECIMEN FOR TISSUE

PROCESSING
Specimens submitted to the histopathology laboratory must be entered into the
surgical pathology database via accessioning process. The receptionist records or numbers the
tissue specimen. Tissues are classified based on the type of specimen received, for the purpose
of easy identification, ease in handling and fast retrieving of results in case future reports are
needed. Tissue specimens are prepared by the medical technologists for the pathologists to
describe and cut.
Unlabeled specimens are absolutely unacceptable for accessioning. Proper

identification includes matching the data on the specimen container label with the
accompanying histopathology request form. If there are inconsistencies between the two, the
specimen and request form are returned to the operating room for correction by the staff
concerned. Specimen received in the laboratory, whether tissue or smear, are numbered upon
receipt for eases in handling and easy retrieval when further reports or investigations are needed.
1. OBJECTIVES
1.1 Demonstrate the proper numbering of specimens immediately upon receipt.
1.2 Demonstrate the proper cutting and appropriate size of tissue needed for the
process.
2. MATERIALS
2.1 Tissue specimen: Liver & Lung 2.5 Pencil
2.2 Sharp knife / Dissecting knife / Scalpel blade 2.6 Ruler
2.3 Dental Floss 2.7 Filter paper
2.4 Scissors
3. PROCEDURE
3.1 Cut ½ x 2 inch of filter paper.
3.2 Using a pencil, write the number corresponding assigned to you by the instructor.
3.3 Cut a 2.5 x 2 x 0.5 cm tissue
3.4 Tie the tissue using one end of a dental floss. On the other end
HISTOPATHOLOGIC REPORT SAMPLE:
1
4.1 Draw and label tissue specimen based in the histopathologic report inside a sample container
with fixative and proper label attached to the container.
2
4.2. Copy the histopathologic report attached in this activity sheet.
5. STUDY QUESTIONS
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5.1 Why is pencil used to label the filter paper for tissue processing?
Pencil is used to label the filter paper for tissue processing since it will not create
smudges compared to the ink of ball pens and other labelling pens. Once the labeled filter
paper is also dipped into the melted paraffin, the labels written using a pencil will not be
blurred, erased or removed. Thus, pencil is used given the fact that it is a resistive marker
to reagents or solvents used in processing such as formalin, acetone, xylene, and alcohol.
5.2 Why are the specimen cut before tissue processing?
The specimen is cut before tissue processing for penetration and identification
purposes. The solutions such as fixative and dehydrating agents used for processing will
only penetrate the interior of the tissue to a certain point therefore, the tissue specimen
must be small enough to allow penetration to take place properly. Cutting the specimen
does not only allow the tissue to be penetrated within a reasonably short time but this allows
the specimen to fit into the tissue cassette/receptacle. Moreover, specimen must be of
appropriate size and thickness, which is ideally 4-5 mm, so that it will be easily identified
and examined under the microscope leading to a proper and correct diagnosis done by the
pathologist.
5.3 What are the basic information that should be recorded upon the receipt of
tissue specimen?
The basic information that should be recorded upon the receipt of tissue specimen
includes the following; patient's name, birthdate of patient, patient's age and gender,
patient's location that comprises hospital number and ward, receipt or request number,
collection date and time, specimen type or specimen source, test required wherein there
should be note of special handling requirements if applicable and name of requesting
physician.
5.4 Enumerate the 5 criteria for rejection of gross specimen.
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The five criteria for a gross specimen to be rejected involves the following:
a) Discrepancies or inconsistency between requisition form and specimen

container labels
b) Incorrectly labelled/mislabeled or inadequately labeled or unlabeled specimen
and/or specimen container
c) When the specimen container is leaking
d) Absent/missing clinical data or history
e) Insufficient or unsuitable or incomplete specimen for procedure
5.5 Enumerate 4 pre-analytical factors that impact tissue diagnosis, prognostication

and theranostic.
The four pre-analytical factors that impact tissue diagnosis, prognostication and
theranostic include the following:
a) Tissue Degradation – in warm ischemia, the tissue degradation starts as soon

as its blood supply is interrupted which then lead to poor tissue quality; in
cold ischemia, factor that affects tissue is due to the lack of oxygen and
happens before all metabolic processes are stopped by fixation
b) Fixation of the tissue – properly preserved tissues are more resistant to
processing artifacts; proper fixative to tissue ratio should be followed (10
portions fixative to 1 tissue or 10:1 ratio)
c) Properly filled-up surgical pathologic requisition form
d) Proper accessioning procedure
Student’s Name MENCHAVEZ, ESTELLE A. Group& Sec 3-G Date JULY 1, 2021
5
Exercise No. 2: ACCESSIONING & CUTTING OF SPECIMEN FOR TISSUE PROCESSING

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
Preceptor’s Signature: ____________________________
6
EXERCISE 3
FIXATION
Histopathologic technique is the process wherein tissue sections of good quality are
prepared so that the pathologist can diagnose the presence or absence of disease. The first and
most critical step in histotechnology involves fixation. Classically, it is defined as the killing
and hardening of tissues. Killing is a necessary part of fixation to stop the metabolic process
that continue to alter the state of the tissue to be examined. Fixation is currently define as the
alteration of tissues by stabilizing protein so that the tissues become resistant to further changes.
The reagents used for fixation are called fixatives. The volume of the fixative should be 10 to
20 times the volume of the tissue. Formalin is the common fixative used in histopathology
because it forms additive compounds and complexes by the development of links or methylene
bridges between adjacent protein molecules.
1. OBJECTIVES
1.1 Accurately prepare 10% neutral buffered formalin.
1.2 Demonstrate the proper fixing of tissues by using a manual method.
2. MATERIALS
2.1 10 ml Reagent bottle / vial 2.7 Funnel
2.2 Serologic pipette 2.8 Masking Tape / Gum label
2.3 Calculator 2.9 Marker pen
2.4 Stirring rod 2.10 Dental Floss
2.5 Aspirator 2.11 1000 ml Flask
2.6 2.5 x 2 x 0.5 cm Kidney, Liver, Lung & Bone Tissues

3. REAGENTS
3.1 40% Formaldehyde (Stock solution) ……….. 100 ml

3.2 Distilled water ………….. 900 ml
3.3 Sodium phosphate, monobasic ………….. 4g
3.4 Sodium phosphate, dibasic …………. 6.5 g
4. PROCEDURE
4.1 Prepare 1 L of 10% Neutral Buffered Formalin by pouring a base of 100 ml distilled
water into a 1000 ml flask.
4.2 Add 4g sodium dihydrogen orthophosphate (monohydrate)
4.3 Add 6.5g disodium hydrogen orthophosphate (anhydrous)
4.4 Add 100 ml stock solution Formaldehyde. Mix thoroughly using the stirring rod
4.5 Add a further 800 ml distilled water and mix thoroughly. Label the bottle correctly.
4.6. Fill 3 10 ml reagent bottles or vials with the prepared 10% Neutral buffered formalin.
This is where the tissue will be fixed. Label the bottle correctly with your group
number.
4.7. Immerse samples and fix for 12 hours for the lung, 24 hours for the kidney, liver and
bone.
4.8. Store the bottle in a clean dry place during the entire process.
1
5.1 Draw and label the set up for fixation and indicate their recommended fixation time.
A. KIDNEY
B. LIVER
2
C. LUNG
6. STUDY QUESTIONS
6.1 What are the factors that hasten and retard fixation time? Explain each.
a) Size and Thickness of Tissue Specimen – these factors are directly proportional
with the amount of fixative and duration of fixation time. That means, larger tissues
require more fixatives and longer fixation time while smaller and thinner ones require
less fixatives and shorter fixation time.
b) Presence of Mucus – prevents complete fixative penetration, hence, tissues

containing mucus are fixed slowly and poorly
c) Presence of Fats – lipids/fats allow fixatives to penetrate at a slower pace thus, fatty
tissues should be cut in thin sections and should be fixed longer
d) Temperature – cold temperatures inactivates enzymes while increase in temperature

accelerates fixation but also increases the rate of autolysis and enzyme destruction.
Further, temperature also affects the specimen morphology.
3
e) Agitation – fixation is accelerated when automatic or mechanical tissue processing
is used
f) Volume Ratio – the ratio of the fixative volume to tissue volume is a critical factor.
Formalin is being absorbed by the tissue and if the ratio is less than the ideal, it will
leave a little or none on the solution affecting the quality of the specimen. Ideal ratio
of fixative volume to specimen volume is 10:1 or 20:1.
g) Time – cold ischemia time has its effect on the fixation process. The longer the tissue
sits in the air without fixative, the more autolytic changes are seen in the section.
Also, some tissues take longer time to fix than the others depending on their structure
like the brain.
h) Hydrogen Ion Concentration – the presence of buffer causes polymerization of the

aldehyde with consequent decrease in its effective concentration. Satisfactory
fixation occurs between pH 6-8.
i) Osmolality – deals with the shrinkage or swelling of the tissue. Tissue must be
slightly hypertonic.
6.2 Explain the actions of fixatives.
The fixatives act by denaturing or precipitating proteins which will form a

meshwork or a sponge which tends to hold other cell constituents together. Insoluble
proteins provide mechanical strength for subsequent procedures. As the primary aim of
fixation, fixatives function to preserve the morphologic and chemical integrity of the
cell in as life-like manner as possible. Additionally, fixatives also inactivate enzymes,
kill bacteria and molds, make tissue more receptive to dyes, modify tissue constituent
for maximum retention of form and stabilize the tissue elements so that the effect of
any subsequent procedures or trauma of future handling will be minimal.
Microorganisms are also fixed or killed which will prevent putrefaction of tissues and
decrease the risk of infection to the person handling it. It also serves as a mordant or
accentuator that will intensify the color of the stain or bind the stain to the tissues.
4
6.3 Enumerate and define the different types of fixatives based on function?
a) Microanatomical Fixatives: permit general microscopic study of tissue

structures without altering its structural pattern and normal intercellular
relationship. Examples include 10% formol saline, 10% buffered neutal
formalin, Heidenhain’s Susa, formol sublimate, Zenker’s solution, Zenker-
Formol, Bouin’s solution, and Brasil’s solution.
b) Cytological Fixatives: those that preserve specific parts and particular

microscopic elements on the cell itself. This type is further divided into
Nuclear, Cytoplasmic and Histochemical fixatives.
Nuclear: preserve nuclear structures and contain glacial acetic

acid as their primary component, has pH 4.6 or less. Examples
are Flemming’s fluid, Newcomer’s fluid, Heidenhain’s Susa
Cytoplasmic: preserve cytoplasmic structures and must never

contain glacial acetic acid; pH greater than 4.6. Examples of
cytoplasmic fixatives are Flemming’s fluid without acetic acid,
Formalin with post-chroming, Regaud’s or Moller’s fluid,
Orth’s fluid; for RNA, the precipitant fixatives are ethanol and
acetone.
Histochemical: those that preserve chemical constituents of

cells and tissues. These are 10% formol saline, absolute ethyl
alcohol, acetone, and Newcomer’s fluid.
6.4 Describe the reaction of cellular components to fixation.
Primary aim of fixation is to preserve the tissue’s morphologic and chemical

integrity. Hence, fixation retains cellular components in their native compartments and
present cells with a distinct and detailed microscopic appearance. These cells are now
permeable to allow antibodies to access intracellular structures. They are also resistant
to damage and distortion caused by the hypotonic and hypertonic solution used during
tissue processing. A good fixative is isotonic, therefore, there is only minimal physical
and chemical alteration of cells and their components; it should also make the cellular
components insoluble to hypotonic solutions and render them insensitive to subsequent
processing.
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6.5 What are the signs of incomplete fixation and how will you remedy the problem?
Signs of incomplete fixation include loss of nuclear chromatin, disappearance of

cells, cell shrinkage with artifactual space around the cells, a softened tissue, a smudgy
appearance of the cellular components, cytoplasmic clumping, and indistinct nucleoli.
To fix these, the following must be done:
a) Minimize cold ischemia time
b) Ensure adequate fixative to specimen ratio that is 10:1 or 20:1
c) Increase fixation time
d) Change formalin solution subsequently
e) Ensure that grossed sections should not be more than 0.4 cm
f) Do not pack tissue sections in the cassette
g) Agitate cassettes in formalin holding solutions during or after grossing
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Exercise No. 3: FIXATION

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
7
EXERCISE 4
DEHYDRATION and CLEARING
Dehydration is the process wherein water and fixative are removed from the tissue
and replaced with a dehydrating fluid in preparation of impregnation. Reagents for this
purpose are called dehydrating agents. Water is immiscible with the medium where the tissues
will be embedded, therefore dehydration must be performed prior to subsequent processing.
The most common dehydrating agent is the ascending grades of alcohol. The volume of the
dehydrating agent should not be less than 10 times the volume of the tissue. Ethyl alcohol
mixes with and has affinity for water which allows it to penetrate easily between the tissue
cells.
While clearing or dealcoholization is performed so that the alcohol in the tissues

are removed and replaced a fluid that will dissolve the wax with which the tissue is to be
impregnated or the medium in which the tissue is to be mounted. The clearing agent serves to
mix with alcohol and removes it from the tissues. The volume of the clearing agent should not
be less than 10 times the volume of the tissue. Xylene as a clearing agent removes and
replaces water from the tissues, leaving the tissue with a translucent appearance.
1. OBJECTIVES
1.1 Accurately prepare 70% and 80% ethyl alcohol from 95% (stock) ethyl alcohol.
1.2 Properly dehydrate tissues using manual methods of processing.
1.3 Demonstrate properly manual method of clearing.
2. MATERIALS
2.1 Fixed Liver, Lung & Kidney tissue 2.8 Funnel

2.2 Dental floss 2.9 Calculator
2.3 Pasture pipette 2.10 Marker pen
2.4 Masking tape / gum label 2.11 Ruler
2.5 10ml Reagent bottles / Vials 2.12 Litmus paper
2.6 Serologic pipette 2.13 Aspirator
2.7 Stirring rod
3. REAGENTS
3.1 Absolute ethyl alcohol ……….….. 100 ml
3.2 95% Ethyl alcohol ……………… 100 ml
1.3 Xylene ………………………………….. 1000 ml
1. PROCEDURE
1. DEHYDRATION:
4.1 Prepare 250ml of 70% and 80% ethyl alcohol from 95% ethyl alcohol using the
formula: C1V1 = C2V2. Show your computation to your instructor before you
proceed. Label the container the bottle correctly with your group.
4.2 Immerse the previously fixed and decalcified tissue in 70% for 6 hours, 95% ethyl
alcohol for 12 hours, absolute ethanol for 2 hours and another 2 changes of absolute
ethanol for 2 hours.
2. CLEARING:
2.1. Immerse the previously dehydrated tissue specimen in two changes of xylene for 1
hour each.
1
5.1 Draw and label the set up for dehydration. A. LIVER
1 hour 1 hour 1 hour 1 hour 1 hour 1 hour
B. KIDNEY
2
C. LUNG
5.1 Draw and label the set up for Clearing.
A. LIVER
1 hour 1 hour
3
B. KIDNEY
1 hour 1 hour
C. LUNGS
1 hour 1 hour
4
1.2 Computations:
Compute for the volume of alcohol and distilled water in preparing 70%, 80% and 95% alcohol
from the alcohol stock solution (95%).
5
6. STUDY QUESTIONS
6.1 What are the characteristics of an ideal dehydrating agent?
An ideal dehydrating agent has the following characteristics:
It should dehydrate rapidly without producing considerable shrinkage or distortion

of tissues.
It should not evaporate very fast.
It should not be able to dehydrate even fatty tissues.
It should not harden tissues excessively.
It should not remove stains.
It should not be toxic to the body.
It should not be a fire hazard.
6.2 Give examples of dehydrating agents that also act as clearing agent?
Dehydrating agents that also act as clearing agent are Dioxane (Diethylene
Dioxide) and THF or Tetrahydrofuran. Dioxane has a dual purpose/function; it
dehydrates and clears tissue at the same time, and is miscible with water, alcohol, and
paraffin. THF or Tetrahydrofuran has similar properties with Dioxane and is used during
the embedding process.
6.3 Aside from alcohol, what are the other dehydrating agent which can be used in tissue
processing? Cite their advantages and disadvantages.
Other dehydrating agents which can be used in tissue processing are the following
(with their advantages and disadvantages):
Acetone
• Advantages: It is cheap, rapid-acting dehydrating agent utilized for most urgent

biopsies; dehydrates in ½ to 2 hours; and miscible with most embedding resins.
• Disadvantages: Penetrates tissues poorly and causes brittleness in tissues that

are placed in acetone for prolonged period of time; not recommended for routine
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dehydration purposes because of considerable tissue shrinkage produced;
flammable; absolute acetone is easily contaminated with water, resulting in
complete dehydration.
Diethylene Dioxide (Dioxane)
• Advantage: Excellent dehydrating and clearing agent readily miscible with H2O,
melted paraffin, alcohol and xylol; a universal solvent; produces less tissue
shrinkage as compared to alcohol dehydration; faster dehydrant than ethanol.
• Disadvantage: extremely dangerous, its vapor produces a cumulative and highly

toxic action in man.
Ethylene Glycol Monoethyl Ether (Cellosolve)
• Advantages: Dehydrates rapidly; avoids distortion and does not require graded
dilutions; and tissue may remain in it for months without injury.
• Disadvantages: Combustible at 110 – 120 F and are toxic by inhalation, skin

contact, and ingestion; expensive; rapidly absorbs water from the air; and requires
a clearing agent.
Triethyl Phosphate
• Advantages: It removes H2O very readily and produces very little distortion and
hardening of tissue; soluble in alcohol, benzene, toluene, xylene, ether, and
chloroform; may be used as a dehydrating solution in the staining sequence; and it
is used to dehydrate sections and smears following certain stains and produces
minimum shrinkage.
• Disadvantage: No disadvantage has been noted so far.
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Tetrahydrofuran (THF)
• Advantage: Both dehydrates and clears tissues since it is miscible in both water
and paraffin, it can dissolve many substances including fats, most staining
procedures give improved results with THF
• Disadvantage: Toxic if ingested/inhaled; odorous; and dyes are not soluble in

tetrahydrofuran.
6.4 What are the essential oils that can act as clearing agent? Give their functions.
The essential oils that can act as clearing agents are the following:
Oil of Cedarwood - clears both paraffin and celloidin sections during the
embedding process only; improves cutting of the sections; hard to eliminate from
the tissues in paraffin bath; and more expensive.
Terpineol (Artificial Oil of Lilac) – used for both embedding and mounting steps
in tissue processing; particularly used in the clearing celloidin section in the
mounting procedure; and a working substitute/replace for oil of cedarwood in the
paraffin embedding technique.
Aniline Oil - used only for clearing embryos, insects and very delicate specimens
due to its ability to clear 70% alcohol without excessive tissue shrinkage and
hardening.
Clove Oil – causes minimum shrinkage of tissues; quality is not guaranteed due to
its tendency to become adulterated because wax impregnation after clearing with
clove oil is slow and difficult; tissues become brittle, aniline dyes are removed, and
celloidin is dissolved.
Oil of Bergamot – slow clearing agent but not harmful to the tissue.
Oil of Origanum – aka Spanish Hop Oil or Thyme Oil
Oil of Wintergreen – applicable only when double embedding is required; a

nitrocellulose solvent also known as Methyl Salicylate or Methyl Benzoate.
8
6.3 What will happen to a clearing agent if water is not completely removed from tissues?
The clearing agent turns cloudy or milky or turbid, an indication of incomplete

dehydration. When dehydrating, water and alcohol must be removed since they are
not miscible, or they are immiscible with paraffin and should be replaced with a
miscible substance. If they are not completely removed, inadequate dehydration will
be followed by inadequate clearing. The clearing agent cannot act properly which will
result to mushy tissue blocks that are non-receptive to wax infiltration.
9
Exercise No. 4: DEHYDRATION and CLEARING

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
10
11
EXERCISE 5
IMPREGNATION and EMBEDDING
Impregnation or Infiltration involves removal of the clearing agent from the tissue
and saturating the tissue with a liquid that will fill natural cavities and tissue spaces. This
allows easier handling and cutting of suitably thin sections without any damage or distortion
to the tissue and it cellular components.
Embedding or Blocking or casting is the process by which the impregnated tissue

is placed precisely arranged position in a mold containing melted wax which is allowed to
solidify. The volume of the embedding medium should be at least 25 times the volume of the
tissue.
Impregnating and Embedding medium should be soluble in processing fluid. It

should be easy to section and make ribbons molten between 30 to 60° C. Common medium
used is the paraffin wax that can fill the tissue spaces so that airholes will not form upon
sectioning. After embedding, a paraffin block is produced which will be cut using the
microtome.
1. OBJECTIVES
1.1 Demonstrate the proper infiltration of the tissue with paraffin wax.
1.2 Demonstrate the proper placing of tissue in a precisely arranged position in an

embedding mold using manual method.
2. MATERIALS
2.1 Cleared Kidney, Liver & Lung tissue 2.6 Embedding mold
2.2 Paraffin pitcher with thermal jacket 2.7 Ruler
2.3 Paraffin bath 2.8 Scissors
2.4 Stirring rod 2.9 Thermometer
2.5 Forceps
3. REAGENT
3.1 Paraffin wax pellets ……….….. 1 kg
4. PROCEDURE
4.1 IMPREGNATION / INFILTRATION:
4.1a Dissolve paraffin wax in five separate beakers. The maximum temperature should
be only 2 – 5°C higher than the melting point of the wax which is 60°C.
4.1b While waiting for the wax to melt, prepare the embedding mold.
4.1c Immerse previously cleared specimens in 2 changes of wax 2 hours each,

having 15-minute interval per change for a total of 4 hours impregnation.
4.1d Leave the specimen in the paraffin bath and prepare materials for embedding.
4.2. EMBEDDING:
4.2a Dissolve paraffin wax pellets in the pitcher with thermal jacket. The maximum
temperature should be only 2 – 5°C higher than the melting point of the wax
which is 60°C.
4.2b Prepare the label using a pre-measured strip of paper. Using a pencil, write the
family name, initials of the patient & type of specimen (represented by block
number).
4.2c Seal the label by immersing it in the paraffin pitcher with melted wax.
4.2d Prepare the hinge pop-out mold, making sure that it is clean and dry prior to
use. The size of the mold must fit the tissue with allowance for at least 2 mm
margin of wax at the four sides.
4.2e Fill the mold with melted wax up to the rim. Using a clean and preheated
forceps, transfer the impregnated tissue from the paraffin bath to the mold.
Orient the tissue in the perfect central position.
4.2f. Place the label in such a way that it is facing outside on one-side of the mold,
avoiding the slit part.
4.2g Allow the wax to cool using ice about 3-5 mins for complete cooling and
hardening. Do not over-expose the block in the ice for it may crack or break.
4.2h. Remove the block from the mold.

1
4.2i. Cross-check the block if the tissue was oriented correctly and transfer the label
at the side of the block using the pre-heated paraffin knife.
4.2j. You must redo the procedure if there is any error in the embedded tissue.
4.2k. If everything is done properly, the tissue block is now ready for cutting using
a rotary microtome.
5.1 Draw and label the set up for Infiltration / Impregnation:
Paraffin Wax Pellets
2
5.2 Draw and label the set up for Embedding.
Label Strips
Paraffin Wax Pellets
3
Ice Bath for 3-5 mins.
4
6. STUDY QUESTIONS
6.1 What are the different types of molds used in tissue processing?
Leuckhart’s Embedding Mold – consists of two L-shaped strips of heavy brass or

metal arranged on a flat metal plate and which can be moved to adjust the size of
the mold to the size of the specimen. It is recommended for routine use, although,
too slow and cumbersome for use in a busy laboratory.
Compound Embedding Unit – is made up of a series of interlocking plates resting

on a flat metal base, forming several compartments. It has the advantage of
embedding more specimens at a time, thereby reducing the time needed for
blocking.
Plastic Embedding Rings & Base Mold – consist of a special stainless steel base
mold fitted with a plastic embedding ring, which later serves as the block holder
during cutting.
o Tissue Tek – a model under this type that is equipped with a warm
plate to manage the impregnated specimen, and a cold plate at -5°C
for rapid solidification of the block.
Disposable Embedding Molds
o Peel-Away - disposable thin plastic embedding molds, available in 3

different sizes, are simply peeled off one at a time, as soon as the wax
has solidified, giving perfect even block without trimming. It may be
placed directly in the chuck or block holder of the microtome.
o Plastic Ice Trays – such as those used in ordinary refrigerators may be

recommended for busy routine laboratories. Each compartment may
be utilized for embedding one tissue block, which may then be
removed by bending the plastic tray once the wax has solidified or by
smearing the inner mold with glycerin or liquid paraffin before
embedding.
o Paper Boats – are normally utilized for embedding celloidin blocks

but are equally useful for paraffin wax blocks. Rapid embedding of
small or large volume of individual specimen is possible, since paper
molds can be made to suit any size of tissue.
Hinge Pop-Out Mold
5
6.2 Enumerate substitutes for paraffin wax used in infiltration or impregnation? Give its
advantages and disadvantages
o Paraplast
• More elastic and resilient than paraffin wax thereby permitting large dense
tissue blocks such as bones and brain to be cut easily with the same result
as in double embedding. Blocks obtained are more uniform than with any
other medium, with better ribboning of sections.
• During the summer it may be necessary to use 60 to 63C, although this is
to be avoided if possible in order to not to "cook" the tissue. "Cooked"
tissue does not section well or, if it does, it does not stain well and most
details are destroyed.
o Embeddol
• A synthetic wax substitute similar to Paraplast with a melting point of 56-
58°C.
• It is less brittle and less compressible than Paraplast. Bio/aid is a
semisynthetic wax recommended for embedding eyes. Tissue Mat is a
product of paraffin, containing rubber, with the same property as
Paraplast.
o Ester Wax
• It has a lower melting point (46-48°C), but it is harder than paraffin.
• It is not soluble in water but is soluble in 95% Ethyl Alcohol and other
clearing agents; hence, it can be used for impregnation without prior
clearing of the tissue.
• Needs a heavy-duty microtome; not applicable for routine microtomy.
o Water Soluble Waxes

• These are plastic polymers, mostly polyethylene glycols with melting
points of 38-42°C or 45-56°C.
• Polymer waxes are incorporated in the majority of proprietary histological
paraffin wax blends presently available to improve adhesion, hardness and
plasticity.
• Recommended for histochemical studies.
o Dimethyl sulphoxide (DMSO)

• This is added to proprietary blends of plastic polymer paraffin waxes
reduces infiltration times and facilitates thin sectioning.
• DMSO scavenges residual transition solvent and probably alters tissue
permeability by substituting for or removing bound water thus improving
infiltration.
6
6.3 Aside from paraffin, what are the other media which can be used for impregnation
and embedding? When are these media used?
Celloidin (Collodion)
- a purified form of nitrocellulose soluble in many solvents, suitable for

specimens with large hollow cavities which tend to collapse, for hard and
dense tissues such as bones and teeth and for large tissue sections of the
whole embryo.
- No heat is required, and the resultant block has a rubbery consistency

which gives good support to the tissues. Disadvantages include inability to
cut thin sections, storage of blocks in alcohol and speed of technique
(which can take several weeks or months).
- This issued mainly for preparing soft tissue sections of mixed consistency
such as eyes and brain.
Gelatin
- rarely used except when dehydration is to be avoided and when tissues are
to be subjected to histochemical and enzyme studies. It is water-soluble,
and does not require dehydration and clearing, although fixatives (such as
10% formalin) should still be washed out by running water whenever
indicated. It has a low melting point and does not cause over-hardening of
tissues by heating.
- It is used as an embedding medium for delicate specimens and frozen

tissue sections because it prevents fragmentation of tough and friable
tissues when frozen sections are cut.
Plastic (Resin)
- the introduction of plastic resin embedding media has provided superior

results for light microscopic studies, particularly in hard tissues such as
undecalcified bone and for high resolution light microscopy of tissue
sections thinner than the usual 4-6 µm, such as renal biopsies and bone
marrow biopsies. Plastics are classified into epoxy, polyester, or acrylic,
based on their chemical composition.
7
6.4 Discuss the myriad factors affecting tissue processing.
Tissue Density and Thickness
- Variable tissue density affects infiltration and subsequent sectioning of

tissues. Spongy tissues (like lungs) are usually more rapidly infiltrated
than hard and dense tissues. Thickness of the tissue also influences the rate
of reagent diffusion and, hence processing time.
Agitation
– This increases the flow of fresh fluids in and around the tissues. Without
agitation, tissues tend to settle to the bottom of the processing device or become too
tightly packed, therefore reducing surface area available for fluid exchange.
Temperature
– Temperatures in the range of 37° to 45°C, for a limited time can speed up
fluid penetration and tissue processing protocols. High temperature can cause the
tissue to shrink and to become hard and brittle, while low temperature increases
the viscosity of reagents used in tissue processing, thereby reducing the rate of
diffusion, and increasing processing time.
Vacuum and pressure
– Reduced pressure can increase the infiltration rate and decrease the time
needed to complete steps in tissue processing protocols. High pressure facilitates
infiltration of dense specimens with the more viscous embedding media.
6.5 Discuss the automated tissue processing.
Tissue processing can be performed manually (hand processing), but when

there is a large volume of tissues that need to be processed, it is more convenient
and much more efficient to use an automated tissue processing machine (“tissue
processor”). This machine allows the specimens to be infiltrated with a sequence
of different solvents finishing in molten paraffin wax. The specimens are in an
aqueous environment to start with (water-based) and must be passed through
multiple changes of dehydrating and clearing solvents (typically ethanol and
xylene) before they can be placed in molten wax (which is hydrophobic and
immiscible with water). The duration and step details of the “processing
schedule” chosen for a particular batch of specimens will depend on the nature
and size of the specimens. The older design of an automatic issue processor is a
8
carousel which contains a cage in which the tissue cassettes are placed. This
carousel has a number of glass beakers containing solvents and solutions which
ensure that the tissue is dehydrated and cleared ready for paraffin wax embedding.
The carousel vertically agitates the cage in each solution before moving on to the
next solution in the dehydration/ clearing method. The modern processors have a
chamber in which the specimens are held and the different solutions are pumped
in and out of the chamber. In general, the whole process takes around six hours
and is usually set up to run overnight and tissues that come off the tissue
processor are still in the cassettes.
9
Exercise No. 5: INFILTRATION and EMBEDDING

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
10
EXERCISE 6
TRIMMING and CUTTING OF TISSUE BLOCKS

Trimming is the process wherein excess wax is removed from tissue by the use of
a knife so that it forms a four-sided prism or until the block assumes the shape of a truncated
prism with opposite sides being parallel. After trimming, the tissue should be surrounded with
at least 2 mm of wax. This is done so that a serial ribbon will be produced after section cutting.
In order to produce a series of tissue sections to be placed on a slide, tissue blocks

are cut using a microtome. The microtome consists of 3 essential parts: the block holder, knife
carrier and knife, and the pawl, rachet feed wheel with adjustment screws. Different types of
microtome are available for each specific purpose. The most commonly and routine used in the
laboratory is the rotary microtome.
A spring balanced teeth or pawl is brought into contact with, and turn a rachet feed
wheel connected to a microtome crew which is in turn rotated, moving the tissue block at a
predetermined distance towards the knife for cutting sections at uniform thickness.
1. OBJECTIVES
1.1 Demonstrate the proper trimming of tissue block into a four-sided prism or truncated
pyramid.
1.2 Perform the proper procedure in using rotary microtome.
1.1 Identify the parts along with their functions.
2. MATERIALS
2.1 Fixed Liver, Lung & Kidney tissue block

2.2 Paraffin block trimmer / cutter / scalpel
2.3 Microtome
3. PROCEDURE
A. TRIMMING OF TISSUE BLOCK:
A.1. Prepare materials needed for trimming: White side of the illustration board, paraffin
block trimmer or scalpel and Bucket with Ice.
A.2. On the white side of the illustration board, place the paraffin block trimmer and the
tissue block 45 degree-angle or slanting position against the sharp part of trimmer.
A.3. Gently sliced the side of the tissue block.
A.4. Repeat this procedure with the other sides of the block.
A.5 Trim the 4-sides of the tissue block into 4-sided prism using a trimmer exposing the
tissue. It is recommended that the tissue should be surrounded with at least 2mm of
the wax.
A.6. Option in trimming is the use of scalpel. Follow steps 1-5 in trimming using the
scalpel exposing the tissue and leaving at least 2mm of the wax.
A.7 Placed the trimmed block in the bucket with ice for few minutes prior to sectioning
to harden the tissue block and facilitate easy cutting.
B. CUTTING / SECTIONING OF THE BLOCK:
Before the actual cutting or sectioning of the block, it is important that we should be
familiar with the rotary microtome aside from the other 5 types, namely: Rocking
microtome, Sliding Microtome, Freezing Microtome, Cryostat or Cold Microtome and
Ultrathin Microtome each with specific purpose.
The Rotary Microtome is consisting of 3 essential parts: (1) the block holder, (2) knife
carrier and knife, (3) the pawl, rachet feed wheel with adjustment screws. Together with the
essential parts are other mechanical parts fitted in the instrument. This is the most commonly
and routinely used in the histopathology laboratory.
1. Block holder - This part is comprised of several components including the blade clamp that
holds the blade, the knife tilt for adjusting the knife angle, and the face plate that guides that
ribbons away from the blade and towards the operator. In general, block holder holds the
tissue block in place for proper positioning during sectioning. It moves the block up and
down with each revolution while the blade is stationary. The block holder may have knobs
1
that allow the user to manipulate the block face in various directions to bring the tissue in
alignment with the blade.
2. Knife carrier and knife - This part is comprised of several components including the blade
clamp that holds the blade, the knife tilt for adjusting the knife angle, and the face plate that
guides that ribbons away from the blade and towards the operator. This also controls the
bevel angle and the clearance angle during sectioning. This is also equipped with a knife
guard to enhance the safety feature of the instrument. While the knife, is a disposable blade
made up of high-quality and sharp stainless steel that is used for the actual cutting of the
tissue.
3. Pawl Rachet feed wheel with adjustment screws - A spring balanced teeth or pawl is brought
into contact with, and turn a rachet feed wheel connected to a microtome crew which is in
turn rotated, moving the tissue block at a predetermined distance towards the knife for
cutting sections at uniform thickness.
4. Other Mechanical Parts:
a. Handwheel: Moves the block holder either toward the knife or away from the knife. It
is equipped with a safety lock to prevent the wheel from releasing and having the block
holder come down towards the blade while a block is inserted or removed. The safety
lock should be used anytime the microtomist is not actively sectioning paraffin blocks.
b. Knife holder base: A part that anchors the knife holder to the microtome stage. The knife
holder base can be moved toward or away from the block, but must be stationary and
locked during microtomy.
c. Micron adjustment screw: Micron settings for section thickness can range from 1 to 60
microns on most microtomes but for routine surgical material, it is usually set to 3-5
microns. And here in the laboratory, we will set this to 5 microns.
d. Microtome base plate or stage: A platform which has rails that secure the knife holder
base. It is also equipped with receptacle to collect waste and excess wax during
sectioning.
2
C. MICROTOMY PROPER:
1. Ensure all the safety locks and screws in place.
2. Mount the knife in the knife holder and tighten the screws. Set the clearance angle in a
way that it can make tissue ribbon. Be sure that the knife blade is secure to prevent
uneven sectioning.
3. Advance the block close to the knife by turning the handwheel.
4. Rough cut the paraffin block by advancing the block using the coarse feed mechanism
exposing the tissue.
5. Cut the paraffin tissue block by rotating the handwheel continuously in uniform motion,
not too fast and not to slow to have an even tissue ribbon.
6. As the sections are cut, a ribbon is created because of the successive sections stick edge
to edge due to local pressure with each stroke. Continue to rotate the wheel at a
moderate and even pace until the desired length of tissue ribbon was produced.
7. As the ribbon comes off the knife, the loose edge of the ribbon is held with a camel’s
hairbrush or with your hand simultaneously rotating the handwheel. Stop rotating the
handwheel when you reach the desired length of the tissue ribbon.
8. Place the ribbon on the black side of the illustration and paste both ends to prevent it
from flying off.
9. Set aside the sectioned tissue for the next step that is Mounting.
10. Clean the microtome after use.
3
5.1 Draw and label the set up after Trimming.
5.2 Draw and label the different parts of the microtome.
4
6. STUDY QUESTIONS
6.1 What is the importance of trimming section blocks?
Trimming tissue blocks is important since it allows exposure of the tissue surface,
through cutting off excess wax in preparation for actual cutting or sectioning. Since tissue
is completely surrounded by paraffin, it is useful to uncover the surface of the block to
reveal the tissue. This is done so that a serial ribbon will be produced during section cutting.
6.2 What are the different microtome knives available for cutting? Give its description.
Plane-Concave Knife (usually 25 mm. in length)
– One side of the knife is flat while the other is concave. Less concave sides
are recommended for cutting celloidin-embedded tissue blocks on a sliding
microtome. More concave sides are used to cut paraffin sections on base-sledge,
rotary or rocking microtome.
Biconcave Knife (usually 120 mm. in length)
– With both sides concave, recommended for cutting paraffin - embedded

sections on a rotary microtome.
Plane-Wedge Knife (usually 100 mm. in length)
– have both sides straight, recommended for frozen sections or for cutting
extremely hard and tough specimens embedded in paraffin blocks, using a base
sledge type or sliding microtome.
6.3 What are the different types of microtome? For what specific embedding media are
they used?
Rocking microtome – for cutting serial sections of large blocks of paraffin

embedded tissues.
Rotary microtome - for cutting paraffin embedded sections.
Sliding microtome - for cutting celloidin embedded sections.
Freezing microtome -for cutting unembedded frozen sections.
Cryostat or cold microtome – for cutting frozen sections

5
Ultrathin microtome - for cutting sections for Electron Microscopy.
6.4 What are the two stages of knife sharpening? Give its purpose.
The knife we use now is a disposable knife. However, if you use the conventional
type, it becomes dull and badly nicked hence, we have to sharpen the knives. The two
stages of knife sharpening are:
a) Honing (Coarse Sharpening) - Its purpose is for the removal of gross nicks
on the knife edge (Coarse Honing) to remove blemishes or irregularities and
grinding the cutting edge of the knife on a stone (Honing Proper) to acquire an
even edge.
b) Stropping (Fine Sharpening) - Its purpose is for the removal the "burr"
formed during honing and for the polishing of the cutting edge of the knife. If
the knife has become dull and blunt, but is free from nicks or teeth, it is usually
only necessary to strop it. For delicate work, the knife is stropped before every
object is sectioned.
6.5 What are the different types of hones? Give the usage of each type.
Belgium Yellow – for manual sharpening when cutting edge has been
rendered blunt or nicked. This type usually gives the best result.
Arkansas – gives more polishing effect than the Belgium Yellow.
Fine carborundum – is much coarser than the first two types and is used
only for badly nicked knives followed by either one of the first two knife
sharpeners
6
Exercise No. 6: TRIMMING and CUTTING of TISSUES

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
7
EXERCISE 7
MOUNTING OF TISSUE SECTIONS

Mounting is the process of making permanent slides by attaching into the slides for
staining. After cutting, sections are floated out on a water-bath and transferred to a glass slide.
Bubbles accumulating under the ribbon may then be removed with smooth teasing needle, care
being taken not to tear the section. When the sections chosen have flattened out, the numbered
slide is immersed in the water bath and the section is fished out.
To promote adhesion of sections, adhesives may be spread thinly and evenly on a

clean grease-free slides which is gently approximated to the end of the ribbon, and drawn
upwards in a near vertical position. Common adhesive use is the Mayer’s egg albumin. At the
end, aligned tissue sections must be done.
1. OBJECTIVES
1.1Properly prepare Mayer’s egg albumin adhesive.
1.2 Perform the proper procedure in mounting sections in the glass slides.
2. MATERIALS
2.1 Tissue ribbons of Kidney, Liver, and Lung Tissue

2.2 Glass slides
2.3. Scalpel
2.4 Teasing needles
2.5 Floating Bath
2.6 Pencil
2.5 Illustration board: black side
3. REAGENT
3.1 Egg white …………… 50 ml

3.2 Glycerin …………… 50 ml
3.3 Thymol crystals …………… 1 gram
4. PROCEDURE
Mayer’s Egg Albumin Preparation:
4.1 First, shake the egg whites to break them up and make them slightly frothy.
4.2 Then mix equal parts of egg whites and glycerin. Add a crumb of thymol to prevent
mold.
4.3 Store in a dropper bottle and keep refrigerated when not in use.
Mounting of Tissue sections:
4.1 Prepare all materials and set the Mayer’s egg albumin in room temperature.
4.2. After cutting, the tissue ribbon is positioned on the black side of the illustration
board. Make a pattern of the slide to determine its center.
4.3. Select a portion in the ribbon that contains a flat tissue section.
4.4. Cut the section using a scalpel and float on the floatation bath.
4.5 Using a clean, dust-free slides, apply Mayer’s egg albumin evenly on the surface of
the slide.
4.6. Fish-out the tissue in the floatation bath.
4.7 Position the section at the center of the slides using the teasing needle while removing
excess water in the slides.
4.8. Lay flat the slides and let it dry.
4.9 Repeat procedure for the next slide.
4.10 Store the dry mounted tissue in the slide box for the next step which is staining.
1
5.1 Draw and label the set up after Mounting.
MATERIALS USED IN MOUNTING
MOUNTED SLIDES
2
6. STUDY QUESTIONS
6.1 What is the purpose of adhesive in mounting sections on the slides?
The purpose of adhesive in mounting sections on the slide is that it makes the
sections stick well to the slides once smeared on the slide. They are essential for methods
that require exposure of sections to acids and alkalis (especially ammoniacal silver
solutions) during staining. These special stains, particularly the alkaline reagents, can cause
sections to lift, requiring adhesives.
6.2 Enumerate other adhesives. Give their advantages and disadvantages.
The different adhesives with their advantages and disadvantages are the following:
Mayer’s Egg Albumin
 Advantages: This is very easy to make, convenient, and relatively

inexpensive. This is the most commonly used adhesive.
 Disadvantages: The excess albumin takes up stain and interferes with

diagnosis. Thymol-resistant organisms will grow and contaminate gram-
stained sections.
Dried Albumin
 Advantages: This is easy to prepare, convenient, cheap.
 Disadvantages: This retains some stains and gives a dirty background.
Gelatin (1%)
 Advantages: Adding aqueous gelatin to the water in floating out bath and
mixing it well is the most convenient alternative to direct coating of slides.
 Disadvantages: This adhesive is slow in action; may take up to 24 hours.
3
Gelatin Formaldehyde Mixture
 Advantages: This is not commonly used but is effective due to formaldehyde.
 Disadvantages: This requires use of fume hood for preparation due to

formaldehyde.
Poly-L-Lysine
 Advantages: This is widely used as a section adhesive in

immunohistochemistry.
 Disadvantages: This must be used within few days after they are prepared.
The effectiveness of the adhesive slowly decreases in time.
APES (3-aminopropyl triethoxysilane)
 Advantages: This is very useful in cytology, particularly for cytosine

preparations of proteinaceous or blood materials. This can be stored for a long
time without losing adhesiveness.
 Disadvantages: This is expensive and has a tedious preparation.
6.3 What is the purpose of thymol in Mayer’s egg albumin?
Thymol in Mayer’s egg albumin is added to prevent the growth or formation of molds,
and to avoid bacteria and fungi. Hence, thymol acts as a preservative to sustain the
quality of the adhesive.
6.4 How is the Mayer’s egg albumin applied in the paraffin embedded section? What is
its effect if applied excessively?
A drop of Mayer's egg albumin is usually smeared into the clean glass slide before
sections are oriented. Sections which have been creased on cutting may be stretched
by gentle heating before attaching them into slides. During staining, the excess of
albumin may also take up or absorbs the stain and interfere with diagnosis; hence, it
should be wiped off from the slide to remove any excessive solution.
4
6.5 How is the Mayer’s egg albumin applied in the celloidin embedded section?
For celloidin sections, egg albumin is smeared on the slide. The section is then
transferred from 95% alcohol bath to the slide, pressed flat on the slide with a smooth
filter paper coated with thin celloidin (2-4%) mixture.
5
Exercise No. 7: MOUNTING of TISSUES SECTION:

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
6
EXERCISE 8A
STAINING OF TISSUE SECTIONS

(H&E and Masson’s Trichrome Staining)
The end product of optimal tissue processing and successful microtomy is a thin and
translucent tissue section set on a slide. The only way to render the tissue visible under the light
microscope is to provide color to the cell components through the stains. Differential staining
enables microscopic evaluation of all areas of the tissue. For clinical laboratories, such
evaluation is necessary for the anatomic pathologist to render a histopathologic diagnosis. The
most common routine staining is the Hematoxylin and Eosin (H & E) method, Special stains
are the other alternative staining techniques employed when H & E cannot visualize certain
tissue components under consideration. H & E is a regressive type of staining which consist of
overstaining the nuclei, removal of superfluous and excessive color of the tissue constituent by
acid differentiation.
Dyeing or staining of the tissue sections enables the technologist or pathologist see and
study the physical characteristics and relationships of the tissue and of their constituent cells
under the microscope. And since, H&E has limitations in staining specialized parts in the tissue,
special stains were employed.
Masson's trichrome is a three-color staining protocol used in histopathology suited for

distinguishing cells from surrounding connective tissue. It is also used in paraffin-embedded
sections.
1. OBJECTIVES
1.1 Demonstrate the proper procedure in H & E stains for liver tissue.
1.2 Demonstrate the proper procedure in Masson’s Trichrome stain for kidney tissue.
1.3 Demonstrate the proper procedure in Toluidine Blue-Alizarin Red S Staining of Bone
tissue.
2. MATERIALS
2.1 Paraffin embedded Bone, Liver & Lung tissue

2.2 Staining dish
2.3 Staining rack
2.4 Rag
2.5 Timer
3. REAGENT
3.1 H & E Stain set
3.2 Masson’s Trichrome Stain set
4. PROCEDURE
1. H & E for Liver Tissue: 10 slides
4.1 Xylene 1 ………………………… 5 dips
4.2 Xylene 2 ………………………… 5 dips
4.3 Absolute alcohol …………………. 10 dips
4.4 90% Alcohol ………………… 5 dips
4.5 70 % Alcohol ………………… 5 dips
4.6 Wash in running water
4.7 Harris Hematoxylin ……………… 5 minutes
4.9 1% Acid Alcohol ……………….. 10 – 30 seconds
4.11 Ammonia water
4.13 5% Aqueous Eosin ……………… 5 minutes
4.14 70 % alcohol ……………………. 5 dips
4.15 90% alcohol ……………………. 5 dips
4.16 Absolute alcohol ……………….. 10 dips

1
4.17 Xylene 1 ………………………. 5 dips
4.18 Xylene 2 ………………………. 5 dips
4.19 Mount
2. Masson’s Trichrome for Kidney Tissue: 10 slides
4.1 Xylol ………………………… 2 mins
4.2 Xylol ………………………… 2 mins
4.3 Xylol ……………………………. 10 dips
4.4 Absolute alcohol …………………. 10 dips
4.5 Absolute Alcohol ………………… 10 dips
4.6 Distilled water ……………………. 10 dips
4.7 Weigert’s Iron Hematoxylin ……… 20 minutes
4.8 Wash in running water (overflow technique) … 10 minutes
4.9 Distilled water …………………….. 10 dips
4.10 Biebrich Scarlet-Acid Fuchsin Solution ….. 15 minutes
4.11 Distilled water ………………………. 10 dips
4.12 Aqueous Phosphotungstic acid (5%)….. 15 minutes
4.13 Light green solution ……………… 5 minutes
4.14 Distilled water ……………………. 10 dips
4.15 Acetic water (1%) ……………………. 3 dips
4.16 95% Alcohol …………………….. 10 dips
4.16 Absolute alcohol ……………….. 10 dips
4.17 Absolute alcohol ………………. 10 dips
4.19 Mount
2
5.1 Draw and label the set up after staining.
A. H & E STAINING
slide carrier
10 block 1
(liver) slides placed in
(heat-fixed) a slide
carrier
Staining dish
Block 1 slides
running running
water water
running running
water water
H&E stained
liver specimen
label
10 block 1 (liver)
slides
(stained with H&E)
3
B. MASSON’S TRICHROME STAINING:
slide carrier
10 block 2
(kidney) slides placed in
a slide
(heat-fixed)
carrier
Staining dish Block 1 slides
running
water
label Masson’s Trichrome stained

kidney specimen
10 block 2 (kidney)
slides
(stained with
Masson’s Trichrome)
4
6. STUDY QUESTIONS
6.1 What is the principle of H and E Staining & Masson’s Trichrome Staining?
H & E is composed of hematoxylin stain and eosin stain. Hematoxylin is a basic

cationic (positively charged) thus, stains cell nuclei blue. Meanwhile, eosin is an acid
anionic (negatively charged) hence stains the cytoplasm, connective tissue and other
extracellular substances pink or red. H & E colors otherwise transparent tissue sections and
extracellular components such as the cytoplasm, nucleus and organelles to be clearly
visible under the microscope.
Masson’s Trichrome is a 3-color stain which stains keratin and muscle fibers red,
collagen and bone blue or green, cytoplasm light red or pink and nuclei black. It uses dyes
in acid solution involving nuclear staining with iron hematoxylin, followed by cytoplasmic
staining with a red dye (e.g., Ponceau phosphotungstic acid, phosphomolydic acid or both,
and fixed staining of fibers with a blue or green stain (e.g. aniline blue or light green). In
the exercise, we used Weigert’s iron Hematoxylin solution, Bibebrich-Scarlet acid fuchsin
solution and Light green solution.
6.2 Give the expected color of the following:
TISSUE COMPONENT H&E MASSON’s TRICHROME
Nuclei purple/blue black
Cytoplasm pink to red in color light red/pink
RBC red red
Muscle Fiber pink red
Collagen Fibers pink light green
5
6.3: What are the different staining methods that are commonly employed for
frozen sections?
The staining methods that are commonly employed for frozen sections are the
following:
• Hematoxylin Eosin Method
• Thionine Method
• Polychrome Methylene Blue Method
• Alcoholic Pinacyanol Method (used also for supravital staining of mitochondria

and primarily for color sensitization in photography)
6.4 Define chromophore, chromogen and auxochrome in relation to dyes?
Chromophores are substances with definite atomic groupings that are capable of
producing visible colors. These are groups on the benzene ring that confers color.
Chromophores alters the light resonance properties of the compound so that unequal
absorption occurs when white light is passed through. It has two group: quinoid group and
quinone-imene group.
Chromogens temporarily impart color which is easily removed. It is a benzene

derivative containing chromophoric groups. Examples include stilbene (colorless),
azobenzene (orange) and thiobenzophenone (blue).
Auxochrome is an auxiliary radical that imparts the property of electrolytic

dissociation which alters the shades of dyes by allowing other compounds to form salts and
retaining its colors in the tissue. When added to chromogen, it will allow the chromogen to
retain its color in the tissue therefore, turning it into a dye.
6.5 Differentiate regressive staining to progressive staining. Give examples of each.
In progressive staining, staining solution is applied until desired intensity of the

coloring of the tissue elements is achieved. Once the desired coloring intensity is achieved,
the staining solution is not washed nor decolorized. During this method, the mordant
precedes the stain or with the stain as in H& E staining method. Examples include Cole’s
Hematoxylin and Phosphotungstic Acid Hematoxylin.
6
Regressive staining is a technique done by overstaining the tissue to colorize the
different tissue elements and then treated by decolorization to remove excess stain from
specific tissue elements. Examples include Alum Hematoxylin, Harris Hematoxylin,
Mayer’s Hematoxylin, Iron Hematoxylin, and Heidenhain’s Hematoxylin.
7
Exercise No. 8A: H&E and Masson’s Trichrome:

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
8
EXERCISE 8B
STAINING OF TISSUE SECTIONS:

FITE-FARACO & KINYOUN’S STAINING
Fite-Faraco and Kinyoun Carbol Fuchsin staining, are bacteriological stain used to
identify acid-fast organisms, mainly Mycobacteria. These have cell walls that are resistant to
conventional staining by aniline dyes such as Gram Stain. However methods that promote the
uptake of dyes are available; once stained these organisms are not easily decolorized even with
acid-alcohol or acid-acetone solutions therefore they are described as acid-fast. Their resistance
to destaining is a useful characteristic in differentiating these organisms from contaminating
organisms and host cells. The reagents used for Fite-Faraco staining are – carbol fuchsin,
sulfuric water, and methylene blue. Acid-fast bacilli are bright red after staining.
The Kinyoun staining procedure is often referred to as cold carbolfuchsin because

no heat is applied during the staining process unlike the Ziehl Neelsen procedure. The primary
stain for the Kinyoun procedure is the aniline dye, basic fuchsin, that stains all the cells present
red. The unique ability of mycobacteria to resist decolorization by acid-alcohol is why they are
termed acid-fast, and will keep their red coloration throughout the staining process. The
decolorizer used is a hydrochloric acid-ethanol mixture that will decolorize non-acid-fast
material present. The last step in the staining procedure is the application of a counterstain. If
methylene blue is used other cells and background material present on the slide stain blue; if
brilliant green is used other cells and background material present on the slide stain green.
1. OBJECTIVES
1.1 Demonstrate the proper procedure in Fite-Faraco staining for lung tissue.
1.2 Demonstrate the proper procedure in Kinyoun’s staining for lung tissue.
2. MATERIALS
2.1 Paraffin embedded Lung tissues

2.2 Staining dish
2.3 Staining rack
2.4 Rag
2.5 Timer
3. REAGENT
3.1 Fite-Faraco Stain set
3.2 Kinyoun Stain set
4. PROCEDURE
4.1. FITE-FARACO STAIN FOR LUNG TISSUE (5 SLIDES):
1. Xylene – Peanut oil ……………… 12 minutes
2. Xylene – Peanut oil ……………… 12 minutes
3. Wash in Tap water …….………… 10 dips
4. Drain. Wipe off excess oil on slide carrier. Each member should wipe his/her own
Slide.
5. Ziehl-Neelsen carbol fuchsin solution .. 30 minutes
6. Wash in Tap water…………………. 10 dips
7. 1% Sulfuric water until faint pink
8. Working Methylene Blue …..…….. 3 minutes
9. Rinse in Tap water ……….……… 10 dips
10. Mount
1
4.2. KINYOUN’s ACID FAST STAIN for LUNG TISSUE (5 SLIDES)
1. Xylene …………………………… 2 minutes
2. Xylene …………………………… 2 minutes
3. Xylene …………………………… 10 Dips
4. 95% Alcohol ……………..……… 10 Dips
5. 95% Alcohol ………………….. 10 Dips
6. 95% Alcohol …………………. 10 Dips
7. Distilled water ……………….. 10 Dips
8. Kinyoun’s Carbol Fuchsin ….. 1 Hour
9. Wash well in Tap Water
10. 1% Acid Alcohol ……………. 10 Dips
11. Wash well in Tap water
12. Working Methylene Blue …… 3 minutes
13. 95% Alcohol ………………… 1 Dip
14. Mount
2
5.1 Draw and label the set up after staining.
A. FITE-FARACO STAINING:
3
B. KINYOUN’S STAINING:
4
6. STUDY QUESTIONS
6.1 What is the principle of Fite-faraco and Kinyoun’s Staining?
Fite-Faraco and Kinyoun Carbol Fuchsin staining are bacteriological stain used to
identify acid-fast organisms, mainly Mycobacteria. The Fite-Faraco stain is based on the ability of
the acid-fast organism’s lipoid capsule which has the ability to take up carbol fuchsin and resist
decolorization with dilute miner acids. Xylene-peanut oil is the first two changes in Fite Raraco
because Fite Faraco stain is intended to demonstrate Mycobacterium leprae (leprosy) or Leprae
bacilli. Since leprae bacilli are easily lost during staining, instead of xylol, we add peanut oil to
protect or coat of the microorganism.
Meanwhile, Kinyoun’s staining uses a carbol fuschin primary stain that is applied
to the cells and instead of heating which is used in the Ziehl-Neelsen method, phenol is added to
allow the stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is
removed by 1% acid alcohol and stained with methylene blue which is the counterstain.
6.2 Give the expected color of the following:
TISSUE COMPONENT FITE-FARACO KINYOUN’s
Nuclei blue blue
Cytoplasm light blue blue
Organism bright red red
6.3 What is negative staining?
Negative staining is a method of simple staining for bacteria. An acidic, anionic

dye is used to change the background color which will cause the cells to stand out. This can be
achieved by smearing the sample onto the slide and then applying nigrosin (a black synthetic dye)
or India ink (an aqueous suspension of carbon particles). It turns the background into a dark brown
to black while leaving the clear bright cells unstained and highly visible.
6.4 Differentiate simple staining to differential staining. Give examples of each.
Simple staining employs a single dye so that all the cells will have the same color.
It is categorized into positive staining (cells take up the stain) and negative staining (background
5
take up the stain). Simple stains are single dyes used to stain the organism and it has limited clinical
application. The dye is negative and the bacterium is positively charged and they will get stained
due to its interaction with a negatively charged dye. Before a sample can be stained with a simple
stain, it must be heat fixed to the slide. This process makes staining more effective but can damage
or distort the cells, changing their appearance from a truly natural, free-living state. Simple staining
includes Methylene Blue and Dilute Carbol Fuschin.
On the other hand, differential staining uses multiple dyes so that more than one
color is observed, this is to observe contrast between cells. It takes advantage of the different
physical and chemical properties of different groups of bacteria that will cause them to stain
differently. This includes Gram Staining and Acid-Fast Staining.
6.5 What are the indicators of viral invasion in tissues?
The indicators of viral invasion in tissues include inflammation, necrosis, cellular

damage, ulceration, the tissues’ appearance, formation of intracytoplasmic bodies, margination of
chromatins along nuclear membranes, prominent intranuclear inclusions, granular cytoplasmic
inclusions, and etc.
Specifically the following are observed in different viruses:
• Hepatitis viruses (A, B, C, D) – massive necrosis of liver cells or hepatocytes; some will
have eosinophilic ground glass appearance due to accumulation of hepatitis B surface
antigens
• Molluscum contagiosum virus – appearance of warts in children and young adults;

formation of large eosinophilic intracytoplasmic bodies in maturing keratinocytes of the
skin
• Herpes virus – blistering and ulceration of the skin and mucous membranes; Cowdry Type
A herpes causes margination of chromatin along the nuclear membranes giving an “owl’s
eye” appearance to the cell
• Cytomegalovirus – prominent intranuclear inclusions and sometimes granular

hematoxyphilic cytoplasmic inclusion in infected cells
• Rabies virus – form intracytoplasmic eosinophilic inclusions in the hippocampal neurons

of the brain
• JC virus – form intranuclear hazy hematoxyphilic or slightly eosinophilic inclusions in the

hypertrophic oligodendrocytes.
6
Exercise No. 8B: STAINING of TISSUE: FITE-FARACO & KINYOUN’s STAIN

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
7
EXERCISE 9
COVERSLIP MOUNTING and LABELLING

Mounting is the process of covering the stained slides in order to create a permanent
slide for histopathology. Mounting medium is being used in this process. They hold the
specimen in place, in between the slide and cover slip and allow the specimen to be stored for
years. Mounting media also provide the maximum degree of transparency to stained tissue
sections.
Labelling is very important before submitting the slides for microscopic evaluation
of the pathologist. It maybe written, etched or embossed on each slide. Permanent pens, which
are chemical-resistant should be used. Each slide must bear unique identification and accession
codes, patient names or other accession information. Accession number usually include the year
the specimen was collected with varying prefixes for different types of specimen.
1. OBJECTIVES
1.1 Demonstrate the proper procedure in mounting the coverslips.
1.2 Demonstrate the proper procedure in labelling histopathologic and cytologic slides.
2. MATERIALS
2.1 Stained Liver, Kidney & Lung smear slides

2.2 Coverslips (22mm x 22 mm square)
2.3 Teasing needle (pair)
2.4 Illustration board (1/8 size: white side)
2.4 Gum label (same size of the frosted end of the glass slide)
2.5 Permanent marker
2.6 Tissue paper / gauze
3. REAGENT
3.1 Eukitt mounting medium – synthetic and commercially available and contains
5% Acrylic Resin and 55% Xylenes
3.2 Xylene
4. PROCEDURE
1. Mounting or Cover slipping:
4.1 There are 2 common techniques in mounting: Cover-slip method and Inverted slide
Method.
4.2 Choose one technique for mounting and mount all the slides.
4.3 Each slide will be cleared using xylene 10 dips each before mounting.
4.4 To save time, you can clear and clean the slides simultaneously.
4.5 After dipping the slide in xylene, clean the sides of the tissue using a clean cloth and
do not touch the tissue section, clean only the surrounding by removing the excess stain
and haziness of the slide. Do this for 10 times or until the slide is free of excess stain
and cloudiness or haziness. Do not soak the slide in xylene because it may decolorize
the stain.
4.6 After cleaning, place the slide horizontally on the white side of the illustration board,
with the tissue facing upward for mounting.
A. Mounting using Cover-slip technique:
1. Use a square coverslip with good quality and make sure it is also cleared by xylene.
2. Add few drops of mounting medium at the side of the tissue.
3. Position the coverslip at an angle of the tissue, touching the mounting medium.
4. With the teasing needle, slowly lower the coverslip towards the tissue until it touches
the slide.
5. Through capillary action, wait for the mounting medium to spread evenly then position
the coverslip in a way that it covers the entire tissue with equal distance in 4 sides.
1
6. Using the teasing needle, light pressure is applied on the coverslip to ensure that the
mounting medium was spread entirely and to eliminate air bubbles if present.
7. Remount if air bubble is still present.
8. Set aside to dry.
B. Mounting using Inverted Slide technique:
1. Use a square coverslip with good quality and make sure it is also cleared by xylene.
2. Place the coverslip on top of the lid of the container of the mounting medium.
3. Add few drops of mounting medium at the center of the coverslip.
4. Using the stained slides, invert the slide where the tissue faces down.
5. Slowly touch the mounting medium by the tissue.
6. Through capillary action, wait for the mounting medium to spread evenly then invert
the slide back.
7. Position the coverslip in a way that it covers the entire tissue with equal distance in 4
sides using the teasing needle.
8. Apply light pressure on the coverslip to ensure that the mounting medium was spread
entirely and to eliminate air bubbles if present.
7. Remount if air bubble is still present.
8. Set aside and keep it in flat position to dry.
2. Labelling:
2.1 Before labeling, remove the excess mounting medium that surrounds the coverslip by
dipping it in xylene for several times and wipe it dry with clean cloth or gauze.
2.2 Paste gum labels to all cleaned slides.
2.2 Using a permanent marker, label each slide with your family name, initials of your
first names and accession codes or block number.
2
2.3 Arranged in the slide box per specimen.
2.4 Submit slide box for checking.
5.1 Draw and label the set up after mounting and labelling.
A. MATERIALS:
3
B. FINISH SLIDES:
Menchavez, E. Menchavez, E. Menchavez, E. Menchavez, E.

Block 1 Block 2 Block 3 Block
E. A.3
H and E Masson’s Trichrome Kinyoun’s Fite Faraco
4
6. STUDY QUESTIONS
6.1 What are the properties of a good mounting medium?
The properties of a good mounting medium include:
It should be colorless and transparent.

It should be freely miscible with xylene and toluene.
It should not dry to a non-stick consistency and harden relatively quickly.
It should protect the section from physical damage and chemical activity
(oxidation and changes in pH).
It should be resistant to contamination (particularly microorganism growth).
It should not cause shrinkage and distortion of tissues.
It should not leach out any stain or affect staining.
It should not change in color or pH.
It should be compatible with the adhesive in use.
It should set without crystallizing, cracking or shrinking (or otherwise deform
the tissue being mounted) and not react with, leach or induce fading in stains
and reaction products (including those from enzyme histochemical,
hybridization, and immunohistochemical procedures).
6.2 What are the types of mounting medium? Describe each.
The types of mounting medium include:
Aqueous Mounting Media – This is used for mounting sections from distilled
water when the stains would be decolorized or removed by alcohol and xylene,
which is usually seen in cases with most fat stains or for metachromatic staining
of amyloid. This mounting medium is usually made up of gelatin, glycerin jelly
or gum arabic to solidify the medium.
Resinous Mounting Media – This is used for preparation that have been
dehydrated and cleared in xylene or toluene and are recommended for majority
of staining methods. This may be divided into natural and synthetic resins; the
most important synthetic resins are used for embedding undecalcified bones and
for electron microscopy.
5
6.3 What are the properties of a good coverslip? Describe each type.
The properties of a good coverslip include:
Must be flat
Clear
High spectral transmission
Same refractive index of the glass slide which is 1.518
Good resistance to chemical attacks
Has appropriate thickness
The five types of coverslips are:
Silica glass coverslip – This is pre-cleaned and is used for routine microscopy
procedures. This comes in different sizes (rectangular, square, etc.).
Round glass coverslip – This is used for motility studies in the Microbiology
department. It has a concave center.
Quartz Coverslip – This is used for UV radiation transparency.
Plastic Coverslips – A type of coverslip that is unbreakable and has a refractive
index of 1.54.
Thermanox – This is used for high temperature studies for south culture and
fluorescence microscopy.
6.4 Give the importance of proper labelling.
Labelling is very important before submitting the slides for microscopic evaluation
of the pathologist. Proper labelling is essential for correct and accurate interpretation of
laboratory results, which are relied upon to correctly diagnose patients. Moreover,
accurately identifying patients and correctly labeling specimens are critical to ensure
patient safety as this avoids mismatching of patient results. If a specimen is not accurately
identified, it can lead to delayed or wrong diagnoses, missed or incorrect treatments, blood
transfusion errors, and more. Inaccurate results can also lead to additional laboratory
testing.
6
6.5 What is the advantage of using resinous media over water-based mounting media?
The advantage of using resinous media over water-based mounting media is it

produces a permanent slide that is insoluble in water and may be used for future studies.
Resinous media harden quickly and has a neutral reaction. Xylene can dissolve and it
affects staining less while the water-based media would fade hence it produces temporary
slides and is prone to formation of bubbles.
7
Exercise No. 9: MOUNTING & LABELLING OF SLIDES:

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
8
EXERCISE 10
DECALCIFICATION
Decalcification is the process wherein selected pieces of tissues such as bones teeth
and other calcified tissues are immersed in an agent that will remove calcium and lime salts in
tissues which may interfere with the accurate evaluation and examination of histologic sections.
The reagents for this purpose are called decalcifying agents.
Acid decalcifying agents removed calcium ions or lime salts from the organic
extracellular matrix, calcified collagen and surrounding tissues of the bone. This process makes
the bone easy for histopathological investigation by producing thin sections using a microscope.
1. OBJECTIVES
1.1 Accurately prepare 10% nitric acid from the concentrated solution of nitric acid
(100% nitric acid).
1.2 Properly decalcify bone tissue using an acid decalcifying agent.
2. MATERIALS
2.1 Bone tissue 2.8 Funnel
2.2 Dental floss 2.9 Calculator
2.3 Pasture pipette 2.10 Marker pen
2.4 Masking tape / gum label 2.11 Ruler
2.5 Reagent bottle 2.12 Litmus paper
2.6 Serologic pipette 2.13 Aspirator
2.7 Stirring rod
3. REAGENTS
3.1 Concentrated Nitric acid ……….….. 100 ml

3.3 Concentrated Ammonium hydroxide ……….. 10 ml
3.4 Ammonium oxalate …………. 0.5 ml
4. PROCEDURE
4.1 Prepare 250ml of 10% nitric acid from the concentrated solution using the formula:
C1V1 = C2V2. Show your computation to your instructor before you proceed. Label
the bottle correctly with your group number.
1.2 Cut a ½ x 2 inch piece of filter paper. Using a pencil, write the number corresponding
to the number assigned to you by your instructor.
1.3 Tie the fixed bony tissue using one end of a dental floss. On the other end of the floss,
tie the prepared filter paper containing the specimen number.
1.4 Immerse the bony tissue using one end of the dental floss. On the other end of the
floss, tie the prepared filter paper containing the specimen number.
1.5 Immerse the bony tissue in the prepared reagent. Make sure that the bottle is tightly
covered. Store in a cool dry place for 48 hours, then immerse the tissue in a freshly
prepared decalcifying agent.
1.6 Test for the completeness of decalcification using chemical method.
To Test for the completeness of decalcification by Chemical Method:
1. Pipette 5ml of the discarded decalcifying agent into the test tube. Add a piece of blue
litmus paper. Note the reaction.
2. Using a Pasteur pipette, add concentrated ammonium hydroxide drop by drop until the
fluid is neutralized by the change in color of the litmus paper.
3. If cloudiness is observed, decalcification is incomplete, hence the need for further

decalcification.
4. If the solution remains clear after the neutralization with concentrated ammonium
hydroxide, pipette 5 ml of the discarded decalcifying agent into the test tube. Add 0.5
ml ammonium oxalate. Let stand for 30 minutes.
1
5. If cloudiness is observed after addition of ammonium oxalate, decalcification is still
incomplete, hence the need for further decalcification.
6. If the solution remains clear after 30 minutes, decalcification is considered to be

complete.
5.1 Draw and label the set up for decalcification.
2
1.2 Computation: For preparation of 250ml of 10% nitric acid.
6. STUDY QUESTIONS
6.1 What are the characteristics of a good decalcifying agent?
The characteristics of a good decalcifying agent include the following:
Can completely remove calcium from the specimen

Absence of damage to tissue or fibers
Non-impairment of subsequent staining techniques
Reasonable speed of decalcification
Rapid in action
Permits good nuclear and cytoplasmic staining
Does not distort cells and tissues
Can be easily removed by dehydrating agent
3
6. 2 Enumerate the factors that influence the rate of decalcification?
The factors that influence the rate of decalcification are the following:
Concentration of decalcifying agent – more concentrated solutions

decalcify BM rapidly but more harmful to tissue.
Volume of decalcifying fluid – greater amount of fluid increases the
speed of the process; ratio of fluid to tissue volume is 20:1
Fluid access – ready access to all surfaces of the specimen affect diffusion
& penetration; facilitate solution, ionization & removal of calcium.
Size, thickness and consistency of tissue being decalcified – increase in
these will require longer periods for complete decalcification.
Compactness and density of the bone or tissue being decalcified – very
hard or dense bone blocks take much longer for complete decalcification to
take place
Agitation – gentle agitation will increase decalcification rate.
Temperature – increase in temperature will hasten decalcification but also
give way to damaging effects of acid on tissue.
6.2 What are the methods of measuring the extent of decalcification? Which among
them is the best and why?
The methods of measuring the extent of decalcification are:
Physical tests / mechanical methods – test for its pliability; requires

manipulation such as bending, probing or trimming of the specimen to feel
for remaining calcified areas which makes the method unreliable; may cause
mechanical damage to the tissue specimen. Using a thin needle, prick the
tissue, if the needle enters into the tissue easily, it shows that it is now
decalcified.
Chemical tests - the fluid that held the tissue specimen is tested by using
blue litmus paper to 5mL of the discarded fluid to test for the acidity of the
fluid which causes the blue litmus paper to turn red; strong ammonia may
4
also be added drop by drop until fluid is neutralized which is detected by the
change of litmus paper color from red to blue indicating alkalinity; presence
of cloudiness = presence of calcium; chemical methods are cumbersome and
useless in everyday practice
X-ray method – best method since it is sensitive and reliable but very
expensive; sensitive because it can detect even the smallest focus or spicules
of calcium which appears opaque in an x-ray plate
6.3 What is the most suitable fixative for bone tissue and why?
The most suitable fixative for bone tissue is 10% Buffered Neutral Formalin
since it penetrates and renders the soft tissue that is attached to the bone resistant to
acids. It adequately protects the cellular and fibrous elements of bone from damage
caused by the acids used as decalcifying agents.
6.4 What is the treatment of bone following decalcification?
Prior to the processing of bone tissues after decalcification, acid must be

removed from the tissues by immersing the decalcified bone in saturated lithium
carbonate solution for several hours or immersing the bone tissue into 5 -10% aqueous
sodium bicarbonate solution. A short, effective wash in tap water should also be
sufficient as any remaining acid will be removed during processing. Adequate water
rinsing can be accomplished in 30 minutes for small samples and 1 – 4 hours for larger
specimens. Another would be washing it in 70% alcohol for 12-18 hours.
Acid decalcified tissues for frozen sections must be thoroughly washed in water
or stored in formol-saline containing 15% sucrose or phosphate-buffered saline (PBS)
with 15-20% sucrose at 4°C before freezing. Tissues decalcified in EDTA solutions
should not be placed directly into 70% alcohol, because this will cause residual EDTA
to precipitate in the alcohol and within the tissue. Rinsing the decalcified tissue with
water or storing overnight in formol-saline or phosphate buffered saline (PBS) will
prevent the formation of crystalline precipitate.
5
Exercise No. 10: DECALCIFICATION

(5) (10) (15) (20)
the required items
in the activity .
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE
Pr eceptor’s Signature: __________________
6
EXERCISE 11
CYTOLOGIC TECHNIQUE: PAP’s STAIN
Cytology plays a major role in determining the etiology of the disease of the patient.
It involves analyzing the interpretation of exfoliated or abraded cells. It is widely accepted
because of its cost-effectiveness, simplicity, quick turnaround time, low complication rate and
high diagnostic accuracy. Common specimens for cytological studies include cervico-vaginal
smear, bronchial washing/brushing, bronchoalveolar lavage, sputum, urine sediment, bladder
washing/brushing, peritonial, pleural, cerebrospinal fluids, and discharge and scrape cytology.
Papanicolaou staining (PAP’s) method is the routine staining procedure used in

cytopathology laboratory. It is a polychrome staining reaction that results in a well stained
nuclear chromatin, differential cytoplasmic counterstaining and cytoplasmic transparency.
Cytoplasmic stains are OG-6 and EA-36. Both are synthetic stains and OG-6 is a monochrome
stain while EA-36 is a polychrome stain.
1. OBJECTIVES
1.1 Demonstrate the proper smear preparation for PAP’s staining.
1.2 Demonstrate the proper procedure in PAP’s staining for cytological studies.
2. MATERIALS
2.1 Buccal smears (10 slides)

2.2 Cytobrush
2.2 Staining dish
2.3 Staining rack
2.4 Rag
3. REAGENT
3.1 PAP’s Stain set
4. PROCEDURE
1. Buccal Smear Preparation
4.1 Choose a partner before collection. One will act as a collector and the other will be
the patient. Exchange places when the procedure is done.
4.2 Let the patient rinse mouth with water prior to sample collection.
4.3 The person taking the sample will prepare the cytobrush and slides to be used.
4.4 Label the slides with patient’s Initials and code assigned by the instructor using a
pencil.
4.1 With the brush in between the forefinger and thumb, insert the brush into one side of
the mouth between the inside of the cheek and the upper gum. Press firmly and twirl
the cytobrush against the inner cheek using an up and down motion from front to back
and back to front. Use a stopwatch to time the collection for at least 30 seconds per
swab to collect cells. Collect samples from maximum mucosal surfaces using both sides
of the cheek in alternate collection.
4.6 Collect five slides from the right and the other 5 slides from the left cheek.
4.7 Allow the swab to air dry for at least 5 minutes before fixation.
4.8 Fix the smear with 95% Ethanol.
4.8 Proceed to PAP’s staining.
2. PAPANICOLAOU STAINING (10 SLIDES):
2.1 96% Ethyl alcohol ……………… 15 seconds
2.4 Wash in Distilled water ……………… 10 dips
2.5 Harris hematoxylin ………………….. 6 minutes
2.6 Wash in Distilled water…………... 10 dips
1
2.7 0.5 % Hydrochloric acid……………. 1-2 quick dips
2.8 0.1 % Ammoniated water ……….. 5 dips
2.9 50% Ethyl alcohol ……..……….. 15 seconds
2.12 Orange G-6 …………………….. 2 minutes
2.13 96% Ethyl alcohol ……………… 10 dips
2.14 96% Ethyl alcohol ……………… 10 dips
2.15 EA 50 Eosin yellowish ………… 3 minutes
2.16 96% Ethyl alcohol …………….. 10 dips
2.17 100% Ethyl alcohol …………… 10 dips
2.18 Xylene ……………………….. 10 dips
2.10 Mount
5.1 Draw and label the set-up of Pap’s staining.
MATERIALS
2
PAP’S STAINING
MOUNT
3
6. STUDY QUESTIONS
6.1 What are the clinical significance of doing cytological procedures?
Doing cytological procedures can help in the following:
Detection of malignant cells in body fluids, mainly used for staging cancer.
Detection of precancerous cervical lesions in women (cervicovaginal smear/Pap
smear).
Assessment of female hormonal status in case of sterility and endocrine disorders.
This is achieved by microscopic evaluation for determination of maturation index
(MI), based on examination of smears taken from the lateral vaginal walls.
Determination of genetic sex -most of the nuclei of females exhibit conglomeration
of chromatin, representing XX chromosomes (Barr body), which may be
demonstrated in the smears from buccal or vaginal mucosa.
Detection of infectious agents
Assess hormonal changes, endocrine disorders
6.2 Differentiate exfoliative cytology from aspiration cytology.
Exfoliative cytology deals and involves with the microscopic study of cells that
have desquamated from epithelial surfaces. The exfoliated cells are found in smears which
may have been spontaneously shed or physically removed from the epithelial and mucous
membranes. Exfoliative cytology includes cervicovaginal smears, washing, discharge
cytology, and scrape cytology.
On the other hand, aspiration cytology involves the studying of cells from
specimens that do not spontaneously shed cells which is the exact opposite of exfoliative.
It is used to diagnose any palpable lesions (e.g. Fine Needle Aspiration Biopsy (FNAB))
and deep-seated or non-palpable lesions (Image Guided Biopsy).
4
6.3 What are the cells that can be seen in a vaginal smear for cytopathologic studies?
The cells that can be found in the vaginal smear for cytopathologic studies are the
following:
Mature superficial cells - These polygonal squamous cells measure 45-50

µm in diameter and are usually identified by the presence of pale, pink-
staining cytoplasm and dark pyknotic nuclei, less than 6 µ in diameter. True
acidophilia is characteristic of superficial vaginal cells under estrogen
influence
Intermediate cells - are medium sized polyhedral or elongated cells with

basophilic vacuolated cytoplasm.
Parabasal cells - are round to oval cells with small dense basophilic
cytoplasm, and a total cell diameter of 15-30 µm. They are smaller than
intermediate cells and have a larger vesicular nucleus. They are normally
found from two weeks of age to puberty, after childbirth, with abortions and
after menopause.
Navicular cells - are boat-shaped intermediate cells with strong tendency

to fold or curl on edges. Their presence suggests a combined estrogen
progesterone effect. They are found in the latter half of the menstrual cycle,
during pregnancy and menopause.
Pregnancy cells - are round, oval or boat-shaped cells with translucent

basophilic cytoplasm observed greatest at the center of the cell, due to
glycogen accumulation, pushing the nucleus to the side or towards the cell
membrane. This appearance is characteristic due to a deeper blue stain of
the cytoplasm at the periphery.
Endometrial cells - are small cells, slightly cylindrical with less basophilic
cytoplasm, occurring in tightly packed groups of 3 or more. They are found
during and 1-10 days after menstruation and are shed in response to ovarian
hormones.
Endocervical glandular cells - occur in large groups or small sheets. The

cytoplasm is usually stained pale blue/gray and is finely vacuolated, often
with indistinct cell borders and nuclei with finely granular chromatin. They
may present a honeycomb appearance when viewed on end.
5
6.4 Give the adhesive agents that can be used for cytologic studies?
The adhesive agents that can be used for cytologic studies are:
Pooled Human serum or Plasma

Celloidin Ether Alcohol
Leukonostroc Culture
6.5 Enumerate the 5 techniques in exfoliative cytology and describe each.
The 5 techniques in processing fluids for cytological examination are as follows:
Direct Smear Technique – This technique is based on the sample’s

viscosity. The smears should be made from fresh material. The smear is the
product of a diagnostic technique in which cells and other components are
spread out thinly in a clean glass slide.
Membrane Filter Technique – The principle of this procedure is that the

cells are filtered from a fluid through a porous membrane/filter. Afterward,
they are subsequently fixed and stained. The pore size is important for
collecting cells of interest. This method is used for cytodiagnosis in urine,
spinal fluid bronchial washing, and substances with low cellular content.
Cell Block Technique/Preparation – This is the technique wherein cell

button or dot fluids, such as pleural fluid, ascetic fluid, CSF, urine, and
exudates, are processed and sectioned on a rotary microtome while utilizing
the paraffin embedding technique. This is mainly used in smears as an
adjunct for establishing a more definitive cytopathologic diagnosis.
Liquid Based Cytology – In this method, the smears are taken with a
specially designed spatula and cells are suspended in a fixative solution.
The cells are able to spread forming a monolayer and improve the sensitivity
of screening of cervico-vaginal smears. Overall, this improves cell
representation.
Concentration Technique – This technique makes use of a cytospin and

sediment preparations. Cells are isolated via a series of centrifugation steps
to concentrate cells into a small suspension.
6
Exercise No. 11: CYTOLOGIC TECHNIQUE: PAP’s STAINING

(5) (10) (15) (20)
the required items
in the activity.
facts based on
knowledge of the
subject.
support the
answers.
Ideas were
presented in order
and spontaneous.
time. date.
TOTAL SCORE

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SAFETY IN HISTOPATHOLOGIC LABORATORY

Hands must be washed:

 Whenever there is visible contamination with blood and body fluids

 Before and after work

 After gloves are removed and between glove changes

 Before leaving the laboratory

3. Sharps should be disposes in a puncture-resistant container. This should be leak proof.

4. Gloves must be worn when handling biologic specimens.

6. Any accident must be reported immediately to instructor so that appropriate prophylactic

7. Mixing of chemicals must be done inside the fume hood.

8. Properly dispose all contaminated supplies in the appropriate container.

9. Disinfect and clean biohazard spills immediately.

SUPPLIES & EQUIPMENT IN HISTOPATHOLOGY LABORATORY

for histopathologic and cytologic examinations.

equipment to ensure their usability and functionality.

2.1 Laboratory manual

2.2 Ball pen

3.1 Identify the material or equipment presented in the picture.

3.2 Give its uses, purpose, or application in histopathologic or cytologic techniques.

4.1 Label and provide required information provide.

Name: Rotary Microtome

Uses/Application: It is used in obtaining sections from

Maintenance: The equipment is cleaned daily from

Name: Cryostat or Cryostat Machine

Uses/Application: It is used for cutting frozen samples and

Maintenance: Microtomy debris must be removed from

Name: Tissue Embedding Center

Uses/Application: It is used in moderate to heavy

Maintenance: Maintain in a clean condition and use only

Uses/Application: It is used to prepare tissue samples for

Maintenance: For the maintenance of the equipment,

Uses/Application: Microscope is used to magnify small

Maintenance: Lenses should be kept away from anything

Name: Microtome Knife or Blade Holder

Uses/Application: This is used to accommodate

Maintenance: This equipment is less susceptible to nicks

Uses/Application: These tissue/embedding cassettes hold

Maintenance: Such equipment is soaked in xylene to

Name: Glass Slides and Cover Slips

Uses/Application: These are used for microscope

Maintenance: These should be clean and fingerprint-free

Name: Teasing Needle

Uses/Application: This is used to straighten tissue section,

Maintenance: Teasing needle must be cleaned after each

Uses/Application: It is used to assist with the handling of

Maintenance: The equipment must be stored in a clean

Name: Glass Staining Dish

Uses/Application: It is used to hold microscope slides

Maintenance: It should be cleaned properly for

Name: Coplin Jar

Uses/Application: Coplin Jar is used to stain specimens in

Maintenance: To maintain its functionality, filter out

Name: Electric Paraffin Pitcher or Electric Paraffin Wax

Uses/Application: This equipment is used for wax melting

Maintenance: Ensure that there is no debris left in the

Uses/Application: This equipment is used for immersing

Maintenance: The accuracy of temperature is vital since it

Name: Electric Paraffin Knife

Uses/Application: Electric Paraffin Knife is used to pick up