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Exercise 3

The document discusses fixation in histopathology. Fixation is the process of killing and hardening tissues to preserve their structure for microscopic examination. It stops metabolic processes and alters tissues by stabilizing proteins. The key steps are preparing 10% neutral buffered formalin fixative and fixing tissue specimens manually in it for various time periods. Common fixatives like formalin work by cross-linking proteins to preserve tissue structure and prevent autolysis. Proper fixation is important for accurate histopathological diagnosis.

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Gerlie Antolijao
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0% found this document useful (0 votes)
160 views6 pages

Exercise 3

The document discusses fixation in histopathology. Fixation is the process of killing and hardening tissues to preserve their structure for microscopic examination. It stops metabolic processes and alters tissues by stabilizing proteins. The key steps are preparing 10% neutral buffered formalin fixative and fixing tissue specimens manually in it for various time periods. Common fixatives like formalin work by cross-linking proteins to preserve tissue structure and prevent autolysis. Proper fixation is important for accurate histopathological diagnosis.

Uploaded by

Gerlie Antolijao
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Name: _______________________________

Gerlie M. Antolijao Date: ______________


July 2, 2021
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
At the end of the laboratory exercise, the students should be able to:

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.

5. RESULT/OBSERVATION

5.1 Draw and label the set up for fixation and indicate their recommended fixation time.

A. KIDNEY

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B. LIVER

C. LUNG

6. STUDY QUESTIONS

6.1 What are the factors that hasten and retard fixation time? Explain each.

• Size and Thickness of Tissue Specimen – larger tissues require more fixatives
and longer fixation time, while smaller and thinner tissues require less fixatives
and shorter fixation

• Tissue covered by Mucus or blood – may slow down fixative penetration

• Presence of Fat – fatty tissues are fixed slowly

• Temperature – low or cold temperature inactivates enzymes while high


temperature accelerates fixation but also increases the rate of autolysis.

2
• Agitation – fixation is accelerated when automatic or mechanical tissue
processing is used

6.2 Explain the actions of fixatives.

Fixatives work by denaturing or precipitating proteins, that result to the formation


of a meshwork or sponge that tends to hold other cell constituents. Proteins that are
insoluble provide mechanical strength for subsequent procedures. Fixatives also
inactivate enzymes, kill bacteria and molds, make tissue more receptive to dyes,
modify tissue components for maximum form retention, and stabilize tissue elements
so that the effect of any subsequent procedures is minimal. Microorganisms are also
fixed, preventing tissue putrefaction, and lowering the risk of infection to the person
handling it.

6.3 Enumerate and define the different types of fixatives based on function?

• Microanatomical Fixatives - permit general microscopic study of tissue


structures without altering its structural pattern and normal intercellular
relationship of tissues.

• Cytological Fixatives - those that preserve specific parts and particular


microscopic elements on the cell itself; further divided into 1.) Nuclear fixatives -
contain glacial acetic acid, has pH 4.6 or less, 2.) Cytoplasmic fixatives - must
never contain glacial acetic acid; pH greater than 4.6, and 3.) Histochemical
fixatives - those that preserve chemical constituents of cells and tissues.

6.4 Describe the reaction of cellular components to fixation.

Fixation changes the chemical nature of the cells and tissues to which it is
applied, and fixatives also cause physical changes to cellular and extracellular
components. Surface membranes of viable cells are impermeable to large, hydrophilic
molecules. Fixation allows relatively large molecules to penetrate and escape,
especially in organic liquids that dissolve or disrupt the lipids of the cell membrane.
Furthermore, the cytoplasm becomes permeable to macromolecules, forming a
proteinaceous network that is porous enough to allow large molecules (includes
paraffin wax, for immunostaining antibodies, and larger dye molecules) to penetrate
further.

Different fixatives produce varying degrees of porosity. Non-coagulant fixatives,


such as formaldehyde, glyoxal, or glutaraldehyde, result in larger pore sizes than
coagulants such as mercuric chloride, picric acid, or zinc sulfate. Some fixatives have
both coagulant and non-coagulant properties. Coagulant fixatives can also increase

3
antigenic site exposure as a result of the deformation of macromolecular shapes that
occurs during coagulation. The addition of fixative molecules, with or without cross-
linking, can alter antigenic sites and suppress immunostaining. The majority of them
contain both coagulant and non-coagulant ingredients.

6.5 What are the signs of incomplete fixation and how will you remedy the problem?

Signs of incomplete fixation:

• loss of nuclear chromatin

• disappearance of cells

• cell shrinkage with artifactual space around the cells

Remedies:

• Minimize cold ischemia time

• Ensure adequate ratio of fixative to specimen

• Increase fixation time

• Change formalin solution frequently

• Ensure that grossed sections should not be more than 0.4 cm

• Do not pack tissue sections in the cassette

• Agitate cassettes in formalin holding solutions during or after grossing

4
Student’s Name __________________________________
Gerlie M. Antolijao Group& Sec ____
1-F Date July
_________
2, 2021
Exercise No. 3: FIXATION

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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