Exercise 3
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:
2. MATERIALS
3. REAGENTS
4.1 Prepare 1 L of 10% Neutral Buffered Formalin by pouring a base of 100 ml distilled
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
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• Agitation – fixation is accelerated when automatic or mechanical tissue
processing is used
6.3 Enumerate and define the different types of fixatives based on function?
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.
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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?
• disappearance of cells
Remedies:
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Student’s Name __________________________________
Gerlie M. Antolijao Group& Sec ____
1-F Date July
_________
2, 2021
Exercise No. 3: FIXATION