Chapter 3: Fixation and Fixatives a.

Formaldehyde 10%
b. Glutaraldehyde 3%
Fixation c. Glutaraldehyde (immune electron
- first and most critical step in microscopy) 0.25%
histotechnology 6. Duration of fixation
- Involves fixing or preserving fresh a. Primary fixation (buffered formalin)
tissue for examination 2-6 hours
b. Electron microscopy 3 hours
Primary aim of fixation:
To preserve the morphologic and chemical Practical Considerations of Fixation
integrity of the cell in as life-like manner as 1. Speed specimen should be placed in
possible fixative as soon as it is removed from
the body to prevent autolysis and
Secondary goal of fixation: putrefaction
2. Penetration 1mm per hour
To harden and protect the tissue from the
3. Volume the amount of fixative used
trauma of further handling
has been 10-25 times the volume of
tissue to be fixed; maximum
Notes:
effectiveness is 20 times (20:1)
Postmortem decomposition autolysis 4. Duration of Fixation
Decomposition putrefaction
Effects of Fixatives in General
Two basic mechanisms involved in fixation 1. Harden soft and friable tissues and
1. Additive fixation chemical constituent make the handling and cutting sections
of the fixative is taken and becomes easier
part of the tissue 2. Make cells resistant to damage and
2. Non-additive fixation not incorporated distortion
into the tissue, but alters the tissue 3. Inhibit bacterial decomposition
composition and stabilizes tissue 4. Increase optical differentiation of cells
and tissue components
Main factors involved in fixation 5. Acts as mordants and accentuators to
1. Hydrogen Ion concentration pH 6 and promote and hasten staining
8 6. Reduce risk of infections during
2. Temperature handling
a. Surgical specimens Room temp.
b. Tissue processors 40oC Types of Fixative According to Action and
c. Electron microscopy 0-4oC Composition
d. Formalin (urgent biopsies) - 60oC
According to Composition
e. Formalin (tuberculosis) - 100oC
A. Simple Fixatives only one component
3. Thickness of section
a. Electron microscopy 1 to 2 mm2 substance
b. Light microscopy 2cm2 1. Aldehydes
c. Light microscopy (thin cut) - a. Formaldehyde
<0.4cm b. Glutaraldehyde
d. Large solid tissues (Uterus) 2. Metallic Fixatives
a. Mercuric chloride
opened sliced thinly
b. Chromate fixatives
e. Brain suspended in 10% buffered
Potassium dichromate
formalin for 2-3 weeks
Chromic acid
4. Osmolality
c. Lead fixatives
a. Hypertonic cell shrinkage
Picric acid
b. Isotonic & hypotonic cell swelling
Acetic acid
c. Best results slightly hypertonic sol
Acetone
400-450mOsm; isotonic 340mOsm Alcohol
5. Concentration Osmium tetroxide (osmic acid)
 d. Heat c. Bouins fluid
B. Compound Fixatives two or more d. Newcomers fluid
fixatives e. Heidenhains Susa
2. Cytoplasmic Fixatives
According to Action a. Flemmings w/o acetic acid
A. Microanatomical Fixatives permit b. Kellys fluid
general microscopic study c. Formalin with post chroming
1. 10% Formol Saline d. Regauds fluid (Mullers fluid)
2. 10% Neutral buffered formalin e. Orths fluid
3. Heidenhains Susa 3. Histochemical Fixatives preserve
4. Formol sublimate (formol corrosive) chemical constituents of cells and
5. Zenkers solution tissues
6. Zenkers formol (Kelly solution) a. Formol saline 10%
7. Bouins solution b. Absolute Ethyl Alcohol
8. Brasils solution c. Acetone
B. Cytological Fixatives preserve specific d. Newcomers Fluid
parts and particular microscopic
elements of the cell itself Lipid Fixation
1. Nuclear Fixatives nuclear Used: cryostat or frozen sections
structures (chromosomes) contains
glacial acetic acid
a. Flemmings fluid
b. Carnoys fluid