NAME:
SECTION: BSMT-3E
FIXATIVE FUNCTION AND USES
Fixative of choice in routine
histopathology;
1. 10% Neutral Buffered Best for nervous tissue
Formalin preservation, pigments with
iron, and elastic
fibers;
Simple fixative;
70-100% concentrations are
needed to prevent lysis;
2. Ethanol Giver better results if
incorporated with compound
fixatives;
Enzyme Histochemistry
Fixes and stains conjugated
fat and lipids, fixes
3. Osmium Tetroxide
neurological tissues
Electron microscopy
Rapid fixation;
Enzyme Histochemistry;
4. Glutaraldehyde
Electron Microscopy;
Cellular and fluid proteins
5. Microwave Heat Increases diffusion rate of
molecules = rapid fixation;
Useful for staining,
DATE: 22 AUGUST 2020
STORAGE AND
HANDLING
Fixation time: “Overnight”(12-
24 hours)
Wash hands before and after
handling the reagent. Always
keep container closed. Avoid
breathing fumes.
Fixation time: 18-24 hours
Keep in the original container
in a well-ventilated place
(fireproof place). Discarded
with a good-grade, medium-
speed filter (Whatman No. 1),
tested with a hydrometer
before reuse.
Kept in dark-colored,
chemically clean bottle to
prevent evaporation.
Kept in cool or refrigerated
place (prevent deterioration). If
swallowed, seek immediate
assistance.
Fixation time: 2-24 hours
(depending on the tissue size;
Used only under a chemical
fume hood. Do not breathe or
ingest. Keep refrigerated to
retard polymerization.
Fixation time: up to 12 hours;
Optimum temperature: 45-55
C;
OTHER
INFORMATION
pH 7.0;
formation of precipitate of
paraformaldehyde (10%
methanol prevents this);
fixes lipids for frozen
sections;
slow to fix
Can also be a dehydrating
agent;
Can be used in PCR;
Preserves but not fix
glycogen
Pale yellow powder that
dissolves in water (6% at
20C)
Very expensive, poor
penetration; causes
conjunctivitis and
blindness, inhibits
hematoxylin, volatile
Penetrates poorly and
deforms the alpha helix
structure in protein;
Similar with formalin;
Small specimens (tissue
fragments and needle
biopsies): 2.5 %;
Large specimens: 4%;
Expensive, less
Irritating, less stable
Replaced the used of direct
flaming;
Able to penetrate tissue
 decalcification, electron
microscopy, immune-
thickness of 10-15 mm;
cytochemical techniques,
Dehydrating agent:
and enzyme histochemistry; Avoid inhaling vapors. Wear
Methanol or Ethanol
Non-chemical technique appropriate PPE for all.
Clearing Agent:
useful to preserve
Isopropanol
neurochemical substances in
brain (Acetylcholine)

STRUCTURE RECOMMENDED FIXATIVE NOT RECOMMENDED FIXATIVE

Formalin; Glutaraldehyde; 10% Neutral Although choice of fixative is not that

Formal Saline critical in demonstrating amyloid in tissue,
there are some unrecommended ones:
Alcohol based fixatives: tend to denature
1. Amyloid proteins
10% Formal Saline: negative effects on the
staining (metachromasia)
Mercuric Chloride: reacts to amino acids

10% Neutral buffered formalin; Ethanol; Formalin: distorts cell structure

Orth’s Fluid; Glutaraldehyde Carnoy’s fixative: lyses erythrocytes
2. Bacteria

Osmium tetroxide; 10% Formal saline;
Fixatives with mercuric chloride and
3. Fats and Lipids
potassium dichromate (Formol-corrosive
(Formol-Sublimate))
Osmium tetroxide; Zenker’s Solution;
Acetone; Carnoy’s Fixative
4. Negri Bodies
Formaldehyde: reacts to fatty acids and
lipids negatively
Carnoy’s Fixative: dissolves fats and lipids
Formalin: could promote gradual loss and
bacterial contamination

Formalin Regaud’s (Muller’s) Fluid: prolonged

fixation could cause darkened melanin
5. Melanin Pigment

10% Neutral Buffered Formalin; Ethanol: causes RBC hemolysis and doesn’t
Osmium tetroxide; Glutaraldehyde preserve leukocytes
6. Nervous Tissue

Rossman’s fluid or cold absolute alcohol Regaud’s (Muller’s) Fluid: poor glycogen
penetration, not suitable for carbohydrates
7. Polysaccharide

Osmium tetroxide; Carnoy’s fixative; Coagulant fixatives: not suitable to preserve

cell structures
8. Nissl Bodies

Zenker’s fluid 10% Neutral buffered formalin

Helly’s fluid
9. Bone Marrow
B-5

10. Samples for Electron
Microscopy
Osmium tetroxide, glutaraldehyde, and Formaldehyde and methyl alcohol
paraformaldehyde;
For electron histochemistry and electron
immunocytochemistry, Karnovsky's
paraformaldehyde-glutaral-dehyde is
used.

REFERENCES:
Gregorios-Bruce, J. Histopathologic Techniques 2nd ed. Quezon City: JMC Press, 2006.

MSDS
http://www.labchem.com/tools/msds/msds/VT450.pdf
http://www.labchem.com/tools/msds/msds/VT230.pdf
https://www.fishersci.com/shop/msdsproxy?productName=BP25471&productDescription=GLUTARALDEHYDE
https://www.fishersci.com/store/msds?partNumber=AC319010050&productDescription=OSIUM+TETROXIDE+4+WT
%25+SO+5ML&vendorId=VN00032119&countryCode=US&language=en