FIXATIVES

ALDEHYDES
FORM ALEDEHYDE 10% Formol Saline 10% Neutral Buffered Formol-Corrosive Alcoholic
(Formalin) Formalin (Formol-sublimate) Formalin(Gendre’s)
Fixative

× most widely × 40% saturated × is the optimal choice × recommended for × faster fixation
used Fixative is formaldehyde for many reasons. routine post- times
10% diluted with  fast penetration mortem tissues × can be used
× pure stock 10%NaCl  cheap & stable × penetrates small for rapid
available is 37- × Recommended  prevents alterations pieces of tissues diagnosis ;
40%; for fixation of during processing rapidly fixes and
(Unsatisfactory central  less shrinkage than × excellent for many dehydrates at
for routine nervous other fixatives staining the same time.
fixation: tissues and  not osmotically active procedures × used to fix
should be general post × hardens tissue better including silver sputum, since
diluted to mortem × tissue can be stored in reticulin methods it coagulates
1:10 ratio ) tissues formalin indefinitely × cytological mucus.
× Formaldehyde × Fixation times: × fixative of choice for structures and × good for
= colorless 24 hrs at 35C Immunohistochemistry blood cells are well preservation of
gas commonly 48 hrs at 20- & molecular tests preserved glycogen and
obtained in 25C × Most widely used × No need of for micro-
histopathology solution for routine washing –out, can incineration
laboratory as a formalin fixation. be transfer directly technique.
37% to 40% × Has a pH of from fixative to × Disadvantage:
solution in approximately 6.8 and alcohol -gross
water. it is hypotonic in the hardening of
× Commercial × buffer ions present tissues
solutions are (approximately 165 -causes partial
37% to 40% mOsm). lysis of RBC
formaldehyde -preservation
but are of iron-containing
considered to pigments is poor
be 100%
formalin.

DISADVANTAGE: forms
mercuric chloride
deposits; doesn’t allow
frozen tissue sections
to be made; tissue
section should not be
more than 1cm thick

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GLUTARALDEHYDE
 Made up of two formaldehyde residue
 More stable effect on tissue especially on central nervous tissue
 Preserves plasma CHON better
 Produces less tissue shrinkage
 Preserves cellular structures better; recommended for enzyme histochemistry and electron microscopy.
 Less irritating to nose
 Doesn’t cause dermatitis
DISADVANTAGES:
 expensive and less stable
 Reduces PAS positivity of reactive mucin
 Specimen vial/container must be kept refrigerated during fixation
 Solution may be changed several times during fixation by swirling

CHROMATE FIXATIVES
Potassium Dichromate REGAUD’S (MULLER’S) ORTH’S FLUID Chromic acid
FLUID
 used in a 3% aqueous  Recommended for  Recommended for  used in 1-2% aqueous
solution demonstration of study of early solution
 Preserves lipid and chromatin, degenerative  Precipitates all
mitochondria mitochondria, mitotic processes and tissue proteins and
figures, Golgi bodies, necrosis adequately preserves
RBC and colloid  Demonstrates carbohydrates
containing tissue rickettsiae and other  A strong oxidizing
bacteria agent

DISADVANTAGES: Disadvantages:  DISADVANTAGE:

 needs freshly  same in Muller’s formaldehyde must
prepared be added to chrome-
 does not preserve fats containing fixatives
 prolonged fixation before use
blackens tissue
pigments
 should be thoroughly
washed in running
water before
dehydration

Lead Fixatives
 Used in 4% aqueous solution of basic lead acetate
 Recommended for acid mucopolysaccharides
 Fixes connective tissue mucin

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PICRIC ACID FIXATIVES

 Normally used in strong saturated aqueous solution
 Also dyes the tissues but the yellow color may be removed by treatment with another acid dye or lithium
carbonate
 Preserves glycogen well but causes considerable tissue shrinkage
 Allows brilliant staining with the Trichrome method
Disadvantages:
 causes RBC hemolysis
 reduces the amount of demonstrable ferric iron in tissue
 not suitable for frozen sections
 tissues must never washed in water before dehydration; must be first rendered insoluble by
direct immersion in 70% alcohol
 interferes with azure eosin method of staining; should be washed thoroughly with alcohol
 Alters and dissolves lipids
 Highly explosive when dry; must be kept moist with distilled water or saturated alcohol during
storage
BOUIN’S SOLUTION BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE

 Recommended for fixation of embryos and pituitary  Excellent fixative for glycogen
biopsy
 An excellent fixative for preserving soft and delicate
structures(e.g. Endometrial curettings)
 Does not need “washing out”
 Preserves glycogen
 Preferred fixative for tissues to be stained by
MASSON’S TRICHOME for collagen, elastic and
connective tissue
Disadvantages: Disadvantage:
 penetrates large tissues POORLY  Utilizes 3-4 changes of Brasil’s fixative (30-2 hrs
 soluble in water; needs to be directly transferred to each); succeeded directly by Absolute aclcohol
70% alcohol when using Automatich processing technique.

Glacial acetic acid

 Precipitates chromosomes and chromatin material; very useful in study of NUCLEAR COMPONENTS OF THE CELL
Disadvantage:
 contraindicated for cytoplasmic fixation; - destroys mitochondria and Golgi elements of cells.

ACETONE
 it is used in fixing brain tissues for the diagnosis of rabies
 Recommended for study of water diffusible enzymes especially phosphatases and lipases.
DISADVANTAGES:
 dissolves fat and evaporates rapidly

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Alcohol fixatives

 Used in concentrations ranging from 70-100%

 Rapidly denatures and precipitates proteins
 Used both as fixative and dehydrating agent
 Excellent for glycogen preservation
Disadvantage:
 if tissues left in alcohol too long will shrink; making it difficult to cut
METHYL ALCOHOL ISOPROPYL ETHYL ALCOHOL CARNOY’S FLUID NEWCOMER’S FLUID
100% ALCOHOL 95%

 Excellent for  Used for fixing  Used at  Recommended  Recommended for

fixing dry and touch concentrations for fixing fixing
wet smears, preparations of 70-100% chromosomes, mucopolysaccharides
blood smears  Lower lymph glands and nuclear protein
and bone concentration and urgent  Acts both as nuclear
marrow causes RBC’s to biopsy. and histochemical
tissues. be hemolyzed  Also used to fix fixative
 Fixes and and WBC’s are brain tissue for
hydrates at inadequately the diagnosis of
the same time preserved. rabies.
 Excellent
fixative for
glycogen

OSMIUM TETRAOXIDE
 Preserves cytoplasmic structures well
 Used extensively for neurological
 Fixes myelin and peripheral nerves
 Produces brilliant nuclear staning with safranin
 Adequately fixes materials for ultrathin sectioning in electron microscopy
 DISADVANTAGE:
very expensive, extremely volatile and inhibits hematoxylin(makes staining difficult)
FLEMING’S SOLUTION W/ACETIC ACID FLEMING’S SOLUTION W/O ACETIC ACID
 most common chrome-osmium acetic acid fixative  recommended for cytoplasmic structures,
 excellent fixative for nuclear structure particularly mitochondria
 Permanently fixes fat.
 DISADVANTAGE:
very expensive, forms artefact(needs washing-out),
poor penetrating agent

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