Fixatives

Best General Tissue Fixative

Formaldehyde (Formalin)

Produced by oxidation of methanol

Formaldehyde (Formalin)

Formaldehyde (Formalin) usually used as

10% formalin

T/F: Formaldehyde (Formalin) is soluble in water

True

Formaldehydes concentrates =

Overharden

In Formaldehyde (Formalin), phosphate buffer maintain pH at

7

T/F: Formaldehyde is a gas and formalin is a liquid

True

In Formaldehyde (Formalin), stabilizing agent:

10-15% methanol

Chemical composition of concentrated formalin:

* 37-38% of formaldehyde
* 10-15% of methanol
* 48-53% water

A fixative labeled as 10% buffered formalin is actually only a ___ solution of formaldehyde

4%

Formaldehyde (Formalin) fixation Time

24 hours

Preserved fats, mucins, glycogen

Formaldehyde (Formalin)

Recommended for colored tissue photography

Formaldehyde (Formalin)

T/F: Require washing out

False

May cause lung and nasopharyngeal CA

Formaldehyde (Formalin)

If formaldehyde (Formalin) is unbuffered ______________ will accumulate

Formic acid

Removal of Formalin Pigments

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* Kardasewitch - ammonia water + ethyl alcohol
* Lillie’s method - ammonia water + alcohol + acetone + hydrogen peroxide
* Picric acid

Made up of saturated formaldehyde (37-40%) diluted to 10% with sodium chloride (HCHO + NSS)

10% Formol-Saline

10% Formol-Saline pH

6\.8

It is recommended for fixation of CNS (brain) tissues and general post mortem tissues for histochemical examination

10% Formol-Saline

It is also recommended for the preservation of lipids, especially phospholipids

10% Formol-Saline

10% Formol-Saline fixation Time

12-24 hours; 24 hours at 35℃

48 hours at 20-25℃

It is ideal for most staining techniques, including silver impregnation

10% Formol-Saline

It allows natural tissue color to be restored upon immersion in 70% alcohol

10% Formol-Saline

Formal-saline fixed tissues tend to __________ during alcohol dehydration

Shrink

Most widely used fixative for routine histology

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin pH

7

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin fixation time

4-24 hours; 24 hours at RT

It prevents precipitation of acid formalin pigments on post-mortem tissue

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin

It is the best fixative for tissues containing iron pigments and for elastic fibers which do not stain well after Susa, Zenker and chromate fixation

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin

It requires no post treatment after fixation and goes directly into 80% alcohol for processing

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin

Reactivity of Myelin to Weigert’s iron hematoxylin stain is reduced

10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin

Alternative to mercuric chloride fixatives (metallic fixatives)

Zinc formalin (Unbuffered)

HCHO + Mercuric Chloride

Formal-Corrosive (Formal-Sublimate)

Formal-Corrosive (Formal-Sublimate) Fixation time

3-24 hours

It is excellent for many staining procedures including silver reticulum methods

Formal-Corrosive (Formal-Sublimate)

Cytological structures and blood cells are well preserved

Formal-Corrosive (Formal-Sublimate)

It forms mercuric chloride deposits

Formal-Corrosive (Formal-Sublimate)

It does not allow frozen tissue sections to be made

Formal-Corrosive (Formal-Sublimate)

It inhibits the determination of the extent of tissue decalcification

Formal-Corrosive (Formal-Sublimate)

Polymerized form of formaldehyde (white powder that causes turbidity)

Paraformaldehyde

Suitable for paraffin embedding and sectioning, and for immunocytochemical analysis

Paraformaldehyde

For long term tissue storage and good tissue penetration

Paraformaldehyde

Mixture of paraformaldehyde and glutaraldehyde

Karnovsky’s Fixative

Suitable for preparing samples for light microscopy in resin embedding and sectioning, and for electron microscopy

Karnovsky’s Fixative

It is made up of two formaldehyde residues, linked by three carbon chains

Glutaraldehyde

Has a more stable effect on tissues

Glutaraldehyde

It preserves plasma proteins better and produces less tissue shrinkage

Glutaraldehyde

More pleasant and less irritating to the nose and does not cause dermatitis

Glutaraldehyde

It reduces PAS positivity of reactive mucin

Glutaraldehyde and 10% NBF

It is excellent for fixing dry and wet smears, blood smears and bone marrow tissues

100% Methyl Alcohol (Methanol)

It fixes and dehydrates at the same time

100% Methyl Alcohol (Methanol)

If left in fixative (100% Methyl Alcohol (Methanol)) for more than __________, the tissues maybe over hardened and difficult to cut

48 hours

Used for fixing touch preparations, although some touch preparations are air dried and not fixed, for certain special staining procedures such as Wright-Giemsa

95% Isopropyl Alcohol

Ethyl Alcohol is used at concentrations of

70-100%

If ethyl Alcohol used at lower concentrations, the RBCs become ___________ and WBCs are inadequately preserved

Hemolyzed

It may be used as a simple fixative but more frequently incorporated into compound fixatives for better results

Ethyl Alcohol

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Ethyl Alcohol Fixation time

18-24 hours

It preserved but does not fix glycogen

Ethyl Alcohol

It preserves nucleoproteins and nucleic acids

Ethyl Alcohol

Alcohol containing fixatives are contraindicated when ________ are to studied

lipids

Absolute alcohol + chloroform + GAA

Carnoy’s Fixative

Most rapid fixative

Carnoy’s Fixative

Carnoy’s Fixative Fixation time

1-3 hours

It permits good nuclear staining and differentiation because of GAA

Carnoy’s Fixative

Carnoy’s Fixative preserves _______________________ well

Nissl granules and cytoplasmic granules

Used for small pieces only

Carnoy’s Fixative

Ethanol + chloroform + GAA

Clarke’s solution

Has been used on frozen section and smears

Clarke’s solution

It preserves nucleic acids but extracts lipids

Clarke’s solution

Clarke’s solution Fixation time

3-4 hours

Ethanol + HCHO

Alcoholic formalin

Acts as fixative and dehydrating agent at the same time

Alcoholic formalin

Alcoholic formalin clears and extracts lipids and therefore can be used to visualize ___________________

Lymph nodes in breast tissues

Alcoholic formalin Fixation time

12-24 hours

HCHO +  Ethanol + HCHO + GAA

Formol-acetic alcohol

Formol-acetic alcohol is a faster acting agent than an alcoholic formalin due to the presence of

Acetic acid

Formol-acetic alcohol Fixation time

1-6 hours

Post-fixation with phenol-formalin for 6 hours enhance immunoperoxidase studies on the tissues and electron microscopy

Gendre’s Fixative (Alcoholic Bouin’s)

Micro-incineration technique

Gendre’s Fixative (Alcoholic Bouin’s)

Used to fix sputum (coagulates mucus)

Gendre’s Fixative (Alcoholic Bouin’s)

Causes partial lysis of RBC

Gendre’s Fixative (Alcoholic Bouin’s)

What is the main component in Newcomer’s Fluid?

Isopropyl alcohol

It is recommended for fixing mucopolysaccharides and nuclear proteins

Newcomer’s Fluid

It produces better reaction in Feulgen stain (stain for DNA) than Carnoy’s fluid

Newcomer’s Fluid

It acts both as a nuclear and histochemical fixative

Newcomer’s Fluid

Newcomer’s Fluid Fixation time

12-18 hours at 3℃

Most common metallic fixative frequently used in saturated aqueous solutions of 5-7%

Mercuric Chloride Fixatives

It has greater affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining

Mercuric Chloride Fixatives

It is the routine fixative of choice for the preservation of cell detail in tissue photography

Mercuric Chloride Fixatives

It leads to the formation of black granular deposits in the tissues. 

Mercuric Chloride Fixatives

Remedy for formation of black granular deposits in the tissues when using Mercuric Chloride Fixatives

Use saturated iodine solution in 95% alcohol (alcoholic iodine)

Zenker’s Solution Composition

1. HgCl2
2. Glacial acetic acid
3. K2Cr2O2
4. NaSO2

Zenker’s Solution Fixation time

12-24 hours

It permits brilliant staining of nuclear and connective tissue fibers

Zenker’s Solution

Act as mordant to make certain special staining directions possible

Zenker’s Solution

It causes lysis of RBCs and removes iron from hemosiderin

Zenker’s Solution

It does not permit cutting of frozen sections

Zenker’s Solution

Zenker’s Solution has the tendency to form __________________ deposits or precipitate

Mercuric pigment

In Zenker’s Solution, Mercuric deposits may be removed by immersing tissues in alcoholic iodine solution prior to staining through a process known as

Dezenkerization