Chemical Fixatives-Continuation

FIXATION

VI. Osmium Tetroxide/Osmic Acid – yellow discoloration (picric acid)

- This is a pale yellow powder which dissolves in water to form strong oxidizing solution. (up to 6% @
20 deg. C)
- adequately fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small
pieces of tissues and aids in their staining (Other stains: methylene blue, safranin, congo red)
- disadvantage: inhibits hematoxylin and makes counterstaining difficult

1. Flemming’s Solution (contains Glacial Acetic acid which preserves nuclear elements and structure)
- recommended for nuclear preparation of sections
2. Flemming’s Solution without Acetic Acid (because glacial acetic acid destroys mitochondria, Golgi
bodies)
- recommended for cytoplasmic structures

Flemming’s solution:
 The most common Chrome-Osmium acetic Acid used, recommended for nuclear preparation of such sections.
 Excellent for chromosomes (demonstration)
 Permanently fixes fats
 Less amount is required for fixation (because it is expensive) one of disadvantages
 Fixation time: 24-48 hours (because it penetrates well) one of advantages

Disadvantages:
 Very expensive
 Poor penetrating medium
 Extremely volatile
 Prolonged exposure to acid vapors causes eye irritation (conjunctivitis) or black osmic deposition in the cornea
(blindness) for greater than 6 months

Flemming’s Solution without Acetic Acid

- Recommended for mitochondria, golgi bodies, endoplasmic reticulum
- The removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell.
- Fixation time: 24-48 hours

• Trichloroacetic acid
-Sometimes incorporated into compound fixatives
-it precipitates proteins & nucleic acid
-causes marked swelling effect on tissues (disadvantage)
-can be used as a weak decalcifying agent.

• Acetone
- used at such temperature range Red: - 5 0C to 4 0C and Blue: 00C to 40C
- advantages: recommended for the study of water diffusible enzymes especially lipases or
phosphatases used in fixing brain tissues for diagnosis of RABIES (negri bodies)
- disadvantages: - produces inevitable shrinkage and distortion
-dissolves fat
-preserves glycogen poorly
-evaporates rapidly

VII. Heat Fixation

- this procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for
frozen tissue sections and preparations of bacteriologic smears.
 Question:
What are the two tissue constituents usually dissolved by heat fixation?

Answer:
Starch
Glycogen (main) liver: readily available in case of starvation, muscle: last to give
The 2 most commonly used fixatives for general use:
A. 10% Formol saline
B. Zenker’s Fluid All enzymes are
proteins but not
Best general tissue fixative: 10% BNF (Buffered Neutral Formalin) all proteins are
enzymes.
Special Factors Affecting Fixation:
A. Retarded by: (slowed down)
1. size & thickness– larger tissues require more fixations and longer fixation time
2. presence of mucus - prevents complete penetration of fixative, hence, tissues that contain mucus are fixed
slowly and poorly which coats tissues (Mucus seen in Wharton’s Jelly)
- excess mucus may be washed away with saline
3. presence of fats– fatty tissues should be cut in thin sections and should be fixed longer.
4. presence of blood-– tissues containing large amount of blood (e.g. Blood vessels and spleen) should be
flushed out with saline arterial cannulization before fixing (requires 6-8 washes)
5. cold temperature – inactivates enzymes. The colder the temperature the longer the fixation.
6. hot temperature- denatures enzymes (if greater than 560C) The higher the temperature the faster the
fixation.
B. Enhanced by:
1. size & thickness – smaller and thinner tissues require less fixative and shorter fixation time
2. agitation – fixation is accelerated when automatic or mechanical tissue processing is used.
3. moderate heat ( 37 – 56 0C) – accelerates fixation but hastens autolytic changes and enzyme destruction
(Hastens autolytic changes in the deeper parts of the tissue block before the fixing agents gain access to these
regions.)
Compatibility & Incompatibility

Few fixatives permit the use of all stains. Some fixatives may act as a mordant for one group of dyes and an
inhibitor for another set of stains. Some may inhibit, some may act as a mordant, some may promote

Secondary fixation (application of fixative for the purpose of fixing completely)

- Not recommended when primary fixation for specific observations is possible in the first instance.
- This is done before dehydration.
We can use the same
10% Formalin/ common primary
fixative for primary
10% formol saline fixative used
and secondary
Post-chromatization fixation
- a.k.a. post-chroming
post-mordanting

- a second fixation whereby a primary fixed tissue is placed in aqueous solution of 2.5 – 3.0% potassium
dichromate for 24 hours, to act as mordant for better staining effects and to aid in cytologic preservation of tissues.

- some labs use this technique for special purposes (i.e. when chromation (oxidation) is desired in tissue that have
already been fixed in chromaffin medium.

Washing out
- the process of removing excess fixative from the tissue after fixation in order to improve staining and remove
artifacts from the tissue.
 Solutions that may be used:
1. tap water – used to remove: (CFO)
a. Excess chromic acid from tissues fixed in Helly’s,Zenker’s and Flemming’s solution
b. Excess formic acid (10% formalin is extracted more rapidly in 70% alcohol than in water)
c. Excess osmic acid
(Washing out:)
2. 50-70% roh– used to wash out excess amount of picric (bouin’s)
3. alcoholic iodine - used to remove excessive mercuric chloride
Practice questions:
1. Glacial acetic acid is added in Zenker’s fluid which contains mercuric chloride. What disadvantage may it pose?
a) Swelling of the cellular components.
b) Shrinkage of the cellular components.
c) Poor penetration and cell lysis
d) Solution instability

2. What is the best fixative for a frozen section?

a) 10 % BNF
b) Glutaraldehyde
c) Zenker-Formol
d) Carnoy’s fluid

3. After the usage of Heidenhain’s susa fixative, where should you subject the tissue?
a) Water
b) NSS
c) 96% ethanol
d) Absolute alcohol
e) A and b

4. Which of the following is used as a diluent for the Wright’s stain?

a) Potassium dichromate
b) Mueller’s fluid
c) Alcoholic formalin
d) Methanol
e) None of these

5. Which of the following fixatives may cause blindness through its deposition in the cornea if exposure is
prolonged?
a) 10 % Formalin
b) 10 % BNF
c) Heidenhain’s Susa
d) Osmium tetroxide
e) Chromic acid

Decalcification (soften bony materials & calcified organs)

De -remove, calci - calcium

- is a procedure where calcium or lime salts are removed from tissues (most especially, bones and teeth)
- done after fixation and before impregnation
* best done at room temperature
* ideal time required for decalcifying tissue: 24 hours
* volume: greater than or equal to 20x the volume of the tissue
 Decalcification is the removal of calcium ions from a bone or calcified tissues through a histological process that
makes them flexible and easier to cut and to prevent damage to the microtome knife (nicks).
 Decalcification adjusts the hard substance of bones to the softness of paraffin embedding medium.
  Bones are the main object of decalcification in a surgical pathology laboratory, but other specimens, such as teeth,
calcified organs and calcified heart valves (atherosclerosis) also require this procedure. Lungs can also be
decalcified.
 One alternative is to apply “surface decalcification” to a paraffin block, allowing sections to be obtained where the
presence of calcium was not anticipated when the specimen was processed. Use 2% HCl / 1% HCl in 70% alcohol.
 In choosing a technique and processing method, consideration must be given to the type of investigation being
carried out.
 For example, if a metabolic bone disease is being investigated and it is necessary to differentiate mineralized bone
from osteoid (bone-like cartilage), it may be necessary to retain and demonstrate the mineral content by producing
sections of “undecalcified” bone.

The rate of decalcification will depend upon the: The bigger = faster fixation
a. structure Increase in temp = decrease in decal time
b. temperature (directly proportional for speed)
c. volume of decalcifying
Increase volume = increase speed of fixation
Temperature = decalcifying time (inversely proportional) (directly proportional)
Temperature = speed (directly proportional)
(too rapid removal of CA++2 salts may produce complete digestion of the tissue specimen and poor staining capacity of
the tissue prep)
* Macerated – tissue will be melted with the solution
Decalcifying Agents:
1. acids
2. Ion exchange resins
3. chelating agents (Versene – EDTA)
4. electrophoresis

There are three main types of decalcifying agents:

 Those based on strong mineral acids
 Those based on weak organic compounds
 Those composed of chelating agents.
 The acids make up a solution of calcium ions while the chelating precipitate the calcium ions agents (precipitate
the calcium ions) take up the calcium ions.
 Dilute mineral acids (hypochloric acid (inferior) or nitric acid) or formic acid (better nuclear staining substitute)
can be used effectively if the end point of decalcification is monitored carefully.
 nuclear and cytoplasmic details are compromised if specimens are exposed for too long to acidic decalcifying
agents, which can extract RNA *(tigroid bodies – nissl granules) and remove the purine and pyrimidine bases from
DNA *(Feulgen reaction is inhibited)
 It is also imperative to wash the acid out of the tissue.
Almost all the methods commonly used for decalcification involve the use of acids, in which the bone salts are
dissolved. All such acids are injurious to the organic ground substance of the bone or other tissue, which must,
therefore, be protected by adequate fixation before decalcification is begun.

For routine purposes, only acids are recommended (formic & nitric acids) (commonly uutilized)
nitric – almost 2X as fast as formic acid (fastest most /rapid)
formic– provides better tissue preservation and staining (slow but better)
hydrochloric - inferior

1 ounce / gram
- the volume of acid decalcifying solution used
- should be changed once or twice a day until decalcification is completed

ACID DECALCIFYING AGENTS

1. nitric acid
- most common decalcifying agent used
- recommended for routine purpose
 - advantage: very rapid decalcifying agent
produces minimum tissue distortion

* The end point has to be carefully watched for, otherwise, progressive tissue damage occurs and
staining is severely impaired.
* Nitric acid undergoes spontaneous yellow discoloration (impair proper staining) owing to formation of
nitrous acid (HNO2).
 this accelerates decalcification
 also stain and damage the tissues
 deleterious effect in tissues
a. 10% Aqueous nitric acid
Decal time: 12 – 24 hours
adv: rapid in action
produces minimum distortion
produces good nuclear staining
easily removed by 70% alcohol (subject to dehydration)
recommended for urgent biopsies

disadv:
*prolonged decalcification could lead to distortion
*imparts a yellow color, thereby impairing the staining reaction
*Old nitric acid solution and strong acids are damaging to the tissue which cause destruction/maceration
b. Formol Nitric Acid
-with formalin
- Decal time: 1 – 3 days (24-72 hours)
- less tissue destruction than 10% aq. nitric acid
- For urgent biopsies with good nuclear staining.
- This should be used inside a fumehood (because it is irritating to the nostrils)
- (remedy) yellow color imparted by nitrous acid is removed through neutralization with 50% sodium sulfate and
running tap water for 12 hours/ overnight.

c. Perenyi’s Fluid

Decal time: 2 - 7 days

It is recommended for routine purposes.
It decalcifies and softens the tissues at the same time.

maceration is avoided due to the presence of chromic acid and alcohol.

* maceration – tissue is dissolved & become a part of the solution rendering it insoluble

 Complete decalcification cannot be determined by chemical test because of precipitate formation after addition of
ammonia (turbid which causes false positive)
 Remedy:
(add 0.5 mL of saturated ammonium oxalate)
 Result: Reappearance of a white precipitate within 30 minutes reaffirms the presence of Ca=incomplete
decalcification
3 ways
1. Physical / Mechanical
2. Chemical – most common way to determine the completion of decalcification
3. Radiologic / X-rays
 d. Phloroglucin nitric acid
– most rapid decalcifying agent, recommended for urgent works.
- Decal time: 12 – 24 hours
- Has poor nuclear staining
-Yellow color formation
-completion of decalcification cannot be determined by chemical test.
* Use 0.5 ml of sat aqueous ammonia oxalate

 When decalcification is complete, the acid must be removed by threechanges of 70 % to 90% ethanol, since
washing in watery solutions will lead to excessive swelling and deterioration of tissue.
 When the sections are cut, the slides are brought to water and placed in 1 %
 1% aqueous lithium carbonate for 1 hour, mwashed in water for 30 minutes, and then stained.

2. Hydrochloric acid

 is inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater
distortion of tissue produced on the decalcified section.
 However, it produces good nuclear staining and if used in 1% solution with 70% alcohol, may be recommended
for surface decalcification of the tissue blocks.
 Rapid proprietary solutions usually contain hydrochloric acid, whereas slow proprietary mixtures contain buffered
formic acid or formalin/formic acid.
 Dilution of a proprietary HCl is not deleterious for effective decalcification or staining, and this is an option if a
strong mixture is considered too concentrated.
a. Von Ebner’s fluid
– Recommended for teeth and small pieces of bones and extent of decalcification cannot be measured by Chemical test.

Advantages:
 1. It permits relatively good cytologic staining.
 2. It is a moderately rapid decalcifying agent.
 3. It does not require washing-out before dehydration.

3. Formic acid (slower compared to nitric)

- moderate-acting decalcifying agent
Adv:
*produces better nuclear staining with less tissue distortion
*recommended for routine decalcification of post-mortem research tissues

Disadv:
*not suitable for urgent examinations (slow decalcification speed)

*Tissues are not ruined if they remain in formic acid a day beyond the completion of decalcification.

* It is safer to handle than nitric acid.

a. Aqueous Formic Acid
b. Formic Acid-Sodium Citrate solution

Advantages: Formic acid

1. It may be used both as a fixative and decalcifying agent.
2. It permits excellent nuclear and cytoplasmic staining.
3. It is recommended for small/minutes pieces of bones and teeth.
4. It is suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed.
Disadvantages: Formic acid
1. It is relatively slow; hence, is not suitable for urgent specimens.
 2. Decalcification may be hastened by increasing the proportion of formic acid to 25 ml. However, such
concentration may make the solution opaque, thereby interfering with the staining results.
3. It requires neutralization with 5% sodium sulfate, and washing out to remove the acid from the tissue.
4. DECALCIFICATION TIME: 2-7 days

4. Trichloroacetic acid
Advantages:
 1. It permits good nuclear staining.
 2. It does not require washing-out
; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration. (directly place
on dehydrating agent)

Disadvantages:
1. It is a weak decalcifying agent, not used for dense tissues, and is suitable only for spicules of bones.
2. It is very slow-acting; hence, is not recommended for urgent examinations.
*DECALCIFICATION TIME: 4- 8 days

5. Sulfurous acid – weakest decalcifying agent, only for minute pieces of bone.

6. Osmic acid + Chromic acid (Flemming’s Solution)

Advantages:
 1. It may be used both as a fixative and decalcifying agent.
 2. It may be used for decalcifying minute bone spicules.

Disadvantages:
 1. Nuclear staining with hematoxylin is inhibited.
 2. It tends to undergo reduction and forms precipitates at the bottom of the container thus requiring frequent
changes of solution.
 3. Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol; hence, tissues must be
washed out prior to dehydration.
 4. Degree of decalcification cannot be measured by the routine chemical test.

7. Citric acid-citrate buffer– does not produce cell or tissue distortion

Advantages:
 1 It permits excellent nuclear and cytoplasmic staining.
 2. It does not produce cell or tissue distortion.
Disadvantage:
 Its action is too slow (not for urgent) for routine purposes.
DECALCIFICATION TIME: 6 days

II. Chelating agents - subs. which combine with calcium ions and other salts (iron and magnesium salts) to
form weakly dissociated complexes and facilitate removal of Calcium salt
EDTA– the most common chelating agent
- commercial name: versene (K2 EDTA)
EDTA+Ca insoluble non-ionic complex (Removes Ca) precipitate
- precipitate at the bottom
- used as an anticoagulant and water softeners.
- 2 ml (lavender, pink, royal blue top tubes)

III. Ion exchange resins (IER’s)

- removes Ca+2 salts from formic acid, thereby increasing
solubility from the tissue.
- not recommended for fluids containing mineral acids such as nitric or hypochloric acid (cause unduly
action and explosion)
 IV. Electrophoresis
- process whereby positively charged (anode) Calcium ions are attracted to a negatively charged (cathode)
electrode and subsequently removed from the decalcifying solution (calcium 2+ will migrate to cathode (negatively
charge).
- the time required for decalcification is thereby shortened due to the heat and electrolytic reaction produced in this
process.
- the principle applied is similar to that of chelating agents; with the main difference that this process utilizes
electricity and is dependent upon a supply of direct current to remove Ca deposits.

Solutions used:
1. Formic acid (88% conc or greater)
2. Conc. HCl
3. Distilled water

3 ways by which the extent of decalcification may be measured:

A. Physical / mechanical
- by touching or bending the tissue with the finger to determine the consistency of tissues
- by pricking the tissue with a fine probe/needle

B. Radiological / X-rays (based on principle of opacity)

- expensive
- most ideal
- most reliable
- good but not always convenient

C. Chemical
- presence of cloudiness indicates the presence of Ca
- (still) favored
- decalcifying fluid is changed every 24-48 hrs
Solutions used:
saturated aqueous ammonium oxalate (calcium oxalate)
aqueous / conc. Ammonium hydroxide (calcium hydroxide)

Other tissue softeners:

1. Perenyi’s– act both as a decalcifying agent and tissue softener
2. Molliflex
3. 2% HCl
4. 1% HCl in 70% Alcohol
5. 4% Phenol

Board exam question:

Which of the following methods of the determination of the extent of decalcification cannot be used when radiopaque
metallic salts such as HgCl2 has been used in fixation?

Answer:
_____________