Leyte Normal University Tacloban City

Presented by: Jenelyn P. Cadion BS Biology 4

A. Acids B. Chelating Agents C. Ion Exchange Resins D. Electrical Ionization (Electrophoresis)

Nitric Acid Hydrochloric Acid Formic Acid Trichloroacetic Acid Sulfurous Acid

Aqueous Nitric Acid Formol-Nitric Acid Perenyis Fluid Phloroglucin-Nitric Acid

Formic Acid-Sodium Citrate Solution

Chromic Acid Citric Acid-Citrate Buffer Solution Von Ebners Fluid

 Most widely used agents Selected pieces of tissues are placed in a gauze bag and suspended in decalcifying solution by means of thread Thread is dipped in melted paraffin wax

Rate of Decalcification
Optimum Temperature Room Temperature 25oC Ideal Time 24-48hours for less dense tissues 14days or longer for dense bones

Advantage:
-Most common and very rapid decalcifying agent Disadvantage: -Inhibits nuclear stains and destroys tissues especially in concentrated solutions.

+ +

mandible area containing new bone formation decalcified in 5% nitric acid

stained tooth (Masson Trichrome) and decalcified by nitric acid 5% showing the good preservation of the dentinal tubules (1), inflammatory cells (2), pulp tissue (3).

Formula: Conc. Nitric Acid Distilled Water

10mL 100mL

Advantages: Rapid in action Minimum distortion of tissues Produces good nuclear staining Easily removed by 70% Alcohol

12- 24 hours

Disadvantages: Prolonged decalcification may lead to tissue distortion Imparts yellow color (Nitrous Acid) impairing the staining reaction of the tissue

Formula: Conc. Nitric Acid Strong Formaldehyde (40%) Distilled Water

10mL 5mL 85mL

Advantages: Rapid action Good nuclear staining Lesser tissue destruction than 10% Aqueous Nitric Acid

1-3 days

Disadvantages: Imparts yellow color (Nitrous Acid) impairing the staining reaction of the cell Can be prevented by neutralizing the tissue with 5% Na2SO4 and washing in running tap water for at least 12 hours. Addition of 0.1%Urea to pure concentrated Nitric acid

Formula: 10% Nitric Acid 40mL 0.5% Chromic Acid 30mL Absolute Ethyl Alcohol 30mL Advantages: Decalcifies & softens tissues at the same time Good nuclear and cytoplasmic staining Maceration is avoided due to the presence of chromic acid and alcohol

 2-7 days

Disadvantages: Slow decalcifying agent for dense bones and so is not recommended for urgent works Complete decalcification cannot be determined by chemical test
Dissolve precipitate by adding Glacial Acetic Acid and about 0.5mL of Saturated Aqueous Ammonium Oxalate

Formula: Conc. Nitric Acid Phloroglucin Nitric Acid 10%

10mL 1g 100mL

Advantages: Most rapid decalcifying agent;hence recommended for urgent works

12-24 hours

Disadvantages: Nuclear staining is poor Yellow color must be neutralized with 5% Na2SO4 and thoroughly wash with running tap water for at least 24 hours. Complete decalcification cannot be determined by chemical tests.

Advantage: Produces good nuclear staining and if used in 1% solution with 70% alcohol may be recommended for surface decalcification of tissue blocks Disadvantage: Inferior compared to Nitric Acid due to its slower action and greater distortion produced on tissues

This is a low power Haematoxylin and Eosin stained section of bone that was processed undecalcified and surface decalcified before cutting a section.

Formula: Formic Acid (Sp. Grav. 1.20) Formol-Saline 10%

10mL 90mL

Advantages: Can be used as fixative and decalcifying agent Excellent nuclear and cytoplasmic staining Recommended for small pieces of bones and teeth

2-7 days

Disadvantages: Slow action, but can be hastened by increasing the Formic Acid to 25mL, however it may interfere with the staining results. Requires neutralization with 5% Na2SO4 and washing out

formic acid decalcified rat knee joints

Formula: Aqueous Sodium Citrate 20% 50mL Formic Acid 45% 50mL Advantages: Better nuclear staining than Nitric Acid Recommended for autopsy materials, bone marrow, cartilage and other tissues

3-14 days

Disadvantages: Relatively slow and not recommended for dense tissues Requires neutralization with 5% Sodium Sulfate

Formula: Trichloroacetic Acid Formol Saline 10%

5g 95mL

Advantages: Permits good nuclear staining Does not require washing out; excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration.

4-8 days

Disadvantages: Weak decalcifying agent and not used for dense tissues Very slow-acting; hence, is not recommended for urgent examinations.

References
http://www.rowleybio.com/pdfs/F93%20Formic%20AcidSodium%20Citrate%20Method..pdf Luna, L.G. (ed,) Manual of Histologic Staining Methods of the AFIP, 3rd edition,McGraw-Hill, N.Y. pg. 8, c1968 http://www.ihcworld.com/_protocols/histology/de calcification_solution.htm http://books.google.com.ph/books?id=Dhn2Kispfd QC&pg=PA339&lpg=PA339&dq=perenyi's+fluiddecalcifying&source=bl&ots=JyjBbtWxH6&sig=Oy2u arBZMStg0FKz13YqnGzN5C4&hl=tl&ei=m8QzTrWJ EY_4mAXC_vXwCg&sa=X&oi=book_result&ct=result &resnum=5&ved=0CCsQ6AEwBA#v=onepage&q&f=f alse Histophatologic Techniques. M.D. Bruce-Gregorios, Jocelyn. 2nd ed.2006.Goodwill Trading Co.,Inc.pg 53-59