EMS26108


Components:

Harris Haematoxylin

Hucker’s Crystal Violet

Basic Fuchsin Stock Sol. 0.1%

Gram’s Iodine Solution

Lithium Carbonate Solution

Acid Alcohol, 1%

Acetone-Alcohol, 1:1

Acetone

Picric Acid-Acetone Sol., 0.1%

Acetone-Xylene I

Acetone-Xylene II


Fixation:

10% Buffered Neutral Formalin


Section:

Paraffin @ 6µm


Staining Procedure:

Use Control Slide

  1. Deparaffinise and hydrate to distilled water.
  2. Harris Haematoxylin for 5-10 minutes.  Wash in running distilled water 1 minute.
  3. Differentiate in Acid Alcohol.  Wash in running water 3 minutes.
  4. Wash in Lithium Carbonate Solution to intensify blue  ***From this point carry only one slide at a time.
  5. Wash in running water, 5 minutes. Hucker's Crystal Violet two minutes.  Wash quickly in water.
  6. Mordant in Gram's Iodine1 minute.  Wash in water. Blot with filter paper moistened with water, but do not allow to dry.
  7. Decolorize in Acetone:Alcohol until no more blue comes off. Blot with filter paper moistened with Acetone-Alcohol, but do not allow to dry.
  8. Basic Fuchsin Working (or 5.0ml Basic Fuchsin Stock and 60ml Distilled water) for 3 minutes. Wash in water. Blot with filter paper moistened with water, but do not allow to dry.
  9. Dip in Acetone until section begins to decolourise.
  10. Differentiate in Picric Acid-Acetone, (A-108-9) until section becomes reddish-brown-yellow (15 seconds).
  11. Pass quickly through Acetone-Xylene I, then Acetone-Xylene II
  12. Clear in Xylene, two changes. Mount.


Results:

Gram-Positive Organisms

blue to blue-black

Gram-Negative Organisms

bright red

Nuclei

brownish red

Erythrocytes

red to yellow-green

Necrotic Tissue

yellow-green

Cytoplasm

yellow

Connective Tissue

red


References:

AFIP Manual of Histological Staining Methods, 3rd ed., Ed. L. Luna:  New York:  McGraw Hill Publications, c. 1968, p. 226.

Clark, G.:  Staining Procedures, Williams and Wilkins Company, Baltimore, 3rd Ed., c. 1973, p. 320

Taylor, R.D., Amer. J. Clin. Path., 46:472 (1966).