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MICROWAVE COLLOIDAL IRON METHOD
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FIXATION: 10% buffered neutral formalin. Avoid the use of dichromate fixatives.
TECHNIQUE: Paraffin sections cut at 5 μm.
SOLUTIONS:
20% Ferric Chloride Solution
Ferric chloride, FeC13 6H2O --------------------------------------------------- 2.0 gm
Distilled water ------------------------------------------------------------------- 10.0 ml
Colloidal Iron Solution (Stock)
Distilled water ------------------------------------------------------------------- 250.0 ml
Ferric chloride, 20% ------------------------------------------------------------ 10.0 ml
Bring the distilled water to a boil and then add 10.0 ml of 20%
ferric chloride. When the solution turns dark red remove from
the heat and cool. This solution is stable for at least one year.
Colloidal Iron-Acetic Acid Solution (Working)
Colloidal iron (stock) ----------------------------------------------------------- 20.0 ml
Distilled water ------------------------------------------------------------------- 15.0 ml
Acetic acid, glacial -------------------------------------------------------------- 5.0 ml
12% Acetic Acid Solution
Acetic acid, glacial -------------------------------------------------------------- 12.0 ml
Distilled water ------------------------------------------------------------------- 88.0 ml
2% Hydrochloric Acid Solution
Hydrochloric acid, concentrated ------------------------------------------------- 2.0 ml
Distilled water ------------------------------------------------------------------- 98.0 ml
10% Triton X-100
Triton X-100 -------------------------------------------------------------------- 10.0 ml
Distilled water ------------------------------------------------------------------- 90.0 ml
1% Potassium Ferrocyanide Solution
Potassium ferrocyanide --------------------------------------------------------- 1.0 gm
Distilled water ------------------------------------------------------------------- 96.0 ml
10% Triton X-100--------------------------------------------------------------- 4.0 ml
Hydrochloric Acid-Potassium Ferrocvanide Solution
4% Hydrochloric acid ---------------------------------------------------------- 20.0 ml
2% Potassium ferrocyanide ----------------------------------------------------- 20.0 ml
10% Triton X-100 -------------------------------------------------------------- 0.4 ml
Prepare just before use.
Nuclear Fast Red Solution
Dissolve 0.1 gm of nuclear fast red in 100 ml of 5% aqueous solution of aluminum sulfate with the aid of heat. Cool, filter and add a few grains of thymol as a preservative.
1 . Deparaffinize and hydrate to distilled water.
2. Place in 12% acetic acid for I minute.
3. Place in working colloidal iron-acetic acid solution in a glass Coplin jar and
microwave at power level 1 (60W) for 3 minutes. Dip the slides up and down several times and allow them to remain in the warm solution for 10 minutes.
4. Rinse in 12% acetic acid, four changes, 1 minute each.
5. Place in the hydrochloric acid-potassium ferrocyanide solution in a glass Coplin jar and microwave at power level 1 (60W) for 2 minutes. Dip the slides up and down several times and allow them to remain in the warm solution for 2 minutes.
6. Wash in running tap water for 1 minute.
7. Rinse in two changes of distilled water.
8. Nuclear fast red solution for 3 minutes.
9. Rinse in three changes of distilled water.
10. Debydrate through graded alcohols.
11. Clear in three or four changes of xylene.
12. Mount with synthetic resin.
Acidic mucopolysaccharides -------------------------------------- deep blue
Acidic epithelial mucus -------------------------------------------- deep blue
Cryptococcus ---------------------------------------------------- deep blue
Nuclei ------------------------------------------------------------ red
The colloidal iron method stains essentially the same acidic mucopolysaccharides that are stained with alcian blue, except that the color with colloidal iron is a more intensive blue.
It appears that cryptococcus is the only fungus which can be demonstrated by methods for acid mucopolysaccharides such as Mowrys alcian blue, Southgates and Mayers mucicarmine and Mowrys colloidal iron. Most texts recommend Southgates or Mayers mucicarmine for demonstrating cryptococcus but, based on our experience, this colloidal iron method gives the best results.
When heating a solution with microwave irradiation the top portion of the solution is warmer by l 0- 15° C than that near the bottom of the Coplin jar. Therefore in order to equalize the temperature of the solutions the slides are dipped up and down in steps #3 and #5 of the staining procedure. This assures uniformity of staining throughout the tissue sections.
The stain may be performed at room temperature by staining the sections for l hour in the colloidal iron-acetic acid working solution and for 30 minutes in the hydrochloric acid-potassium ferrocyanide solution.
Mowry, R.W.: Improved procedure for the staining of acidic polysaccharides by Mullers colloidal-hydrous-ferric oxide and its combination with the Feulgen and the periodic acid-Schiff reactions. Lab. Invest. 7:566-576, 1958.
McManus, J.F.A. and Mowry, R.W.: Staining Methods Histologic and Histochemical, New York, Paul B. Hoeber, 1960, pp. 133-136.
Image Examples:
Diagnostic Application: