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MICROWAVE WOLBACHS GIEMSA METHOD
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FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 μm.
SOLUTIONS:
0.5% Hydrochloric Acid-Alcohol
Alcohol, 70% --------------------------------------------------------------------- 995.0 ml
Hydrochloric acid, concentrated --------------------------------------------------- 5.0 ml
Giemsa Solution, Stock
Azure A, C.I. 52005 ------------------------------------------------------------ 0.50 gm
Eosin Y, C.I. 45380 ------------------------------------------------------------- 0.44 gm
Phloxine B, C.I. 45410 ---------------------------------------------------------- 0.06 gm
Glycerol ------------------------------------------------------------------------- 60.00 ml
Methyl alcohol ------------------------------------------------------------------- 40.00 ml
Pour the glycerol in a beaker and add the dyes. Place on a hot plate stirrer, apply gentle heat and mix for one hour. Do not allow the solution to exceed 55o C. Allow the solution to cool to 30 o C and add the methanol and mix on a stirrer for five minutes. Store in a tightly stoppered amber bottle. The solution is stable for at least one year.
10% Triton X-100
Triton X-100 ---------------------------------------------------------------------- 10.0 ml
Distilled water --------------------------------------------------------------------- 90.0 ml
Add a few grains of thymol to prevent the growth of fungi.
Giemsa Solution, Working
Giemsa solution, stock -------------------------------------------- ------------- 4.0 ml
Methyl alcohol ----------------------------------------------------------------- 1.0 ml
Distilled water ------------------------------------------------------------------ 40.0 ml
10% Triton X-100 ------------------------------------------------------------- 0.4 ml
0.5% Acetic Acid Solution
Acetic acid----------------------------------------------------------------------- 0.5 ml
Distilled water-------------------------------------------------------------------- 99.5 ml
1. Deparaffinize and hydrate to distilled water.
2. Place in acid-alcohol for 3 minutes.
3. Wash well in tap water and rinse in two changes of distilled water.
4. Place in the working Giemsa solution in a glass Coplin jar. Place in a microwave oven and microwave at power level 1 (60W) for 3 minutes. Allow the slides to remain in this warm solution (55˚ C) for 10 minutes.
5. Rinse in distilled water.
6. Place in 0.5% acetic acid for 3 seconds. When staining for helicobacter, one quick dip.
7. Rinse in distilled water.
8. Rinse in three changes of 95% alcohol and four changes of absolute alcohol.
9. Clear in three of four changes of xylene.
10. Mount with synthetic resin.
Nuclei ---------------------------------------------------------------- blue
Collagen and other tissue elements ----------------------------------- pink to rose
Bacteria -------------------------------------------------------------- blue
Mast cells ------------------------------------------------------------ purple
Eosinophils ---------------------------------------------------------- pink
Rickettsia ------------------------------------------------------------ purple
Helicobacter (formerly campylobacter) ---------------------------- - pale blue
We have found some variation in the quality of commercial Giemsa stains in both solution and powder form. The Giemsa formula used in this method compares favorable with the best commercial Giemsa stains and can be used in any Giemsa staining method.
The use of the microwave oven has greatly reduced the time required to perform this method. Also, the staining results appear to be better than the original overnight staining method.
Triton X-100 is added to the working Giemsa solution because it keeps the solution stable when it is heated with microwave irradiation. If the Triton X-100 is omitted a fine precipitate will form in the solution. This precipitate also forms when Triton X-100 is omitted when the solution is prepared with commercial Giemsa stains.
The stain may be performed at room temperature by staining the sections overnight in the working Giemsa solution.
Lillie, R.D. and Fullmer, H.M.: Histopathologic Technic and Practical Histochemistry, 4th ed., New York, McGraw-Hill, 1976, p. 195.
Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., New York, McGraw-Hill, 1968, pp. 119-120.
Churukian, C.J. and Listinsky, C.M.: A new Giemsa stain for undecalcified bone marrow sections. Lab. Med. 20:162-165, 1989
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