TECHNIQUE:        Undecalcified glycol methacrylate embedded bone marrow sections cut at 3-4 μm.

SOLUTIONS:

0.5% Hydrochloric Acid-Alcohol

Alcohol, 70% -----------------------------------------------------------------                1000.0 ml

Hydrochloric acid, concentrated ----------------------------------------------                5.0 ml

10% Triton X-100

Triton X-100 --------------------------------------------------------------------        10.0 ml

Distilled water -------------------------------------------------------------------        90.0 ml

Phosphate Buffer, pH 6.6 – Triton X-100

Potassium phosphate, monobasic, KH2PO4 ------------------------------------                6.15 gm

Sodium phosphate, dibasic, Na2HPO4 ----------------------------------------                3.85 gm

Distilled water -----------------------------------------------------------------                1000.00 ml

10% Triton X-100 -------------------------------------------------------------                10.00 ml

       Store in a refrigerator at 3-5o C.

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

Glycerin ------------------------------------------------------------------------                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 30o 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.

       

Giemsa Solution, Working

Giemsa solution, stock ----------------------------------------------------------                4.0 ml

Phosphate buffer, pH 6.6 – Triton X-100 ------------------------------------                40.0 ml

1% Acetic Acid

1. Place in 0.5% hydrochloric acid-alcohol for 10 minutes.
2. Wash well in running tap water.
3. Rinse in two changes of distilled water.
4. Place slides in the working Giemsa solution in a glass Coplin jar and microwave at power level 1 (60 W) for 2 minutes.  Allow the slides to remain in the warm solution for 60 minutes.
5. Rinse in distilled water, three dips.
6. Place in 1% acetic acid for 5 seconds.
7. Rinse in distilled water, three dips.
8. Dehydrate in three changes of 95% alcohol and four changes of absolute alcohol, three dips in each.
9. Clear in xylene, three or four changes.
10. Mount with synthetic resin.

All the colors of the hemopoietic bone marrow are similar to the colors of bone marrow air dried aspirates stained with Giemsa.

Eosinophil granules -----------------------------------------------                round red refractile granules

Erythrocytes, mature ----------------------------------------------                red

Erythrocytes, immature -------------------------------------------                small purple (azuraphilic)

                                           granules

Mast cells -----------------------------------------------------------                deep purple

Megakaryocyte cytoplasm ---------------------------------------                pale purple as are the platelets

Megakaryocytes, immature --------------------------------------                eosinophilic cytoplasm

Neutrophils, mature -----------------------------------------------                tan to pink

Nuclear chromatin -------------------------------------------------        blue

Plasma cells ---------------------------------------------------------        purple with the characteristic

                                           perinuclear clear region

The use of the microwave oven reduces the time required to perform the method.  Also, the staining results appear to be better than when the stain is done at room temperature.  The final temperature of the solution should be approximately 50o C.

We have found considerable variation in the quality of commercial Giemsa stains in both solution and powder form.  The Giemsa formula used in this method compares favorably with the best commercial Giemsa stains and can be used in any Giemsa staining method.

The addition of the detergent Triton X-100 to the staining solution results in more distant staining of the various cell types found in hemopoietic bone marrow.  Because morphological details are more distinct microscopical detection and identification of the cells is facilitated.  Another advantage gained by the addition of Triton X-100 is that it stabilizes the working Giemsa solution.  If the Triton X-100 is omitted a fine precipitate forms in the solution.  This precipitate also forms when Triton X-100 is omitted from solutions prepared with commercial Giemsa stains.

The stain may be done at room temperature by staining in the working solution for 2 hours.

       Boon, M.E., et al.  Microwave-stimulated staining of plastic embedded bone marrow sections with the Romanowsky-Giemsa stain:  Improved staining patterns.  Stain Technol. 62:257-266, 1987.

Lillie, R.D. and Fullmer, H.M.:  Histopathologic Technic and Practical Histochemistry, 4th ed., New York, McGraw-Hill, 1976.

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.

Melvin, D.M. and Brooke, M.M.:  Triton X-100 in Giemsa staining of blood parasites.  Stain Technol. 55:269-275, 1955.