May-Grunwald Giemsa Staining: A Method to Stain Bone Marrow Cells

- May-Grunwald Giemsa, or MGG staining, is a two-step procedure for the differential staining of bone marrow cells, or BMCs. To begin staining, obtain a concentrated mono-layered smear of BMCs on a glass slide. Allow the smear to air dry. Treat the cells first with May-Grunwald stain containing eosin and methylene blue dissolved in methanol.

The solvent methanol initially fixes the cells. Methylene blue, a basic dye with a net positive charge, stains acidic cellular components like nucleic acids. Eosin, an acidic dye with a net negative charge, stains basic cellular components like cytoplasmic cationic proteins. Treat the cells with a buffer solution of pH 6.8 to enable the dye to precipitate and bind well with the cellular materials.

Additionally, treat the cells with Giemsa stain, also containing acidic and basic dyes, to enhance the overall staining effect. The basic dyes, azure and methylene blue, intensify acidic components' stain while the acidic dye, eosin, augments basic components' stain.

Under a microscope, erythrocytes appear pinkish-brown and platelets as small purple dots. Granulocytes display purple-blue multi-lobed nucleus and red granulated cytoplasm. Agranulocytes appear clear blue with a large purple-pink nucleus. In the following protocol, we show the MGG staining of bone marrow cells.

- To prepare the bone marrow cells for staining, first place slides into disposable chambers with pre-attached filter cards and place the chambers into a cytocentrifuge. Add 100 microliters of FACS buffer to each filter card chamber and briefly centrifuge the slides. At the end of the spin, add 100 microliters of cells to each chamber and centrifuge the cells.

Then, carefully remove the slides from the chambers for air drying. To stain the bone marrow cells, immerse the slides in a Coplin jar containing May-Grunwald solution for 5 minutes. Next, transfer the slides into a jar of buffer solution with a pH of 6.8 for 1 minute. Then transfer the slides into Giemsa R solution for 10 minutes, followed by a 10-second wash with pH-neutral water.

After the wash, allow the slides to air dry and apply one drop of mounting medium to each sample. Then, place one edge of a cover glass onto the slide and carefully lower the cover glass onto the cells, pressing gently to remove any air bubbles.