TECHNIQUE:        Unfixed smears and touch preparations.

SOLUTIONS:

Fixative Solution

Methyl alcohol -----------------------------------------------------------------                500.000 ml

Toluidine blue O, C.I. 5204 ----------------------------------------------------                0.002 gm

Phosphate Buffer, pH 6.5

Sodium phosphate, dibasic -----------------------------------------------------                1.63 gm

Potassium phosphate, monobasic -----------------------------------------------                3.37 gm

Distilled water ------------------------------------------------------------------                500.00 ml

Phosphate Buffer, pH 6.7

Sodium phosphate, dibasic -----------------------------------------------------                2.21 gm

Potassium phosphate, monobasic -----------------------------------------------                2.79 gm

Distilled water ------------------------------------------------------------------                500.00 ml

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.

Solution I (Eosin Y)

Eosin Y, C.I. 45380 -------------------------------------------------------------        0.625 gm

Phosphate buffer, pH 6.5 --------------------------------------------------------        500.000 ml

Sodium azide ---------------------------------------------------------------------        0.050 gm

Solution II (Azure A-Azure B -Methylene Blue)

Azure A, C.I. 52005 -------------------------------------------------------------        0.250 gm

Azure B, C.I. 52010 -------------------------------------------------------------        0.125 gm

Methylene blue, C.I. 52015 ------------------------------------------------------        0.250 gm

Phosphate buffer, pH 6.7 --------------------------------------------------------        495.000 ml

10% Triton X-100 ---------------------------------------------------------------        5.000 ml

1.        Place slides in fixative solution for 30 seconds.

2.        Rinse in three changes of distilled water.

3.        Place in Solution I (eosin Y) for 20 seconds.

4.        Rinse in three changes of distilled water.

5.        Place in Solution II (azure A-azure B-methylene blue) for 40 seconds.  If a more intense nuclear stain is desired, stain for 1 minute.

6.        Rinse quickly in three changes of distilled water.

7.        Blot slides or stand on end and allow to dry.

8.        Dip in xylene and mount with synthetic mounting media.

Nuclei ----------------------------------------------------------------        bluish purple

Cytoplasm -----------------------------------------------------------        pink, blue or sky blue, depending on the cell type

Erythrocytes ---------------------------------------------------------        pink

Bacteria --------------------------------------------------------------        blue

The staining results with this method are about the same as those obtained with the Giemsa or Wright-Giemsa method.

The sodium azide in the eosin Y and azure A, azure B and methylene blue solutions helps to retard the growth of microorganisms and the 10% Triton X-100 in the azure A-methylene blue solution gives it greater stability.  These solutions have a shelf-life of about one year when stored at room temperature.

Diff-Quick stain for the rapid, differential staining of hematological smears yielding qualitative results similar to Wright-Giemsa stain.  Baxter Healthcare Corporation, Dade Division, Miami, Florida 33153-0672, 1989.