May-Grünwald Giemsa

The coloring of May-Grünwald Giemsa is a method of coloring used in Hématologie to differentiate the cells from the Sang during the cellular preparations (Cytologie).

Principle of coloring

It rests on the action complementary to two neutral dyes and on the affinity of the cellular elements for the dyes Acide S or Basique S. These two dyes are:

the Neutral May-Grünwald , , containing an acid dye, the éosine, and a basic dye, the Methylene blue (in the form of éosinate of methylene blue).
the Giemsa , neutral, container him also of the éosine, and a basic dye, the Azure of methylene (in the form of éosinate of methylene azure)

Those two dyes are solubilized in the methyl alcohol and are of this fact inactive: it is the contact of the water which gives them a coloring capacity. Salts dissociate then while coloring acid (the éosine) and basic (azure and methylene blue).

the acid cellular elements, will be coloured selectively by the basic dyes. These elements are described as basophilic (DNA, cytoplasm of the Lymphocyte S)
the basic cellular elements, will be coloured selectively by the acid dyes. These elements are described as acidophiles or of eosinophilic (cytoplasm of the Hématie S)
the elements neutrophiles is coloured at the same time by the acid and basic dyes.

It colors in:

blue, the residues Basophilic S
, the residues eosinophilic S
beige-pink, the residues Neutrophile S
crimson, the residues Azurophile S

Characteristics

Colorings are orthochromatic: the coloured elements do not change conformation and take the same color as that of the dye. There exists however a particular case: the elements azurophiles, fixing the blue methylene azure become red because of a change of conformation. The Water used to activate to them dyes and to rinse the blades must be nearest possible to neutrality (pH ranging between 6.8 and 7.4) not to accentuate colorings (a basic pH accentuates the methylene blue with depend on the éosine, and conversely). To obtain water in these values of pH one can

use a plugged water has pH 7 with a mixture of hydrogénophosphate and dihydrogénophosphate
Mélanger water distilled (acid) with tap water (basic) in the presence of BBT.

In all the case this water is not preserved and must be prepared at the last time.

Technique

Fixing

It is initially necessary to fix the blood cells present on the smear. For that horizontally to place the smear in a box of coloring and to pour 15 have 20 drops of May-Grünwald dye in order to cover the smear completely. To wait 3 minutes so that the Méthanol fixes the cells.

May-Grünwald coloring

To add as much water than there was of dye, to let act two minutes and to rinse the blade.

Coloring in Giemsa

To prepare the dye by putting 20 ml of neutral water with 30 drops of dye in a oil-can. The dye must remain on the surface. To pour the contents in a box of Laveran as soon as the blade is ready and to mix while agitating gently (the capacity of the dye is maximum at the time of the mixture). To pose the blade, smear in contact with the dye, in the box of Laveran. To let act 20 min. and rinse with neutral water.

Drying

To let the blade dry with the air after having wiped it with filter paper. To await complete drying before observation under the microscope.

Observation

See too

Other techniques of coloring:

Coloring of Papanicolaou of Georgios Papanicolaou.

Others

technical May-Grunwald-Giemsa video

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