History^88-92
In 1879 Ehrlich described the use of 'neutral' dyes (mixtures of acidic and basic dyes) for the differentiation of cells in peripheral blood smears. In 1891 Romanowsky and Malakowsky independently developed a method which used mixtures of Eosin Y and 'ripened' Methylene Blue that not only differentiated blood cells, but also demonstrated the nuclei of malarial parasites. A number of 'ripening' (oxidation or polychroming) techniques were investigated by different groups (Unna 1891, Nocht 1898) but the aqueous dye solutions produced were unstable and precipitated rapidly. Subsequently, methanol was introduced as a solvent for the dye precipitate (Jenner 1899, England; May and Grünwald 1902, Germany) and techniques were developed that utilised the fixative properties of the methanolic solution, prior to aqueous dilution for staining (Leishman 1901, England; Wright 1902, Germany). Giemsa (1902) further improved these techniques by using more controlled methods of oxidation with measured amounts of known dyes, and adding glycerol to the methanol solvent to increase the solubility and stability of the dyes.

Traditional formulae and mechanism of staining
Traditional Romanowsky-type dyes are produced by oxidation (polychroming) of Methylene Blue in aqueous solution, using heat and alkali. The resulting solution contains a mixture of Azure A, Azure B, Methylene Violet and Methylene Blue. A measured quantity of Eosin Y is then added to produce a 'neutral' dye. The precipitate formed is dissolved in methanol, or a mixture of equal volumes of methanol and glycerol, to produce a stable stock solution. Working solutions are prepared by diluting the stock solution with distilled water, or an aqueous buffer, to allow ionisation of the dyes. Typical staining results obtained using air-dried, methanol-fixed smears are:

The full range of colours obtained cannot be accounted for by ionic binding alone⁸⁸. The 'red' staining of the nuclei of parasitic protozoa is thought to be produced by the azure dye and eosin binding to the basic protein protamine, rather than the nuclear chromatin, to produce an imino base of the azure dye (which is red). Metachromasia also contributes to the staining pattern observed. Mixtures of pure azure B and eosin Y alone can also produce the same staining pattern as traditional Romanowsky-type dye mixtures⁹³.

Current practice
Romanowsky-type stains are used routinely in haematology and cytology but are not commonly applied to tissue sections. The examination of smears or imprints and thin (1-2 µm) sections stained with both H&E and a Romanowsky-type stain is recommended for optimal evaluation of bone marrow^94,95 and lymphoid tissues^89,96. Advantages of Romanowsky-type staining over haematoxylin and eosin include: demonstration of mast cell granules; differentiation of mucosubstances; and identification of blood and marrow cells of different lineages and stages of maturation^97,98. Numerous modifications of the Romanowsky method have been described, particularly for application to tissue sections and, although the various techniques may produce slight differences in staining, the basic mechanisms and staining effects are the same. The staining obtained in tissue sections is more variable than in smears because the protocols used require additional steps (differentiation, dehydration, clearing) and tissue sections contain more stainable components⁹⁸. Some stains are used in combination to produce the desired colouring of cell and tissue component as for example with the Jenner-Giemsa and May Grünwald-Giemsa techniques⁹⁹.

Specimen preparation
Bone marrow cores require fixation and decalcification before processing. Zenker's fluid, which simultaneously fixes and decalcifies tissue, is recommended, but formalin fixation followed by mild acid or EDTA decalcification can be used. However, formalin fixation alters the expected staining pattern with the nuclei staining dark blue and the cytoplasm pale blue¹⁰⁰, although the staining of cytoplasmic granules remains unchanged. Decalcification causes some loss of cellular detail95 and can also affect the staining pattern^98,101. It may also be difficult to obtain good quality, thin (1-2 µm) sections of paraffin-embedded tissue. Alternatively, undecalcified tissue can be embedded in resin and thin (2 µm) sections prepared, to provide improved cellular morphology. One disadvantage of resin sections is the extended staining times required, but these can be significantly reduced when microwave-assisted techniques are used⁹⁷. These also produce more intense staining with improved contrast, however, the characteristic staining pattern is altered, with RNA-rich sites such as nucleoli and 'basophilic' cytoplasm staining purple rather than blue^97,102. A 24 hour protocol for processing and staining (H&E, Giemsa, reticulin) resin sections without microwaves has been described¹⁰³.

Lymphoid tissue can be processed routinely to paraffin wax or resin and thin (1-2 µm) sections prepared. Smears and imprints should be air-dried and fixed in methanol for 1 to 5 minutes before staining, depending upon the technique.

FACTORS AFFECTING STAINING^{89,91,100,104}
The Stain
Stock stains
Stains are available commercially as dry powders or stock solutions. For storage, powders are most stable, and stock solutions in a methanol/glycerol solvent mixture are more stable than those in methanol alone. Different brands and batches can produce variable staining and therefore reagents should be carefully selected and the stain quality continually monitored¹⁰⁵. Stock stains should be stored in tightly capped polyethylene containers, in the dark at 4°C to prolong shelf life, as they are affected by moisture, contact with metals, exposure to light and variation in temperature.

Working solutions
Working stain solutions are prepared by diluting the stock with distilled water or an aqueous buffer. The buffer composition, and the concentration and pH of the working solution (6.0 to 8.5) all affect staining. The colour of erythrocytes, in particular, is altered by changes in the pH (pH 6.0 - red, pH 7.5 - colourless, pH 8.5 - bluish green)92. Working solutions are stable for only a few hours and should be freshly prepared in small volumes. Deterioration of dye solutions is evidenced by loss of red staining due to eosin precipitation¹⁰⁰.

The staining protocol
The staining time must be long enough to achieve differential staining^97,106, but if excessive, then effective differentiation may not be possible⁹⁸. The best differential staining is achieved using a more dilute staining solution with a longer incubation time¹⁰¹. Differentiation is usually performed in two steps: 1) dilute acetic acid to remove excess blue staining and; 2) 95% ethanol to remove excess Eosin. These steps are difficult to standardise and sections must be processed individually under microscopical control.

Jenner Giemsa²⁸ for bone marrow sections
SPECIMEN PREPARATION
Cut 2 µm thick paraffin sections from tissue fixed in Zenker's fluid or 10% neutral buffered formalin. Bone marrow or tonsil are used as control tissue.

REAGENT PREPARATION
1 Jenner stock solution:
Jenner stain powder 1 g
Methanol 400 ml
(A commercial stock solution can be used).
Jenner working solution:
Jenner stock solution 25 ml
Distilled water 25 ml

2 Giemsa stock solution: Giemsa powder 1 g
Glycerol 66 ml
Methanol 66 ml
Mix the powder with the glycerol and place at 60°C for 2 hours. Add the methanol and mix well. (A commercial stock solution can be used).
Giemsa working solution:
Giemsa stock solution 2.5 ml
Distilled water 50 ml

3 1% acetic acid
Glacial acetic acid 0.5 ml
Distilled water 50 ml

METHOD
1 Dewax and rehydrate sections.
2 Remove mercuric pigment with iodine/thiosulphate if necessary.
3 Rinse in distilled water.
4 Rinse in two changes of methanol.
5 Place sections in working Jenner solution for 6 minutes.
6 Transfer to working Giemsa solution for 45 minutes.
7 Rinse rapidly in distilled water.
8 Differentiate in 1% acetic acid (control microscopically).
9 Rinse rapidly in distilled water.
10 Dehydrate rapidly, clear and mount.

TECHNICAL NOTE
May and Grünwald's solution can be used in place of Jenner's but requires a longer staining time (45 minutes).
Lennert's Giemsa⁹⁶ for lymph nodes

SPECIMEN PREPARATION
Cut 2 µm thick paraffin sections from tissue fixed in Zenker's fluid or 10% neutral buffered formalin. Lymph node or tonsil are used as control tissue.

REAGENT PREPARATION
1 Working Giemsa solution
Giemsa stock (Merck) 10 ml
Distilled water 40 ml

2 Acetic acid solution
Add 1-2 drops of glacial acetic acid to 50 ml distilled water.

METHOD
1 Dewax and rehydrate sections.
2 Remove mercuric pigment with iodine/thiosulphate if necessary.
3 Place sections in working Giemsa solution for 1 hour.
4 Rinse briefly in acetic acid solution.
5 Place in 96% ethanol until differentiated (control microscopically.
6 Rinse in two changes of isopropanol each of 2 minutes.
7 Clear and mount.

RESULTS (for both methods)
Nuclear chromatin - dark blue
Cytoplasm of lymphocytes and monocytes - pale blue
Neutrophil granules - purple
Eosinophil granules - pink/orange
Basophil granules - purple/black
Nucleoli - blue
Erythrocytes - pink
Connective tissue - pink to light purple
Mast cell granules - dark purple