Dyes are generally defined along the lines of being coloured, aromatic compounds that can ionise. They are thus able to interact with compounds that are oppositely charged. This includes other dyes. In other words, acid dyes can form compounds with basic dyes. Compounds so formed are called neutral dyes. This is not to imply that solutions of these compounds have a pH of 7, merely that both anion and cation are coloured.
Many acid and basic dyes can form neutral dyes, but the commonest are probably those that make up the Romanowsky stains. These are derived from the homologues of methylene blue, the azures, and eosins. Particularly azure A or azure B as the cation, and eosin Y or eosin B as the anion. However, most of the dyes in both groups will form neutral dyes, but with inferior staining characteristics.
Other neutral dyes have been recommended from time to time, such as Bowie's stain for juxtaglomerular granules and Twort's Gram counterstain using neutral red and light green. The Romanowsky type are the only ones to have gained much popularity.
Making a neutral dye
Generally, neutral dyes are insoluble in water. However, one of the difficulties with making them is that they are often soluble in an excess of either of the initial dyes used to make them. Before making a large amount of a particular neutral dye, it is advisable to determine the amount of the acid dye needed to combine with the basic dye. Remember that many dyes are impure, and may contain substantial amounts of extraneous materials. Some of these materials may also be dyestuffs and could form neutral dyes of their own, but much is inert and merely serves to expand the volume.
To determine how much of each dye to use, begin with the procedure below. Keep in mind that it is a starting point, and the ratios may need to be changed, as may the dye concentrations.
Compounding the neutral dye
Using the neutral dye
Neutral dyes are not usually soluble in water, but they are soluble in alcohols. For the Romanowsky type of neutral dye, methanol is the alcohol that has been commonly used. This can also be used for non Romanowsky neutral dyes. Ethanol is also quite suitable. For each new dye the optimum concentration has to be determined empirically. A good starting point is to dissolve 0.1 g of the dye into 100 mL of methanol. Trituration may be necessary.
Once the dye has been dissolved into the methanol, do a Romanowsky type stain of a blood smear to get an indication of the staining characteristics. Based on the results, and on subsequent trials, adjust the dye concentration, the times, the degree of dilution and the pH of the diluent as necessary to obtain the optimal stain. Repeat this using tissue sections. Appendix is a suitable tissue. Remember that dehydration may, or may not, remove the stain, so try blotting to dehydrate and clear. Also, try out one of the more complicated, dehydratable Romanowsky techniques, they may be suitable.