The saturation points of ammonium alum and potassium alum are 142 grams and 139 grams per litre respectively in water. In modern practice alum solutions of about 50 grams to a litre of solvent, mostly water, is fairly typical. In addition, the question of purity is no longer a concern. Examples are aluminum acetate inįrom these examples, it would appear that the source of the aluminum is not that critical in practice. Other aluminum salts have also been specified. Of course, the second metal in the double sulphate should not be capable of acting as a mordant. It was therefore a simple convenience to use them in preference to other sources of aluminum. The usual explanation for the use of alums is that they were available in good purity in the late 1800s when these solutions were being introduced. Ammonium or potassium aluminum sulphates are the most common, although there is no reason at all why sodium aluminum sulphate should not be used. The usual mordant for nuclear staining with hemalum is an alum, or aluminum double sulphate. This can be shown easily enough by adding increasing amounts of a ripened 10% alcoholic hematoxylin solution to 5 mL of a 10% solution of alum, then adjusting the volume to 10 mL with water. In general, however, if the concentration of mordant remains constant, a solution with a lower dye content will likely be more nuclear selective than that with a higher content. Both the amount of mordant present and the pH also have an influence. However, the dye content alone does not determine this characteristic. Most progressive solutions have about 1 gram per litre or a little more, and the regressive solutions have 4 or more grams per litre. Obviously, the actual dye content must have an affect on the staining properties, and it can be observed that the more concentrated the dye content the more likely it is that the solution stains regressively. Mayer's solution, to twenty times that in The amount of hematoxylin in the various solutions varies widely from about 1 gram per litre in A more complete view is that the attachment, at least to DNA, is also by means of covalent and co-ordinate bonding. The simplest view is that it reacts as a cation and attaches to tissue anions, such as phophate groups of DNA and carboxyl groups of proteins. It is the lake which is the staining component of hemalum solutions. However, since a dye is involved, it is more commonly called a lake. In this way the aluminum is firmly attached to the molecule by a process called chelation, therefore the compound is sometimes called a chelate. This page will focus on the relationship between the dye and the mordant, and the affect of adding acid.Ĭo-ordination complexes of this kind are formed with a covalent bond between the aluminum and the oxygen of the hydroxyl group, and a co-ordinate bond between the carbonyl oxygen and the same aluminum atom. The principles involved in the attachment of mordants to some dyes is also the subject of a separate discussion, although the specific application to hematein is mentioned briefly here. Hematoxylin to hematein is the subject of a separate discussion. Additions to the solvent are also common, often to inhibit evaporation or precipitation.ĭue to its importance, the oxidation, or ripening, of.Stabilizers to inhibit further oxidation once the hematein has been formed, since this can shorten staining life.The solution, also affect the selectivity of the staining. Acids to adjust pH, primarily for extending the useful life of the solution but which can, depending on.Oxidizing agents to convert hematoxylin (the dye precursor), to hematein, (the dye).These are not essential but modify the behaviour in some fashion. In addition to these three items, other ingredients may be added. The variations are, at times, almost contradictory and make the underlying principles on which these solutions are based difficult to make clear.Īt its minimum, there are three items needed to produce an effective nuclear staining alum hematoxylin. They vary in the amount of hematoxylin, the amount and type of aluminum salts, solvents, oxidizing agents and stabilisers. This site, for instance, lists more than 50 hemalum solutions. Such solutions usually contain hematoxylin and an alum, and are called hemalum or alum hematoxylin solutions. Probably the single most commonly used dye in histotechnology is hematoxylin or hematein, mostly used to demonstrate nuclear morphology.
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