Ripening Hematoxylin

Hematoxylin itself is not a dye, and it has to be oxidised to hematein, which is a dye, before it can be used. This process is called ripening, and can be accomplished in two distinct ways. Simple alcoholic or aqueous solutions made with hematoxylin are usually pale yellow-brown in colour. On oxidation the colour changes to a deep, mahogany brown. When combined with an aluminum salt such as aluminum potassium sulphate, the colours are pale, transparent violet (unripened) and deep opaque purple (ripened). They may also be combined with iron salts. In these the colour is deeper, usually a very dark violet.

Hematoxylin

Hematein

Natural ripening
It was common practice in the past to use natural oxidation in the belief that it gave a more reliable and longer lasting solution. Natural ripening is accomplished by putting the solution in an oversize flask, so it can be shaken, with the top plugged loosely with cotton wool, allowing air to enter. This is left in a warm, light and airy place (a windowsill) for oxidation to take place. The flask is shaken periodically. Oxidation may take several months, and is determined by testing the solution from time to time. When the solution gives a satisfactory depth of staining, it is transferred to a brown bottle and tightly stoppered for storage in the dark to retard further oxidation.

 

Chemical ripening
The other way to ripen hematoxylin is to use chemical oxidising agents. The most commonly used is sodium iodate, at about 200 mg for each gram of hematoxylin. Others have also been suggested for particular formulas, but sodium iodate can be substituted for just about all of them if used at the stated amount.

Mercuric oxide was often recommended as an oxidant in the past. It is now deprecated. It is very toxic, and should be avoided whenever possible. If it must be used, then full safety precautions should be taken (refer to an MSDS), and the used solution must be disposed of in compliance with government regulations to avoid contamination of the environment.

Boiling a solution of hematoxylin with an oxidant invariably causes rapid oxidation and such solutions may be used as soon as cooled. Boiling is not always necessary as it will take place at room temperature over a few days with most oxidising agents, including sodium iodate. When a solution is needed rapidly, it does enable it to be made available with no negative effects on its staining.

Half oxidation is a term used to describe partial oxidation of hematoxylin to hematein. The rationale is that during natural ripening the solution becomes usable when a significant portion (i.e. half) of the hematoxylin has been oxidised. These solutions have an extended life because they are usable before full conversion takes place. Throughout their life, oxidation of hematoxylin and further oxidation of already converted hematein continues. They do not become unusable until oxidation proceeds so far that insufficient hematein remains to give acceptable staining.

Chemical half oxidation seeks to emulate this process by adding sufficient oxidant to convert only half of the hematoxylin to hematein. Oxidation will then continue as for a naturally ripened solution. In practice, the limiting factor for use of a solution is more likely to be carry over of alkaline tap water into the solution, neutralising the acid, and allowing the lake to precipitate. This is why alum hematoxylin solutions can often be rejuvenated by the addition of a small amount of acetic acid. In routine anatomic pathology staining half oxidation is hardly needed.

 

Oxidant per gram of dye
Chemical Formula Maximum Recommended Source
Sodium iodate NaIO3 200 mg 40-150 mg Lillie, Lynch
Mercuric oxide HgO 500 mg 100 mg Lillie
Potassium permanganate KMnO4 177 mg 175 mg Lillie
Potassium periodate KIO4 50 mg 50 mg Lillie, Lynch
Hydrogen peroxide USP H2O2 2.0 mL   Lynch

 

In addition, calcium hypochlorite (bleach) was used by Anderson, who includes 4 grams to oxidise 2.5 grams hematoxylin in one formula (1.6 grams per gram of hematoxylin), 40 grams to oxidise 5 grams hematoxylin in another (8 grams per gram of hematoxylin), and 5 grams to oxidise 0.5 gram hematoxylin in a third, iron hematoxylin formula (10 grams per gram of hematoxylin). Debiden suggested sodium hypochlorite, using 2 mL of a 5.25% solution of commercial Javex bleach (105 mg chemical) to oxidise 5 grams dye.

Potassium ferricyanide is also included in de Groot's solution but, although it is a mild oxidant, it is not clear whether this is its function since the solution also contains hydrogen peroxide.

Iron hematoxylin soloutions are rarely oxidised directly. Usually the ferric salts present, being oxidising agents themselves, do double duty as both mordant and ripener. Nevertheless, some formulae do call for a ripened alcoholic solution to be used. This is particularly so when the mordant and dye are applied seperately. It is not an uncommon practice when iron hematoxylin solutions are made by combining two stock solutions just before use, to wait a short time before using the resulting solution and allow the hematoxylin to be oxidised to some extent by the ferric salt mordant first.

 

Hematoxylin versus Hematein
The question arises, if hematein is the dye and if hematoxylin needs to be oxidised to hematein before it can stain, why not use hematein in the first place?

The answer lies in the difficulty of obtaining satisfactory hematein. It is quite easy to obtain good hematoxylin, and most companies' products are perfectly OK. Only very rarely is a poor quality hematoxylin provided.

Hematein is another story, however. It is quite common to purchase unsatisfactory dye, even from reputable providers. Many hematein samples contain only small amounts of the dye, the rest being something else. This is most likely the oxidation products of hematein. These are not dyes and do not stain satisfactorily. They do sometimes give objectionable brown or other off-colour tinges to the staining.

The problem is that hematein continues to oxidise to unusable materials. It seems to do this as a powder, and it certainly does it in solution. If we start with hematein, oxidation will start to diminish staining capacity right from the start, shortening the working life of the solution. However, if you get a good sample of hematein, alum-hematein solutions made from it do work well and can be used immediately. The formulations below specify hematein as the dye.

    Baker        Launoy        Mann        Martinotti        Masson        Rawitz   

 

Keep in mind that almost all solutions could be made with hematein, if the oxidant were omitted. Similarly, solutions specifying hematein can be made with hematoxylin if an appropriate amount of oxidant is added.

If naturally ripened alum hematoxylin solutions are wanted, but the time for ripening is too inconvenient, then a strong alcoholic solution might be the answer. Hematoxylin saturates in ethanol at about 3% and Hematein does so at about 7%.

In practice, experiences from very large numbers of histotechnologists have shown that the most convenient and reliable approach is to start with hematoxylin and oxidise it to hematein, either completely or partially. With sodium iodate, this can be so fast and easy that hematein is scarcely needed at all.

 

References
Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.

Lynch M.J. et. al.
Medical Laboratory Technology
W. B. Saunders Company, Toronto, Canada

Horobin R W & Kiernan J A, (2002)
Conn's Biological Stains, 10th ed.
BIOS Scientific Publishers, Oxford, UK

 


 

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