STAINING, PRACTICAL AND THEORETICAL 



Solutions of haematoxylin must be " ripened " — that is, 

 partially oxidized to produce the dye, haematein — before they 

 are ready for use. This ripening occurs gradually, through 

 spontaneous atmospheric oxidation over a period of weeks and 

 months, after the solutions have been made up and left to stand. 

 However, the solutions may be ripened artificially so that they 

 are ready for immediate use, by adding minute quantities of 

 oxidizing agents such as hydrogen peroxide, mercuric oxide, 

 potassium permanganate or potassium iodate. 



Haematoxylin will not attach itself to tissue-element unless a 

 mordant is used. A mordant is a substance, such as alum, em- 

 ployed to make certain dyes " bite " and hold on to tissue- 

 elements. A mordant acts as the link between the dye with which 

 it unites to form a " lake ", and the tissue-elements with which 

 it also combines. It appears, however, that a conventional 

 mordant is not required when haematoxylin is used with certain 

 acid dyes; e.g. light green. The latter not only stains basic tissue- 

 elements when used in conjunction with haematoxylin, but they 

 also act as mordants between acidic tissue-elements and haema- 

 toxylin (see Rafferty's stain, page 389). 



Haematein is a feeble reddish-yellow dye with a negative 

 charge, which according to Baker (1956, 1958), quoting Seki 

 (1933), has an isoelectric point of about 6'5. When used without 

 a mordant, haematein has no affinity for tissue components and 

 it is quite useless as a stain, although it is used in this form as a 

 hydrogen ion indicator. Therefore, mordanting in one form or 

 another is essential when haematoxylin (haematein) solutions are 

 used for staining. The mordants most commonly used are am- 

 monia alum, potash alum and iron alum (ferric ammonium 

 sulphate). The lakes formed with these and other mordants 

 carry a strong positive charge. Accordingly they behave as 

 strongly basic dyes. In some cases the mordant is incorporated in 

 the stock solution of the haematoxylin: e.g. Delafield, and Ehrlich. 

 In other cases the mordant is kept as a separate stock solution, 

 e.g. Heidenhain, and Weigert. 



Other natural colouring matters of importance are orcein and 

 carmmic acid. 



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