284 METHODS AND FORMULAS DS 11. 113-DS 11.12 



11.113 Schultze 1904 23632, 21 :5 



HEAGENTs REQUIRED: A. F 1600.0010 Flemming 1882; B. sat. sol. hematoxylin in 70% 



ale; C. 1% potassium dichromate in 50% ale; D. bergamot oil 

 method: [sections of material fixed, or mordanted, in /I]— » 50% ale. -^ B, 24 hrs. — > 



wash — > C, 1 hr. —♦95% ale. -^ D, till differentiated — > balsam 



11.113 Welters 1890 see DS 11.113 Kulschitsky 1889 (note) 



23632,7:466 



11.12 DIRECT HEMATOXYLIN STAINING 



Hematoxylin cannot be used in direct staining unless some mordant is incorporated with 

 the solution for the purpose of fixing the stain on the material to be colored. The term direct 

 staining is used in this case in contrast to mordant staining and must not be confused with 

 the term direct staining as opposed to indirect staining. The former indicates merely that the 

 object is stained in a relatively strong solution for a length of time sufficient to impregnate 

 the whole and that it is subsequently exposed either to an acid solution or to a solution of the 

 mordant with a view to extracting it from those objects which it is desired to bring into con- 

 trast. The term indirect staining, as used in this same sense, indicates the employment of a 

 very weak solution in order to permit a differential absorption of the stain by those parts 

 of the object to be stained (usually the more dense) which it is intended to bring out. As a 

 generality it may be said that direct staining, in this sense, is usually applied to sections 

 while indirect staining is better for the preparation of wholemounts, provided that one has 

 the leisure to wait for the somewhat lengthy process to finish. 



The direct-staining formulas are divided into four classes according to the mordant which 

 is incorporated. The first group, incorporating iron mordants, is used almost exclusively for 

 staining the central nervous system in sections; their use and variations are more fully 

 described in Chapter 21. It is doubtful that these stains could ever be employed for indirect 

 staining of wholemounts, but for staining nuclei they are far better than the other three 

 classes, though less widely employed. 



The next two divisions include these formulas containing alum mordants and acid-alum 

 mordants, the separation of these two being necessitated by the large number of formulas 

 to be found in each. Both are employed for sections and for wholemounts, the best known 

 being unquestionably the formula of Delafield (1885). This reagent has the advantage of 

 being almost foolproof, but it has to be ripened for a considerable period before it can be 

 employed: Watson 1945 (11360, 63:21) recommends barium peroxide for ripening these 

 solutions. The formula of Carazzi 1911 is almost identical but may be used as soon as it is 

 prepared. It is strongly recommended to the attention of those whose staining has previously 

 been confined to Delafield. The formula of Mayer 1896 was once very widely employed for 

 staining wholemounts, but it has nowadays fallen somewhat into disuse. It also required 

 ripening for a considerable period before employment. 



The formulas incorporating an acid, usually acetic, in addition to the alum mordant are 

 among the best of the general-purpose stains. The formula of Ehrlich 1886 is the most widely 

 known, though any of the others can be recommended. 



The alum-mordant formulas are the only ones which can be employed in great dilution for 

 indirect staining. It is usually a waste of time to employ acid-alum formulas for this purpose. 

 The diluent to be employed should have the same composition as the formula itself, without 

 the inclusion of hematoxylin. It is a mistake to follow the very wide recommendation that 

 0.1% hydrochloric acid be employed. This reagent is difficult to remove from the object 

 before its final mounting and leads ultimately to the breakdown of the color. 



In all cases hematoxylin stains should be "blued" after they have been differentiated, in 

 some alkaline solution, preferably containing free ions of an alkali metal. Lithium carbonate 

 is widely used, though a weak solution of calcium chloride, adjusted with ammonium hydrox- 

 ide to a pH of about 8, is more satisfactory. The old exhortation to use tap water originated 

 in Europe where most of the tap waters are alkaline. The majority of city tap waters in the 

 United States are worthless for this purpose. 



The chrome-hematoxylins and copper-hematoxylins, which form the fourth class, are of 

 comparatively recent introduction or, at least, of comparatively recent acceptance. The 

 formulas of Hanson 1905 and of Liengme 1930 are, however, excellent reagents and should 

 be tried for sections in those instances in which the more customary formulas do not yield 



