390 Journal of Applied Microscopy. 



Delajield's HcBtnatoxylin. — "To 100 cc. of a saturated solution of ammonia 

 alum add, drop by drop, a solution of 1 g. of haematoxylin dissolved in G cc. of 

 absolute alcohol. Expose to air and light for one week. Filter. Add 25 cc. of 

 glycerine and 25 cc. of methyl alcohol. Allow to stand until the color is suffi- 

 ciently dark. Filter, and keep in a tightly stoppered bottle " {Stirling and Lee). 



The solution should stand for at least two months before it is ready for using. 

 This " ripening " is brought about by the oxidation of haematoxylin into haematin, 

 a reaction which may be secured in a few minutes by a judicious application of 

 peroxide of hydrogen. 



Transfer to the stain from 35 per cent, alcohol or from water. The length 

 of time required is exceedingly variable. Sometimes sections will stain deeply 

 in three minutes, but it is often necessary to stain for thirty minutes. The length 

 of time required will be fairly uniform for all material taken from the same 

 bottle. This fact indicates that the washing process, which follows killing and 

 fixing, is an important factor ; if the washing has been thorough, the material 

 will stain readily, but if the washing has been insufficient, the material may stain 

 slowly or not at all. The washing is particularly important when the fixing 

 agent contains an acid. Transfer from the stain to water. Distilled water is 

 neither necessary nor desirable. Precipitates are often formed when slides are 

 transferred directly to alcohol from this stain ; otherwise, it would be better to 

 transfer from the stain to 35 per cent, alcohol. Pass through the alcohols to 70 per 

 cent, alcohol and then give the slide a few dips (two seconds is often sufficient) in 

 acid alcohol, (1 cc. HCl. to 100 cc. of 70 per cent, alcohol.) This extracts the 

 stain more rapidly from other parts than from the nuclei and hence gives a good 

 nuclear stain. Some prefer to stain for a very short time and use no acid 

 alcohol, but, as a rule, it seems best to overstain and then differentiate in this 

 way. Transfer from acid alcohol to 70 per cent, alcohol and leave here until a 

 rich purple color replaces the red due to the acid. Since small quantities of the 

 acid alcohol are carried over into the 70 per cent, alcohol, it is well to add a 

 drop of ammonia now and then to neutralize the effect of the acid. Too much 

 ammonia is to be avoided, for it gives a disagreeable bluish color with poor 

 differentiation, probably on account of the precipitation of alumina. The slide 

 may now be passed through the alcohols, cleared in xylol, and mounted in 

 balsam ; or, if a double stain be preferred, treat for thirty seconds to one minute 

 with eosin, erythrosin, or some other stain affording a good contrast ; rinse in 70 

 per cent, alcohol and proceed as usual. 



Delafield's haematoxylin is the most generally useful stain in the haematoxylin 

 group. It brings out cellulose walls very sharply, and consequently is a good 

 stain for embryos and the fundamental tissue system in general. With safranin 

 it forms a good combination for the vascular system, the safranin giving the 

 lignified elements a bright red color, while the haematoxylin stains the cellulose a 

 rich purple. It is a good stain for chromatin and the achromatic structures show 

 up fairly well, but can be brought out much better by special methods. Arche- 

 sporial cells and sporogenous tissue are very well defined if proper care be taken. 

 Whenever you are in doubt as to the selection of a stain for general purposes, 

 we should advise the use of Delafield's haematoxylin. 



