Dissolve 1 grm. of hæmatein in 50 cc. of 90 per cent alcohol and warm. Add this solution to a solution of 50 grms. of potassium alum in 1,000 cc. of distilled water dissolved by heating. Mix warm, cool, and filter. With hæmatein no ripening is required, and the solution can be used at once. Hæmalum is a more precise nuclear stain, but stains more slowly than the formulæ given above.
It may be prepared directly from hæmatoxylin crystals by dissolving 1 grm. of hæmatoxylin in boiling water; add water up to 1 litre, and cool. Add 0.2 grm. sodium iodate and 50 grms. potassium alum, dissolving at room temperature; 50 grms. chloral hydrate and 1 grm. citric acid may be added to make solution keep better.
f. Mayer’s Acid-haemalum.
Add 2 cc. glacial acetic acid to 100 cc. of hæmalum solution. It stains the nuclei more precisely than hæmalum.
g. Weigert’s Iron-haematoxylin.
Dissolve 1 grm. of hæmatoxylin in 100 cc. of 96 per cent alcohol. Allow to ripen several days, but this solution should not be kept longer than six months. Make a second solution of 4 cc. of liq. ferri. sesquichlor. (German Pharmak. IV, sp. gr. 1,124), 1 cc. of concentrated hydrochloric acid and 100 cc. of water. Mix equal parts of each solution just before staining. The mixture stains well for 5-8 days, so that a quantity of stain sufficient for this time only should be made up. The two stock solutions are easily made and keep well. The nuclei stain quickly and deeply, and differentiation and long washing are unnecessary when hydrochloric acid is included in the second solution, as given above. After-staining with eosin, picric acid or the Van Gieson’s mixture gives better results with Wiegert’s iron-hæmatoxylin than with any other hæmatoxylin.
Method of Staining with Haematoxylin.
1. Stain 1-15 minutes, controlling progress of stain by examination of section in water, on slide, using low-power.
2. If sections are over-stained, differentiate in ½-1 per cent potassium-alum or in acid alcohol.
3. Wash thoroughly in tap water, until a good blue is obtained. Exposure to ammonia vapor or washing for a few seconds in lithium-carbonate solution will hasten the development of the blue color. If these reagents are used the section should afterwards be thoroughly washed in water.