STAINS AND STAINING 49 



with iron-alum haematoxylin for protoplasmic connections, 24 hours 

 in 4 per cent iron-alum, with 1 or 2 minutes washing in water, 2 days 

 in haematoxylin, 5 minutes washing in water, and 1 minute in 1 per 

 cent iron-alum may be a good schedule to start with. The times must 

 be determined for each case. Many put the slide into iron-alum in the 

 morning and finish the process in the afternoon. These short schedules 

 are not likely to prove satisfactory with mitotic figures. A plan which 

 has proved convenient and very successful is to put the slide into the 

 iron-alum in the morning, wash in water for an hour at some con- 

 venient time in the afternoon, leave it in the | per cent haematoxylin 

 overnight, and finish the preparation the next morning. It is a long 

 process, requiring care, patience, and judgment, but it is worth the 

 effort. 



Chromosomes, centrosomes, and pyrenoids take a brilliant black; 

 or, if the second treatment with iron-alum be more prolonged, a blue 

 black or purple. Achromatic structures stain purple, but the stain can 

 be extracted while it is still bright in the chromosomes. Lignified, 

 suberized, and cutinized structures stain lightly or not at all. Cellu- 

 lose does not stain so deeply as with Delafield's haematoxylin. Arche- 

 sporial cells and early stages in sporogenous tissue stain gray. Many 

 details which are not so brilliantly colored often show good defini- 

 tion. 



If a counter-stain is desired, anything which gives a serviceable 

 contrast may be used. In any case, the haematoxylin stain must be 

 complete and the washing thorough before the second stain is applied. 

 An aqueous stain should be applied just after the final washing in 

 water; an alcoholic stain should be applied during the process of pass- 

 ing the slides through the alcohols, staining in a solution of safranin in 

 50 per cent alcohol from the alcohol of a concentration nearest that of 

 the stain; and staining after the final absolute alcohol, if the stain is 

 dissolved in clove oil. 



A stain of 3 or 4 minutes in safranin adds an excellent differentiation 

 in case of many algae and does not obscure nuclear details. The exine 

 of pollen grains may take a brilliant red with safranin in 5 or 10 min- 

 utes, contrasting sharply with the mouse gray of the intine. Orange G, 

 in clove oil, often gives a pleasing contrast. 



Delafield's haematoxylin. — "To 100 c.c. of a saturated solution of 

 ammonia alum add, drop by drop, a solution of 1 g. of haematoxylin 

 dissolved in 6 c.c. of absolute alcohol. Expose to air and light for one 



