400 BLOOD AND GLANDS 



an eosinate — not of methylen blue, but^ — of Methylenazur. The 

 method, only vaguely indicated by Romanowsky, has under- 

 gone, at the hands of Ziemann, Zettnow, Nocht, Reuter, 

 MicHAELis, RuGE, Maurer, Leishman, Giemsa and others, 

 numerous modifieations which have culminated in the establish- 

 ment of a process worked out by Giemsa as perhaps the most 

 trustworthy and efficient of " Romanowsky " stains. This is 

 as follows : 



875. Giemsa's Azur-eosin Process. You start with a mixture 

 of eosin with methylenazur (instead of methylen blue). This 

 mixture is very troublesome to prepare, and is best obtained 

 ready from Griibler and Hollborn (their " Giemsa'sche Loesung 

 fiir Romano wskyfaerbung." * Air-dried films {Deutsch. med. 

 Wochenschr., 1907, No. 17) are fixed in alcohol or in methyl 

 alcohol (two to three minutes), and dried with blotting paper. 

 They are treated for ten to fifteen minutes with a dilution of 

 1 drop of the stock mixture to 1 c.c. of water, washed under a tap, 

 dried with blotting paper, and again dried in the air and mounted 

 in balsam, or (preferably) preserved unmounted. All reagents, 

 especially the balsam, must be strictly /r^e/rom acid. 



876. Wet films {ibid., 1909, p. 1751) are treated as follows : 

 Fix them for twelve to twenty-four hours in a mixture of 2 parts 

 saturated aqueous solution of sublimate with 1 of absolute alcohol. 

 Wash and treat for five to ten minutes with a mixture of 2 parts 

 of iodide of potassium, 100 of water and 3 of Lugol's solution. 

 Wash and treat for ten minutes with 0-5 per cent, solution of 

 sodium thiosulphate. Wash, and stain as above (changing the 

 stain for fresh after half an hour) for one to twelve hours. Then 

 pass through mixtures of acetone with first 5, then 30, then 50 

 parts per cent, of xylol into pure xylol, and mount in cedar oil. 

 This process is applicable to sections. 



Or {ibid., 1910, p. 2476) a slide is placed in a Petri dish and 

 covered with a mixture of equal parts of methyl alcohol and stock 

 mixture. After half a minute this is poured off and enough 

 distilled water poured in to cover the slide, and the whole is rocked 

 to mix the two. After three to five minutes, wash in running 

 water, dry, and mount in cedar oil. 



By any of these processes nuclei (red) are demonstrated not 

 only in hsematozoa, but in many bacteria, spirochsetae, coccidia, 

 sarcosporidia, etc. 



877. Giemsa's Method for Sections. Paraffin Sections. Next 

 to haematoxylin and eosin, Giemsa's stain is perhaps the most 

 useful for biologists and pathologists alike. The former, however, 

 are slow to appreciate its value. Not only is it helpful for blood 



* To make this up from Grubler's powders, dissolve 3 grm. of 

 Azur Il-eosin and 8 decigrammes of Azur II. in 125 c.c. of glycerin 

 and 375 c.c. of methyl-alcohol. 



