384 BLOOD AND GLANDS. 



water (not under the tap), dry, and mount in balsam. Erythrocytes 

 red, all nuclei blue, parasites blue, but with unstained nuclei. 



The methods of MAY and GRUNWALD are closely similar to this. 



ASSMANN (Munch, med. Wochenschr., 1906, No. 28 ; " Das 

 eosinsaure Methylenblau," Leipzig, 1908, p. 35) treats fresh films 

 for half a minute to three minutes in a Petri dish with a few drops 

 of Jenner's solution (from Griibler and Hollborn), then pours on 

 20 c.c. of distilled water with 5 drops of JQ per cent, solution of 

 lithium carbonate, leaves for five minutes, rinses in distilled water, 

 dries with blotting paper, and mounts in neutral balsam. 



The foregoing mixtures give a stain seemingly due to the forma- 

 tion of an eosinate of methylen blue in which the nuclei of blood- 

 cells are blue and their plasma red to violet. It was made out by 

 ROMANO WSKY (St. Petersburger med. Wochenschr., 1891) that 

 under certain conditions mixtures of these two dyes give a stain 

 which is in some respects the inverse of this, blood-cells being 

 stained in divers hues, according to their kinds, and any protozoan 

 parasites that may be present showing red nuclei and blue plasma, 

 which greatly facilitates their detection and diagnosis. This 

 reaction appears to be due to the formation of an eosinate not 

 of methylen blue, but of Methylenazur ( 377). The method, 

 only vaguely indicated by Romanowsky, has undergone, at the 

 hands of ZIEMANN, ZETTNOW, NOCHT, REUTER, MICHAELIS, RUQE, 

 MAURER, LEISHMAN, GIEMSA and others, numerous modifications 

 which have culminated in the establishment of a process worked 

 out by GIEMSA as perhaps the most trustworthy and efficient 

 of " Romanowsky " stains. This is as follows : 



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 made from 

 Griibler and Hollborn (their " Giemsa'sche Loesung f iir Romano wsky- 

 faerbung "*). 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) pre- 

 served unmounted. All reagents, especially the balsam, must be 

 strictly free from acid. 



* To make this up from Griibler's powders, dissolve 3 grins, of Azur 

 II-eosin and 8 decigrammes of Azur II in 125 grms. of glycerin and 375 

 of methyl-alcohol. 



