544 METHODS FOE INVERTEBRATES. 



(6) Wash thoroughly in several changes of 70 per cent, alcohol to 

 remove mordant. 



(7) Dehydrate and mount in Canada balsam. 



1024. DOBELL'S Haematein Method (see Arch. f. Protistinkunde, 1914, 

 p. 144.) Films or sections are transferred from 70 per cent, alcohol 

 into 1 per cent, solution of iron alum in 70 per cent, alcohol. This is 

 most easily made in the way described by HICKSON (Quart. Journ. 

 Micr. Sci. 44, 1901, p. 470), 1 grm. of salt dissolved in 23 c.c. of warm 

 water, then add 77 c.c. of 90 per cent, alcohol after cooling. They are 

 mordanted in this for ten minutes, rinsed in 70 per cent, alcohol and 

 transferred to a 1 per cent, solution of haematein in 70 per cent, alcohol. 

 They are left in this for ten minutes and then differentiated, either in 

 the iron alum or in acidified 70 per cent, alcohol (0-6 per cent. HC1 in 

 70 per cent, alcohol). After differentiation wash in several changes of 

 70 per cent, alcohol and then pass up through the alcohols into any of 

 the usual mounting media. Any alcoholic counterstain can be used, 

 light green in 90 per cent, alcohol being very good. By this method the 

 organism is stained a purple grey, and hard black and white contrasts 

 are not obtained. The various nuclear and cytoplasmic constituents 

 may be stained with individual intensity. 



(The haematein method is good, when it works, but often it is a 

 complete failure ; certain amoebae, for instance, are not stained at all 

 by it.) 



1025. DOBELL'S Modification of Mann's Methyl Blue, Eosin Stain. 



(1) Stain with Mann's methyl blue eosin mixture (see Lancet, p. 196) 

 in distilled water till everything is overstained (two to eight hours). 



(2) Einse in distilled water and differentiate in 70 per cent, alcohol 

 dried from water, containing a small quantity of Orange G just 

 enough to colour it. It is best to keep Orange G in concentrated solu- 

 tion in 90 per cent, alcohol ; add sufficient of this with a glass rod to 

 clear 70 per cent, before differentiating. This weak Orange G cannot 

 be used often as it gets discoloured very quickly. 



(3) Dehydrate in absolute alcohol (not too long) and transfer to 

 xylol (also not too long, as eosin comes out in time if left). 



(4) Mount in balsam. 



By this method permanent and pretty results are obtained, often 

 quite as good or better than those got with Giemsa. The method is 

 especially good after Bonin fixation, but it is important that all the 

 picric acid is removed before staining. 



1026. Giemsa Stain (MINCHIN'S Method, Q. J. M. S., Ix, 1915, 

 p. 510). Slides are washed in tap water and put in dilute Lugol 

 solution (1 c.c. Lugol to 25 c.c. distilled H 2 0) for ten minutes. 

 After this, rinsed in tap water and put into a -5 per cent, watery 

 solution of hyposulphite of soda for ten minutes. Next wash in 

 a current of water for five minutes and then put into the stain. 

 The distilled water used to dilute the Giemsa stain has to be 

 neutralised in the way prescribed by Giemsa. 



