532 METHODS FOR INVERTEBRATES. 



spread themselves out on the agar and many are in states of division. 

 The slide is quickly removed and placed in a larger tube containing 

 wool saturated with 2 per cent, osmic acid. The vapour fixes the 

 organisms and the slide is removed in two to five minutes, and at 

 once carefully placed in 50 per cent, alcohol. It should remain in 

 this for fifteen minutes and is then transferred to 70 per cent., 

 90 per cent, and absolute aicohol fifteen minutes in each. It is then 

 brought down to distilled water by passage through the alcohols, 

 and is stained by Heidenhain's iron hsematoxylin method. The 

 secret of success in this method is to prepare the agar coated slides 

 with as thin a film as possible, otherwise it will peel off in the 

 alcohol. 



Amoebae may be mixed with 1 per cent, serum water, and spread in a 

 thin layer over slides, which are then fixed in Schaudinn's fluid, passed, 

 as before, through the upgraded alcohols and back again to water, 

 and stained with iron alum. Dobell's alcoholic iron hsematin method 

 may be used for staining the free forms of amoebae, but is not so good 

 for the cysts. A modification of Mann's stain by Dobell is also an 

 excellent stain for amosbfe and cysts, and also sections of intestinal 

 ulcers and tissues. The stain consists of Mann's methyl blue-eosin, 

 which is made up in the usual way. Differentiation is carried out in 

 dilute orange G in 70 per cent, alcohol. A simple staining in Lugol's 

 iodine solution is also of use, especiaUy in the routine examination of 

 faeces. 



1001. Coccidia.- -These parasites stain rather badly and conse- 

 quently may be examined by adding a drop or two of 1 per cent, 

 aqueous eosin to the material containing them. The coccidia stand 

 out as unstained bodies on a pink ground. Tissues may be fixed in 

 the following solution :- 



10 per cent, cobalt chloride in distilled water 20 c.c. 

 2 per cent, chromic acid in distilled water . 5 

 Formic acid ...... 1 drop. 



Schaudinn's alcoholic sublimate is also a good fixative. Bertarelli's 

 method consists in fixing in saturated perchloride of mercury and 

 staining in Grenadier's hsematoxylin and differentiating in acetic 

 alcohol. 



B oriel' s method is of considerable utility. 



Tissues are fixed in the following solution for twenty -four hours : 

 Osmic acid . . . . . . . 2 gms. 



Platinum chloride . . . . 2 



Chromic acid . . . . . 3 ,, 



Acetic acid ..... 20 c.c. 



Distilled water ... . 350 



They are then washed in water for an hour or two, and passed through 



