APPENDIX ON PROTOZOOLOGY 749 



Bouin's Fluid, or modifications thereof, is also very useful for wet 

 fixation. Bouin's picro-formol solution consists of : 



Saturated aqueous solution of picric acid ... 30 volumes 



Formalin, 40 per cent. ... ... ... 10 ,, 



Acetic acid, glacial ... ... ... 2 ,, 



The best-known modification is one due to Duboscq and Brasil, 

 and often known as Bouin-Duboscq Fluid. Its formula is as follows : 



Alcohol, 80 per cent. ... ... ... 150 c.c. 



Formalin, 40 per cent. ... ... ... 60 ,, 



Acetic acid, glacial ... ... ... 15 ,, 



Picric acid ... ... ... ... i grm. 



Thorough washing of the smear or cover-slip preparation with 

 70 per cent, alcohol until the yellow colour disappears is necessary 

 to remove excess of fixative. 



Other fixatives, which may be of use, more especially for fixing 

 small pieces of tissue for sectioning, are the solutions of Flemming 

 (chromo-aceto-osmic acids) and of Zenker (sublimate-bichromate- 

 acetic, with sodium sulphate). 



Regarding the time of fixation, there is much difference of opinion. 

 Usually, exposure to or contact with the fixative for five minutes is 

 sufficient in the case of films or smears. Material for sections 

 should be cut into small cubic pieces, of a thickness of about 

 5 mm. (1 in.). One or two hours should be sufficient time for the 

 fixation of such pieces of tissue, though some, as Langeron, prefer 

 a longer time of fixation. On the other hand, Gustav Mann 1 recom- 

 mends a short fixation period. The excess of fixative should be 

 thoroughly washed out of the tissue in the manner appropriate to 

 the particular fixative used. If it is desired to keep the tissue for some 

 time before sectioning and staining, it should be transferred to 70 per 

 cent, alcohol. 



When fluid fixatives are employed, large quantities of the fixing 

 media are necessary. The volume of the fixative should be at least 

 ten to twenty times that of the object, and the latter should be sus- 

 pended in the middle of the fixative. The tissue should be fixed as 

 soon as possible after the death of the host. 



For sectioning tissue parasitized by Protozoa, embedding in 

 paraffin is generally recommended. Microtome sections should not, 

 if possible, exceed 5 //, in thickness. Details of special procedures 

 must be sought in larger works. 



Staining. Here, as with fixatives, much choice is presented. The 

 various modifications of the Romanowsky stain have aided greatly in 



i Physiological Histology," 1902, Clarendon Press, Oxford. 



