FILARIA BANCROFTI 395 



Preservation of Living Larvce. Blood from the vein (or finger 

 puncture) is shaken up with twenty times its volume of sterile o'9 per 

 cent, salt solution, and kept in an ice cupboard (Fulleborn). 



Concentration of Larvce. (a) The above mixture is haemolysed with 

 water and then sufficient salt solution added to make up to 0*9 per 

 cent. The solution is allowed to stand or can be centrifugalized. 

 (6) The blood is mixed with sodium citrate and centrifugalized ; the 

 larvae are found in the leucocytic layer (Bahr). (c) Allow blood to 

 clot in a small tube ; the larvae appear on the surface of the clot and 

 are so got in pure serum. A drop of blood may also be allowed to 

 clot on the slide ; the larvae are found in the clear areas of serum. 

 (d) Haemolyse blood with water or acetic acid. Centrifugalize, make 

 smears from, or examine the sediment. 



Removal of Red Corpuscles. The blood film is allowed to stand for 

 some minutes in a moist atmosphere. The staining solution is sucked 

 through with blotting paper : the larvae stick to the slide, while the 

 corpuscles are washed out. 



Morphology of Larvce. Wet staining: Azur II one part, 0-9 per 

 cent., salt solution 3,000, or very dilute Giemsa or ripened methylene 

 blue or neutral red solutions. Place a drop on the slide and add a 

 drop of blood to this. The larvae remain alive for one or more days ; 

 it sometimes takes twenty- four hours to stain some particular structure. 

 Differentiation by drawing through weak eosin solution is often 

 useful. This method is the best for finest details. The excretory 

 pore, anal pore, excretory cell, and chief " genital " cell stain first, 

 then the matrix cells and finally the column of nuclei. 



Wet fixation and staining : The blood is spread on a large cover- 

 glass floated on the surface of 70 per cent, alcohol heated to about 

 70 C. Wash in water, (i) overstain with i in 1,000 azur II solution, 

 warming slightly ; (2) differentiate with (a) absolute alcohol (con- 

 taining, if necessary, a trace of HC1), or (6) with absolute alcohol 

 96 per cent, ninety parts, anilin oil ten parts ; (3) clear in origanum, 

 bergamot or cajeput oil ; (4) mount in balsam. Or stain with haema- 

 toxylin, e.g., Mayer's glycerine alumhaematein, heating till slightly 

 steaming. Differentiate with acid (2 per cent. HC1) alcohol if over- 

 stained. Clear and mount as above. 



Dry fixation and staining: (i) With azur II as above, or (2) with 

 haematein (warm). Examine the dried films in the usual way without 

 a cover-glass. The azur stains the excretory and genital cells clearly. 



Thick films : (i) The blood is smeared out fairly thickly over an 

 area as big as a sixpence. 



(2) Dry quickly to prevent shrinking, using carefully a spirit lamp 

 in a moist climate. 



(3) Place films downwards in water for a few minutes. 



