DEMONSTRATION OF SPIRONEMA 613 



cent, potassium carbonate solution.) The preparations may also 

 be stained in the undiluted Giemsa stain for half to six hours. 

 Leishman's solution may also be used for the Giemsa method 

 described under " Malaria." 



Fontana's method is probably the best. Three solutions are 

 required : 



1. Fixing Fluid : 



Acetic Acid, 1 c.c. 

 Formalin, 20 c.c. 

 Distilled water, 100 c.c. 



2. Mordant : 



Tannic Acid, 5 grm. 



Aqueous Carbolic Acid (1 per cent.) 100 c.c. 



3. Silver Solution : 



Prepare a 0-25 per cent, solution of silver nitrate in dis- 

 tilled water and add just sufficient ammonia to it to 

 cause a slight permanent opalescence. 



Prepare films and allow them to dry spontaneously. Fix by 

 flooding with the fixing solution for a few seconds and then pouring 

 it off and repeating the process several times for not less than a 

 minute. Wash well with distilled water. Flood the film with 

 the mordant, heat until it steams and treat for half a minute. 

 Wash thoroughly with distilled water. Flood with the silver 

 solution and warm gently for half a minute. Wash with distilled 

 water, blot and dry. The spirochaetes are stained jet-black. 



Sections may be stained by Levaditi's method : 



(1) Fix pieces of tissue about 1 mm. thick in 10 per cent, 

 formalin for twenty-four hours. 



(2) Wash in water, and harden in 96 per cent, alcohol for 

 twenty-four hours. 



(3) Wash in distilled water for some minutes (until pieces sink). 



(4) Place in 3 per cent, silver nitrate solution at 37 C. for three 

 to five days in the dark. 



(5) Wash in distilled water for some minutes, and then place 

 in the following solution at room temperature for twenty-four to 

 forty-eight hours. 



Pyrogallic acid ...... 2-4 grm. 



Formalin ....... 5 c.c. 



Distilled water 100 c.c. 



