110 MICROSCOPIC METHODS 



(2) They are washed for an hour in water, and then brought into 96 per 

 cent, alcohol for twenty-four hours. 



(3) They are then placed in 1'5 per cent, solution of nitrate of silver in 

 a dark bottle, and are kept in an incubator at 37° C. for three days. 



(4) They are washed in water fof about twenty minutes, and are 

 thereafter placed in the following mixture, namely : — 



Pyrogallic acid, 4 parts. 



Formalin, 5 parts. 



Distilled water up to 100 parts. 



They are kept in this mixture in a dark bottle for forty-eight hours at 

 room temperature. 



(5) They are then washed in water for a few minutes, taken through 

 increasing strengths of alcohol, and embedded in paraffin in the usual 

 way. The sections ought to be as thin as possible. In satisfactory 

 preparations the spirochetes appear of an almost black colour against the 

 pale yellow background of the tissues. The latter can be contrast- 

 stained by weak carbol-fuchsin or by toluidin blue. 



(6) Levaditi's Newer Pyridin Method. 



(1) The tissues are fixed in formalin as in the previous method, are 

 hardened in alcohol for twelve to sixteen hours, and then washed in water. 



(2) They are then impregnated with a 1 per cent, solution of silver 

 nitrate, to which 10 per cent, of pyridin puriss. is added at the time of 

 use. The tissues are placed in the solution in a well-stoppered bottle, and 

 are kept for two to three hours at room temperature and four to six hours 

 at about 50° C. They are thereafter washed quickly in 10 per cent, 

 pyridin solution. 



(3) Reduction is then carried out in the following mixture, namely, a 

 4 per cent, solution of pyrogallic acid to which are added, at the time of 

 use, 10 per cent, pure acetone and 15 per cent, pyridin. 



(4) The tissues are then put through alcohol and xylol, and embedded 

 in paraffin. The sections can be stained with toluidin blue or Unna's 

 polychrome blue. 



Examination of Spirochetes in Films. — The following 

 methods may be recommended : — 



(1) Fontanel's Method. — This is a silver impregnation method, and three 

 solutions are necessary — a fixing fluid, a mordant, and a. silver solution. 

 They are as follows : — 



(a) Acetic acid 1 c.c, formalin 20 c.c, and water 100 c.c. 



(6) 5 per cent, tannic acid in a 1 per cent, watery solution of carbolic 

 acid. 



(c) J per cent, solution of silver nitrate in distilled water. For use a 

 small quantity of this is put in a test-tube, and a minute amount of 

 ammonia solution is added till there is distinct turbidity. (If too much 

 ammonia is added the fluid becomes clear again.) 



Dried films, which should not be fixed by heat, are fixed in solution 

 (a) for about a minute, the fluid being dropped on the film and renewed 

 once or twice. The preparation is then washed thoroughly in running 

 water, solution (ft) is dropped on the film, heated till steam rises, and 

 allowed to remain for about half a minute. It is again washed in water, 



