112 MICROSCOPIC METHODS 



Although the results obtained by this method are sometimes excellent, 

 they vary considerably. Frequently both the organisms and flagella 

 appear of abnormal thickness. This is due to the fact that the process 

 on which the method depends is a precipitation rather than a true 

 staining. The pictures on the whole are less faithful than in the first 

 method. 



Staining of Spirochsetes in Sections. The following im- 

 pregnation methods have been applied for this purpose by 

 Levaditi, and give excellent results : 



(a) Levaditi's Original Method. 



(1) The tissues, which ought to be in thin slices, about 1 mm. in 

 thickness, are best fixed in 10 per cent, formalin solution for twenty-four 

 hours. 



(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 for 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 spirochsetes 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. 



(b) 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. 



(For the staining of spirochoetes in films, see p. 115.) 



