MICROSCOPIC EXAMINATION. 481 



7. Neisser's Stain for Diphtheria Granules. The requirements 

 for good success with the stain, according to Neisser, are as follows: 



The preparation is stained one to three seconds with acetic acid 

 methylene-blue, rinsed off, counter-stained two to five seconds with 

 Bismarck brown. 



1. The cultures must be grown upon Loffler's serum, solidified at 

 100. 



2. The cultures should not be under nine nor over twenty to 

 twenty-four hours old. 



3. The cultures must be kept in an incubator at 34-35, not above 

 36. 



8. Stain for the Organisms of Syphilis. According to Lustgarten 

 the preparations are stained for twenty-four hours in anilin gentian- 

 violet, placed a few seconds in 10 % permanganate of potassium solu- 

 tion, and quickly decolorized in dilute sulphuric acid. The syphilis 

 organisms are dark violet to steel blue; the tissues are decolorized. 



(B) Preparation of Sections. 



1. Universal method according to Loftier, adapted to almost all 

 bacteria: 



The sections are carried upon a German silver or glass spatula from 

 alcohol to Loffler's alkaline methylene-blue solution, where they re- 

 main for five to thirty minutes. They are then placed for a few 

 seconds in 1 % acetic acid, and after differentiation are passed through 

 alcohol and xylol and mounted in Canada balsam. It must be deter- 

 mined how long the acetic acid may be allowed to act, and the dehy- 

 dration in alcohol must not be prolonged any more than is essential. 

 The bacteria should be blackish-blue, the nuclei blue, the protoplasm 

 bluish. 



2. Nicolle states that by the following method he has obtained 

 satisfactory staining in sections in the case of bacteria which stain 

 with difficulty; for example, glanders, typhoid, etc.: 



Joiner's blue one to three minutes; wash in water; 10% solution 

 of tannic acid a few seconds; wash in water; absolute alcohol, oil of 

 cloves, xylol, Canada balsam. 



3. Gram's method: (1) Ehrlich's solution, three minutes; (2) 

 Gram's solution of iodin, two minutes ; (3) alcohol, one-half minute ; 

 (4) alcohol containing 3% hydrochloric acid, ten seconds ; (5) alcohol 

 several minutes until maximum decolorization ; (6) xylol, mount in 

 Canada balsam. 



If it is desirable to have a contrast stain of the tissue, after max- 

 imum decolorization in alcohol, the sections are placed in an aqueous 

 solution of Bismarck brown (10 : 100) for a few minutes, then again 

 for fifteen to twenty seconds in absolute alcohol, then in xylol, and 

 finally mounted in Canada balsam. 



4. Botkin maintains that Gram's stain is facilitated by washing 

 preparations which are stained with anilin gentian-violet in anilin 

 water. The preparations, when removed from the iodin solution, sub- 

 sequently bear the action of alcohol much better. In this way it is 

 possible to stain the Bac. oedematis maligni and Bacterium pneumonias 

 Friedlaiider. 



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