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APPENDIX B. 



A sketch of methods especially adapted to the detec- 

 tion and recognition of bacteria is added. 



The basic aniline pigments Bismarck brown, genti- 

 ana violet, methyl blue and violet, and fuchsin are 

 especially valuable for this purpose, because readily ab- 

 sorbed and retained by bacteria (Weigert). These may 

 be kept as simple filtered solutions (one to two per 

 cent.) in distilled water, though the addition of a little 

 alcohol (ten per cent.) is desirable to secure permanence 

 and to prevent the growth of bacteria in the liquid a 

 possible source of error which must be always borne in 

 mind. 



Sections may be prepared from fresh or from hardened 

 tissues, but must be thin, in order to permit satisfactory 

 inspection of these minute objects ; a microtome is 

 therefore an extremely desirable, indeed almost essential, 

 means for the preparation of such sections. For harden- 

 ing tissues absolute alcohol should be used ; small pieces 

 cubes not more than one-half or three-fourths of an 

 inch in each dimension, for example are sufficiently 

 hard after immersion for one or two days in several (four 

 or five) ounces each of alcohol. 



After immersion for three to ten minutes in the two 

 per cent, aqueous solution of the aniline color, a section 

 exhibits an intense, diffused staining; it is then trans- 

 ferred for a few minutes to alcohol, which abstracts the 

 color from all morphological elements (with certain oc- 

 casional exceptions) except the nuclei of cells, and bac- 

 teria ; it is usually desirable to transfer the sections to a 

 second and even a third dish of alcohol, in order to wash 

 off the coloring matter dissolved out of the section by 

 the alcohol in the first dish. For permanent preserva- 

 tion the sections should be passed through oil of cloves 

 and mounted in Canada balsam after the usual method ; 



