TECHNICAL APPENDIX. 171 



times after cautious warming). By means of a drop 

 of Canada balsam the dry cover-glass is finally fixed 

 to the slide with the bacterial layer downward. 

 2. Gram's Stain. 



(1) Making the smear preparation as above. 



(2) Staining with Ehrlich's solution three minutes. 



(3) Washing off with water. 



(4) Differentiation with iodine-potassium iodide so- 

 lution one minute. 



(5) Decolorizing with absolute alcohol up to color- 

 lessness (usually one to two minutes). 



(6) Drying and mounting. 



For the species which are adapted to Gram's stain, 

 vide the table. In our experience the common 

 opinion that every variety of bacteria may be pre- 

 pared invariably either well or not at all according 

 to this method is erroneous. For example, we ob- 

 served among the fluorescents, which are usually 

 described in literature as unstainable, that three 

 varieties out of twelve stained very beautifully after 

 twenty-four hours' culture. Indeed, according to 

 Zimmermann, all fluorescents may be stained in 

 young cultures. 



In like manner we were able to stain the bacillus 

 of symptomatic anthrax which has often been re- 

 garded as incapable of staining. The contradictory 

 statements may be explained in part by the fact that 

 the material employed has varied greatly in age, and 

 also that the differentiation with alcohol was effected 

 in different ways. But tyrothrix tenuis, which has 

 been regarded as unstainable by Gram's method, was 

 found to stain very well on a subsequent test of the 

 same culture with the same technique. At all events 



