TECHNICAL APPENDIX. 173 



per cent soda lye is added drop by drop until the 

 clear fluid just begins to grow opaque) and heating 

 for one minute until steam is evolved. 



(6) Washing off in water, drying, mounting in 

 Canada balsam. 



The manipulations must be carried out with the 

 most scrupulous cleanliness, and the cover-glasses 

 must be especially well cleaned with acids and alco- 

 hol. The cultures must be young, although it is not 

 necessary, as some authors maintain, to make the 

 staining only in cultures that are twenty-four hours 

 old. We have often obtained very good preparations 

 even at the end of twelve days. The mordants are 

 usually prepared fresh. 



According to Loffler, it is necessary, in the case of 

 the majority of bacteria, to add a definite amount of 

 acid or alkali to the mordant in order to obtain well- 

 stained flagella. Loffler advises that to 16 c.c. of the 

 mordant there be added for : 



Drops. Soda lye. 



Cholera vibrios i to 1 1 per cent 



Spirillum rubrurn 9 1 " 



Bacterium typhi 20 to 22 1 



Bacillus subtilis 28 to 38 1 



Bacillus oadematis maligni. .. 36 to 37 1 " 



Bacterium pyocyaneum 5 to 6 Equivalent sul- 

 phuric acid. 



Oar results show that in the majority of cases we 

 obtain very useful pictures with the unchanged mor- 

 dant and that the addition of alkali or acid is by no 

 means material. Similar experiences have been had 

 by other writers, for example, Lucksch, Giinther, A. 

 Fischer, Nicolle and Morax, but our investigations 

 have not been concluded. 



