208 STUDY OF INDIVIDUAL BACTERIA-STAINING 



stain which are kept free from precipitate, (4) gentle heating. 

 The author's students are furnished only stock materials and 

 make their own cultures, mordants and stains. 



The slides are cleaned with pumice in the usual way. An 

 agar slope culture of the organism to be stained from six to 

 twenty-four hours old is selected. A bit of the culture is 

 removed and placed in a watch-glass of water. The bac- 

 teria are allowed to diffuse of themselves without stirring. 

 After several minutes a loop of this water is removed and 

 three streaks are made across the slide, one in the middle 

 and one on each side of this about one-quarter of an inch 

 from it. This gives well scattered bacteria in one of the 

 three streaks at least and very little other material on the 

 slide to cause precipitates. The slide is carefully dried and 

 fixed and then covered with an abundance of the mordant 

 by filtering through a small filter onto the slide so that the 

 mordant shows transparent on the slide. The preparation 

 is then gently warmed and cooled three times, adding mor- 

 dant if necessary. Do not heat to steaming. After mordant- 

 ing for about five minutes the excess is washed off under the 

 tap. It is a good plan to hold the slide level and allow the 

 water to run into the center of the mordant and flow it off. 

 Inclining the slide is apt to cause the film on the surface of 

 the mordant to settle down on the slide and spoil the prepa- 

 ration. After the mordant is washed off and all traces of it 

 removed with a clean cloth if necessary the stain is applied 

 and gently heated and cooled the same way for from three 

 to five minutes. The preparation is then washed, dried and 

 examined. 



The mordant used is a modification of Lofller's, which is 

 somewhat simpler in preparation, since the stock solution of 

 FeCls is more permanent than FeS04 solution. 



Mordant sufficient for one student: 



5 per cent solution of FeCls 20 . cc 



25 per cent solution of tannic acid 20.0 cc 



Anilin f uchsin 4 . cc 



Normal NaOH 1.5 cc 



The solution of FeCls is made up in the cold and must 

 be perfectly clear. The tannic acid solution must be thor- 

 oughly boiled and filtered until clear. The iron and the acid 



