104 STUDY OF BACTERIA 



Pitfield's Method. 



This is the simplest stain and the easiest to use, but does not 

 give the good results that the previous one does. But one solution 

 is needed, this is made in two parts and mixed. 



A. Tannic acid i gram. 



Water 10 c.c. 



B. Saturated watery solution alum (old) 10 c.c. 



Saturated alcoholic solution gentian violet i c.c. 



Mix. 



A heavy precipitate is formed by this process which is useful in the stain- 

 ing. The stain is almost a saturated solution of alum and tannic acid, and 

 when it becomes supersaturated by evaporation and heat, staining takes place. 

 After this the process is very simple. The cover-slip is carefully flooded with 

 the stain and warmed for a minute over the flame of a bunsen burner, turned 

 very low, until steam arises. Not too much stain should be run over the 

 cover-slip. After steaming occurs, the stain should remain for a minute, then 

 the preparation is washed, dried and mounted. It will be found that the best 

 stained flagella are on those bacteria nearest to the edges where the evapora- 

 tion has been most intense. If the preparation is not equally stained, Wei- 

 gert's aniline gentian violet can be run on for a minute to deepen the color. 



Loffler's Method. 



This is the original flagella stain and is a very good one. 

 It is made as follows : 



A. Mordant 



20 percent watery solution tannic acid 10 c.c. 



Sat. solution ferrous sulphate 5 c.c. 



Fuchsin sat. alcoholic solution i c.c. 



Mix. 



B. Stain 

 Carbol-fuchsin. 



Proceed as in the previous methods. 



The most important steps in flagella staining are to clean the 

 cover-slips thoroughly, to mix the culture with water and have no 

 culture media with it, to fix gently, and not to overheat the stain. 

 Even in expert practised hands it is not always easy to demon- 

 strate flagella readily. 



