STAINING 209 



are carefully mixed, boiled and filtered clear. The anilin 

 fuchsin must be added slowly with constant stirring and the 

 mixture boiled and filtered. The NaOH is added in the 

 same way and this mixture boiled and filtered. The final 

 mordant should not leave a film on a clean slide when 

 poured on and allowed to run off. Unless the mordant is in 

 this condition and perfectly clear, it should not be used, but 

 a new one must be made up. Time and care in the prepa- 

 ration of the mordant are essential. 



The 2jH of the mordant is most important. An excellent 

 mordant for staining the flagella of Proteus vulgaris has a yYi 

 of 1.03 as determined with a potentiometer. 



The stain to follow this mordant is anilin fuchsin. 



Staining of Metachromatic Granules.— iVm^er'^ Method.— 

 Prepare the film in the usual way. Stain with Neisser's 

 stain a few seconds only. Wash and stain with Bismarck 

 brown a few seconds only. 



Neisseria Stain: 



Saturated alcoholic solution of methylene blue . . 1.0 part 



Glacial acetic acid 2.5 parts 



Distilled water 50 . parts 



Bismarck Brown: 



Bismarck brown (dry dye) 2 parts 



Distilled water 1000 parts 



By the use of the hanging drop slide and the methods of 

 staining just described all the various morphological feat- 

 ures of the bacterial cell may be ascertained. 



It is necessary when cell groupings as characteristic of 

 definite modes of division are to be determined to make 

 slides from a liquid culture as broth. Place a drop of the 

 material, preferably from the bottom of the tube in most 

 instances, from the top in case a pellicle or scum is formed 

 on the surface, on the slide and allow this to dry without 

 spreading it out, fix, wash gently with water, then stain 

 lightly with Lofiler's blue. Such slides also show charac- 

 teristic cell forms as well. Slides should be made from solid 

 media to show variations in form and size and involution 

 forms. These latter are especially apt to occur on potato 

 media. 

 14 



