STAINING METHODS 27 



3. Wash with a gentle stream of distilled water, and follow steps 4-6 of 

 above schedule (Casares-Gil method). 



Results: Same as with Casares-Gil method. 



Leif son's Stain^ 

 Leifson (1930) 



KA1(S04)2-12H20, or NH4A1(S04)2-12H20 (sat aqu solution) 20 ml 



Tannic acid (20 % aqu solution) 10 ml 



Distilled water 10 ml 



Ethyl alcohol, 95 % 15 ml 



Basic fuchsin (sat solution in 95 % ethyl alcohol) 3 ml 



Mix ingredients in order named. Keep in tightly stoppered bottle, and the stain 

 may be good for a week. 



Staining schedule: 



1. Prepare slides as for the preceding methods. 



2. Flood sHdes with the above solution, and allow to stand 10 min at room 

 temperature in warm weather or in an incubator in cold weather. 



3. Wash with tap water. (If a counterstain is desired, borax methylene 

 blue may be applied, without heat, followed by another washing. See 

 page 29.) 



4. Dry and examine. 



Results: When no counterstain is used, same as with the two above 

 procedures; with methylene blue counterstain, flagella red, cells blue. 



Bailey Method 



Bailey {1929) 



Modified by Fisher and Conn (1942) 



This method is specially recommended for bacteria on which flagella 

 are difficult to stain (as is frequently the case with soil and Vv^ater non- 

 sporeformers and with plant pathogens) because of slime production, 

 unusually fine flagella or flagella that are readily lost. 



Mordant: Solution A 



Tannic acid (10 % aqu solution) 18 ml 



FeCls-eHsO (6 % aqu solution) 6 ml 



Solution B 



Solution A 3.5 ml 



Basic fuchsin; (0.5 % in ethyl alcohol) 0.5 ml 



HCl, concentrated 0.5 ml 



Formalin 2.0 ml 



Staining schedule: 



1. Prepare smears of young cultures, following carefully the procedure 

 recommended on page 25 under ''Methods of handling cultures." 



2. Filter the above solution A onto the slide and allow it to remain 

 33^ min without heating. 



^This stain, already mixed, is now available commercial!}' in powder form. 



